An in vitro grafting method to quantify mechanical forces of adhering tissues

AbstractsGrafting is an indispensable agricultural technology for propagating useful tree varieties and obtaining beneficial traits of two varieties/species—as stock and scion—at the same time. Recent studies of molecular events during grafting have revealed dynamic physiological and transcriptomic changes. Strategies focused on specific grafting steps are needed to further associate each physiological and molecular event with those steps. In this study, we developed a method to investigate the tissue adhesion event, an early grafting step, by improving an artificial in vitro grafting system in which two pieces of 1.5-mm thick Nicotiana benthamiana cut stem sections were combined and cultured on medium. We prepared a silicone sheet containing five special cutouts for adhesion of cut stem slices. We quantitatively measured the adhesive force at these grafting interfaces using a force gauge and found that graft adhesion started 2 days after grafting, with the adhesive force gradually increasing over time. After confirming the positive effect of auxin on grafting by this method, we tested the effect of cellulase treatment and observed significant enhancement of graft tissue adhesion. Compared with the addition of auxin or cellulase individually, the adhesive force was stronger when both auxin and cellulase were added simultaneously. The in vitro grafting method developed in this study is thus useful for examining the process of graft adhesion.

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The depressing (negative) effect of oestradiol benzoate on the in vitro secretion of LH and FSH by the pituitary gland of the LRH-pretreated long-term ovariectomized rat changes into the augmentative (positive) effect after discontinuation of the LRH-pretreatment

Acta Endocrinologica ◽

10.1530/acta.0.1100329 ◽

1985 ◽

Vol 110 (3) ◽

pp. 329-337 ◽

Cited By ~ 2

Author(s):

G. A. Schuiling ◽

H. Moes ◽

T. R. Koiter

Keyword(s):

Depressing Effect ◽

Ovx Rats ◽

Gonadotrophin Secretion ◽

Oestradiol Benzoate ◽

Negative Effect ◽

Positive Effect ◽

Perifusion System

Abstract. The effect of pretreatment in vivo with oestradiol benzoate on in vitro secretion of LH and FSH was studied in long-term ovariectomized (OVX) rats both at the end of a 5-day continuous in vivo pretreatment with LRH and 4-days after cessation of such LRH pretreatment. Rats were on day 0 sc implanted with osmotic minipumps which released LRH at the rate of 250 ng/h. Control rats were implanted with a piece of silicone elastomer with the dimensions of a minipump. On days 2 and 4 the rats were injected with either 3 μg EB or with oil. On day 5 part of the rats were decapitated and the in vitro autonomous (i.e. non-LRH-stimulated) and 'supra-maximally' LRHstimulated release of LH and FSH was studied using a perifusion system. From other rats the minipumps were removed on day 5 and perifusion was performed on day 9. On the 5th day of the in vivo LRH pretreatment the pituitary LH/FSH stores were partially depleted; the pituitaries of the EB-treated rats more so than those of the oil-injected rats. EB alone had no significant effect on the content of the pituitary LH- and FSH stores. On day 9, i.e. 4 days after removal of the minipumps, the pituitary LH and FSH contents had increased in both the oil- and the EB injected rats, but had not yet recovered to control values. In rats not subjected to the 5-days pretreatment with LRH EB had a positive effect on the supra-maximally LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. EB had no effect on the non-stimulated secretion of FSH. After 5 days of in vivo pretreatment with LRH only, the in vitro non-stimulated and supra-maximally LRH-stimulated secretion of both LH and FSH were strongly impaired, the effect correlating well with the LRH-induced depletion of the pituitary LH/FSH stores. In such LRH-pretreated rats EB had on day 5 a negative effect on the (already depressed) LRH-stimulated secretion of LH (not on that of FSH). EB had no effect on the non-stimulated LH/FSH secretion. It could be demonstrated that the negative effect of the combined LRH/EB pretreatment was mainly due to the depressing effect of this treatment on the pituitary LH and FSH stores: the effect of oestradiol on the pituitary LRH-responsiveness (release as related to pituitary gonadotrophin content) remained positive. In LRH-pretreated rats, however, this positive effect of EB was smaller than in rats not pretreated with LRH. Four days after removal of the minipumps there was again a positive effect of EB on the LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. The positive effect of EB on the pituitary LRH-responsiveness was as strong as in rats which had not been exposed to exogenous LRH. The non-stimulated secretion of FSH was again not affected by EB. The results demonstrate that the effect of EB on the oestrogen-sensitive components of gonadotrophin secretion consists of two components: an effect on the pituitary LRH-responsiveness proper, and an effect on the pituitary LH/FSH stores. The magnitude of the effect of EB on the LRH-responsiveness is LRH dependent: it is very weak (almost zero) in LRH-pretreated rats, but strong in rats not exposed to LRH as well as in rats of which the LRH-pretreatment was stopped 4 days previously. Similarly, the effect of EB on the pituitary LH and FSH stores is LRH-dependent: in the absence of LRH, EB has no influence on the contents of these stores, but EB can potentiate the depleting effect of LRH on the LH/FSH-stores. Also this effect disappear after cessation of the LRH-pretreatment.

