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This study aimed to determine the effect of sulfated xylans on the growth of probiotics. Xylan (Xyl) was chemically modified using the sulfamic acid-N,N-dimethylformamide method to produce sulfated xylan (SXY). The SXY was characterized by Fourier infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and Congo red experiment. The Xyl and SXY were tested for their in vitro prebiotic effects using four strains of intestinal microflora, including Lactobacillus delbrueckii subsp. bulgaricus (GIM1.155), L. plantarum (GIM1.191), L. brevis (GIM1.773), and Streptococcus thermophilus (GIM1.540). From the recorded infrared spectra, vibrational bands for SXY were observed at 894, 1052 and 1243 cm−1, they could be assigned to the C—O—S vibration of a C—O—SO3, C—O stretched and S＝O stretching vibrations. The results of scanning electron microscopy showed that the surface structure of SXY increased smoothness. Congo red experiment revealed that SXY had a triple helix structure. The substitution degree of SXY measured using a BaCl2-gelatin turbidimeter was 0.341. In vitro experiment showed that the optimum concentration of SXY for the in vitro probiotic growth was 2.0%, rapid growth and increased proliferation were observed after 10 h of culture. The results obtained suggest that SXY could promote probiotic growth. It is an alternate source of prebiotics for maintaining gut health.

Periodontitis is a gingiva disease sustained by microbially associated and host-mediated inflammation that results in the loss of the connective periodontal tissues, including periodontal ligament and alveolar bone. Symptoms include swollen gingiva, tooth loss and, ultimately, ineffective mastication. Clinicians utilize regenerative techniques to rebuild and recover damaged periodontal tissues, especially in advanced periodontitis. Human periodontal ligament stem cells (hPDLSCs) are considered an appealing source of stem cells for regenerative therapy in periodontium. hPDLSCs manifest the main properties of mesenchymal stem cells, including the ability to self-renew and to differentiate in mesodermal cells. Significant progress has been made for clinical application of hPDLSCs; nevertheless, some problems remain, including the small number of cells isolated from each sample. In recent decades, hPDLSC ex vivo expansion and differentiation have been improved by modifying cell culture conditions, especially with the supplementation of cytokines' or growth factors' mix, chemicals, and natural compounds, or by using the decellularized extracellular matrix. Here, we analyzed the changes in stemness properties and differentiation potential of hPDLSCs when culturing in alternative media. In addition, we focused on the possibility of replacing FBS with human emoderivates to minimize the risks of xenoimmunization or zoonotic transmission when cells are expanded for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Abstract Background Speckle-type POZ protein(SPOP), a substrate adaptor of Cul3 ubiquitin ligase, plays crucial roles in solid neoplasms by promoting the ubiquitination and degradation of substrates. Limited studies have shown that SPOP is overexpressed in human renal cell carcinoma (RCC) tissue. However, the exact role of SPOP in RCC remains unclear and needs to be further elucidated. The present study showed that SPOP was expressed at different levels in different RCC cell lines. The purpose of this study was to explore the roles of SPOP in the biological features of RCC cells and the expression levels of SPOP in human tissue microarray (TMA) and kidney tissues. Methods Here, SPOP was overexpressed by lentiviral vector transfection in ACHN and Caki-1 cells, and SPOP was knocked down in Caki-2 cells with similar transfection methods. The transfection efficiency was evaluated by quantitative PCR and western blotting analyses. The role of SPOP in the proliferation, migration, invasion and apoptosis of cell lines was determined by the MTT, wound-healing, transwell and flow cytometry assays. Moreover, the cells were treated with different drug concentrations in proliferation and apoptosis assays to investigate the effect of sunitinib and IFN-α2b on the proliferation and apoptosis of SPOP-overexpressing cells and SPOP-knockdown RCC cells. Finally, immunohistochemical staining of SPOP was performed in kidney tissues and TMAs, which included RCC tissues and corresponding adjacent normal tissues. Results Overexpression of SPOP inhibited cell proliferation, migration and invasion and increased cell apoptosis. Interestingly, sunitinib and IFN-α2b at several concentrations increased the proliferation inhibitory rate and total apoptosis rate of cells overexpressing SPOP. The findings of the present study showed that the SPOP protein was significantly expressed at low levels in most clear cell RCC (ccRCC) tissues and at relatively high levels in the majority of adjacent normal tissues and kidney tissues. Kaplan–Meier survival analysis showed that there was no statistically significant difference in cumulative survival based on the data of different SPOP expression levels in TMA and patients. Conclusions In contrast to previous studies, our findings demonstrated that overexpression of SPOP might suppress the progression of RCC cells, which was supported by cell experiments and immunohistochemical staining. SPOP could be a potential tumour inhibitor in RCC.

