Increasing protein identifications in bottom-up proteomics of T. castaneum - Exploiting synergies of protein biochemistry and bioinformatics.

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant. Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications. A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant's Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis. Our work with Tribolium castaneum larvae demonstrates that sometimes - depending on matrix and research question - more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.

Active and passive microrheology with large tracers in hard colloids.

The dynamics of a tracer particle in a bath of quasi-hard colloidal spheres is studied by Langevin dynamics simulations and mode coupling theory (MCT); the tracer radius is varied from equal to up to seven times larger than the bath particles radius. In the simulations, two cases are considered: freely diffusing tracer (passive microrheology) and tracer pulled with a constant force (active microrheology). Both cases are connected by linear response theory for all tracer sizes. It links both the stationary and transient regimes of the pulled tracer (for low forces) with the equilibrium correlation functions; the velocity of the pulled tracer and its displacement are obtained from the velocity auto-correlation function and the mean squared displacement, respectively. The MCT calculations give insight into the physical mechanisms: At short times, the tracer rattles in its cage of neighbours, with the frequency increasing linearly with the tracer radius asymptotically. The long-time tracer diffusion coefficient from passive microrheology, which agrees with the inverse friction coefficient from the active case, arises from the transport of transverse momentum around the tracer. It can be described with the Brinkman equation for the transverse flow field obtained in extension of MCT, but cannot be recovered from the MCT kernel coupling to densities only. The dynamics of the bath particles is also studied; for the unforced tracer the dynamics is unaffected. When the tracer is pulled, the velocity field in the bath follows the prediction of the Brinkman model, but different from the case of a Newtonian fluid.

Impact of Ergonomic Posture on the Chemical Exposure of Workers in the Petroleum and Chemical Industry.

OBJECTIVES: Despite a rise in automation, workers in the petroleum refining and petrochemical manufacturing industry are potentially exposed to various chemicals through inhalation while performing routine job duties. Many factors contribute to the degree of exposure experienced in this setting. The study objective was to characterize the impact of workplace conditions, anthropometric variability, and task orientation on exposure for a simulated routine operations task. METHODS: A chemical exposure laboratory simulation study was designed to evaluate the dependent variable of chemical exposure level in the breathing zone for methane and sulfur hexafluoride. The independent variables were (i) posture of the worker, (ii) worker anthropometry, (iii) process configuration, and (iv) gas density. RESULTS: Pipe height was a significant predictor of gas concentration measured in the breathing zone when located in a position that encouraged the gas to enter the breathing zone of the worker. Worker anthropometry had a major impact; tall worker's (male) chemical concentrations exceeded those of the short worker (female) for methane simulations but the opposite resulted for sulfur hexafluoride. Also, worker posture had a significant impact on gas exposure where nonneutral postures were found to have higher levels of chemical concentration. CONCLUSIONS: The study findings indicate that the breathing zone location is altered by posture and worker height, which changes the exposures relative to the emission source depending on the gas density of the chemicals that are present. As a result, qualitative risk assessment cannot be performed accurately without accounting for these factors. Practically, controls may need to account for worker size differences and posture adaptations.

[Effect of low-dose PDT on perfusion of the choriocapillaris in cCSCR : An OCT-angiography-based study]. / Effekt der "low-dose PDT" auf die choriokapilläre Perfusion bei cCRCS : Eine OCT-Angiographie-gestützte Studie.

BACKGROUND: In patients with chronic central serous chorioretinopathy (cCSCR) it is assumed that low-dose photodynamic therapy (PDT) induces short-term hypoperfusion of the choriocapillaris, which leads to long-term choroidal vascular remodeling and subsequent reduction of vascular hyperpermeability and leakage; however, it remains unclear whether the hypoperfusion completely returns to normal. The main aim of the retrospective study was to analyze the choroidal flow signal after treatment with low-dose PDT by optical coherence tomography-angiography (OCT­A). PATIENTS AND METHODS: In this study 16 eyes with a total of 19 treated areas were included. Aside from visual acuity and metamorphopsia checks, all patients underwent enhanced depth imaging-OCT (EDI-OCT) and OCT­A in the areas treated with low-dose PDT. In the present study, the flow signal in the choriocapillaris and choroidal thickness were measured retrospectively both in the PDT-treated area and in a non-treated reference area located in close proximity with similar eccentricity to the center of the fovea. RESULTS: The study found a mean reduction of 33% (p < 0.001) of the choroidal flow signal in the treated area compared to the reference area and an average decrease in choroidal thickness of 71 µm (p = 0.001). In the long term 7 out of 10 patients suffered from metamorphopsia despite the low-dose PDT; however, visual acuity was improved in almost half of the patients by the treatment. CONCLUSION: Using OCT­A it could be shown that at the choriocapillaris level, hypoperfusion persists in spite of low-dose PDT. Thus, the choroidal vascular remodeling is not able to completely compensate for the hypoperfusion induced by thrombosis.

