Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H⁺-F_OF₁-ATP synthase couples proton flow through its membrane portion, F_O, to the synthesis of ATP in its headpiece, F₁. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.     

Skeleton binding protein-1-mediated parasite sequestration inhibits spontaneous resolution of malaria-associated acute respiratory distress syndrome

Malaria is a hazardous disease caused by Plasmodium parasites and often results in lethal complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Parasite sequestration in the microvasculature is often observed, but its role in malaria pathogenesis and complications is still incompletely understood. We used skeleton binding protein-1 (SBP-1) KO parasites to study the role of sequestration in experimental MA-ARDS. The sequestration-deficiency of these SBP-1 KO parasites was confirmed with bioluminescence imaging and by measuring parasite accumulation in the lungs with RT-qPCR. The SBP-1 KO parasites induced similar lung pathology in the early stage of experimental MA-ARDS compared to wildtype (WT) parasites. Strikingly, the lung pathology resolved subsequently in more than 60% of the SBP-1 KO infected mice, resulting in prolonged survival despite the continuous presence of the parasite. This spontaneous disease resolution was associated with decreased inflammatory cytokine expression measured by RT-qPCR and lower expression of cytotoxic markers in pathogenic CD8⁺ T cells in the lungs of SBP-1 KO infected mice. These data suggest that SBP-1-mediated parasite sequestration and subsequent high parasite load are not essential for the development of experimental MA-ARDS but inhibit the resolution of the disease.     

Individual variation in pup vocalizations and absence of behavioral signs of maternal vocal recognition in Weddell seals (Leptonychotes weddellii)

Individually stereotyped vocalizations often play an important role in relocation of offspring in gregarious breeders. In phocids, mothers often alternate between foraging at sea and attending their pup. Pup calls are individually distinctive in various phocid species. However, experimental evidence for maternal recognition is rare. In this study, we recorded Weddell seal (Leptonychotes weddellii) pup vocalizations at two whelping patches in Atka Bay, Antarctica, and explored individual vocal variation based on eight vocal parameters. Overall, 58% of calls were correctly classified according to individual. For males (n= 12) and females (n= 9), respectively, nine and seven individuals were correctly identified based on vocal parameters. To investigate whether mothers respond differently to calls of familiar vs. unfamiliar pups, we conducted playback experiments with 21 mothers. Maternal responses did not differ between playbacks of own, familiar, and unfamiliar pup calls. We suggest that Weddell seal pup calls may need to contain only a critical amount of individually distinct information because mothers and pups use a combination of sensory modalities for identification. However, it cannot be excluded that pup developmental factors and differing environmental factors between colonies affect pup acoustic behavior and the role of acoustic cues in the relocation process.     

Desflurane consumption during automated closed-circuit delivery is higher than when a conventional anesthesia machine is used with a simple vaporizer-O2-N2O fresh gas flow sequence

Background

Current analgesics have drawbacks such as delays in acquisition, lag-times for effect, and side effects. We recently presented a preliminary report of a new analgesic method involving a two-minute sciatic nerve press, which resulted in immediate short-term relief of pain associated with dental and renal diseases. The present study investigated whether this technique was effective for pain associated with other disease types, and whether the relief was effective for up to one hour.

Methods

This randomized, placebo-controlled, parallel-group trial was conducted in four hospitals in Anhui Province, China. Patients with pain were sequentially recruited by participating physicians during clinic visits, and 135 patients aged 15 – 80 years were enrolled. Dental disease patients included those with acute pulpitis and periapical abscesses. Renal disease patients included those with kidney infections and/or stones. Tumor patients included those with nose, breast, stomach and liver cancers, while Emergency Room patients had various pathologies. Patients were randomly assigned to receive a "sciatic nerve press" in which pressure was applied simultaneously to the sciatic nerves at the back of both thighs, or a "placebo press" in which pressure was applied to a parallel region on the front of the thighs. Each fist applied a pressure of 11 – 20 kg for 2 minutes. Patients rated their level of pain before and after the procedure.

Results

The "sciatic nerve press" produced immediate relief of pain in all patient groups. Emergency patients reported a 43.5% reduction in pain (p < 0.001). Significant pain relief for dental, renal and tumor patients lasted for 60 minutes (p < 0.001). The peak pain relief occurred at the 10 – 20^th minutes, and the relief decreased 47% by the 60^th minutes.

Conclusion

Two minutes of pressure on both sciatic nerves produced immediate significant short-term conduction analgesia. This technique is a convenient, safe and powerful method for the short-term treatment of clinical pain associated with a diverse range of pathologies.