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In Vitro Effect of Chlorquine and Picroliv on Plasmodium Berghei Induced Alterations in the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and Malondialdehyde in spleen Explant Culture

Infectious Disorders - Drug Targets ◽

10.2174/1871526520666200630124419 ◽

2020 ◽

Vol 20 ◽

Author(s):

Anchal Trivedi ◽

Aparna Misra ◽

Esha Sarkar ◽

Anil K. Balapure

Keyword(s):

Drug Resistance ◽

Plasmodium Berghei ◽

Adenosine Triphosphatase ◽

Explant Culture ◽

Need To Evaluate ◽

Mda Content ◽

High Level ◽

Vitro Effect ◽

Positive Effect

Background: In recent years, great progress has been made in reducing the high level of malaria suffering worldwide. There is a great need to evaluate drug resistance reversers and consider new medicines against malaria. There are many approaches to the development of antimalarial drugs. Specific concerns must be taken in to account in these approaches, in particular there requirement for very in expensive and simple use of new therapies and the need to limit drug discovery expenses. Important ongoing efforts are the optimisation of treatment with available medications, including the use of combination therapy. The production of analogs of known agents and the identification of natural products, the use of compounds originally developed against other diseases, the assessment of overcoming drug resistance and the consideration of new therapeutic targets. Liver and spleen are the important organs which are directly associated with malarial complications. Aim: An analysis the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and Malondialdehyde in spleen Explant Culture. Objective: To determine in-Vitro Effect of Chlorquine and Picroliv on Plasmodium Berghei Induced Alterations in the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and Malondialdehyde in spleen Explant Culture. Material and method: 1-Histological preparation of spleen explants for paraplast embedding 2-Biochemicalstudies (Enzymes (Atpase, ALP&GST) and the level of protein, Malondialdehyde (MDA). Result: Splenomegalyis one of the three main diagnostic parameters of malaria infection besides fever and anaemia. Many enzymes present in the liver and spleen may also be altered or liberated under different pathological conditions. Enzymes (ATPase, ALP&GST) and the level of protein, Malondialdehyde (MDA) content was found to increase in the liver and spleen explants during malarial infection. In the liver and spleen derived from parasitized CQ treated animals, the activity of all the above enzymes (ATPase, ALP&GST) and the level of protein & MDA of liver/spleen reversed towards the normal for all the 4or3 days of incubations. Picroliv efficacy decreased with the increment of parasitaemia and at 60%parasitaemia. Conclusion: Alkalinephosphatase (ALP) was found to increase with increasing parasitaemia. After the addition of Picroliv to the medium, a decrement in the activity was observed up to day 4 of culture.A similar positive effect of Picroliv was observed on the ATPase and ALP activity of spleen explants.DNA and protein contents also increased in the parasitized liver cultured in the presence of picroliv.On the contrary, in the spleen explants DNA, protein and MDA content were found to decrease after Picroliv supplementation to the culture medium.