[Biomarkers in full-thickness and lamellar defects of the macula]. / Biomarker bei durchgreifenden und lamellären Defekten der Makula.

BACKGROUND: Biomarkers are characteristic biological measurable signs and objective parameters to detect the state of health. Besides functional and temporal factors, imaging biomarkers play an increasingly important role. In full-thickness and lamellar defects of the macula numerous imaging parameters have been described. This knowledge resulted in new classifications for both clinical pictures, which are based on important biomarkers of these clinical pictures and characteristic features of described subtypes. METHODS: Imaging biomarkers for full-thickness and lamellar macular defects are described with respect to the classification and their functional prognostic importance. The importance of these biomarkers is presented. RESULTS: The current classification of full-thickness and lamellar macular defects is based on structural biomarkers. Biomarkers are important for prognostic and therapeutic evaluation and they have an impact on the surgical strategy. There are various surgical strategies for treatment of full-thickness macular holes depending on the size of the foramen and other biomarkers. The inverted ILM flap technique improves the closure rate of large macular holes. In lamellar macular holes showing signs of progression an early surgical intervention results in a good anatomical and functional prognosis. CONCLUSION: Multimodal diagnostics provide important preoperative, intraoperative, and postoperative features for macular holes and lamellar macular holes, which are essential biomarkers for the exact classification, the therapeutic strategy and for assessment of the prognosis.

[Optical coherence tomography biomarkers in epimacular membranes and vitreomacular traction syndrome]. / Optische Kohärenztomographie-Biomarker bei epimakulären Membranen und vitreomakulärem Traktionssyndrom.

Using high-resolution imaging, such as optical coherence tomography (OCT), the different layers of the retina as well as the vitreoretinal interface and its alterations can be very clearly differentiated. This includes the morphological characteristics of tractive maculopathies, such as epiretinal gliosis and vitreomacular traction syndrome. Additionally, structural alterations of the various layers of the neurosensory retina as a result of traction due to these pathologies can be demarcated. The latter have been investigated in clinical trials and evaluated as OCT biomarkers with respect to their prognostic and predictive value. In this review we would like to present and discuss various OCT biomarkers in the context of epimacular membranes and vitreomacular traction syndrome.

[Large macular hole-Always a poor prognosis?] / Großes Makulaforamen ­ immer eine schlechte Prognose?

BACKGROUND: Age, preoperative visual acuity and macular hole size have been reported to be prognostic markers for the postoperative outcome in patients with full thickness macular holes (FTMH). OBJECTIVE: A retrospective observational clinical study was performed in order to compare the postoperative course after conventional internal limiting membrane peeling (c-ILM) and inverted flap ILM peeling (i-ILM). Patients with i­ILM peeling had preoperatively a significantly larger macular hole MATERIAL AND METHODS: In this study 45 consecutive patients with a full thickness macular hole (FTMH) were divided into two groups (c-ILM vs. i ILM) and evaluated with respect to the postoperative best corrected visual acuity (BCVA) and retinal morphology. Using spectral domain optical coherence tomography (SD-OCT) the integrity of the outer retinal layers, external limiting membrane (ELM), ellipsoid zone (EZ) and outer photoreceptor outer segments (OS) were analyzed postoperatively for at least 6 months. RESULTS: The preoperative aperture in the i­ILM group was significantly larger (i-ILM = 408.4 µm, SD = 157.5 µm; c­ILM = 287.4 µm, SD = 104.9 µm; p = 0.01). The preoperative BCVA and postoperative BCVA after 1 month were significantly better in the group with c­ILM peeling (p = 0.03 and p = 0.001). The postoperative BCVA after at least 6 months showed no significant difference between the two groups (p = 0.24). The ELM was the first of the outer retinal layers to show postoperative recovery in both groups. CONCLUSION: Using the i­ILM peeling technique in this consecutive series it seemed to be possible to achieve a similar postoperative visual outcome in patients with a large FTMH as for patients with a smaller FTMH operated on with the c­ILM peeling technique.

[MEK inhibitor-associated retinopathy under binimetinib treatment for cutaneous malignant melanoma]. / MEK-Inhibitor-assoziierte Retinopathie unter Binimetinib-Therapie bei kutanem malignem Melanom.

The treatment options for patients with metastatic melanoma (MM) have been dramatically expanded in recent years with the approval of new drugs. The MEK (mitogen-acitvated protein kinase kinase) and BRAF (serine/threonine-protein kinase B-Raf coding gene) inhibitor combination therapy is currently part of the standard of care for stage IIIC/IV of BRAF mutant melanoma. The MEK inhibitor-associated retinopathy (MEKAR) is observed in patients with MM who are treated (or have been treated) with such a combination therapy. This article reports the case of a 72-year-old male patient, who suffered from such a pathological condition under treatment with binimetinib in combination with nivolumab. This case study illustrates the importance of interdisciplinary collaboration in the treatment of MM patients.

