Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.     

Behavioural and genetic interactions between an endangered and a recently-arrived hummingbird

The invasion or expansion of non-native species into new geographic areas can pose a major threat to the conservation of biodiversity. These threats are augmented when the newly-arrived species interacts with native species that are already threatened by other ecological or anthropogenic processes. Potential interactions can include both competition for scarce resources and reproductive interference, including hybridisation. Understanding the dynamics of these interactions forms a crucial component of conservation management strategies. A recent contact zone occurs in the north of Chile between the endangered Chilean woodstar (Eulidia yarrellii) and the closely-related and recently-arrived Peruvian sheartail (Thaumastura cora), which expanded its range from Peru into Chile during the 1970s. We characterised the interactions between the species by combining population size estimates with molecular, morphological and behavioural data. We show that a low degree of hybridisation, but not introgression, is occurring between the two species. Despite interspecific morphological similarities, behavioural observations indicate that food niche overlap between the species is relatively low, and that the dietary breadth of sheartails is larger, which may have aided the species’ range expansion. Finally, woodstars dominate the sheartails in male–male territorial interactions. However, potentially increased energetic costs for woodstars associated with frequent territorial chases and courtship displaying with sheartails may exacerbate the effects of other threats on woodstar viability, such as human-induced habitat modification. This study highlights the value of implementing multidisciplinary approaches in conservation biology to gain a more complete understanding of interactions between recently-arrived and endangered species.     

Insights into the complex formed by matrix metalloproteinase-2 and alloxan inhibitors: molecular dynamics simulations and free energy calculations

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S(1)' specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S(1)' subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC(50) = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases.     

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3 pM for TREM-1, 1.1 nM for MMP-9 and 1.4 nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody-antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent proteasome and immunoproteasome inhibitors

This paper describes the design, synthesis, and biological evaluation of 2-thioxoimidazolidin-4-one derivatives as inhibitors of proteasome and immunoproteasome, potential targets for the treatment of hematological malignancies. In particular, we focused our efforts on the design of noncovalent inhibitors, which might be a promising therapeutic option potentially devoid of drawbacks and side-effects related to irreversible inhibition. Among all the synthesized compounds, we identified a panel of active inhibitors with K_i values towards one or two chymotrypsin-like activities of proteasome (β5c) and immunoproteasome (β5i and β1i subunits) in the low micromolar range. Docking studies suggested a unique binding mode of the molecules in the catalytic site of immunoproteasome proteolytic subunits.     

Toward High‐Temperature Stability of PTB7‐Based Bulk Heterojunction Solar Cells: Impact of Fullerene Size and Solvent Additive

The use of fullerene as acceptor limits the thermal stability of organic solar cells at high temperatures as their diffusion inside the donor leads to phase separation via Ostwald ripening. Here it is reported that fullerene diffusion is fully suppressed at temperatures up to 140 °C in bulk heterojunctions based on the benzodithiophene‐based polymer (the poly[[4,8‐bis[(2‐ethylhexyl)oxy]‐benzo[1,2‐b:4,5‐b′]dithiophene‐2,6‐diyl][3‐fluoro‐2‐[(2‐ethylhexyl)carbonyl]‐thieno[3,4‐b]thiophenediyl]], (PTB7) in combination with the fullerene derivative [6,6]‐phenyl‐C71‐butyric acid methyl ester (PC₇₀BM). The blend stability is found independently of the presence of diiodooctane (DIO) used to optimize nanostructuration and in contrast to PTB7 blends using the smaller fullerene derivative PC₇₀BM. The unprecedented thermal stability of PTB7:PC₇₀BM layers is addressed to local minima in the mixing enthalpy of the blend forming stable phases that inhibit fullerene diffusion. Importantly, although the nanoscale morphology of DIO processed blends is thermally stable, corresponding devices show strong performance losses under thermal stress. Only by the use of a high temperature annealing step removing residual DIO from the device, remarkably stable high efficiency solar cells with performance losses less than 10% after a continuous annealing at 140 °C over 3 days are obtained. These results pave the way toward high temperature stable polymer solar cells using fullerene acceptors.     

Benzimidazole-based derivatives as privileged scaffold developed for the treatment of the RSV infection: a computational study exploring the potency and cytotoxicity profiles

Respiratory syncytial virus (RSV) has been identified as a main cause of hospitalisation in infants and children. To date, the current therapeutic arsenal is limited to ribavirin and palivizumab with variable efficacy. In this work, starting from a number of in-house series of previously described anti-RSV agents based on the benzimidazole scaffold, with the aim at gaining a better understanding of the related chemical features involved in potency and safety profiles, we applied a computational study including two focussed comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The results allowed us to derive useful suggestions for the design of derivatives and also to set up statistical models predicting the potency and selectivity index (SI?=?CC₅₀/EC₅₀) of any new analogue prior to synthesis. Accordingly, here, we discuss preliminary results obtained through the applied exhaustive QSAR analyses, leading to design and synthesise more effective anti-RSV agents.