Digging the New York City Skyline: Soil Fungal Communities in Green Roofs and City Parks

In urban environments, green roofs provide a number of benefits, including decreased urban heat island effects and reduced energy costs for buildings. However, little research has been done on the non-plant biota associated with green roofs, which likely affect their functionality. For the current study, we evaluated whether or not green roofs planted with two native plant communities in New York City functioned as habitats for soil fungal communities, and compared fungal communities in green roof growing media to soil microbial composition in five city parks, including Central Park and the High Line. Ten replicate roofs were sampled one year after planting; three of these roofs were more intensively sampled and compared to nearby city parks. Using Illumina sequencing of the fungal ITS region we found that green roofs supported a diverse fungal community, with numerous taxa belonging to fungal groups capable of surviving in disturbed and polluted habitats. Across roofs, there was significant biogeographical clustering of fungal communities, indicating that community assembly of roof microbes across the greater New York City area is locally variable. Green roof fungal communities were compositionally distinct from city parks and only 54% of the green roof taxa were also found in the park soils. Phospholipid fatty acid analysis revealed that park soils had greater microbial biomass and higher bacterial to fungal ratios than green roof substrates. City park soils were also more enriched with heavy metals, had lower pH, and lower quantities of total bases (Ca, K, and Mg) compared to green roof substrates. While fungal communities were compositionally distinct across green roofs, they did not differentiate by plant community. Together, these results suggest that fungi living in the growing medium of green roofs may be an underestimated component of these biotic systems functioning to support some of the valued ecological services of green roofs.     

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P2₁2₁2) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C222₁ with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.     

Pulmonary biocompatibility assessment of inhaled single-wall and multiwall carbon nanotubes in BALB/c mice

With the widespread application of carbon nanotubes (CNTs) in diverse commercial processes, scientists are now concerned about the potential health risk of occupational exposures. In this study, CNT-induced pulmonary toxicity was investigated by exposing BALB/c mice to aerosolized single-wall (SW) CNT and multiwall (MW) CNT (5 μg/g of mice) for 7 consecutive days in a nose-only exposure system. Microscopic studies showed that inhaled CNTs were homogeneously distributed in the mouse lung. The total number of bronchoalveolar lavage polymorphonuclear leukocytes recovered from the mice exposed to SWCNT and MWCNT (1.2 × 10(6) ± 0.52 and 9.87 × 10(5) ± 1.45; respectively) was significantly greater than control mice (5.46 × 10(5) ± 0.78). Rapid development of pulmonary fibrosis in mice that inhaled CNT was also confirmed by significant increases in the collagen level. The lactate dehydrogenase levels were increased nearly 2- and 2.4-fold in mice that inhaled SWCNT and MWCNT, respectively, as compared with control mice. In addition, exposure of CNTs to mice showed a significant (p < 0.05) reduction of antioxidants (glutathione, superoxide dismutase, and catalase) and induction of oxidants (myloperoxidase, oxidative stress, and lipid peroxidation) compared with control. Apoptosis-related proteins such as caspase-3 and -8 activities were also significantly increased in mice that inhaled CNT than in control mice. Together, this study shows that inhaled CNTs induce inflammation, fibrosis, alteration of oxidant and antioxidant levels, and induction of apoptosis-related proteins in the lung tissues to trigger cell death.     

MLST based serotype prediction for the accurate identification of non typhoidal Salmonella serovars

Molecular Biology Reports - Traditional serotyping based on the phenotypic variation of O- and H-antigen remains as the gold-standard for the identification and classification of Salmonella...     

Biofilm inhibitory effect of alginate lyases on mucoid P. aeruginosa from a cystic fibrosis patient

Chronic mucoid Pseudomonas aeruginosa infections are a major scourge in cystic fibrosis patients. Mucoid P. aeruginosa displays structured alginate-rich biofilms that are resistant to antibiotics. Here, we have assessed the efficacy of a panel of alginate lyases in combating mucoid P. aeruginosa biofilms in cystic fibrosis. Albeit we could not demonstrate alginate degradation by alginate lyases in sputum, we demonstrate that the endotypic alginate lyases, CaAly (from Cellulophaga algicola) and VspAlyVI (from Vibrio sp. QY101) and the exotypic alginate lyases, FspAlyFRB (from Falsirhodobacterium sp. alg1), and SA1-IV (from Sphingomonas sp. A1), indeed inhibit biofilm formation by a mucoid P. aeruginosa strain isolated from the sputum of a cystic fibrosis patient with comparative effect to that of the glycoside hydrolase PslG, a promising candidate for biofilm treatment. We believe that these enzymes should be explored for in vivo efficacy in future studies.     

Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²⁺ in an autaptic neuron culture system

Neurotransmitter release is triggered by membrane depolarization, Ca(2+) influx and Ca(2+) sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca(2+) dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca(2+) concentrations via UV photolysis of caged Ca(2+). We developed a culture protocol for Ca(2+) uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca(2+)-chelator nitrophenyl-EGTA and Ca(2+) indicators, and a UV flash is used to trigger Ca(2+)-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.     

Mechanism of nucleotide sensing in group II chaperonins

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.     

Regulation of phosphorylation at Ser of GluN2B receptor in the postsynaptic density

Neuronal N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) that plays essential roles in excitatory synaptic transmission is regulated by phosphorylation. However, the kinases and phosphatases involved in this regulation are not completely known. We show that the GluN2B subunit of NMDAR is phosphorylated at Ser¹³⁰³ by protein kinase C (PKC) and is dephosphorylated by protein phosphatase 1 (PP1), but not protein phosphatase 2A (PP2A) in isolated postsynaptic density (PSD). Although PSD is known to harbor PKC, PP1 and PP2A, their ability to regulate phosphorylation of GluN2B-Ser¹³⁰³ would depend on the accessibility of GluN2B-Ser¹³⁰³ to these proteins. Since PSD preparation is likely to maintain the organization of its component proteins as inside neurons, accessibility of kinases and phosphatases to GluN2B-Ser¹³⁰³in vivo would be addressed by experiments using this system. Using an antibody specific for the phosphorylated state of GluN2B-Ser¹³⁰³ we demonstrate that PP1 is the major phosphatase in rat brain PSD that can dephosphorylate the GluN2B-Ser¹³⁰³ endogenous to PSD. We also show that PKC present in PSD can phosphorylate GluN2B-Ser¹³⁰³. The events reported here might be important in regulating GluN2B-Ser¹³⁰³ phosphorylation in vivo.