Antigenic Variation: Investigation of GPI-Anchoring of the Variant Surface Glycoprotein in Trypanosoma brucei

Der Parasit Trypanosoma brucei exprimiert ein variantes Oberflächen-glycoprotein (VSG), das ihn vor einer Lyse durch das Immunsystem des Wirtes schützt. Um trypanosomen-spezifische post-translationale Modifikationen von VSG zu untersuchen, wurde durch limitierte Lyse der Blutform von Trypanosoma brucei ein zell-freies System entwickelt. N-Glycosidase F, Biotin-Lysin-tRNAlys und anti-CRD Antiköper wurden für den Nachweis der N-Glycane und GPI-Anker im neu sythesierten VSG erfolgreich verwendet. Das Erscheinen von mfVSG durch Hemmung der endogenen GPI-PLC mit ZnCl2 und Markierung des VSG mit UDP-[14C]-Galactose stellen eindeutig Beweise für die Verbindung zwischen in vitro Translation und post-translationalen Modifikationen in unserem zellfreiem System dar. Dieser Tranamidase-Mechanismus der GPI-Verankerung wurde durch Kultivierung von Blutformen in Medium mit Hydrazin oder biotinyliertem Hydrazin untersucht. In Gegenwart von Hydrazin kann neu gebildetes VSG nicht vom anti-CRD Antiköper detektiert und nicht effizient an die Zelloberfläche transportiert werden; in Gegenwart von biotinyliertem Hydrazin kann neu gebildetes VSG von Streptavidin detektiert werden. Untersuchung der Trasamidase Aktivität in Zell-lysat durch Ac-S-V-L-N-AMC zeigt sich auch ein transamidase Reaktionsmechanismus und ein Sulfhydryl Rest in dem aktiven Zentrum. TbGpi8 wurde kloniert und in E. coli exprimiert. Die Homologie zwischen Gpi8 und anderen Cys- Proteinasen, z.B. Caspasen, deutet auf Cys192 und His150 als potientiellen Rest im aktiven Zentrum hin. Heterogene exprimiertes TbGpi8 führte zur Spalte des Modellsubstrate Ac-S-V-L-N-AMC. Das Ergebnis zeigt, daß TbGPI8 direkt an der proteolytischen Entfernung des C-Terminalen Peptids beteiligt ist. Die intrazellulare Lokalisation von TbGpi8 innerhalb des Golgi-Apparats wurde durch Anti-TbGpi8 Antiköper beobachtet. Western-blot Analyse zeigt 3 Proteinband von 37, 35 und 33 kDa.The parasitic protozoan Trypanosoma brucei expresses a variant surface glycoprotein (VSG), which protects it from lysis by host serum components. To study trypanosome-specific post-translational modifications of VSG, a cell-free system was produced by 'limited lysis' of bloodstream forms of Trypanosoma brucei. N-glycosidase F, Biotin-lysine-tRNAlys and specific anti-CRD antibodies were used successfully to demonstrate the existence of the N-glycans and the GPI anchor on newly formed VSG. Appearance of mfVSG by inhibition of endogenous GPI-PLC with ZnCl2 and labeling of VSG with UDP-[14C]galactose provided conclusive evidences for the linkage between in vitro translation and post-translational modification in our cell free system. The transamidase mechanism of GPI anchoring was studied by culture of bloodstream form Trypanosoma brucei in media containing hydrazine or biotinylated hydrazine. In the presence of hydrazine, newly formed VSG was not detected with anti-CRD antibodies and was not transferred to the cell surface efficiently; in the presence of biotinylated hydrazine, newly formed VSG was detected by streptavidin. Study of the transamidase in lysate using Ac-S-V-L-N-AMC showed also a transamidation reaction mechanism and a functionally sulfhydryl residue in the active centre. TbGpi8 was cloned and expressed in E. coli. Like LmGpi8, TbGpi8 has no C-terminal hydrophobic region. Homology between Gpi8 and other Cys proteinases, such as caspases, pointed to Cys192 and His150 as potential active site residues. Enzyme activity assays using heterologously expressed TbGpi8 and Ac-S-V-L-N-AMC showed cleavage of the substrate, indicating that Gpi8p is indeed directly involved in the proteolytic removal of the GPI anchoring signal. Intracellular localization of the TbGpi8 within the Golgi apparatus was observed by using specific anti-TbGpi8 antibodies. Western blotting analysis demonstrated 3 protein bands of 37, 35 and 33 kDa

Study On Acoustic Characteristics Of Internal Leakage Of Micro-start Spring Safety Valve

To investigate the noise radiation in the leakage of the safety valve. The acoustic fluid-solid coupling simulation analysis of the leakage process of the safety valve is used to study the flow field when the safety valve is leaking. The sound source characteristics of different inlet pressures and different leakage holes when the internal leakage of the safety valve occurs are analyzed, and the noise source is analyzed in combination with the flow field simulation. The results show that when the internal leakage of the safety valve occurs, the noise is mainly dominated by the quadrupole sound source caused by the jet, accompanied by the dipole sound source. At the same time, the noise sound pressure level is positively correlated with the working medium pressure of the safety valve and is quadratically correlated with the inner leakage hole

