Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Rediscovery of Polypodium calirhiza (Polypodiaceae) in Mexico

This study addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The tetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. Our analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum, and we confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico: P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands, whereas P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.     

Characterizing the interactions between the two subunits of the p101/p110gamma phosphoinositide 3-kinase and their role in the activation of this enzyme by G beta gamma subunits.

Recently, we have reported the purification and cloning of a novel G protein betagamma subunit-activated phosphoinositide 3-kinase from pig neutrophils. The enzyme comprises a p110gamma catalytic subunit and a p101 regulatory subunit. Now we have cloned the human ortholog of p101 and generated panels of p101 and p110gamma truncations and deletions and used these in in vitro and in vivo assays to determine the protein domains responsible for subunit interaction and activation by betagamma subunits. Our results suggest large areas of p101 including both N- and C-terminal portions interact with the N-terminal half of p110gamma. While modifications of the N terminus of p110gamma could modulate its intrinsic catalytic activity, binding to the N-terminal region of p101 was found to be indispensable for activation of heterodimers with Gbetagamma.     

Phylogenetic relationships of the enigmatic fern families Hymenophyllopsidaceae and Lophosoriaceae: Evidence fromrbcL nucleotide sequences

Nucleotide sequences fromrbcL were used to infer relationships of Lophosoriaceae and Hymenophyllopsidaceae. The phylogenetic positions of these two monotypic fern families have been debated, and neither group had been included in recent molecular systematic studies of ferns. Maximum parsimony analysis of our data supported a sister relationship betweenLophosoria andDicksonia, and also betweenHymenophyllopsis andCyathea. Thus, both newly-examined families appear to be part of a previously characterized and well-supported clade of tree ferns. The inferred relationships ofLophosoria are consistent with most (but not all) recent treatments. However,Hymenophyllopsis includes only small delicate plants superficially similar to filmy ferns (Hymenophyllaceae), very different from the large arborescent taxa. Nevertheless, some synapomorphic characteristics are shared with the tree fern clade. Further studies on gametophytes ofHymenophyllopsis are needed to test these hypotheses of relationship.     

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

The evolution of chloroplast genes and genomes in ferns

Most of the publicly available data on chloroplast (plastid) genes and genomes come from seed plants, with relatively little information from their sister group, the ferns. Here we describe several broad evolutionary patterns and processes in fern plastid genomes (plastomes), and we include some new plastome sequence data. We review what we know about the evolutionary history of plastome structure across the fern phylogeny and we compare plastome organization and patterns of evolution in ferns to those in seed plants. A large clade of ferns is characterized by a plastome that has been reorganized with respect to the ancestral gene order (a similar order that is ancestral in seed plants). We review the sequence of inversions that gave rise to this organization. We also explore global nucleotide substitution patterns in ferns versus those found in seed plants across plastid genes, and we review the high levels of RNA editing observed in fern plastomes.     

Characterization of X-ray Dose in Murine Animals Using microCT,a New Low-Dose Detector and nanoDot Dosimeters

Background

Dose continues to be an area of concern in preclinical imaging studies, especially for those imaging disease progression in longitudinal studies. To our knowledge, this work is the first to characterize and assess dose from the Inveon CT imaging platform using nanoDot dosimeters. This work is also the first to characterize a new low-dose configuration available for this platform.

Methodology/Principal Findings

nanoDot dosimeters from Landauer, Inc. were surgically implanted into 15 wild type mice. Two nanoDots were placed in each animal: one just under the skin behind the spine and the other located centrally within the abdomen. A manufacturer-recommended CT protocol was created with 1 projection per degree of rotation acquired over 360 degrees. For best comparison of the low dose and standard configurations, noise characteristics of the reconstructed images were used to match the acquisition protocol parameters. Results for all dose measurements showed the average dose delivered to the abdomen to be 13.8 cGy±0.74 and 0.97 cGy±0.05 for standard and low dose configurations respectively. Skin measurements of dose averaged 15.99 cGy±0.72 and 1.18 cGy±0.06. For both groups, the standard deviation to mean was less than 5.6%. The maximum dose received for the abdomen was 15.12 cGy and 0.97 cGy while the maximum dose for the skin was 17.3 cGy and 1.32 cGy. Control dosimeters were used for each group to validate that no unwanted additional radiation was present to bias the results.

Conclusions/Significance

This study shows that the Inveon CT platform is suitable for imaging mice both for single and longitudinal studies. Use of low-dose detector hardware results in significant reductions in dose to subjects with a >12x (90%) reduction in delivered dose. Installation of this hardware on another in vivo microCT platform resulted in dose reductions of over 9x (89%).