AMP-activated protein kinase subunit interactions: beta1:gamma1 association requires beta1 Thr-263 and Tyr-267

AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK alpha1beta1gamma1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian alphabetagamma complex shows that beta binds alpha and gamma. Here we show that a beta1(186-270)gamma1 complex can form in the absence of detectable alpha subunit. Moreover, using alanine mutagenesis we show that beta1 Thr-263 and Tyr-267 are required for betagamma association but not alphabeta association.     

Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.     

Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.     

Determination of water permeability of paramagnetic liposomes of interest in MRI field

The water permeability of various liposome membranes has been determined at 298 K by measuring the NMR longitudinal water proton relaxation rate of vesicles encapsulating the clinically approved Gd-HPDO3A complex (HPDO3A = 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). Two basic formulations based on DPPC (dipalmitoylphosphatidylcholine) and POPC (palmitoyl-oleylphosphatidylcholine) phospholipids were selected and investigated. Furthermore, the permeability changes caused by the membrane incorporation of amphiphiles like cholesterol and/or metal complexes of interest for designing improved liposome-based MRI contrast agents, were also investigated. The incorporation of cholesterol and metal complexes bearing C18 saturated chains in POPC-based liposomes reduces the water diffusivity across the membrane bilayer. On the contrary, the incorporation of a macrocyclic metal complex bearing four C12 alkylic chains, one for each coordination arm of the ligand, considerably enhances the water permeability in DPPC-based liposomes. Finally, it is reported that the permeability of POPC-based bilayer is increased when the liposomes are subjected to an osmotic stress.     

Why students do not turn on their video cameras during online classes and an equitable and inclusive plan to encourage them to do so

Enrollment in courses taught remotely in higher education has been on the rise, with a recent surge in response to a global pandemic. While adapting this form of teaching, instructors familiar with traditional face‐to‐face methods are now met with a new set of challenges, including students not turning on their cameras during synchronous class meetings held via videoconferencing. After transitioning to emergency remote instruction in response to the COVID‐19 pandemic, our introductory biology course shifted all in‐person laboratory sections into synchronous class meetings held via the Zoom videoconferencing program. Out of consideration for students, we established a policy that video camera use during class was optional, but encouraged. However, by the end of the semester, several of our instructors and students reported lower than desired camera use that diminished the educational experience. We surveyed students to better understand why they did not turn on their cameras. We confirmed several predicted reasons including the most frequently reported: being concerned about personal appearance. Other reasons included being concerned about other people and the physical location being seen in the background and having a weak internet connection, all of which our exploratory analyses suggest may disproportionately influence underrepresented minorities. Additionally, some students revealed to us that social norms also play a role in camera use. This information was used to develop strategies to encourage—without requiring—camera use while promoting equity and inclusion. Broadly, these strategies are to not require camera use, explicitly encourage usage while establishing norms, address potential distractions, engage students with active learning, and understand your students’ challenges through surveys. While the demographics and needs of students vary by course and institution, our recommendations will likely be directly helpful to many instructors and also serve as a model for gathering data to develop strategies more tailored for other student populations.     

Role of saprophagous fly biodiversity in ecological processes and urban ecosystem services

1. Direct consumption of organic matter by the saprophagous larvae provides the ecosystem with a fundamental service by recycling nutrients and reducing exposure to decomposing matter. The present study aimed to assess the functional role of saprophagous flies in the mass loss of different types of decomposing organic matter. 2. Two types of common urban waste were used to measure the role of flies in reducing organic matter: chicken viscera (chicken) and a mixture of flour and uncooked eggs (flour and eggs), representing leftover food. Ten traps baited with each substrate, under field conditions, allowed fly access (exposed to flies) and three traps from each substrate did not (unexposed controls); adult flies entering the traps or emerging from the substrates and substrate mass loss were recorded. 3. Species from Calliphoridae, Sarcophagidae, Muscidae, and Fanniidae families were collected mainly in traps baited with chicken, with Phoridae being the most abundant in traps with flour and eggs as bait. A significantly richer (P < 0.05) assemblage of fly species accessed the traps baited with chicken viscera (21 species) compared with those emerging (11 species), whereas similar numbers of species accessed (n = 5) or emerged (n = 1) from traps baited with flour and eggs (average richness accessing 7.97, emerging 2.83). Chicken substrate mass loss and species richness were positively related (r = 0.56, P = 0.001). In traps where richness was larger than 10 species, the substrates were reduced by more than 85% of their initial weight compared with unexposed controls, which lost 30%. Substrate mass loss significantly increased with the abundance of flies (r = 0.73, P < 0.0001). 4. The results of the present study support the functional role of saprophagous species diversity on the decomposition rates of organic matter, reinforcing the negative consequences of loss or gain of species in modified landscapes and for ecosystem function.     

Flurbiprofen release from eudragit RS and RL aqueous nanosuspensions: a kinetic study by DSC and dialysis experiments

The present work investigated the release of Flurbiprofen (FLU) from Eudragit RS100 (RS) and Eudragit RL100 (RL) nanosuspensions to a biological model membrane consisting of Dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV). This release was compared with those observed from solid drug particles as well as with dialysis experiments. Nanosuspensions were prepared by a modification of Quasi-Emulsion Solvent Diffusion technique. Drug release was monitored by the Differential Scanning Calorimetry (DSC). FLU dispersed in MLV affects the transition temperature (T(m)) of DMPC liposomes, causing a shift towards lower values. The temperature shift is modulated by the drug fraction present in the aqueous lipid bilayer suspension. DSC was also performed, after increasing incubation periods at 37 degrees C, on suspensions of blank liposomes added to fixed amounts of unloaded and FLU-loaded nanosuspensions, as well as to powdered free drug. T(m) shifts, caused by the drug released from the polymeric system or by free-drug dissolution during incubation cycles, were compared with those caused by free drug increasing molar fractions dispersed directly in the membrane during their preparation. These results were compared with the drug release and were followed by a classical dialysis technique. Comparing the suitability of the 2 different techniques in order to follow the drug release as well as the differences between the 2 RL and RS polymer systems, it is possible to confirm the efficacy of DSC in studying the release from polymeric nanoparticulate systems compared with the "classical" release test by dialysis. The different rate of kinetic release could be due to void liposomes, which represent a better uptaking system than aqueous solution in dialysis experiments.     

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.     

Immunolocalization of alpha-fetoprotein in the ovary and hypophysis of immature female rats

Summary Alpha-fetoprotein (AFP), an oestrogen-binding protein, has been localized in the ovary and hypophysis of prepuberal rats. In the ovary, AFP was localized in secondary follicles, its distribution being limited to the liquor folliculi and the zona pellucida. In the hypophysis, AFP was only found in blood vessels. The intra-ovarian, localization of the protein is consistent with our previous hypothesis that AFP might act as a regulator of ovarian activity by decreasing the local free oestradiol level.