In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.     

S-100 B Concentrations Are a Predictor of Decreased Survival in Patients with Major Trauma,Independently of Head Injury

Background

Major trauma remains one of the principle causes of disability and death throughout the world. There is currently no satisfactory risk assessment to predict mortality in patients with major trauma. The aim of our study is to examine whether S-100 B protein concentrations correlate with injury severity and survival in patients with major trauma, with special emphasis on patients without head injury.

Methods

Our retrospective data analysis comprised adult patients admitted to our emergency department between 1.12. 2008 and 31.12 2010 with a suspected major trauma. S-100 B concentrations were routinely assessed in major trauma patients.

Results

A total of 27.7% (378) of all patients had major trauma. The median ISS was 24.6 (SD 8.4); 16.6% (63/378) of the patients died. S-100 B concentrations correlated overall with the ISS (p<0.0001). Patients who died had significantly higher S-100 B concentrations than survivors (8.2 μg/l versus 2.2 μg/l, p<0.0001). Polytraumatised patients with and without head trauma did not differ significantly with respect to S-100 B concentration (3.2 μg/l (SD 5.3) versus 2.9 μg/l (SD 3.8), respectively, p = 0.63) or with respect to Injury Severity Score (24.8 (SD 8.6) versus 24.2 (SD 8.1), respectively, p = 0.56). S-100 B concentrations correlated negatively with survival (p<0.0001) in all patients and in both subgroups (p = 0.001 and p = 0.006, respectively)

Conclusions

S-100 concentrations on admission correlate positively with greater injury severity and decreased survival in major trauma patients, independently of the presence of a head injury. S-100 B protein levels at admission in patients with major trauma may therefore be used to assess outcome in all polytraumatised patients. These measurements should be subject to further evaluation.     

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.     

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis

Comparison of Arabidopsis thaliana (Arabidopsis) gene expression induced by Myzus persicae (green peach aphid) feeding, aphid saliva infiltration and abscisic acid (ABA) treatment showed a significant positive correlation. In particular, ABA‐regulated genes are over‐represented among genes that are induced by M. persicae saliva infiltration into Arabidopsis leaves. This suggests that the induction of ABA‐related gene expression could be an important component of the Arabidopsis–aphid interaction. Consistent with this hypothesis, M. persicae populations induced ABA production in wild‐type plants. Furthermore, aphid populations were smaller on Arabidopsis aba1‐1 mutants, which cannot synthesize ABA, and showed a significant preference for wild‐type plants compared with the mutant. Total free amino acids, which play an important role in aphid nutrition, were not altered in the aba1‐1 mutant line, but the levels of isoleucine (Ile) and tryptophan (Trp) were differentially affected by aphids in wild‐type and mutant plants. Recently, indole glucosinolates have been shown to promote aphid resistance in Arabidopsis. In this study, 4‐methoxyindol‐3‐ylmethylglucosinolate was more abundant in the aba1‐1 mutant than in wild‐type Arabidopsis, suggesting that the induction of ABA signals that decrease the accumulation of defence compounds may be beneficial for aphids.     

The deubiquitinase Usp27x stabilizes the BH3‐only protein Bim and enhances apoptosis

Bim is a pro‐apoptotic Bcl‐2 family member of the BH3‐only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non‐malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK‐dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK‐dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK‐dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA‐stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim‐degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non‐small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti‐apoptotic effects of ERK activity, and therefore acts as a tumour suppressor.     

A stochastic model of input effectiveness during irregular gamma rhythms

Gamma-band synchronization has been linked to attention and communication between brain regions, yet the underlying dynamical mechanisms are still unclear. How does the timing and amplitude of inputs to cells that generate an endogenously noisy gamma rhythm affect the network activity and rhythm? How does such ”communication through coherence” (CTC) survive in the face of rhythm and input variability? We present a stochastic modelling approach to this question that yields a very fast computation of the effectiveness of inputs to cells involved in gamma rhythms. Our work is partly motivated by recent optogenetic experiments (Cardin et al. Nature, 459(7247), 663–667 2009) that tested the gamma phase-dependence of network responses by first stabilizing the rhythm with periodic light pulses to the interneurons (I). Our computationally efficient model E-I network of stochastic two-state neurons exhibits finite-size fluctuations. Using the Hilbert transform and Kuramoto index, we study how the stochastic phase of its gamma rhythm is entrained by external pulses. We then compute how this rhythmic inhibition controls the effectiveness of external input onto pyramidal (E) cells, and how variability shapes the window of firing opportunity. For transferring the time variations of an external input to the E cells, we find a tradeoff between the phase selectivity and depth of rate modulation. We also show that the CTC is sensitive to the jitter in the arrival times of spikes to the E cells, and to the degree of I-cell entrainment. We further find that CTC can occur even if the underlying deterministic system does not oscillate; quasicycle-type rhythms induced by the finite-size noise retain the basic CTC properties. Finally a resonance analysis confirms the relative importance of the I cell pacing for rhythm generation. Analysis of whole network behaviour, including computations of synchrony, phase and shifts in excitatory-inhibitory balance, can be further sped up by orders of magnitude using two coupled stochastic differential equations, one for each population. Our work thus yields a fast tool to numerically and analytically investigate CTC in a noisy context. It shows that CTC can be quite vulnerable to rhythm and input variability, which both decrease phase preference.     

Comparison of methods to determine the centre of resistance of teeth

In orthodontic treatment, the locations of the centre of resistance (CR) of individual teeth and the applied load system are the major determinants for the type of tooth movement achieved. Currently, CR locations have only been specified for a relatively small number of tooth specimen for research purposes. Analysing cone beam computed tomography data samples from three upper central incisors, this study explores whether the effort to establish accurate CR estimates can be reduced by (i) morphing a pre-existing simplified finite element (FE) mesh to fit to the segmented 3D tooth-bone model, and (ii) individualizing a mean CR location according to a small parameter set characterising the morphology of the tooth and its embedding. The FE morphing approach and the semi-analytical approach led to CR estimates that differ in average only 0.04 and 0.12 mm respectively from those determined by very time-consuming individual FE modelling (standard method). Both approaches may help to estimate the movement of individual teeth during orthodontic treatment and, thus, increase the therapeutic efficacy.     

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi‐chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division‐replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.