The Effects of Polyphenol, Tannic Acid, or Tannic Acid in Combination with Pamidronate on Human Osteoblast Cell Line Metabolism

Background: This study investigates the effect of tannic acid (TA) combined with pamidronate (PAM) on a human osteoblast cell line. Methods: EC50 for TA, PAM, and different combination ratios of TA and PAM (25:75, 50:50, 75:25) were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The combination index value was utilized to analyze the degree of drug interaction, while trypan blue assay was applied to analyze the cells proliferation effect. The mineralization and detection of bone BSP and Osx genes were determined via histochemical staining and PCR test, respectively. Results: The EC50 of osteoblasts treated with TA and a 75:25 ratio of TA and PAM were more potent with lower EC50 at 0.56 µg/mL and 0.48 µg/mL, respectively. The combination of TA and PAM (75:25) was shown to have synergistic interaction. On Day 7, both TA and PAM groups showed significantly increased proliferation compared with control and combination groups. On Day 7, both the TA and combination-treated groups demonstrated a higher production of calcium deposits than the control and PAM-treated groups. Moreover, on Day 7, the combination-treated group showed a significantly higher expression of BSP and Osx genes than both the TA and PAM groups. Conclusion: Combination treatment of TA and PAM at 75:25 ameliorated the highest enhancement of osteoblast proliferation and mineralization as well as caused a high expression of BSP and Osx genes.

Morphological Characteristics of the Lacrimal Apparatus in its Obstruction of Various Genesis

Purpose: morphological assessment of the lacrimal ducts at various anatomical levels in patients with primary (PANDO) and secondary (SALDO) obstruction after radioactive iodine therapy.Methods. The material was obtained during endoscopic dacryocystorinostomy with revision of Hasner's valve in patients with PANDO (n=7) in the distal segments of the nasolacrimal duct and in patients with SALDO (n=7) after radioactive iodine therapy. During the surgery, a biopsy of Hasner's valve, as well as a biopsy of the lacrimal sac wall were performed. The resulting material was stained with hemotoxylin and eosin, alcyan blue and by Masson method. Morphological and morphometric analyses were performed in semi-automatic mode. The results of histochemical staining of sections were translated into points taking into account the area and optical density (chromogenicity) in relative units: 1 – weak (0 – 0.3); 2 – moderate (0.3 – 0.6); 3 – significant (>0.6). The nonparametric Mann-Whitney criterion was used for statistical analysis. The differences were considered significant at p<0.05.Results. The comparative morphological study both confirmed the available information concerning the radiation nature of the obstruction and allowed to quantify the fibrosis level of the stromal component and other lacrimal ducts structures.Conclusion. It was shown that the nasolacrimal duct sclerosis is significantly lower (p=0.029) in patients with SALDO than in patients with PANDO while fibrosis in the lacrimal sac is the same in patients of the compared groups.

Mobile Host mRNAs Are Translated to Protein in the Associated Parasitic Plant Cuscuta campestris

Cuscuta spp. are obligate parasites that connect to host vascular tissue using a haustorium. In addition to water, nutrients, and metabolites, a large number of mRNAs are bidirectionally exchanged between Cuscuta spp. and their hosts. This trans-specific movement of mRNAs raises questions about whether these molecules function in the recipient species. To address the possibility that mobile mRNAs are ultimately translated, we built upon recent studies that demonstrate a role for transfer RNA (tRNA)-like structures (TLSs) in enhancing mRNA systemic movement. C. campestris was grown on Arabidopsis that expressed a β-glucuronidase (GUS) reporter transgene either alone or in GUS-tRNA fusions. Histochemical staining revealed localization in tissue of C. campestris grown on Arabidopsis with GUS-tRNA fusions, but not in C. campestris grown on Arabidopsis with GUS alone. This corresponded with detection of GUS transcripts in Cuscuta on Arabidopsis with GUS-tRNA, but not in C. campestris on Arabidopsis with GUS alone. Similar results were obtained with Arabidopsis host plants expressing the same constructs containing an endoplasmic reticulum localization signal. In C. campestris, GUS activity was localized in the companion cells or phloem parenchyma cells adjacent to sieve tubes. We conclude that host-derived GUS mRNAs are translated in C. campestris and that the TLS fusion enhances RNA mobility in the host-parasite interactions.