ABSTRACT Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3′-untranslated region (3′UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3′UTRs. Compared with rMM 1775, rMM-CQ3′UTR (recombinant MM 1775 virus carrying the 3′UTR of CQW1) exhibited enhanced proliferation in vitro, with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3′UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3′UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3′UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3′UTRSLI displayed a 15- or 4-fold increase in vitro, and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3′UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3′UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3′UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3′-untranslated region (3′UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3′UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3′UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3′UTR in viral host switching and pathogenicity changes in mice.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica. Key message: An efficient regeneration method has been developed in T. indica. Histology and SEM indicated somatic embryogenesis based morphogenesis. 2C genome size and SCoT marker revealed genetic homogeneity of the regenerated population. [ABSTRACT FROM AUTHOR]

[Display omitted] • Perkinsus spp. infects mollusks from South America without causing mortality. • P. olseni was detected in first in Uruguay, then Brazil and Argentina. • P. qugwadi-like was only detected in Argentina. • P. marinus, P. beihaiensis and P. chesapeaki was only detected in Brazil. • Epizootiology, phylogeny, in vitro proliferation and immune responses were studied. Marine mollusk production is increasing worldwide, and this trend is being evidenced in South American countries, where several species of bivalves are produced, exploited, and traded. This activity brings benefits either for the ecosystem, as it is a less impactful and polluting than other aquaculture practices, and to coastal human communities, as it provides food and income. However, emergence of outbreaks by pathogens is a major concern and can put an entire developing sector at risk. Perkinsosis is a disease caused by Perkinsus spp. protozoans that affect mollusks worldwide. In this review we provide information on Perkinsus spp. among bivalves from South America. Infections by these parasites were only reported to date among coastal Atlantic bivalves of Argentina, Uruguay, and Brazil. The vast majority of cases and studies are reported from Brazil. We comprehensively review those results here. Finally, we suggest some considerations for future investigations that may expand our knowledge of these parasites. [ABSTRACT FROM AUTHOR]

Periodontitis is a gingiva disease sustained by microbially associated and host-mediated inflammation that results in the loss of the connective periodontal tissues, including periodontal ligament and alveolar bone. Symptoms include swollen gingiva, tooth loss and, ultimately, ineffective mastication. Clinicians utilize regenerative techniques to rebuild and recover damaged periodontal tissues, especially in advanced periodontitis. Human periodontal ligament stem cells (hPDLSCs) are considered an appealing source of stem cells for regenerative therapy in periodontium. hPDLSCs manifest the main properties of mesenchymal stem cells, including the ability to self-renew and to differentiate in mesodermal cells. Significant progress has been made for clinical application of hPDLSCs; nevertheless, some problems remain, including the small number of cells isolated from each sample. In recent decades, hPDLSC ex vivo expansion and differentiation have been improved by modifying cell culture conditions, especially with the supplementation of cytokines’ or growth factors’ mix, chemicals, and natural compounds, or by using the decellularized extracellular matrix. Here, we analyzed the changes in stemness properties and differentiation potential of hPDLSCs when culturing in alternative media. In addition, we focused on the possibility of replacing FBS with human emoderivates to minimize the risks of xenoimmunization or zoonotic transmission when cells are expanded for therapeutic purposes.

