In Vitro Interactions of Amphotericin B Combined with Non-antifungal Agents Against Rhodotorula mucilaginosa Strains

Rhodotorula species are emerging as opportunistic pathogens, causing catheter-associated fungemia in patients with compromised immunity. R. mucilaginosa is considered the most common species involved in human infections. Correct identification and susceptibility testing of Rhodotorula isolates recovered from the blood stream or central nervous system are essential to determine the best management of this unusual infection. The antifungal susceptibility tests showed that Rhodotorula was susceptible to low concentrations of amphotericin B (AMB) but was less susceptible to voriconazole. Combinations of AMB plus several non-antifungal medications were evaluated against 35 susceptible (Rm AMB-S) and resistant (Rm AMB-R) clinical Rhodotorula isolates using the broth microdilution checkerboard technique. We showed that in vitro exposure to increasing concentrations of AMB changed the susceptibility profile to these strains, which were named the Rm AMB-R group. The most synergistic interactions were AMB?+?simvastatin, followed by AMB?+?amlodipine and AMB?+?warfarin. Synergism and antagonism were observed in both groups for the combination AMB?+?cyclosporine A. AMB combined with a fluoroquinolone (AMB?+?levofloxacin) also demonstrated antagonism for the Rm AMB-S strains, but a high percentage of synergistic interactions was observed for the Rm AMB-R group. A combination drug approach can provide a different strategy to treat infections caused by AMB-resistant R. mucilaginosa.

     

Shorebirds as important vectors for plant dispersal in Europe

Shorebirds (Charadriiformes) undergo rapid migrations with potential for long‐distance dispersal (LDD) of plants. We studied the frequency of endozoochory by shorebirds in different parts of Europe covering a broad latitudinal range and different seasons. We assessed whether plants dispersed conformed to morphological dispersal syndromes. A total of 409 excreta samples (271 faeces and 138 pellets) were collected from redshank Tringa totanus, black‐winged stilt Himantopus himantopus, pied avocet Recurvirostra avosetta, northern lapwing Vanellus vanellus, Eurasian curlew Numenius arquata and black‐tailed godwit Limosa limosa in south‐west Spain, north‐west England, southern Ireland and Iceland in 2005 and 2016, and intact seeds were extracted and identified. Godwits were sampled just before or after migratory movements between England and Iceland. The germinability of seeds was tested. Intact diaspores were recovered from all bird species and study areas, and were present in 13% of samples overall. Thirteen plant families were represented, including Charophyceae and 26 angiosperm taxa. Only four species had an ‘endozoochory syndrome’. Four alien species were recorded. Ellenberg values classified three species as aquatic and 20 as terrestrial. Overall, 89% of seeds were from terrestrial plants, and 11% from aquatic plants. Average seed length was higher in redshank pellets than in their faeces. Six species were germinated, none of which had an endozoochory syndrome. Seeds were recorded during spring and autumn migration. Plant species recorded have broad latitudinal ranges consistent with LDD via shorebirds. Crucially, morphological syndromes do not adequately predict LDD potential, and more empirical work is required to identify which plants are dispersed by shorebirds. Incorporating endozoochory by shorebirds and other migratory waterbirds into plant distribution models would allow us to better understand the natural processes that facilitated colonization of oceanic islands, or to improve predictions of how plants will respond to climate change, or how alien species spread.     

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔG_bind) obtained for the Tam16 lead molecule was of ?51.43 kcal/mol. When comparing this result with the ΔG_bind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC₅₀ for Tam16 is the smallest necessary to inhibit the Pks13 (IC₅₀ = 0.19 μM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.     

Latitudinal‐diversity gradients can be shaped by biotic processes: new insights from an eco‐evolutionary model

The processes involved in shaping latitudinal‐diversity gradients (LDGs) have been a longstanding source of debate and research. Climatic, historical and evolutionary factors have all been shown to contribute to the formation of LDGs. However, meta‐analyses have shown that different clades have LDG slopes that may vary in more than one order of magnitude. Such large variation cannot be explained solely by climatic or historical factors (e.g. difference in surface area between temperate and tropical zones) given that all clades within a geographic region are subject to the same conditions. Therefore, biotic processes intrinsic to each taxonomic group could be relevant in explaining rate differences in diversity decline across latitudinal gradients among groups. In this study, we developed a model simulating multiple competing species subjected (or not) to a demographic Allee effect. We simulated the range expansion of these species across an environmental gradient to show how these two overlooked factors (competition and Allee effects) are capable of modulating LDGs. Allee effects resulted in a steeper LDG given a higher probability of local extinction and lower colonization capacity compared to species without Allee effects. Likewise, stronger competition also led to a steeper decline in species diversity compared to scenarios with weaker species antagonistic interactions. This pattern occurred mostly due to the strength of priority effects, wherein scenarios with strong competition, species that dispersed earlier in the landscape were able to secure many patches whereas late‐arriving species were progressively precluded from expanding their ranges. Overall, our results suggest that the effect of biotic processes in shaping macroecological patterns could be more important than it is currently appreciated.     

MAPK/ERK pathway inhibition is a promising treatment target for adrenocortical tumors

Unraveling molecular mechanisms that regulate tumor development and proliferation is of the utmost importance in the quest to decrease the high mortality rate of adrenocortical carcinomas (ACC). Our aim was to evaluate the role of two of the mitogen-activated protein kinase (MAPK) signaling pathways (extracellular signal-regulated protein kinases [ERKs 1/2] and p38) in the adrenocortical tumorigenesis, as well as the therapeutic potential of MAPK/ERK inhibition. ERKs 1/2 and p38 activation were evaluated in incidentalomas (INC; n = 10), benign Cushing's syndrome (BCS; n = 12), malignant Cushing's syndrome (MCS; n = 6) and normal adrenal glands (NAG; 8). ACC cell line (H295R) was used to evaluate the ability of PD184352 (0.1, 1, and 10 µM), a specific MEK-MAPK-ERK pathway inhibitor, to modulate cell proliferation, viability, metabolism, and steroidogenesis. ERKs 1/2 activation was significantly higher in MCS (2.83 ± 0.17) compared with NAG (1.00 ± 0.19 “arbitrary units”), INC (1.20 ± 0.13) and BCS (2.09 ± 0.09). Phospho-p38 expression was absent in all the MCS analyzed. MAPK/ERK kinase (MEK) inhibition with PD184352 significantly decreased proliferation as well as steroidogenesis and also increased the redox state of the H295R cells. This data suggests that MEK-MAPK-ERK signaling has a role in adrenocortical tumorigenesis that could be potentially used as a diagnostic marker for malignancy and targeted treatment in ACC.     

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L -cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G ₀ and G ₂ phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.     

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.     

A superposition free method for protein conformational ensemble analyses and local clustering based on a differential geometry representation of backbone

Here a differential geometry (DG) representation of protein backbone is explored on the analyses of protein conformational ensembles. The protein backbone is described by curvature, κ, and torsion, τ, values per residue and we propose 1) a new dissimilarity and protein flexibility measurement and 2) a local conformational clustering method. The methods were applied to Ubiquitin and c-Myb-KIX protein conformational ensembles and results show that κ\τ metric space allows to properly judge protein flexibility by avoiding the superposition problem. The d_max measurement presents equally good or superior results when compared to RMSF, especially for the intrinsically unstructured protein. The clustering method is unique as it relates protein global to local dynamics by providing a global clustering solutions per residue. The methods proposed can be especially useful to the analyses of highly flexible proteins. The software written for the analyses presented here is available at https://github.com/AMarinhoSN/FleXgeo for academic usage only.