Neurotensin Attenuates Nociception by Facilitating Inhibitory Synaptic Transmission in the Mouse Spinal Cord

Neurotensin (NT) is an endogenous tridecapeptide in the central nervous system. NT-containing neurons and NT receptors are widely distributed in the spinal dorsal horn (SDH), indicating their possible modulatory roles in nociception processing. However, the exact distribution and function of NT, as well as NT receptors (NTRs) expression in the SDH, have not been well documented. Among the four NTR subtypes, NTR2 is predominantly involved in central analgesia according to previous reports. However, the expression and function of NTR2 in the SDH has not yet been directly elucidated. Specifically, it remains unclear how NT-NTR2 interactions contribute to NT-mediated analgesia. In the present study, by using immunofluorescent histochemical staining and immunohistochemical staining with in situ hybridization histochemical staining, we found that dense NT- immunoreactivity (NT-ir) and moderate NTR2-ir neuronal cell bodies and fibers were localized throughout the superficial laminae (laminae I-II) of the SDH at the light microscopic level. In addition, γ-aminobutyric acid (GABA) and NTR2 mRNA were colocalized in some neuronal cell bodies, predominantly in lamina II. Using confocal and electron microscopy, we also observed that NT-ir terminals made both close contacts and asymmetrical synapses with the local GABA-ir neurons. Second, electrophysiological recordings showed that NT facilitated inhibitory synaptic transmission but not glutamatergic excitatory synaptic transmission. Inactivation of NTR2 abolished the NT actions on both GABAergic and glycinergic synaptic release. Moreover, a behavioral study revealed that intrathecal injection of NT attenuated thermal pain, mechanical pain, and formalin induced acute inflammatory pain primarily by activating NTR2. Taken together, the present results provide direct evidence that NT-containing terminals and fibers, as well as NTR2-expressing neurons are widely distributed in the spinal dorsal horn, GABA-containing neurons express NTR2 mainly in lamina II, GABA coexists with NTR2 mainly in lamina II, and NT may directly increase the activity of local inhibitory neurons through NTR2 and induce analgesic effects.

Iron treatment induces defense responses and disease resistance against Magnaporthe oryzae in rice

Background: Iron is an essential micronutrient required for plant growth and development. The impact of iron in plant-pathogen interactions is also well recognized. However, the molecular basis underlying the effect of plant iron status and immune function in plants is poorly understood. Here, we investigated the impact of treatment with high iron in rice immunity at the cellular and molecular level. Results: We show that treatment with high iron confers resistance to infection by the blast fungus M. oryzae in rice. Histochemical staining of M. oryzae-infected leaves revealed that iron and Reactive Oxygen Species (ROS) accumulate at high levels in cells in the vicinity of the infection site. During pathogen infection, a stronger induction of defense-related genes occurs in leaves of iron-treated plants. Notably, a superinduction of phytoalexin biosynthetic genes, both diterpene phytoalexins and sakuranetin, is observed in iron-treated plants during pathogen infection. As a consequence, phytoalexin accumulation was higher in iron-treated plants compared with control plants. Transcriptional alterations of iron homeostasis-related genes and a reduction in apoplastic iron content were observed in leaves of Fe-treated rice plants. Conclusions: These results illustrate that the iron status plays a key role in the response of rice plants to pathogen infection, while reinforcing the notion that iron signaling and defense signaling must operate in a coordinated manner in controlling disease resistance in plants. This information provides a basis to better understand the molecular mechanisms involved in rice immunity.

The Sorghum (Sorghum bicolor) Brown Midrib 30 Gene Encodes a Chalcone Isomerase Required for Cell Wall Lignification

In sorghum (Sorghum bicolor) and other C4 grasses, brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. The brown midrib 30 (Bmr30) gene, identified using a bulk segregant analysis and next-generation sequencing, was determined to encode a chalcone isomerase (CHI). Two independent mutations within this gene confirmed that loss of its function was responsible for the brown leaf midrib phenotype and reduced lignin concentration. Loss of the Bmr30 gene function, as shown by histochemical staining of leaf midrib and stalk sections, resulted in altered cell wall composition. In the bmr30 mutants, CHI activity was drastically reduced, and the accumulation of total flavonoids and total anthocyanins was impaired, which is consistent with its function in flavonoid biosynthesis. The level of the flavone lignin monomer tricin was reduced 20-fold in the stem relative to wild type, and to undetectable levels in the leaf tissue of the mutants. The bmr30 mutant, therefore, harbors a mutation in a phenylpropanoid biosynthetic gene that is key to the interconnection between flavonoids and monolignols, both of which are utilized for lignin synthesis in the grasses.

Chondrocytes isolation from hyaline cartilage by continuous monitoring method

Background: Articular cartilage has poor regenerative capacities. Numerous cartilage repair techniques are known, including implantation of autologous chondrocytes. Material and methods: From 18 rabbits pieces of cartilage were harvested from femoral condyle. Minced cartilage was treated with 0.25% trypsin-EDTA. In the 1st group (n=9) the cartilage was digested with 0.6% collagenase in 15 ml tubes by shaking in incubator at 37°C, 5%CO2 . In the 2nd group (n=9) digestion was performed in 25cm2 cell culture flasks placed on the lateral side, monitoring the process under a microscope after 120 minutes. The isolated cells were cultured to a 80-90% confluence. The chondrocytes were identified using histochemical staining after culturing for 16 days in overconfluence. Results: Chondrocytes isolation in the 1st group lasted a fixed 360 minutes, in the 2nd group – 140±10 minutes. In the 1stgroup were isolated 9.2x104 ±3.1x104 chondrocytes with a viability of 85.36±16.41%, but in the 2nd group – 1.6x105 ±3.4x104 chondrocytes with a viability of 98.09±3.85%. The mean period of cell culture in the 1st group was 15±2 days, in the 2nd group – 11±3 days. In first passage of the 1st group were obtained – 1.2x106 ±4.3x105 chondrocytes and in the 2nd group – 2.92x106 ±3.6x105 chondrocytes. The secreted extracellular matrix by chondrocytes was stained specifically for cartilaginous tissue. Conclusions: The method used for chondrocytes isolation has a direct impact on the number of isolated cells, their viability, but also upon the culture period and the number of cells obtained during the first passage.

