EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single‐pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR–DOZI‐aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S‐transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore‐forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM.     

Prevention of postoperative bleeding after complex pediatric cardiac surgery by early administration of fibrinogen,prothrombin complex and platelets: a prospective observational study

Some retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis. Estrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects. Sevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell. Sevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.     

Laminin 332 processing impacts cellular behavior

Laminin 332, composed of the α3, β3 and γ2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external forces, including skin and stratified squamous mucosa. After being secreted and deposited in the extracellular matrix, laminin 332 undergoes physiological maturation processes consisting in the proteolytic processing of domains located within the α3 and the γ2 chains. These maturation events are essential for laminin 332 integration into the basement membrane where it plays an important function in the nucleation and maintenance of anchoring structures. Studies in normal and pathological situations have revealed that laminin 332 can trigger distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and structural characteristics of laminin 332 domains are presented and we discuss whether they trigger specific functions.     

Early Re-Do Surgery for Glioblastoma Is a Feasible and Safe Strategy to Achieve Complete Resection of Enhancing Tumor

Background

Complete resection of enhancing tumor as assessed by early (<72 hours) postoperative MRI is regarded as the optimal result in glioblastoma surgery. As yet, there is no consensus on standard procedure if post-operative imaging reveals unintended tumor remnants.

Objective

The current study evaluated the feasibility and safety of an early re-do surgery aimed at completing resections with the aid of 5-ALA fluorescence and neuronavigation after detection of enhancing tumor remnants on post-operative MRI.

Methods

From October 2008 to October 2012 a single center institutional protocol offered a second surgery within one week to patients with unintentional incomplete glioblastoma resection. We report on the feasibility of the use 5-ALA fluorescence guidance, the extent of resection (EOR) rates and complications of early re-do surgery.

Results

Nine of 151 patients (6%) with glioblastoma resections had an unintentional tumor remnant with a volume >0.175 cm³. 5-ALA guided re-do surgery completed the resection (CRET) in all patients without causing neurological deficits, infections or other complications. Patients who underwent a re-do surgery remained hospitalized between surgeries, resulting in a mean length of hospital stay of 11 days (range 7-15), compared to 9 days for single surgery (range 3-23; p=0.147).

Conclusion

Our early re-do protocol led to complete resection of all enhancing tumor in all cases without any new neurological deficits and thus provides a similar oncological result as intraoperative MRI (iMRI). The repeated use of 5-ALA induced fluorescence, used for identification of small remnants, remains highly sensitive and specific in the setting of re-do surgery. Early re-do surgery is a feasible and safe strategy to complete unintended subtotal resections.     

High Environmental Ozone Levels Lead to Enhanced Allergenicity of Birch Pollen

Background

Evidence is compelling for a positive correlation between climate change, urbanisation and prevalence of allergic sensitisation and diseases. The reason for this association is not clear to date. Some data point to a pro-allergenic effect of anthropogenic factors on susceptible individuals.

Objectives

To evaluate the impact of urbanisation and climate change on pollen allergenicity.

Methods

Catkins were sampled from birch trees from different sites across the greater area of Munich, pollen were isolated and an urbanisation index, NO₂ and ozone exposure were determined. To estimate pollen allergenicity, allergen content and pollen-associated lipid mediators were measured in aqueous pollen extracts. Immune stimulatory and modulatory capacity of pollen was assessed by neutrophil migration assays and the potential of pollen to inhibit dendritic cell interleukin-12 response. In vivo allergenicity was assessed by skin prick tests.

Results

The study revealed ozone as a prominent environmental factor influencing the allergenicity of birch pollen. Enhanced allergenicity, as assessed in skin prick tests, was mirrored by enhanced allergen content. Beyond that, ozone induced changes in lipid composition and chemotactic and immune modulatory potential of the pollen. Higher ozone-exposed pollen was characterised by less immune modulatory but higher immune stimulatory potential.

Conclusion

It is likely that future climate change along with increasing urbanisation will lead to rising ozone concentrations in the next decades. Our study indicates that ozone is a crucial factor leading to clinically relevant enhanced allergenicity of birch pollen. Thus, with increasing temperatures and increasing ozone levels, also symptoms of pollen allergic patients may increase further.     

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.     

Copeptin as a Marker for Severity and Prognosis of Aneurysmal Subarachnoid Hemorrhage

Background

Grading of patients with aneurysmal subarachnoid hemorrhage (aSAH) is often confounded by seizure, hydrocephalus or sedation and the prediction of prognosis remains difficult. Recently, copeptin has been identified as a serum marker for outcomes in acute ischemic stroke and intracerebral hemorrhage (ICH). We investigated whether copeptin might serve as a marker for severity and prognosis in aSAH.

Methods

Eighteen consecutive patients with aSAH had plasma copeptin levels measured with a validated chemiluminescence sandwich immunoassay. The primary endpoint was the association of copeptin levels at admission with the World Federation of Neurological Surgeons (WFNS) grade score after resuscitation. Levels of copeptin were compared across clinical and radiological scores as well as between patients with ICH, intraventricular hemorrhage, hydrocephalus, vasospasm and ischemia.

Results

Copeptin levels were significantly associated with the severity of aSAH measured by WFNS grade (P = 0.006), the amount of subarachnoid blood (P = 0.03) and the occurrence of ICH (P = 0.02). There was also a trend between copeptin levels and functional clinical outcome at 6-months (P = 0.054). No other clinical outcomes showed any statistically significant association.

Conclusions

Copeptin may indicate clinical severity of the initial bleeding and may therefore help in guiding treatment decisions in the setting of aSAH. These initial results show that copeptin might also have prognostic value for clinical outcome in aSAH.     

A Cohort Study of the Impact of Tooth Loss and Periodontal Disease on Respiratory Events among COPD Subjects: Modulatory Role of Systemic Biomarkers of Inflammation

Background

In COPD patients, fatal and non-fatal respiratory-related events are influenced by age, severity of respiratory disease, and comorbidities.

Objectives

Analyze the effects of edentulism, periodontal disease and systemic biomarkers of inflammation on the occurrence of serious fatal and non-fatal respiratory-related events among subjects with COPD.

Methods

Cases were identified from Dental Atherosclerosis Risk in Communities study. Edentulism was defined as study participants without any natural teeth or implants. Participants with one or more natural teeth (comprising 11,378 subjects) were studied as dentate subjects. Periodontal disease status among dentate individuals was determined using the consensus definitions published by the joint Center for Disease Control/American Association of Periodontology working group). Adjusted Hazard Models are developed to evaluate the relationship between edentulism/periodontal disease and COPD Related Events. Models were then stratified by GOLD Stage I, II and III/IV. Serum biomarkers were also evaluated to explore the effect of systemic inflammation.

Results

A statistically significant association was found between oral health status and COPD-related events, even adjusting for conditions such as hypertension, smoking and diabetes. Edentulous individuals who had been diagnosed with COPD had a higher incidence and were at greater risk of having a COPD related event (hospitalization and death) than individuals who had teeth and whose mouths had healthy periodontal status. However, being edentulous did not convey excess risk for COPD-related events for those study participants who were classified as GOLD III/IV at baseline. Finally, we showed that individuals who had levels of serum IL-6 in the highest two quartiles were at even higher risk for COPD-related events.

Conclusions

These findings suggest that the risk for COPD-related events after adjusting for potential confounders may be attributable to both edentulism and elevated serum IL-6 levels.     

Bioavailability and Efficacy of a Gap Junction Enhancer (PQ7) in a Mouse Mammary Tumor Model

The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.