Primary and Immortalized Cultures of Human Proximal Tubule Cells Possess Both Progenitor and Non-Progenitor Cells That Can Impact Experimental Results.

Studies have reported the presence of renal proximal tubule specific progenitor cells which co-express PROM1 and CD24 markers on the cell surface. The RPTEC/TERT cell line is a telomerase-immortalized proximal tubule cell line that expresses two populations of cells, one co-expressing PROM1 and CD24 and another expressing only CD24, identical to primary cultures of human proximal tubule cells (HPT). The RPTEC/TERT cell line was used by the authors to generate two new cell lines, HRTPT co-expressing PROM1 and CD24 and HREC24T expressing only CD24. The HRTPT cell line has been shown to express properties expected of renal progenitor cells while HREC24T expresses none of these properties. The HPT cells were used in a previous study to determine the effects of elevated glucose concentrations on global gene expression. This study showed the alteration of expression of lysosomal and mTOR associated genes. In the present study, this gene set was used to determine if pure populations of cells expressing both PROM1 and CD24 had different patterns of expression than those expressing only CD24 when exposed to elevated glucose concentrations. In addition, experiments were performed to determine whether cross-talk might occur between the two cell lines based on their expression of PROM1 and CD24. It was shown that the expression of the mTOR and lysosomal genes was altered in expression between the HRTPT and HREC24T cell lines based on their PROM1 and CD24 expression. Using metallothionein (MT) expression as a marker demonstrated that both cell lines produced condition media that could alter the expression of the MT genes. It was also determined that PROM1 and CD24 co-expression was limited in renal cell carcinoma (RCC) cell lines.

Methanol, ethyl acetate and n-hexane extracts of Tragia involucrata L. leaves exhibit anxiolytic, sedative and analgesic activity in Swiss albino mice.

INTRODUCTION: Tragia involucrata L. have been utilized as traditional medicine in Indian subcontinent for the treatment of numerous illnesses such as inflammation, pain and skin infection. In this current study we sought to assess the anxiolytic, sedative and analgesic activity of Tragia involucrata L. leaves extract. MATERIALS AND METHODS: We first performed a phytochemical screening test of the leaves extracts following standard phytochemical screening protocols. We next examined the anxiolytic and sedative activity of crude methanol (TIME), ethyl acetate (TIEAE) and n-Hexane (TIHE) extract of Tragia involucrata L. leaves using mouse behavioral models such as elevated plus-maze test and pentobarbital-induced sleeping time test, respectively. Likewise, we evaluated the analgesic activity using acetic acid induced writhing test and formalin induced paw licking test. Additionally, we performed a quantitative analysis of heavy metals content of Tragia involucrata L. leaves by overnight digestion in concentrated nitric acid (HNO3). RESULTS: Phytochemical screening demonstrated that TIME, TIEAE and TIHE contain flavonoids, alkaloids, tannins, phenols, terpenoids and sterols. Administration of these extracts resulted in higher number of open arm entry, lower number of close arm entry and higher time spent in open arm compared to control treatment (p < 0.05). Moreover, these treatments decreased the onset of sleep time and increased the duration of sleep compared to control treated mice (all p < 0.05). Likewise, extracts treated mice exhibited decreased number of writhing as well as lower acute phase and late phase duration compared to control treatment (all p < 0.05). The average level of As and Fe in Tragia involucrata L. leaves was 5.16 ± 0.012 ppm and 2.76 ± 0.015 ppm, respectively. CONCLUSION: Results from this study support that Tragia involucrata L. leaves extracts exhibit an anxiolytic, sedative and analgesic activity in mice.

First report of Pythium ultimum causing damping-off of Sugar beet (Beta vulgaris. L) in Montana, USA.

Sugar beet (Beta vulgaris L.) is a globally important crop for sugar. In May 2019, sugar beet seedlings were observed with wilting, lodging and a few were dead in Glendive (46.970170, -104.838204), Montana. Symptoms appeared near the soil line as the stem (hypocotyl) turned dark brown to black with characteristic thread-like infections which resembled Pythium damping-off. It affected approximately 10% of the growing seedlings. Diseased sugar beet root tissues were excised with a sterile scalpel and small pieces (10 mm²) were surface sterilized with 70 % ethanol for 30 seconds, rinsed twice with autoclaved water, air-dried and transferred to potato dextrose agar (PDA) media amended with pimaricin-vancomycin-PCNB (Conway, 1985). Four plates were incubated at 25° C in the dark (Masago et al., 1977) and two weeks later white, dense colony was observed (Zhang et al., 2018). The terminal smooth, globose oogonia (average 18.5 µm in diameter) and antheridia (average 14.5 × 9.5 µm) extended below the oogonium were observed via VWR N. A. 0.30 microscope. The morphological features of the four isolates were consistent with Pythium ultimum Trow (Watanabe, 2002). Genomic DNAs (NORGEN BIOTEK CORP, Fungi DNA Isolation Kit #26200) of four isolates were used for polymerase chain reaction (PCR) with the ITS6-ITS7 primers (Taheri et al., 2017). Subsequently, PCR products were flushed by E.Z.N.A ®Cycle Pure Kit, OMEGA and four samples were sent for Sanger sequencing to GenScript (GenScript, Piscataway, NJ). The sequences were identical and submitted to GenBank, NCBI (accession no. MN398593). The NCBI Blast analysis showed 100% sequence homology to Pythium ultimum with the following GenBank accessions; KF181451.1, KF181449.1 and AY598657.2. Pathogenicity test was done on sugar beet with the same isolates in the greenhouse. Two week old, pythium culture was mixed with vermiculite and perlite mixer (PRO-MIX FLX) in the plastic trays (24´´ x 15´´× 3˝), (22 °C, 75% Relaive Humidity). Sterile water (500 ml/each tray) was added in the mixer to provide sufficient moisture. Twenty seeds of cv. Hilleshog 4302 were sown in the tray, and the trays were replicated thrice with inoculated and mock treatments. Plants were watered as needed to maintain adequate soil moisture conducive for plant growth and disease development. Seven days after sowing, 50% and 100% germination was observed in the inoculated and control treatments, respectively. At the beginning of the second week, 30% post-emergence damping-off was observed in the inoculated treatments. Diseased seedlings were gently pulled out from the pots where similar symptoms were observed in the sugar beet seedlings as described previously. No incidence of disease was observed in mock-treated seedlings. Consistent reisolation of Pythium ultimum was morphologically and molecularly confirmed from the diseased seedlings, thus fulfilling Koch's postulates. Pythium spp identification is prerequisite to develop effective management of pre and post-emergence damping-off. Pythium ultimum was previously reported in Nebraska to cause sugar beet seed rot and pre-emergence damping-off (Harvenson 2006). To our knowledge, this is the first report of Pythium ultimum causing damping-off on sugar beet in the Sidney factory district in Montana.