In this study, the effects of different hormones and their doses on the micropropagation and rooting of two strawberry cultivar candidates (291 and 299) selected from last step of breeding program carried out by Horticulture Department of Cukurova University (Adana, Turkey) and their parents (Rubygem and Kaşka cultivars developed by Horticulture Department of Cukurova University) were investigated using shoot-tip culture. Nodal segments of strawberry runners were sterilized through keeping in 7% sodium hypochlorite solution containing 1–2 drops of Tween-20 for 10 minutes. Five different Murashige ve Skoog (MS) nutrient media combinations containing 6-Benzylaminopurine (BAP) and Thidiazuron (TDZ) at the ratios 0.75 and 1.50 mg L–1 were tested (Medium 1: MS + 30 g L–1 sucrose, 7 g L–1 agar, Medium 2: MS + 30 g L–1 sucrose, 7 g L–1 agar + 0.75 mg L–1 BAP, Medium 3: MS + 30 g L–1 sucrose, 7 g L–1 agar + 1.50 mg L–1 BAP, Medium 4: MS + 30 g L–1 sucrose, 7 g L–1 agar + 0.75 mg L–1 TDZ, Medium 5: MS + 30 g L–1 sucrose, 7 g L–1 agar + 1.50 mg L–1 TDZ). During the study, darkening, infection and rooting rates (%), shoot number per plant in both first and second sub-culture, root number per plant and root length (cm) parameters were examined. Use of four genotypes/cultivars and five nutrient media, this study was planned according to the factorial design of randomized plots with three replications. JMP package program and LSD test were used for statistical evaluation of data obtained. It was found that the media including TDZ was generally more effective than BAP in terms of shoot proliferation in both first and second sub-culture. The number of shoots per explant varied from 0 to 4.18 shoots per explant in the first sub-culture. Regarding to mean of genotypes and media, Kaşka cultivar (2.53 shoots per explant) and medium 5 (2.88 shoots per explant) gave the best results, respectively. In the second sub-culture, the number of shoots per explant changed between 0.33 and 4.83 shoots per explant. According to mean of genotypes and media, Kaşka cultivar (3.23 shoots per explant) and medium 4 (3.63 shoots per explant) were found to be the best material and medium, respectively. Regarding to rooting parameters, it was determined that the hormone-free MS medium was highly efficient. The number of root per in vitro plant varied from 0 to 30 roots per in vitro plant. According to mean of genotypes and media, Rubygem cultivar (9.60 roots per in vitro plant) and medium 1 (22.30 roots per in vitro plant) gave the best results, respectively. Root lenght changed between 0 and 5.04 cm. Regarding to mean of genotypes and media, the best values were obtained from Kaşka cultivar (2.10 cm) and medium 1 (3.89 cm), respectively. The value of 0 obtained from rooting parameters refers to in vitro plants that can not be rooted. Infection rate was low in both sub-cultures. Especially in the first sub-culture, it was observed only in the genotype 299 cultured in the medium 3. In the second subculture, infection was detected at low rates in the genotypes 291 and 299 and Rubygem cultivar cultured media 2, 3 and 5. No darkening was observed in the explants cultured in both sub-cultures. Based on genotypes, Kaşka cultivar developed in the Horticulture Department of Cukurova University (Turkey) was found to be superior in terms of both shooting and rooting criteria. Achieving successful results of this cultivar will speed up its use in breeding studies. Based on the study result and literature, it was concluded that the selection of the appropriate hormone type and dose are genotype-dependent.

In this study, the effects of different hormones and their doses on the micropropagation and rooting of two strawberry cultivar candidates (291 and 299) selected from last step of breeding program carried out by Horticulture Department of Cukurova University (Adana, Turkey) and their parents (Rubygem and Kaşka cultivars developed by Horticulture Department of Cukurova University) were investigated using shoot-tip culture. Nodal segments of strawberry runners were sterilized through keeping in 7% sodium hypochlorite solution containing 1-2 drops of Tween-20 for 10 minutes. Five different Murashige ve Skoog (MS) nutrient media combinations containing 6-Benzylaminopurine (BAP) and Thidiazuron (TDZ) at the ratios 0.75 and 1.50 mg L-1 were tested (Medium 1: MS + 30 g L-1 sucrose, 7 g L-1 agar, Medium 2: MS + 30 g L-1 sucrose, 7 g L-1 agar + 0.75 mg L-1 BAP, Medium 3: MS + 30 g L-1 sucrose, 7 g L-1 agar + 1.50 mg L-1 BAP, Medium 4: MS + 30 g L-1 sucrose, 7 g L-1 agar + 0.75 mg L-1 TDZ, Medium 5: MS + 30 g L-1 sucrose, 7 g L-1 agar + 1.50 mg L-1 TDZ). During the study, darkening, infection and rooting rates (%), shoot number per plant in both first and second subculture, root number per plant and root length (cm) parameters were examined. Use of four genotypes/cultivars and five nutrient media, this study was planned according to the factorial design of randomized plots with three replications. JMP package program and LSD test were used for statistical evaluation of data obtained. It was found that the media including TDZ was generally more effective than BAP in terms of shoot proliferation in both first and second subculture. The number of shoots per explant varied from 0 to 4.18 shoots per explant in the first subculture. Regarding to mean of genotypes and media, Kaşka cultivar (2.53 shoots per explant) and medium 5 (2.88 shoots per explant) gave the best results, respectively. In the second subculture, the number of shoots per explant changed between 0.33 and 4.83 shoots per explant. According to mean of genotypes and media, Kaşka cultivar (3.23 shoots per explant) and medium 4 (3.63 shoots per explant) were found to be the best material and medium, respectively. Regarding to rooting parameters, it was determined that the hormone-free MS medium was highly efficient. The number of root per in vitro plant varied from 0 to 30 roots per in vitro plant. According to mean of genotypes and media, Rubygem cultivar (9.60 roots per in vitro plant) and medium 1 (22.30 roots per in vitro plant) gave the best results, respectively. Root lenght changed between 0 and 5.04 cm. Regarding to mean of genotypes and media, the best values were obtained from Kaşka cultivar (2.10 cm) and medium 1 (3.89 cm), respectively. The value of 0 obtained from rooting parameters refers to in vitro plants that can not be rooted. Infection rate was low in both subcultures. Especially in the first subculture, it was observed only in the genotype 299 cultured in the medium 3. In the second subculture, infection was detected at low rates in the genotypes 291 and 299 and Rubygem cultivar cultured media 2, 3 and 5. No darkening was observed in the explants cultured in both subcultures. Based on genotypes, Kaşka cultivar developed in the Horticulture Department of Cukurova University (Turkey) was found to be superior in terms of both shooting and rooting criteria. Achieving successful results of this cultivar will speed up its use in breeding studies. Based on the study result and literature, it was concluded that the selection of the appropriate hormone type and dose are genotype-dependent. [ABSTRACT FROM AUTHOR]