Evaluation of Seed Dormancy, One of the Key Domestication Traits in Chickpea

Legume seed dormancy has been altered during the domestication process, resulting in non-dormant seeds with a testa that is readily permeable for water. Ultimately, this provides fast and uniform germination, in contrast to dormant seeds of the wild progenitor. To date, germination and seed dormancy were studied mostly in relation to two types of cultivated chickpea: kabuli and desi. We studied seed dormancy, from physiological and anatomical perspectives, in chickpea crops and compared cultivated chickpeas to the wild chickpea progenitor and set of recombinant inbred lines (RIL). There was significant difference in the macrosclereid length of parental genotypes. Cultivated chickpea (C. arietinum, ICC4958) had mean of 125 µm, while wild C. reticulatum (PI48977) had a mean of 165 µm. Histochemical staining of the seed coat also showed differences, mainly in terms of Sudan Red detection of lipidic substances. Imbibition and germination were tested and several germination coefficients were calculated. Cultivated chickpea seeds imbibed readily within 24 h, while the germination percentage of wild chickpea at various times was 36% (24 h), 46% (48 h), 60% (72 h) and reached 100% only after 20 days. RIL lines showed a broader distribution. This knowledge will ultimately lead to the identification of the underlying molecular mechanism of seed dormancy in chickpea, as well as allowing comparison to phylogenetically related legumes, such as pea, lentil and faba bean, and could be utilized in chickpea breeding programs.

Histochemical Study of Human Placental Tissues in Gestational Diabetic Mellitus

Gestational diabetes mellitus (GDM) is a serious pregnancy complication in which a woman who has never had diabetes develops chronic hyperglycemia during her pregnancy. Normal placental function is essential for optimal fetal growth. The transport of glucose from the mother to the fetus is critical for fetal nutrient demands and can be stored as glycogen in the placenta. However, the function of this glycogen deposition is unknown: It may well be a source of fuel for a placenta itself or the storage reservoir for the later use by the fetus in times of need. While the significance of the placental glycogen remains unknown, the mounting evidence indicates that the altered glycogen metabolism and/or deposition is associated with many pregnancy complications, such as gestational diabetes, that adversely affect fetal development. The aim of this study is to assess glycogen deposition using Histochemical staining of Periodic Acid Schiff (PAS) stain. The placenta tissue collected from 50 women were enrolled in this study (25 women with no complications) and (25 women with gestational diabetes). The placentas of the two groups were compared in this study based on glycogen deposition with periodic acid-Schiff stain. The results of a histochemical investigation using PAS stain revealed a significant increase in the glycogen deposition (p≤0.001) in diabetic women's placentas within the intervillous core, around fetal vessels, and the basement membranes.

VCN-01 disrupts pancreatic cancer stroma and exerts antitumor effects

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplastic stroma that limits the delivery of anticancer agents. VCN-01 is an oncolytic adenovirus designed to replicate in cancer cells with a dysfunctional RB1 pathway and express hyaluronidase. Here, we evaluated the mechanism of action of VCN-01 in preclinical models and in patients with pancreatic cancer.MethodsVCN-01 replication and antitumor efficacy were evaluated alone and in combination with standard chemotherapy in immunodeficient and immunocompetent preclinical models using intravenous or intratumoral administration. Hyaluronidase activity was evaluated by histochemical staining and by measuring drug delivery into tumors. In a proof-of-concept clinical trial, VCN-01 was administered intratumorally to patients with PDAC at doses up to 1×1011 viral particles in combination with chemotherapy. Hyaluronidase expression was measured in serum by an ELISA and its activity within tumors by endoscopic ultrasound elastography.ResultsVCN-01 replicated in PDAC models and exerted antitumor effects which were improved when combined with chemotherapy. Hyaluronidase expression by VCN-01 degraded tumor stroma and facilitated delivery of a variety of therapeutic agents such as chemotherapy and therapeutic antibodies. Clinically, treatment was generally well-tolerated and resulted in disease stabilization of injected lesions. VCN-01 was detected in blood as secondary peaks and in post-treatment tumor biopsies, indicating virus replication. Patients had increasing levels of hyaluronidase in sera over time and decreased tumor stiffness, suggesting stromal disruption.ConclusionsVCN-01 is an oncolytic adenovirus with direct antitumor effects and stromal disruption capabilities, representing a new therapeutic agent for cancers with dense stroma.Trial registration numberEudraCT number: 2012-005556-42 and NCT02045589.