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Optimizing the Process Design of Oil-in-Water Nanoemulsion for Delivering Poorly Soluble Cannabidiol Oil

Processes ◽

10.3390/pr9071180 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1180

Author(s):

Agnieszka Lewińska

Keyword(s):

Process Design ◽

Hacat Keratinocytes ◽

Degree Of Hydration ◽

Skin Cell ◽

Oil In Water ◽

Poorly Soluble ◽

Last Stage ◽

Positive Effect

Process approaches and intensification technological processes are integrated parts of available devices, which have a positive effect on the parameters of the obtained products. Nanoemulsions as delivery carriers are becoming more popular and there is a real need to increase the possibilities of formulation designing and engineering. Therefore, preparations of oil-in-water nanoemulsion with encapsulated cannabidiol (CBD) as oil phase were carried out in two ways: sonication method and two-stage high-pressure homogenization. The provided analysis showed spherical morphology and much larger sizes and polydispersity of nanoemulsions obtained by the sonication approach. The size of nanodroplets was from 216 nm up to 1418 nm for sonication, whereas for homogenization 128–880 nm. Additionally, it was observed that a proportionally higher percentage of surfactin resulted in a higher value of the Zeta potential. The formulations were found to be stable for at least 30 days. The in vitro experiments performed on human skin cell lines (HaCaT keratinocytes and normal dermal NHDF fibroblasts), and in vivo topical tests on probants established the biocompatibility of nanoemulsions with CBD. The last stage exhibits reduced discoloration and a higher degree of hydration by the selected systems with CBD and, thus indicating this nanoformulation as useful in cosmetics applications.

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HopA1 Effector from Pseudomonas syringae pv syringae Strain 61 Affects NMD Processes and Elicits Effector-Triggered Immunity

International Journal of Molecular Sciences ◽

10.3390/ijms22147440 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7440

Author(s):

Shraddha K. Dahale ◽

Daipayan Ghosh ◽

Kishor D. Ingole ◽

Anup Chugani ◽

Sang Hee Kim ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Disease Susceptibility ◽

Null Mutant ◽

In Planta ◽

Host Range Expansion ◽

Unique System ◽

Effector Triggered Immunity ◽

Transcriptomic Changes ◽

Immune Detection

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1′s contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcriptomic changes associated with HopA1pss-elicited ETI are still induced and carry resemblance to PAMP-triggered immunity (PTI) signatures. Enrichment of HopA1pss interactome identifies proteins with regulatory roles in post-transcriptional and translational processes. With our demonstration here that both HopA1 suppress reporter-gene translations in vitro imply that the above effector-associations with plant target carry inhibitory consequences. Overall, with our results here we unravel possible virulence role(s) of HopA1 in suppressing PTI and provide newer insights into its detection in resistant plants.

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The Bone Regeneration Capacity of BMP-2 + MMP-10 Loaded Scaffolds Depends on the Tissue Status

Pharmaceutics ◽

10.3390/pharmaceutics13070979 ◽

2021 ◽

Vol 13 (7) ◽

pp. 979

Author(s):

Patricia Garcia-Garcia ◽

Ricardo Reyes ◽

José Antonio Rodriguez ◽

Tomas Martín ◽

Carmen Evora ◽

...

Keyword(s):