Abstract The FGF/MEK/ERK and Wnt/β-catenin signaling pathways have previously been proved to regulate mouse embryonic stem cell (mESCs) function. However, the relationships between these two pathways, especially their different functions on proliferation and pluripotency of mESCs, were rarely mentioned. Here, we investigated the effects of FGF/MEK/ERK and Wnt/β-catenin pathway regulators and their combinations on the proliferation and pluripotency of mESCs under serum- and feeder-free conditions. We found that MEK inhibitor PD0325901 and FGFR inhibitor SU5402 has paradoxical function on mESCs; one could promote proliferation along with differentiation and the other one could improve pluripotency while impairing cell proliferation. The combination of these two kinds of inhibitors could better regulate FGF/MEK/ERK pathway. Wnt/β-catenin pathway regulators SB216763 led to differentiation while promoting proliferation of mESCs. When we used FGF/MEK/ERK and Wnt/β-catenin pathway regulators in combination, the total expansion fold of mESCs reached 318.78 ± 47.95 and the proportion of SSEA-1-positive cells reached 82.40 ± 2.74% which were significantly higher than using the regulators alone. This finding indicates that regulators of FGF/MEK/ERK and Wnt/β-catenin pathways play different roles in the regulatory networks of mESCs. Their combination can better maintain the undifferentiated state and promote the proliferation of mESCs under serum- and feeder-free conditions.

Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N6-(Δ2-isopentyl) adenine, thidiazuron, zeatin and meta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12 weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06 cm shoot length, 1671 ± 2.82 mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20 µM meta-topolin and 2.25 µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to 5.37 µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8–10 months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid. [ABSTRACT FROM AUTHOR]

Background: New therapeutic approaches are an essential need for patients suffering from colorectal cancer liver metastases. Curcumin, a well-known plant-derived polyphenol, has been shown to play a role in the modulation of multiple signaling pathways involved in the development and progression of certain cancer cells in vitro. This study aims to assess the anti-tumor effect of curcumin on CC531 colorectal cancer cells, both in vitro and in vivo. Methods: On CC531 cultures, the cell viability and cell migration capacity were analyzed (wound healing test) 24, 48, and 72 h after treatment with curcumin (15, 20, 25, or 30 µM). Additionally, in WAG/RijHsd tumor-bearing rats, the total and individual liver lobe tumor volume was quantified in untreated and curcumin-treated animals (200 mg/kg/day, oral). Furthermore, serum enzyme measurements (GOT, GPT, glucose, bilirubin, etc.) were carried out to assess the possible effects on the liver function. Results: In vitro studies showed curcumin’s greatest effects 48h after application, when all of the tested doses reduced cell proliferation by more than 30%. At 72 h, the highest doses of curcumin (25 and 30 µM) reduced cell viability to less than 50%. The wound healing test also showed that curcumin inhibits migration capacity. In vivo, curcumin slowed down the tumor volume of liver implants by 5.6-fold (7.98 ± 1.45 vs. 1.41 ± 1.33; p > 0.0001). Conclusions: Curcumin has shown an anti-tumor effect against liver implants from colorectal cancer, both in vitro and in vivo, in this experimental model.

