Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)¹ (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22).     

Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.     

Development and validation of a liquid chromatography method for simultaneous determination of three process-related impurities: yeastolates, triton X-100 and methotrexate

Yeastolates, triton X-100 (TX-100) and methotrexate (MTX) are common process-related impurities (PRI) in cell-based bioproduction of many active biopharmaceuticals. In this study, a reverse phase high performance liquid chromatography (RP-HPLC) method coupled with ultraviolet (UV) detection was developed for simultaneous determination and quantitation of these impurities. The chromatographic separation was achieved using a Jupiter C4 column and analyses of yeastolates, TX-100 and MTX were monitored at 257, 280 and 302 nm, respectively. The method was further validated with respect to selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The limits of quantitation for yeastolates, TX-100 and MTX were determined to be 27 ppm, 10 ppm and 41 ppb, respectively. Finally, the suitability of the method for analyses of recombinant human hyaluronidase (rHuPH20) in-process (viral inactivation, QFF, PS, APB and CHT filtered, final viral filtrate) and final manufacturing materials was demonstrated, and trace levels of yeastolates, TX-100 and MTX were reliably measured except for three matrices early in the purification process in which TX-100 was not accurately determined due to interfering effects.     

Phylogeny and evolution of the Archaea: one hundred genomes later

Little more than 30 years since the discovery of the Archaea, over one hundred archaeal genome sequences are now publicly available, of which ～40% have been released in the last two years. Their analysis provides an increasingly complex picture of archaeal phylogeny and evolution with the proposal of new major phyla, such as the Thaumarchaeota, and important information on the evolution of key central cellular features such as cell division. Insights have been gained into the events and processes in archaeal evolution, with a number of additional and unexpected links to the Eukaryotes revealed. Taken together, these results predict that many more surprises will be found as new archaeal genomes are sequenced.     

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

Kidney pathology precedes and predicts the pathological cascade of cerebrovascular lesions in stroke prone rats

Introduction

Human cerebral small vessel disease (CSVD) has been hypothesized to be an age-dependent disease accompanied by similar vascular changes in other organs. SHRSP feature numerous vascular risk factors and may be a valid model of some aspects of human CSVD. Here we compare renal histopathological changes with the brain pathology of spontaneously hypertensive stroke-prone rats (SHRSP).

Material and Methods

We histologically investigated the brains and kidneys of 61 SHRSP at different stages of age (12 to 44 weeks). The brain pathology (aggregated erythrocytes in capillaries and arterioles, microbleeds, microthromboses) and the kidney pathology (aggregated erythrocytes within peritubular capillaries, tubular protein cylinders, glomerulosclerosis) were quantified separately. The prediction of the brain pathology by the kidney pathology was assessed by creating ROC-curves integrating the degree of kidney pathology and age of SHRSP.

Results

Both, brain and kidney pathology, show an age-dependency and proceed in definite stages whereas an aggregation of erythrocytes in capillaries and arterioles, we parsimoniously interpreted as stases, represent the initial finding in both organs. Thus, early renal tubulointerstitial damage characterized by rather few intravasal erythrocyte aggregations and tubular protein cylinders predicts the initial step of SHRSPs'' cerebral vascular pathology marked by accumulated erythrocytes. The combined increase of intravasal erythrocyte aggregations and protein cylinders accompanied by glomerulosclerosis and thrombotic renal microangiopathy in kidneys of older SHRSP predicts the final stages of SHRSPs'' cerebrovascular lesions marked by microbleeds and thrombotic infarcts.

Conclusion

Our results illustrate a close association between structural brain and kidney pathology and support the concept of small vessel disease to be an age-dependent systemic pathology. Further, an improved joined nephrologic and neurologic diagnostic may help to identify patients with CSVD at an early stage.     

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

Background  

Cultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain.     

Passage of murine scrapie prion protein across the mouse vascular blood-brain barrier

Prions are the infectious agents associated with transmissible spongiform encephalopathies and are composed mainly of a misfolded form of the endogenous prion protein. Prion protein must enter the brain to produce disease. Previous work has emphasized various mechanisms which partially bypass the blood-brain barrier (BBB). Here, we used the brain perfusion method to directly assess the ability of mouse scrapie protein (PrP(SC)) to cross the mouse BBB independent of the influences of neural pathways or circulating immune cells. We found that PrP(SC) oligomers rapidly crossed the BBB without disrupting it with a unidirectional influx rate of about 4.4microl/g-min. HPLC and capillary depletion confirmed that PrP(SC) crossed the entire width of the capillary wall to enter brain parenchyma. PrP(SC) also entered the cerebrospinal fluid (CSF) compartment. These results show that a prion protein can cross the intact BBB to enter both the parenchymal and CSF compartments of the brain.