Bone Formation ◽

Bone Regeneration ◽

Growth Promotion ◽

Tissue Growth ◽

Bone Morphogenetic Protein 2 ◽

Morphogenetic Protein ◽

Therapeutic Molecules ◽

Positive Effect

Biomaterials-mediated bone formation in osteoporosis (OP) is challenging as it requires tissue growth promotion and adequate mineralization. Based on our previous findings, the development of scaffolds combining bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 10 (MMP-10) shows promise for OP management. To test our hypothesis, scaffolds containing BMP-2 + MMP-10 at variable ratios or BMP-2 + Alendronate (ALD) were prepared. Systems were characterized and tested in vitro on healthy and OP mesenchymal stem cells and in vivo bone formation was studied on healthy and OP animals. Therapeutic molecules were efficiently encapsulated into PLGA microspheres and embedded into chitosan foams. The use of PLGA (poly(lactic-co-glycolic acid)) microspheres as therapeutic molecule reservoirs allowed them to achieve an in vitro and in vivo controlled release. A beneficial effect on the alkaline phosphatase activity of non-OP cells was observed for both combinations when compared with BMP-2 alone. This effect was not detected on OP cells where all treatments promoted a similar increase in ALP activity compared with control. The in vivo results indicated a positive effect of the BMP-2 + MMP-10 combination at both of the doses tested on tissue repair for OP mice while it had the opposite effect on non-OP animals. This fact can be explained by the scaffold’s slow-release rate and degradation that could be beneficial for delayed bone regeneration conditions but had the reverse effect on healthy animals. Therefore, the development of adequate scaffolds for bone regeneration requires consideration of the tissue catabolic/anabolic balance to obtain biomaterials with degradation/release behaviors suited for the existing tissue status.

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The Effect of Platelet-Rich Plasma on the Intra-Articular Microenvironment in Knee Osteoarthritis

International Journal of Molecular Sciences ◽

10.3390/ijms22115492 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5492

Author(s):

Dawid Szwedowski ◽

Joanna Szczepanek ◽

Łukasz Paczesny ◽

Jan Zabrzyński ◽

Maciej Gagat ◽

...

Keyword(s):

Knee Osteoarthritis ◽

Operative Treatment ◽

Animal Studies ◽

Platelet Rich Plasma ◽

Treatment Options ◽

Treatment Protocols ◽

Metabolic Functions ◽

Inflammatory Drugs ◽

Positive Effect

Knee osteoarthritis (KOA) represents a clinical challenge due to poor potential for spontaneous healing of cartilage lesions. Several treatment options are available for KOA, including oral nonsteroidal anti-inflammatory drugs, physical therapy, braces, activity modification, and finally operative treatment. Intra-articular (IA) injections are usually used when the non-operative treatment is not effective, and when the surgery is not yet indicated. More and more studies suggesting that IA injections are as or even more efficient and safe than NSAIDs. Recently, research to improve intra-articular homeostasis has focused on biologic adjuncts, such as platelet-rich plasma (PRP). The catabolic and inflammatory intra-articular processes that exists in knee osteoarthritis (KOA) may be influenced by the administration of PRP and its derivatives. PRP can induce a regenerative response and lead to the improvement of metabolic functions of damaged structures. However, the positive effect on chondrogenesis and proliferation of mesenchymal stem cells (MSC) is still highly controversial. Recommendations from in vitro and animal research often lead to different clinical outcomes because it is difficult to translate non-clinical study outcomes and methodology recommendations to human clinical treatment protocols. In recent years, significant progress has been made in understanding the mechanism of PRP action. In this review, we will discuss mechanisms related to inflammation and chondrogenesis in cartilage repair and regenerative processes after PRP administration in in vitro and animal studies. Furthermore, we review clinical trials of PRP efficiency in changing the OA biomarkers in knee joint.

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In vitro study of human osteoblast proliferation and morphology on orthodontic mini-implants

The Angle Orthodontist ◽

10.2319/100714-717.1 ◽

2015 ◽

Vol 85 (6) ◽

pp. 920-926 ◽

Cited By ~ 2

Author(s):

Ricardo Carvalho Bueno ◽

Roberta Tarkany Basting

Keyword(s):