UCB-1 (Pistacia atlantica × P. integrima) is a commercial rootstock for pistachio in some pistachio plantations across the world. This rootstock is very new in Iran and recently, it has been used commercially in some plantations due to its high growth. Propagation of this rootstock by tissue culture results in many limitations such as shoot tip necrosis (STN) and a low proliferation rate. Therefore, any process that leads to improve the proliferation rate and feature will be used in commercial propagation of this rootstock. Nanotubes are widely used in in vitro cultures. For this reason, we used different concentrations of carbon nanotubes (0, 50, 100, 150, 200 µg/l) and benzyladenine (0, 0.5, 1, 1.5 and 2 mg/l) to improve the proliferation rate and qualitative indices. The results showed that using carbon nanotubes concentration of 200 µg/l with 2mg/l of benzyladenine (BA) led to maximum proliferation (4 microshoots per explant), maximum shoot length (3.68 cm) and minimum STN (8%) and vitrification (this isn’t a word?) (0 %) percentage.

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An in vitro plant tissue culture approach provides a feasible alternative for extensive mass manufacturing and conservation of elite and critically endangered medicinal plant species. The presented work describes a proficient in vitro seed germination and proliferation procedure for the conservation of an endangered high trade medicinal tree, Shorea tumbuggaia Roxb. Given the precarious position of this species, effective conservation is highly indispensable for its continued existence, primarily for ecological reasons and also for forestry purposes as this tree species is a valuable source of timber and oleoresin. In the present study, over 90% in vitro seed germination was achieved. Multiple shoot formation was established from seedlings with a maximum of 4.60 ± 0.30 multiple shoots per explant using MS (Murashige and Skoog) medium supplemented with a combination of 6.66 μM 6-benzylaminopurine and 0.454 μM thidiazuron. Rooting of multiple shoot cultures of 85% was achieved on half strength MS medium supplemented with 2.24 μM indole 3-butyric acid with 6.48 ± 0.58 roots per plantlet. Rooted plantlets showed more than 70% acclimatization in potting conditions and were established successfully in a greenhouse, with morphological similarities to mother plants. Clonal fidelity assessment of micropropagated plants with ten inter-simple sequence repeat primers reproduced a total of 24 monomorphic bands. The presented work represents the first report of in vitro proliferation, conservation, and clonal fidelity assessment of tissue culture generated S. tumbuggaia plantlets. [ABSTRACT FROM AUTHOR]

Although several studies have been reported on the micropropagation of the pistachio and its rootstocks, to date none of them had been efficient on the mass production of these plants in bioreactor systems. Thus, the micropropagation of juvenile pistachio shoot tips and nodal buds was investigated in a temporary immersion bioreactor system (RITA) and on a conventional semi-solid medium. Among the tested immersion conditions, immersion for 24 min every 16 h reduced vitrification and improved proliferation in the pistachio. Interactions were evident in immersion time and frequency in nodal segments. Nodal buds were better than shoot tips as the highest multiple shoot formation was recorded in MS medium containing 4 mg L BA and 0.1 mg L GA in RITA. Although shoot tip necrosis (STN) was observed in shoots proliferated on semi-solid MS medium, such a symptom did not occur in shoots sprouted in the RITA. Additionally, these optimized conditions were applied to nodal buds of mature male pistachio 'Atlı' and Pistacia rootstocks ( P. khinjuk Stocks and P. atlantica Desf.), and the micropropagation in the bioreactor system, in comparison to the semi-solid medium, was also improved. Furthermore, in vitro rooting of pistachio plantlets, despite the lower range (27.5 %), was also achieved in RITA. However, rooting was better on semi-solid medium for all tested species (ranged between 50 and 70 %). The results of this study showed that RITA could be used for the mass propagation of pistachio and its rootstocks, as well as for other woody plant species. [ABSTRACT FROM AUTHOR]

Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3'-untranslated region (3'UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3'UTRs. Compared with rMM 1775, rMM-CQ3'UTR (recombinant MM 1775 virus carrying the 3'UTR of CQW1) exhibited enhanced proliferation in vitro , with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3'UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3'UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3'UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3'UTRSLI displayed a 15- or 4-fold increase in vitro , and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3'UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3'UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3'UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3'-untranslated region (3'UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3'UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3'UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3'UTR in viral host switching and pathogenicity changes in mice.

