Laccases Involved in 1,8-Dihydroxynaphthalene Melanin Biosynthesis in Aspergillus fumigatus Are Regulated by Developmental Factors and Copper Homeostasis

Aspergillus fumigatus produces heavily melanized infectious conidia. The conidial melanin is associated with fungal virulence and resistance to various environmental stresses. This 1,8-dihydroxynaphthalene (DHN) melanin is synthesized by enzymes encoded in a gene cluster in A. fumigatus, including two laccases, Abr1 and Abr2. Although this gene cluster is not conserved in all aspergilli, laccases are critical for melanization in all species examined. Here we show that the expression of A. fumigatus laccases Abr1/2 is upregulated upon hyphal competency and drastically increased during conidiation. The Abr1 protein is localized at the surface of stalks and conidiophores, but not in young hyphae, consistent with the gene expression pattern and its predicted role. The induction of Abr1/2 upon hyphal competency is controlled by BrlA, the master regulator of conidiophore development, and is responsive to the copper level in the medium. We identified a developmentally regulated putative copper transporter, CtpA, and found that CtpA is critical for conidial melanization under copper-limiting conditions. Accordingly, disruption of CtpA enhanced the induction of abr1 and abr2, a response similar to that induced by copper starvation. Furthermore, nonpigmented ctpAΔ conidia elicited much stronger immune responses from the infected invertebrate host Galleria mellonella than the pigmented ctpAΔ or wild-type conidia. Such enhancement in eliciting Galleria immune responses was independent of the ctpAΔ conidial viability, as previously observed for the DHN melanin mutants. Taken together, our findings indicate that both copper homeostasis and developmental regulators control melanin biosynthesis, which affects conidial surface properties that shape the interaction between this pathogen and its host.     

A Phase II,Randomized, Double-Blind,Placebo Controlled,Dose-Response Trial of the Melatonin Effect on the Pain Threshold of Healthy Subjects

Background

Previous studies have suggested that melatonin may produce antinociception through peripheral and central mechanisms. Based on the preliminary encouraging results of studies of the effects of melatonin on pain modulation, the important question has been raised of whether there is a dose relationship in humans of melatonin on pain modulation.

Objective

The objective was to evaluate the analgesic dose response of the effects of melatonin on pressure and heat pain threshold and tolerance and the sedative effects.

Methods

Sixty-one healthy subjects aged 19 to 47 y were randomized into one of four groups: placebo, 0.05 mg/kg sublingual melatonin, 0.15 mg/kg sublingual melatonin or 0.25 mg/kg sublingual melatonin. We determine the pressure pain threshold (PPT) and the pressure pain tolerance (PPTo). Quantitative sensory testing (QST) was used to measure the heat pain threshold (HPT) and the heat pain tolerance (HPTo). Sedation was assessed with a visual analogue scale and bispectral analysis.

Results

Serum plasma melatonin levels were directly proportional to the melatonin doses given to each subject. We observed a significant effect associated with dose group. Post hoc analysis indicated significant differences between the placebo vs. the intermediate (0.15 mg/kg) and the highest (0.25 mg/kg) melatonin doses for all pain threshold and sedation level tests. A linear regression model indicated a significant association between the serum melatonin concentrations and changes in pain threshold and pain tolerance (R² = 0.492 for HPT, R² = 0.538 for PPT, R² = 0.558 for HPTo and R² = 0.584 for PPTo).

Conclusions

The present data indicate that sublingual melatonin exerts well-defined dose-dependent antinociceptive activity. There is a correlation between the plasma melatonin drug concentration and acute changes in the pain threshold. These results provide additional support for the investigation of melatonin as an analgesic agent. Brazilian Clinical Trials Registry (ReBec): (U1111-1123-5109). IRB: Research Ethics Committee at the Hospital de Clínicas de Porto Alegre.     

Novel Pancreatic Cancer Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic Adenocarcinoma: Applications in Diagnosis and Therapy

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras^G12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of Kras^G12D;Trp53^R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. Kras^G12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.     

Modelling potential impacts of climate change on the bioclimatic envelope and conservation of the Maned Wolf (Chrysocyon brachyurus)

Forecasting the influence of climatic changes on the distribution of the Maned Wolf (Chrysocyon brachyurus) is important for the conservation of the species. We explored the environmental characteristics than best explain the current distribution of the species, modelled the past and present distribution, projected the niche model into the future, and identified suitable areas for conservation. Niche modelling was performed using Maxent and 21 environmental variables. For past conditions, we considered the Last Glacial Maximum (LGM) and the mid-Holocene (MH) climates. For future conditions, we used the A2a greenhouse gas emission scenario for 2050. Four General Circulation Models (FGOALS 1.0, HADCM3, IPSL-CM4 and MIROC 3.2) were used. The resulting niche model (AUC = 0.89 ± 0.02) predicts maximum probability of presence at precipitation of 106 mm during the coldest quarter, of 396 mm during the warmest quarter, and in totally flat areas. The suitable area for the Maned Wolf currently covers 4,320,364 km². For the LGM, there were inter-model differences in predicted areas (from 819,324 km² to 6,395,886 km²) and in geographic location. The MH models showed drastic changes with respect to the present and considerable inter-model variation. Predictions for 2050 show significant (at least 33%) reductions in distribution. Only a minor fraction (39%) of the current distribution can be considered stable for the period LGM-2050. The FGOALS model was the best option for projecting species occurrence into the future because it included the three localities known for the Maned Wolf from the late Pleistocene and predicts stable areas that coincide with spatial patterns of genetic diversity. The FGOALS projection for 2050 predicts a 33% reduction in suitable habitats, indicating some stable areas (central South America) that will probably be key sites for the conservation of the species.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Organic Codes: A Unifying Concept for Life

Although the knowledge about biological systems has advanced exponentially in recent decades, it is surprising to realize that the very definition of Life keeps presenting theoretical challenges. Even if several lines of reasoning seek to identify the essence of life phenomenon, most of these thoughts contain fundamental problem in their basic conceptual structure. Most concepts fail to identify either necessary or sufficient features to define life. Here, we analyzed the main conceptual frameworks regarding theoretical aspects that have been supporting the most accepted concepts of life, such as (i) the physical, (ii) the cellular and (iii) the molecular approaches. Based on an ontological analysis, we propose that Life should not be positioned under the ontological category of Matter. Yet, life should be better understood under the top-level ontology of “Process”. Exercising an epistemological approach, we propose that the essential characteristic that pervades each and every living being is the presence of organic codes. Therefore, we explore theories in biosemiotics and code biology in order to propose a clear concept of life as a macrocode composed by multiple inter-related coding layers. This way, as life is a sort of metaphysical process of encoding, the living beings became the molecular materialization of that process. From the proposed concept, we show that the evolutionary process is a fundamental characteristic for life’s maintenance but it is not necessary to define life, as many organisms are clearly alive but they do not participate in the evolutionary process (such as infertile hybrids). The current proposition opens a fertile field of debate in astrobiology, epistemology, biosemiotics, code biology and robotics.