Chemical Composition ◽

In Vitro Study ◽

Chemical Components ◽

Control Group ◽

Osteoblast Proliferation ◽

Mini Implants ◽

Significant Difference ◽

Implant Anchorage ◽

Positive Effect

ABSTRACT Objective: To evaluate the proliferation and morphology of human osteoblasts cultured on two brands of mini-implants after 24, 48, and 72 hours, in addition to the chemical composition found on their surface. Materials and Methods: Two brands of mini-implant (Morelli and Neodent) were evaluated; polystyrene was used as a control group (n = 3). Osteoblasts were cultured on the surface of sterilized mini-implants in a CO2 incubator at different time periods (24, 48, and 72 hours). Osteoblast proliferation was quantified by scanning electron microscopy using up to 5000× magnification, and cell morphology was analyzed by a single observer. For the chemical analysis, spectroscopy X-ray fluorescence was used to identify and quantify chemical components on the surface of the mini-implants. Results: Two-way ANOVA showed no significant interaction between the factors studied (P = 0.686). A Tukey test revealed no significant difference in osteoblast proliferation between the mini-implants at all studied periods; however, a difference in cell proliferation was detected between the Neodent and the control group (P = .025). For all groups, time had a direct and positive effect on osteoblast proliferation (P < .001). The significant elements present in both brands of mini-implants were titanium, aluminum, vanadium, and iron. Conclusions: Osteoblast proliferation was present on the mini-implants studied, which increased over time; however, no significant difference between brands was observed. No difference was seen between the mini-implants evaluated in terms of chemical composition. Cell adhesion after 72 hours suggests that areas of bone remodeling can be achieved, thus initiating the process of mini-implant anchorage.

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Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms

AJP Cell Physiology ◽

10.1152/ajpcell.00216.2009 ◽

2009 ◽

Vol 297 (6) ◽

pp. C1358-C1367 ◽

Cited By ~ 62

Author(s):

Gerald J. Atkins ◽

Katie J. Welldon ◽

Asiri R. Wijenayaka ◽

Lynda F. Bonewald ◽

David M. Findlay

Keyword(s):

Bone Formation ◽

Vitamin K ◽

Type I Collagen ◽

Vitamin K2 ◽

Type I ◽

Vitamin K1 ◽

Vitamin K3 ◽

Positive Effect

The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that γ-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-κB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.

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Persistent Newcastle disease virus infection in bladder cancer cells is associated with putative pro-survival and anti-viral transcriptomic changes

BMC Cancer ◽

10.1186/s12885-021-08345-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lee-Chin Chan ◽

Jeevanathan Kalyanasundram ◽

Sze-Wei Leong ◽

Mas Jaffri Masarudin ◽

Abhi Veerakumarasivam ◽

...

Keyword(s):

Bladder Cancer ◽

Newcastle Disease Virus ◽

Cancer Cells ◽

Disease Virus ◽

Newcastle Disease ◽

Persistent Infection ◽

Signalling Pathways ◽

Bladder Cancer Cells ◽

Transcriptomic Changes

Abstract Background Newcastle disease virus (NDV) is an oncolytic virus with excellent selectivity against cancer cells, both in vitro and in vivo. Unfortunately, prolonged in vitro NDV infection results in the development of persistent infection in the cancer cells which are then able to resist NDV-mediated oncolysis. However, the mechanism of persistency of infection remains poorly understood. Methods In this study, we established persistently NDV-infected EJ28 bladder cancer cells, designated as EJ28P. Global transcriptomic analysis was subsequently carried out by microarray analysis. Differentially expressed genes (DEGs) between EJ28 and EJ28P cells identified by the edgeR program were further analysed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) analyses. In addition, the microarray data were validated by RT-qPCR. Results Persistently NDV-infected EJ28 bladder cancer cells were successfully established and confirmed by flow cytometry. Microarray analysis identified a total of 368 genes as differentially expressed in EJ28P cells when compared to the non-infected EJ28 cells. GSEA revealed that the Wnt/β-catenin and KRAS signalling pathways were upregulated while the TGF-β signalling pathway was downregulated. Findings from this study suggest that the upregulation of genes that are associated with cell growth, pro-survival, and anti-apoptosis may explain the survivability of EJ28P cells and the development of persistent infection of NDV. Conclusions This study provides insights into the transcriptomic changes that occur and the specific signalling pathways that are potentially involved in the development and maintenance of NDV persistency of infection in bladder cancer cells. These findings warrant further investigation and is crucial towards the development of effective NDV oncolytic therapy against cancer.

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