Investigation of Bartonella acquisition and transmission in Xenopsylla ramesis fleas (Siphonaptera: Pulicidae)

Bartonella are emerging and re-emerging pathogens affecting humans and a wide variety of animals including rodents. Horizontal transmission of Bartonella species by different hematophagous vectors is well acknowledged but vertical transmission (from mother to offspring) is questionable and was never explored in fleas. The aim of this study was to investigate whether the rodent flea, Xenopsylla ramesis, can acquire native Bartonella from wild rodents and transmit it transovarially. For this aim, Bartonella-free laboratory-reared X. ramesis fleas were placed on six naturally Bartonella-infected rodents and six species-matched Bartonella-negative rodents (three Meriones crassus jirds, two Gerbillus nanus gerbils and one Gerbillus dasyurus gerbil) for 7 days, 12-14h per day. The fleas that were placed on the Bartonella-positive rodents acquired four different Bartonella genotypes. Eggs and larvae laid and developed, respectively, by fleas from both rodent groups were collected daily for 7 days and molecularly screened for Bartonella. All eggs and larvae from both groups were found to be negative for Bartonella DNA. Interestingly, two of five gut voids regurgitated by Bartonella-positive fleas contained Bartonella DNA. The naturally infected rodents remained persistently infected with Bartonella for at least 89 days suggesting their capability to serve as competent reservoirs for Bartonella species. The findings in this study indicate that X. ramesis fleas can acquire several Bartonella strains from wild rodents but cannot transmit Bartonella transovarially.     

Tyrosine Phosphorylation of Dbl Regulates GTPase Signaling

Rho GTPases are molecular “switches” that cycle between “on” (GTP-bound) and “off” (GDP-bound) states and regulate numerous cellular activities such as gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses. Dysregulation of GTPases is a key feature of many diseases, especially cancers. Guanine nucleotide exchange factors (GEFs) of the Dbl family are activated by mitogenic cell surface receptors and activate the Rho family GTPases Cdc42, Rac1, and RhoA. The molecular mechanisms that regulate GEFs from the Dbl family are poorly understood. Our studies reveal that Dbl is phosphorylated on tyrosine residues upon stimulation by growth factors and that this event is critical for the regulated activation of the GEF. These findings uncover a novel layer of complexity in the physiological regulation of this protein.     

Prolonged and flexible primary moult overlaps extensively with breeding in beach‐nesting Hooded Plovers Thinornis rubricollis

We present the first report of complete overlap of breeding and moult in a shorebird. In southeastern Australia, Hooded Plovers Thinornis rubricollis spend their entire lives on oceanic beaches, where they exhibit biparental care. Population moult encompassed the 6‐month breeding season. Moult timing was estimated using the Underhill–Zucchini method for Type 2 data with a power transformation to accommodate sexual differences in rates of moult progression in the early and late stages of moult. Average moult durations were long in females (170.3 ± 14.2 days), and even longer in males (210.3 ± 13.5 days). Breeding status was known for most birds in our samples, and many active breeders (especially males) were also growing primaries. Females delayed the onset of primary moult but were able to increase the speed of moult and continue breeding, completing moult at about the same time as males. The mechanism by which this was achieved appeared to be flexibility in moult sequence. All moult formulae fell on one of two linked moult sequences, one faster than the other. The slower sequence had fewer feathers growing concurrently and also had formulae indicating suspended moults. Switching between sequences via common formulae is possible at many points during the moult cycle, and three of 12 recaptures were confirmed to have switched sequences in the same moult season. Hooded Plovers thus have a prolonged primary moult with the flexibility to change their rate of moult; this may facilitate high levels of replacement clutches that are associated with passive nest defence and low reproductive success.     

Self-Assembly of MinE on the Membrane Underlies Formation of the MinE Ring to Sustain Function of the Escherichia coli Min System

The pole-to-pole oscillation of the Min proteins in Escherichia coli results in the inhibition of aberrant polar division, thus facilitating placement of the division septum at the midcell. MinE of the Min system forms a ring-like structure that plays a critical role in triggering the oscillation cycle. However, the mechanism underlying the formation of the MinE ring remains unclear. This study demonstrates that MinE self-assembles into fibrillar structures on the supported lipid bilayer. The MinD-interacting domain of MinE shows amyloidogenic properties, providing a possible mechanism for self-assembly of MinE. Supporting the idea, mutations in residues Ile-24 and Ile-25 of the MinD-interacting domain affect fibril formation, membrane binding ability of MinE and MinD, and subcellular localization of three Min proteins. Additional mutations in residues Ile-72 and Ile-74 suggest a role of the C-terminal domain of MinE in regulating the folding propensity of the MinD-interacting domain for different molecular interactions. The study suggests a self-assembly mechanism that may underlie the ring-like structure formed by MinE-GFP observed in vivo.     

