Models of Ternary Complexes for Nonpeptidic Farnesyltransferase Inhibitors: Insights into Structure-Based Screen and Design of Potential Anticancer Therapeutics

Farnesyltransferase (FT) inhibitors can repress tumor cell proliferation without substantially interfering with normal cell growth and are thus promising in cancer treatment. A detailed knowledge of how substrates and inhibitors bind to FT at the atomic level can expedite screening and rational design of improved FT inhibitors. Here we report theoretical models of the FT complexed with FPP and the potent nonpeptidic inhibitor SCH 56580 and other inhibitor-FPP-FT ternary complexes derived from the docking studies prior to any crystal structures of the FT liganded with nonpeptidic inhibitors. On the basis of these models we evaluate the roles of FPP, Zn²⁺ and the zinc-coordinated water molecule in inhibitor binding, and propose the structural determinants of binding of nonpeptidic FT inhibitors. Furthermore, we suggest the use of the FPP-FT binary complex as a novel and effective drug target structure for screening and rational design of improved FT inhibitors.     

Interplay between mobility,multi-seeding and lockdowns shapes COVID-19 local impact

Assessing the impact of mobility on epidemic spreading is of crucial importance for understanding the effect of policies like mass quarantines and selective re-openings. While many factors affect disease incidence at a local level, making it more or less homogeneous with respect to other areas, the importance of multi-seeding has often been overlooked. Multi-seeding occurs when several independent (non-clustered) infected individuals arrive at a susceptible population. This can lead to independent outbreaks that spark from distinct areas of the local contact (social) network. Such mechanism has the potential to boost incidence, making control efforts and contact tracing less effective. Here, through a modeling approach we show that the effect produced by the number of initial infections is non-linear on the incidence peak and peak time. When case importations are carried by mobility from an already infected area, this effect is further enhanced by the local demography and underlying mixing patterns: the impact of every seed is larger in smaller populations. Finally, both in the model simulations and the analysis, we show that a multi-seeding effect combined with mobility restrictions can explain the observed spatial heterogeneities in the first wave of COVID-19 incidence and mortality in five European countries. Our results allow us for identifying what we have called epidemic epicenter: an area that shapes incidence and mortality peaks in the entire country. The present work further clarifies the nonlinear effects that mobility can have on the evolution of an epidemic and highlight their relevance for epidemic control.     

A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes

The efficient activation of p90^rsk by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90^rsk. The MAP kinase p42^mpk1 can associate with p90^rsk in G₂-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90^rsk mutant named D2 constitutively interacts with p42^mpk1. In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42^mpk1 docking site. D2 expression uncouples the activation of p42^mpk1 and p34^cdc2/cyclin B in response to progesterone but does not prevent signaling through p90^rsk. Instead, D2 interferes with a p42^mpk1-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42^mpk1 and Plx1 during oocyte maturation is independent of p34^cdc2/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42^mpk1 can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.     

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.     

Attenuation of induced bronchoconstriction in healthy subjects: effects of breathing depth.

The effects of breathing depth in attenuating induced bronchoconstriction were studied in 12 healthy subjects. On four separate, randomized occasions, the depth of a series of five breaths taken soon (approximately 1 min) after methacholine (MCh) inhalation was varied from spontaneous tidal volume to lung volumes terminating at approximately 80, approximately 90, and 100% of total lung capacity (TLC). Partial forced expiratory flow at 40% of control forced vital capacity (V(part)) and residual volume (RV) were measured at control and again at 2, 7, and 11 min after MCh. The decrease in V(part) and the increase in RV were significantly less when the depth of the five-breath series was progressively increased (P < 0.001), with a linear relationship. The attenuating effects of deep breaths of any amplitude were significantly greater on RV than V(part) (P < 0.01) and lasted as long as 11 min, despite a slight decrease with time when the end-inspiratory lung volume was 100% of TLC. In conclusion, in healthy subjects exposed to MCh, a series of breaths of different depth up to TLC caused a progressive and sustained attenuation of bronchoconstriction. The effects of the depth of the five-breath series were more evident on the RV than on V(part), likely due to the different mechanisms that regulate airway closure and expiratory flow limitation.     

High level of genetic differentiation in the marine isopod Sphaeroma terebrans (Crustacea Isopoda Sphaeromatidae) as inferred by mitochondrial DNA analysis

Sphaeroma terebrans Bate 1866 is a marine isopod belonging to the large family Sphaeromatidae, which normally colonises the aerial roots of the mangrove genus Rhizophora in tropical and subtropical areas. S. terebrans is part of a group of species whose complete life cycle occurs within the same mangrove wood. In this paper, we provide clear evidence of significant genetic differentiation among geographic populations of the taxon S. terebrans. The consistently low internal variation and the large interpopulation distances indicate that almost all the mitochondrial variation (cytochrome oxidase I) in S. terebrans is apportioned among populations rather than within them. The mean haplotype diversity (h) is 0.71%, and the mean nucleotide diversity (π) is 0.34%. The Minimum Spanning Tree (MST) reveals a complex pattern: three principal haplotype groups corresponding to the geographic locations investigated are distributed in a network. This suggests an ancient evolutionary history and very restricted gene flow between populations. The large genetic distances between the populations of S. terebrans could suggest that this taxon is not a single species but a species complex whose taxonomic status must be revaluated.     

Association of inflammatory markers elevation with aggressive behavior in domestic dogs

We tested the hypothesis that elevations of inflammatory markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) could be associated with the presence of aggressive behavior in domestic dogs. Serum concentrations of CRP and IL-6 were determined by ELISA in eighteen adult male German Shepherd dogs showing no clinical signs but aggression. Eighteen healthy male dogs with a negative history of behavioral and neurological disorders were used as controls. Compared with normal dogs, those with aggression had significantly higher levels of CRP (P < 0.05) and IL-6 (P < 0.01) after adjustment for age, body weight, creatinine, blood urea nitrogen, total protein, total bilirubin and cholesterol. Our pilot data suggest for the first time that an activation of systemic inflammatory processes may contribute to the pathophysiology of aggression in domestic dogs. Further investigations are needed regarding the impact of our findings on treatment strategies.