A new score system for predicting response to cardiac resynchronization therapy

Background: The aim of this study was to establish a score system derived from clinical, echocardiographic and electrocardiographic indexes and evaluate its clinical value for cardiac resynchronization therapy (CRT) patient selection. Methods: Ninety-three patients receiving CRT were enrolled. A patient selection score system was generated by the clinical, echocardiographic and electrocardiographic parameters achieving a significant level by univariate and multivariate Cox regression model. The positive response to CRT was a left ventricular end systolic volume decrease of ≥ 15% and not reaching primary clinical endpoint (death or re-hospitalization for heart failure) at the end of follow-up. Results: Thirty-nine patients were CRT non-responders (41.94%) and 54 were responders (58.06%). A 4-point score system was generated based on tricuspid annular plane systolic ex­cursion (TAPSE), longitudinal strain (LS), and complete left bundle branch block (CLBBB) combined with a wide QRS duration (QRSd). The sensitivity and specificity for prediction of a positive response to CRT at a score > 2 were 0.823 and 0.850, respectively (AUC: 0.92295% CI 0.691–0.916, p< 0.001). Conclusions: A patient selection score system based on the integration of TAPSE, LS and CLBBB combined with a wide QRSd can help to predict positive response to CRT effectively and reliably

Automatic Root Cause Analysis via Large Language Models for Cloud Incidents

Ensuring the reliability and availability of cloud services necessitates efficient root cause analysis (RCA) for cloud incidents. Traditional RCA methods, which rely on manual investigations of data sources such as logs and traces, are often laborious, error-prone, and challenging for on-call engineers. In this paper, we introduce RCACopilot, an innovative on-call system empowered by the large language model for automating RCA of cloud incidents. RCACopilot matches incoming incidents to corresponding incident handlers based on their alert types, aggregates the critical runtime diagnostic information, predicts the incident's root cause category, and provides an explanatory narrative. We evaluate RCACopilot using a real-world dataset consisting of a year's worth of incidents from Microsoft. Our evaluation demonstrates that RCACopilot achieves RCA accuracy up to 0.766. Furthermore, the diagnostic information collection component of RCACopilot has been successfully in use at Microsoft for over four years

Parallelism and non-parallelism in diabetic nephropathy and diabetic retinopathy

Diabetic nephropathy (DN) and diabetic retinopathy (DR), as microvascular complications of diabetes mellitus, are currently the leading causes of end-stage renal disease (ESRD) and blindness, respectively, in the adult working population, and they are major public health problems with social and economic burdens. The parallelism between the two in the process of occurrence and development manifests in the high overlap of disease-causing risk factors and pathogenesis, high rates of comorbidity, mutually predictive effects, and partial concordance in the clinical use of medications. However, since the two organs, the eye and the kidney, have their unique internal environment and physiological processes, each with specific influencing molecules, and the target organs have non-parallelism due to different pathological changes and responses to various influencing factors, this article provides an overview of the parallelism and non-parallelism between DN and DR to further recognize the commonalities and differences between the two diseases and provide references for early diagnosis, clinical guidance on the use of medication, and the development of new drugs

Polarization-Flexible and Frequency-Scanning Leaky-Wave HMSIW Antenna for Vehicular Applications

To achieve multifunctional communication and safe driving of a vehicle, a half-mode substrate-integrated waveguide (HMSIW) leaky-wave frequency-scanning antenna with flexible polarization is proposed in this article. It includes two linearly polarized interdigital slot antennas, a compact directional coupler, and microstrip transition lines. It can generate either linear polarization (LP) for base station communication or circular polarization (CP) for satellite navigation by configuring the means of excitation. Its radiation beam can be continuously steered with varying frequency in either the LP or the CP state, which is of benefit to safe vehicular driving. In addition, the use of the HMSIW structure reduces the size of the antenna by almost one-half in comparison with the full SIW structure. Measurements were performed on antenna scattering parameters, radiation patterns, gain, and axial ratio (for CP states); the results show good agreement with the simulated results. With its low profile, low weight, low cost, and capability for continuous frequency scanning and variable polarization states, the multifunctional antenna could be extensively used for adapting to changes in environmental conditions or system requirements

Design and Implementation of A New-type Cloud Computing Examination System

With the rapid development of network technology, large enterprises have established their own online learning and examination system respectively. However, as those network examination systems are dispersive, closed and disconnected, so various resources are unable to be utilized in a balanced way, which may cause substantial waste of enterprise resources. To solve such a problem, the emerging cloud computing technology with the characteristics of service on demand and dynamic expansion capability, provides a possibility of a shared network examination system with lower cost, named as cloud exam support service. A feasible solution for the application of the cloud computing technology in the network examination, which combines the theoretical analysis, system design and technical implementation, is put forward in this paper. The design, development, and pilot application of the cloud examination system described in this paper show that this study is highly practical, operable, and worthy of application and popularization