Trial registration

Current Controlled Trials ACTRN012606000439549     

Genomics of natural bird populations: a gene-based set of reference markers evenly spread across the avian genome

Although there is growing interest to take genomics into the complex realms of natural populations, there is a general shortage of genomic resources and tools available for wild species. This applies not at least to birds, for which genomic approaches should be helpful to questions such as adaptation, speciation and population genetics. In this study, we describe a genome-wide reference set of conserved avian gene markers, broadly applicable across birds. By aligning protein-coding sequences from the recently assembled chicken genome with orthologous sequences in zebra finch, we identified particularly conserved exonic regions flanking introns of suitable size for subsequent amplification and sequencing. Primers were designed for 242 gene markers evenly distributed across the chicken genome, with a mean inter-marker interval of 4.2 Mb. Between 78% and 93% of the markers amplified a specific product in five species tested (chicken, peregrine falcon, collared flycatcher, great reed warbler and blue tit). Two hundred markers were sequenced in collared flycatcher, yielding a total of 122.41 kb of genomic DNA sequence (12096 bp coding sequence and 110 314 bp noncoding). Intron size of collared flycatcher and chicken was highly correlated, as was GC content. A polymorphism screening using these markers in a panel of 10 unrelated collared flycatchers identified 871 single nucleotide polymorphisms (pi = 0.0029) and 33 indels (mainly very short). Avian genome characteristics such as uniform genome size and low rate of syntenic rearrangements suggest that this marker set will find broad utility as a genome-wide reference resource for molecular ecological and population genomic analysis of birds. We envision that it will be particularly useful for obtaining large-scale orthologous targets in different species--important in, for instance, phylogenetics--and for large-scale identification of evenly distributed single nucleotide polymorphisms needed in linkage mapping or in studies of gene flow and hybridization.     

Synthesis and biological evaluation of 11C-labeled β-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled β-galactosyl triazoles 1-(β-d-galactopyranosyl)-4-(p-[¹¹C]methoxyphenyl)-1,2,3-triazole ([¹¹C]-6) and 1-(β-d-galactopyranosyl)-4-(6-[¹¹C]methoxynaphthyl)-1,2,3-triazole ([¹¹C]-13). The precursors for the radiolabeling and the non-radioactive analogues (6 and 13) were synthesized using straightforward ‘click’ chemistry. In vitro incubation experiments of 6 with β-galactosidase in the presence of o-nitrophenyl β-d-galactopyranoside (ONPG) showed that the triazolic compound was an inhibitor of β-galactosidase activity. Radiolabeling of both precursors was performed using [¹¹C]methyl iodide as alkylating agent at 70 °C in DMF in the presence of a small amount of base. The log P values were ?0.1 and 1.4, respectively, for [¹¹C]-6 and [¹¹C]-13, the latter therefore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in normal mice showed a good clearance from blood for both tracers. [¹¹C]-6 was mainly cleared via the renal pathway, while the more lipophilic [¹¹C]-13 was excreted almost exclusively via the hepatobiliary system. Despite the lipophilicity of [¹¹C]-13, no brain uptake was observed. Reversed phase HPLC analysis of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-293T cells showed an increased cell uptake for the more lipophilic [¹¹C]-13, however, there was no statistically higher uptake in LacZ expressing cells compared to control cells.     

The agglutination protein AggA from Shewanella oneidensis MR-1 is a TolC-like protein and forms active channels in vitro

Type I secretion systems (TISS) are associated with the virulence of Gram-negative bacteria and the secretion of pathogenic molecular determinants. The Shewanella oneidensis MR-1 outer-membrane protein AggA is part of a TISS. Recombinant AggA expressed in Escherichia coli as inclusion bodies can be efficiently refolded in vitro, and can form active channel-tunnels as shown by proteoliposome swelling assays and electrophysiological measurements in lipid bilayers. Structure-based sequence alignments identify AggA as a TolC-like protein, and point to a conserved structural framework among such proteins despite their marginal sequence similarity. Phylogenetic analysis reveals that clustering of TolC-like proteins can be correlated with their involvement in TISS, Resistance/Nodulation/Division (RND) or Major Facilitator Superfamily (MFS) complexes. Taken together, our results establish a first set of structure-function relationships for a bacterial outer-membrane protein likely to be exclusively involved in TISS, and may contribute towards a more accurate classification of Outer-Membrane Factor (OMF) family proteins.