[Microstructural retinal changes after pharmacological vitreolysis with ocriplasmin-an SD-OCT supported analysis]. / Mikrostrukturelle retinale Veränderungen nach pharmakologischer Vitreolyse mit Ocriplasmin ­ eine SD-OCT-gestützte Analyse.

PURPOSE: Ocriplasmin (Jetrea®) is a therapeutic option for patients with focal vitreomacular traction (VMT) with or without small full thickness macular holes (FTMH) < 400 µm. Retinal alterations after injection with ocriplasmin have been described. The purpose of this essay was to determine Ocriplasmin-associated side-effects and changes in the retinal microstructure. METHODS: We included 70 patients with ocriplasmin treatment in our study. On all patients SD-OCT (spectral-domain optical coherence tomography) scans were performed prior to injection with Ocriplasmin. If present, adverse events were registered. The OCT scans were then evaluated taking the following into account: macular hole (MH) size, macular edema, subretinal fluid (SRF), changes in the ellipsoid zone (EZ) and the external limiting membrane (ELM). RESULTS: Twenty of the 70 examined patients showed a preoperative FTMH. One week after ocriplasmin IVI (intravitreal injection) 8 of the 20 FTMHs were already closed. Overall 12 patients showed a FTMH closure and 4 patients developed a FTMH after ocriplasmin IVI. Twelve of the 24 MH (macular hole) patients still required an operative closure of the FTMH. We noticed a resolution of the VMT on 51 patients. Three patients developed a retinal detachment. Furthermore, after ocriplasmin IVI we detected changes in the EZ and ELM on 8 patients. CONCLUSIONS: Ocriplasmin is a substantial minimal invasive option in the therapy of VMT with or without small FTMH. Nevertheless, there seem to be some specific ocriplasmin-associated risks, although usually transient. Severe complications like retinal detachment are rare but exist. Therefore, every indication of ocriplasmin should be considered carefully.

[Real-time optical coherence tomography-assisted high-precision vitreoretinal surgery in the clinical routine]. / Echtzeit-optische Kohärenztomographie-assistierte Hochpräzisionsvitreoretinalchirurgie in der klinischen Routine.

The complete integration of spectral domain optical coherence tomography (SD-OCT) into an operating microscope now enables targeted, high-resolution imaging-guided vitreoretinal surgery. This provides real-time visualization of retinal layers, vitreous body and instrument-tissue interactions, which can be used for intraoperative decision making. Compared to conventional surgical microscopes, intraoperative OCT enables a greatly enhanced precision of vitreoretinal surgical maneuvers and is an essential prerequisite for the implementation of real-time guided surgical techniques at the micrometer level.

[Diagnostic criteria: OCT angiography for retinal angiomatous proliferation (RAP lesions, type 3 neovascularization)]. / Diagnostische Kriterien: OCT­Angiographie bei retinalen angiomatösen Proliferationen (RAP­Läsionen, Typ­3-Neovaskularisationen).

BACKGROUND: Multimodal imaging of retinal angiomatous proliferation (RAP) lesions (type 3 neovascularization) and the diagnostic significance of optical coherence tomography angiography (OCT-A). MATERIAL AND METHODS: Confirmation of the diagnosis in six case reports with fundus photography, optical coherence tomography (OCT), fluorescein angiography (FLA), indocyanine green angiography (ICGA) and optical coherence tomography angiography (OCT­A). RESULTS AND CONCLUSION: The use of OCT­A is helpful for the diagnosis and follow-up examinations of RAP lesions (type 3 neovascularization). It enables the detailed visualization of intraretinal and choroidal microcirculation. Furthermore, it is possible to evaluate the progression, classify the stages and comprehend the treatment strategy.

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Homogenization of plasmonic crystals: seeking the epsilon-near-zero effect.

By using an asymptotic analysis and numerical simulations, we derive and investigate a system of homogenized Maxwell's equations for conducting material sheets that are periodically arranged and embedded in a heterogeneous and anisotropic dielectric host. This structure is motivated by the need to design plasmonic crystals that enable the propagation of electromagnetic waves with no phase delay (epsilon-near-zero effect). Our microscopic model incorporates the surface conductivity of the two-dimensional (2D) material of each sheet and a corresponding line charge density through a line conductivity along possible edges of the sheets. Our analysis generalizes averaging principles inherent in previous Bloch-wave approaches. We investigate physical implications of our findings. In particular, we emphasize the role of the vector-valued corrector field, which expresses microscopic modes of surface waves on the 2D material. We demonstrate how our homogenization procedure may set the foundation for computational investigations of: effective optical responses of reasonably general geometries, and complicated design problems in the plasmonics of 2D materials.