Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-β) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.     

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.     

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.     

Growth patterns as deduced from bone microstructure of some selected neotherapsids with special emphasis on dicynodonts: Phylogenetic implications

Osteohistological analysis of the dicynodonts Endothiodon, Diictodon, Lystrosaurus and Wadiasaurus reveals distinctly different growth patterns within a framework of an overall fast growth. The late Permian endemic taxon from India, Endothiodon mahalanobisi and the South African Diictodon feliceps had periodic fast growth. The early Triassic Lystrosaurus murrayi and the middle Triassic Wadiasaurus indicus had an initial fast growth followed by a relatively slow growth later in ontogeny as is observed from the presence of peripheral parallel fibred bone. Although all examined dicynodont genera had an indeterminate growth strategy, the bone microstructure of Wadiasaurus suggests that its growth was much slower than that of other dicynodonts examined. Mapping of osetohistological character states on a cladogram depicting the inter-relationship between available neotherapsid genera shows that fibrolamellar bone tissue, overall fast growth and indeterminate growth strategy were plesiomorphic for the neotherapsids. A considerable reduction in developmental plasticity and evolution of apparently independent growth trajectories from environmental conditions are evident within the non-mammalian cynodonts, with the advanced tritylodontids achieving almost a mammalian growth trajectory.     

Review of inulinase production using solid-state fermentation

The purpose of the present study is to critically analyze the recent literature covering the production of inulinase enzyme from various sources by solid-state fermentation and discuss various approaches to increase its production in solid-state fermentation, purification, and its properties. The review deals with the solid-state fermentative production of inulinase production. Inulinases have many applications in industries, such as for the production of ultra-high fructose syrup, biofuels, lactic acid, citric acid, and single-cell oil. Solid-state fermentation (SSF) is more economic, requires smaller vessels, lowers water intake, reduces wastewater treatments, higher product yield, lesser chance of bacterial contamination, and lowers energy consumption. Furthermore, the crude products obtained from SSF can be directly used as the source of enzyme for biotransformation. Although many reports are available on a wide range of microbes which produces inulinases by SSF, it is important to isolate novel microbes for its production. Also, extensive research is going on to exploit unexplored sources for SSF. Higher yield of inulinases can be achieved by bioreactor modeling and proper monitoring of physical and chemical parameters in SSF.     

Comparative study of metal uptake by Eichhornia crassipes growing in ponds from mining and nonmining areas—a field study

The study deals with metal (Cu, Mn, Pb, Cd) concentrations in sediment, water, and corresponding leaf samples of Eichhornia crassipes obtained from ponds in nonmining (P1) and mining (P2, P3, P4) regions. In spite of significant high metal concentrations in sediments from mining regions rather than from nonmining regions, the unelevated SQG-I (sediment quality guideline index) values proved low levels of toxicity. Irrespective of the wide range of metal concentration in sediments, the levels in water had been nearly consistent in all the ponds. Concentration of metals in leaves decreased with an increase in concentration in the substrate. Mn, Cu, and Cd accumulated within the range of MAC (maximum allowable concentration) for plants, whereas Pb accumulated above the limit. BAF_sl (bioaccumulation factor with respect to sediment) values for Mn (0.20–0.27) were highest, followed by Cu (0.13–0.20) and Pb (0.03–0.20), whereas BAF_wl (bioaccumulation factor with respect to water) was highest for Cu (428–3205), followed by Mn (285–1100), Pb (242–506), and Cd (7–130). This study concludes that E. crassipes plays a very important role in removing the metals from the pond ecosystem, whereas leaves of this plant can be used effectively for biomonitoring surveys. E. crassipes can be used for phytoremediation of polluted wetlands through proper management strategies.     

Mitochondrial Genome Analysis of Primary Open Angle Glaucoma Patients

Primary open angle glaucoma (POAG) is a multi-factorial optic disc neuropathy characterized by accelerating damage of the retinal ganglion cells and atrophy of the optic nerve head. The vulnerability of the optic nerve damage leading to POAG has been postulated to result from oxidative stress and mitochondrial dysfunction. In this study, we investigated the possible involvement of the mitochondrial genomic variants in 101 patients and 71 controls by direct sequencing of the entire mitochondrial genome. The number of variable positions in the mtDNA with respect to the revised Cambridge Reference Sequence (rCRS), have been designated “Segregating Sites”. The segregating sites present only in the patients or controls have been designated “Unique Segregating Sites (USS)”. The population mutation rate (θ = 4N_eμ) as estimated by Watterson’s θ (θ_w), considering only the USS, was significantly higher among the patients (p = 9.8×10⁻¹⁵) compared to controls. The difference in θ_w and the number of USS were more pronounced when restricted to the coding region (p<1.31×10⁻²¹ and p = 0.006607, respectively). Further analysis of the region revealed non-synonymous variations were significantly higher in Complex I among the patients (p = 0.0053). Similar trends were retained when USS was considered only within complex I (frequency 0.49 vs 0.31 with p<0.0001 and mutation rate p-value <1.49×10⁻⁴³) and ND5 within its gene cluster (frequency 0.47 vs 0.23 with p<0.0001 and mutation rate p-value <4.42×10⁻⁴⁷). ND5 is involved in the proton pumping mechanism. Incidentally, glaucomatous trabecular meshwork cells have been reported to be more sensitive to inhibition of complex I activity. Thus mutations in ND5, expected to inhibit complex I activity, could lead to generation of oxidative stress and favor glaucomatous condition.