Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.     

Transient Receptor Potential Channel 6 (TRPC6) Protects Podocytes during Complement-mediated Glomerular Disease

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.     

Draft genome sequence of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure,motility and chemotaxis

Sinorhizobium meliloti of the order Rhizobiales is a symbiotic nitrogen-fixing bacterium nodulating plants of the genera Medicago, Trigonella and Melilotus and hence is of great agricultural importance. In its free-living state it is motile and capable of modulating its movement patterns in response to chemical attractants. Here, the draft genome consisting of a circular chromosome, the megaplasmids pSymA and pSymB and three accessory plasmids of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis, is reported.     

Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum

Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe–2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.     

An Ancestral Recombination Graph for Diploid Populations with Skewed Offspring Distribution

A large offspring-number diploid biparental multilocus population model of Moran type is our object of study. At each time step, a pair of diploid individuals drawn uniformly at random contributes offspring to the population. The number of offspring can be large relative to the total population size. Similar “heavily skewed” reproduction mechanisms have been recently considered by various authors (cf. e.g., Eldon and Wakeley 2006, 2008) and reviewed by Hedgecock and Pudovkin (2011). Each diploid parental individual contributes exactly one chromosome to each diploid offspring, and hence ancestral lineages can coalesce only when in distinct individuals. A separation-of-timescales phenomenon is thus observed. A result of Möhle (1998) is extended to obtain convergence of the ancestral process to an ancestral recombination graph necessarily admitting simultaneous multiple mergers of ancestral lineages. The usual ancestral recombination graph is obtained as a special case of our model when the parents contribute only one offspring to the population each time. Due to diploidy and large offspring numbers, novel effects appear. For example, the marginal genealogy at each locus admits simultaneous multiple mergers in up to four groups, and different loci remain substantially correlated even as the recombination rate grows large. Thus, genealogies for loci far apart on the same chromosome remain correlated. Correlation in coalescence times for two loci is derived and shown to be a function of the coalescence parameters of our model. Extending the observations by Eldon and Wakeley (2008), predictions of linkage disequilibrium are shown to be functions of the reproduction parameters of our model, in addition to the recombination rate. Correlations in ratios of coalescence times between loci can be high, even when the recombination rate is high and sample size is large, in large offspring-number populations, as suggested by simulations, hinting at how to distinguish between different population models.     

Discovery of highly potent and selective D4 ligands by interactive SAR study

A series of thienylmethylphenylpiperazins was synthesized and tested for affinity towards the five subtypes of dopaminergic receptors. Compound 5f showed more than 1000 folds selectivity to D4 receptors; analogue 5e showed the highest affinity to D4 receptors with K_i 3.9 nM. An interactive SAR approach was adopted and lead to compound 14a with K_i (D4) as low as 0.03 nM. Molecular docking studies showed a potential, first to report arene cation interaction between the D4 unique residue Arg-186 and the ligands’ arene moiety, explaining the importance of having a strong negative electrostatic potential at this area of the compound structure.     

Mapping the navigational knowledge of individually foraging ants,Myrmecia croslandi

Ants are efficient navigators, guided by path integration and visual landmarks. Path integration is the primary strategy in landmark-poor habitats, but landmarks are readily used when available. The landmark panorama provides reliable information about heading direction, routes and specific location. Visual memories for guidance are often acquired along routes or near to significant places. Over what area can such locally acquired memories provide information for reaching a place? This question is unusually approachable in the solitary foraging Australian jack jumper ant, since individual foragers typically travel to one or two nest-specific foraging trees. We find that within 10 m from the nest, ants both with and without home vector information available from path integration return directly to the nest from all compass directions, after briefly scanning the panorama. By reconstructing panoramic views within the successful homing range, we show that in the open woodland habitat of these ants, snapshot memories acquired close to the nest provide sufficient navigational information to determine nest-directed heading direction over a surprisingly large area, including areas that animals may have not visited previously.     

Symptoms and Clinical Course of EHEC O104 Infection in Hospitalized Patients: A Prospective Single Center Study

Objectives

Shiga-toxin producing O157:H7 Entero Haemorrhagic E. coli (STEC/EHEC) is one of the most common causes of Haemolytic Uraemic Syndrome (HUS) related to infectious haemorrhagic colitis. Nearly all recommendations on clinical management of EHEC infections refer to this strain. The 2011 outbreak in Northern Europe was the first to be caused by the serotype O104:H4. This EHEC strain was found to carry genetic features of Entero Aggregative E. coli (EAEC) and extended spectrum β lactamase (ESBL). We report symptoms and complications in patients at one of the most affected centres of the 2011 EHEC O104 outbreak in Northern Germany.

Methods

The courses of patients admitted to our hospital due to bloody diarrhoea with suspected EHEC O104 infection were recorded prospectively. These data include the patients’ histories, clinical findings, and complications.

Results

EHEC O104 infection was confirmed in 61 patients (female = 37; mean age: 44±2 years). The frequency of HUS was 59% (36/61) in our cohort. An enteric colonisation with co-pathogens was found in 57%. Thirty-one (51%) patients were treated with plasma-separation/plasmapheresis, 16 (26%) with haemodialysis, and 7 (11%) with Eculizumab. Patients receiving antibiotic treatment (n = 37; 61%) experienced no apparent change in their clinical course. Twenty-six (43%) patients suffered from neurological symptoms. One 83-year-old patient died due to comorbidities after HUS was successfully treated.

Conclusions

EHEC O104:H4 infections differ markedly from earlier reports on O157:H7 induced enterocolitis in regard to epidemiology, symptomatology, and frequency of complications. We recommend a standard of practice for clinical monitoring and support the renaming of EHEC O104:H4 syndrome as “EAHEC disease”.     

Visualisation of Respiratory Tumour Motion and Co-Moving Isodose Lines in the Context of Respiratory Gating,IMRT and Flattening-Filter-Free Beams

Respiratory motion during percutaneous radiotherapy can be considered based on respiration-correlated computed tomography (4DCT). However, most treatment planning systems perform the dose calculation based on a single primary CT data set, even though cine mode displays may allow for a visualisation of the complete breathing cycle. This might create the mistaken impression that the dose distribution were independent of tumour motion. We present a movie visualisation technique with the aim to direct attention to the fact that the dose distribution migrates to some degree with the tumour and discuss consequences for gated treatment, IMRT plans and flattening-filter-free beams. This is a feasibility test for a visualisation of tumour and isodose motion. Ten respiratory phases are distinguished on the CT, and the dose distribution from a stationary IMRT plan is calculated on each phase, to be integrated into a movie of tumour and dose motion during breathing. For one example patient out of the sample of five lesions, the plan is compared with a gated treatment plan with respect to tumour coverage and lung sparing. The interplay-effect for small segments in the IMRT plan is estimated. While the high dose rate, together with the cone-shaped beam profile, makes the use of flattening-filter-free beams more problematic for conformal and IMRT treatment, it can be the option of choice if gated treatment is preferred. The different effects of respiratory motion, dose build-up and beam properties (segments and flatness) for gated vs. un-gated treatment can best be considered if planning is performed on the full 4DCT data set, which may be an incentive for future developments of treatment planning systems.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.