Nitration of Tyrosine 247 Inhibits Protein Kinase G-1α Activity by Attenuating Cyclic Guanosine Monophosphate Binding

The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr²⁴⁷ is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr²⁴⁷ would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [³H]cGMP binding assay. Our study shows that the nitration of Tyr²⁴⁷ and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation.     

Identification of Different Bartonella Species in the Cattle Tail Louse (Haematopinus quadripertusus) and in Cattle Blood

Bartonella spp. are worldwide-distributed facultative intracellular bacteria that exhibit an immense genomic diversity across mammal and arthropod hosts. The occurrence of cattle-associated Bartonella species was investigated in the cattle tail louse Haematopinus quadripertusus and in dairy cattle blood from Israel. Lice were collected from cattle from two dairy farms during summer 2011, and both lice and cow blood samples were collected from additional seven farms during the successive winter. The lice were identified morphologically and molecularly using 18S rRNA sequencing. Thereafter, they were screened for Bartonella DNA by conventional and real-time PCR assays using four partial genetic loci (gltA, rpoB, ssrA, and internal transcribed spacer [ITS]). A potentially novel Bartonella variant, closely related to other ruminant bartonellae, was identified in 11 of 13 louse pools collected in summer. In the cattle blood, the prevalence of Bartonella infection was 38%, identified as B. bovis and B. henselae (24 and 12%, respectively). A third genotype, closely related to Bartonella melophagi and Bartonella chomelii (based on the ssrA gene) and to B. bovis (based on the ITS sequence) was identified in a single cow. The relatively high prevalence of these Bartonella species in cattle and the occurrence of phylogenetically diverse Bartonella variants in both cattle and their lice suggest the potential role of this animal system in the generation of Bartonella species diversity.     

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na⁺/K⁺-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na⁺ form of Tel23 G4, which is the same hybrid form as the K⁺ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na⁺ form to K⁺ form during Na⁺/K⁺ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na⁺/K⁺ spectral conversion. In addition, the K⁺ titration monitored by NMR reveals that the Na⁺/K⁺ exchange in Tel23 G4 is a two-step process. The addition of K⁺ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na⁺/K⁺ exchange and insight into the mechanism of Na⁺/K⁺ structural conversion in human telomeric G4s.     

The molecular path to in vitro shoot regeneration

Plant regeneration through de novo shoot organogenesis in tissue culture is a critical step in most plant transformation and micropropagation procedures. Establishing an efficient regeneration protocol is an empirical process and requires optimization of multiple factors that influence the regeneration capacity. Here, we review the molecular process of shoot induction in a two-step regeneration protocol and focus on the role of auxins and cytokinins. First, during incubation on an auxin-rich callus induction medium (CIM), organogenic callus is produced that exhibits characteristics of a root meristem. Subsequent incubation on a cytokinin-rich shoot induction medium (SIM) induces root to shoot conversion. Through a detailed analysis of the different aspects of shoot regeneration, we try to reveal hinge points and novel candidate genes that may be targeted to increase shoot regeneration capacity in order to improve transformation protocols.     

Altered expression of Aurora kinases in Arabidopsis results in aneu‐ and polyploidization

Aurora is an evolutionary conserved protein kinase family involved in monitoring of chromosome segregation via phosphorylation of different substrates. In plants, however, the involvement of Aurora proteins in meiosis and in sensing microtubule attachment remains to be proven, although the downstream components leading to the targeting of spindle assembly checkpoint signals to anaphase‐promoting complex have been described. To analyze the three members of Aurora family (AtAurora1, ‐2, and ‐3) of Arabidopsis we employed different combinations of T‐DNA insertion mutants and/or RNAi transformants. Meiotic defects and the formation of unreduced pollen were revealed including plants with an increased ploidy level. The effect of reduced expression of Aurora was mimicked by application of the ATP‐competitive Aurora inhibitor II. In addition, strong overexpression of any member of the AtAurora family is not possible. Only tagged or truncated forms of Aurora kinases can be overexpressed. Expression of truncated AtAurora1 resulted in a high number of aneuploids in Arabidopsis, while expression of AtAurora1‐TAPi construct in tobacco resulted in 4C (possible tetraploid) progeny. In conclusion, our data demonstrate an essential role of Aurora kinases in the monitoring of meiosis in plants.