Abstract: The ovarian surface epithelium (OSE) forms a lining around the entire ovary and actively participates in the ovulatory cycle. To investigate how specific growth factors and hormones affect OSE proliferation, the present study used sheep as a model to examine the effects of follicular and luteal products on the proliferation of sheep OSE cells in culture, and to analyse the influences of large antral follicles and corpora lutea (CL) on the expression of gonadotrophin receptors (FSHR and LHR) in the OSE. In the present study, follicular fluids from medium and large follicles, and extracts of corpora lutea stimulated the growth of OSE cells. The results of the present study showed that factors in the follicular fluid can induce OSE proliferative activity, and this stimulation effect could not be attributed to steroids in the follicular fluid since oestrogen and progesterone treatments failed to stimulate OSE cells. The expression of LH and FSH receptors over large follicles (5mm or larger) was two and four times higher than those over stroma and CL, respectively. In conclusion, OSE proliferation in cycling sheep is associated with underlying developing follicles and CL, mediated by, at least in part, the up-regulation of gonadotrophin receptors, and facilitated by the action of mitogenic glycopeptides and growth factors, but not steroids. [Copyright &y& Elsevier]

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The aim of this work was to study the in vitro effects of δ-lactone 1, δ-lactam 3 and their enaminone derivatives 2 and 4, synthesized in our laboratory, on the proliferative responses of human lymphocytes, Th1 and Th2 cytokine secretion and intracellular redox status. Peripheral blood lymphocytes were isolated using differential centrifugation on a density gradient of Histopaque. They were cultured with mitogen concanavalin A (Con A) and with different concentrations of the compounds 1, 2, 3 and 4 (0.1-10 μM). Proliferation (MTT assay), IL-2, INFγ and IL-4 (Elisa kits), oxidative markers (intracellular glutathione, hydroperoxide and carbonyl protein contents) and cytotoxic effect (micronucleus test) were determined. The compounds 1 and 2 are immunosuppressive and decrease IL-2, INFγ and IL-4 secretion with a shift away from Th2 response to Th1 phenotype. The compounds 3 and 4 were immunostimulant and increased cytokine secretion with a shift away from Th1 response to Th2. The introduction of an enamine group to 1 and 3 to provide 2 and 4 seemed to attenuate their immunological properties. These immunomodulatory properties were, however, accompanied by an increase in lymphocyte intracellular oxidative stress, especially with 1 and 2 at high concentrations. In conclusion, the compounds 1, 2, 3 and 4 could be used to provide cell-mediated immune responses for novel therapies in T-cell mediated immune disorders. [ABSTRACT FROM AUTHOR]

n efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L kinetin and 0.3 mg L GA. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity. [ABSTRACT FROM AUTHOR]

Abstract: Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N =7) seeded singly yielded clonal cultures with 253±167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5±4 cells after 21 days. [Copyright &y& Elsevier]

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis. [ABSTRACT FROM AUTHOR]

The therapeutic effects of intravenous immunoglobulins (IVIGs) on different chronic inflammatory and autoimmune diseases is well appreciated, though clinical studies with high-evidence levels are largely missing. Similar to the broad spectrum of diseases and their underlying etiopathogenic background, the mechanisms of action seem heterogenous and multifold. Several studies addressing in vitro and in vivo effects of IVIG on various immunological parameters have been described with partly contradictory results. In this study immunoglobulins and stabilizers present in commercial IVIG preparations were studied in regard to the in vitro proliferation and cytokine production of peripheral blood mononuclear cells when stimulated with phytohemagglutinin (PHA), interleukin 2, and tetanus toxoid. Whereas the immunoglobulins stimulate the proliferation of PBMCs and decrease IFNγ secretion, stabilizers of IVIG seem to inhibit the proliferation of PBMCs while increasing the secretion of IFN γ. These effects have to be taken into account when balancing the impact of IVIG dosage and infusion intervals and relating them to clinical side effects and therapeutic efficacy. [ABSTRACT FROM AUTHOR]