Signal peptide peptidase (SPP) and SPP‐like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II‐oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER‐resident family members SPP and SPPL2c were shown to also cleave tail‐anchored proteins, including selected SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co‐expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail‐anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1‐4 levels in tissues and primary cells of SPPL2a/b double‐deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1‐4 in a cell type‐ and tissue‐dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1‐4 by initiating the degradation of these proteins, which might impact cellular trafficking.
Recent developments in water resource monitoring have increased the demand for the reliable identification of faecal pollution sources, also defined as microbial (faecal) source tracking (MST). Standardized faecal indicator bacteria (SFIB) enumeration does not directly support MST, as SFIB occur in animal and human sources. The aim of this study was to rigorously evaluate the applicability of host-associated faecal genetic MST markers detected by quantitative PCR (qPCR) at representative Austrian water resources (ground-, surface-, raw and treated wastewater, n = 196 samples) with high importance for the water management sector. Groundwater covered a gradient of non- (i.e., deep wells) to surface influenced resources (i.e., karst and shallow wells). In addition, single faecal excreta from humans as well as representative livestock and wildlife species were collected to evaluate the faecal source-specificity and -sensitivity of the MST assays. Genetic MST marker resistance against UV irradiation was evaluated in on-site ground and wastewater treatment installations. Bacteroides-based human- (HF183II, BacHum), ruminant- (BacR), and pig-associated (Pig2Bac) MST marker qPCR quantification was performed in concert with cultivation of E. coli, intestinal enterococci, and Clostridium perfringens (SFIB diagnostics). The selected MST makers revealed high faecal source identification capacity for the Austrian water compartments and quantitatively reflected the selected faecal pollution gradient. The study also demonstrated that SFIB data can efficiently be combined with MST data to solve previously unanswered questions in water safety monitoring and management (e.g., support pollution source-targeted catchment protection, hazard assessment, and health risk management). Further research and development needs are discussed to exploit the full power of MST technology. In conclusion, this study illustrates the capacity of molecular faecal pollution diagnostics to revolutionize water quality testing in the decades to come.
All-atom molecular dynamics (MD) computer simulations of C 60 fullerene patches adsorbed onto graphite are conducted at various coverages for very low temperatures. There appears to be a hindered tumbling rotational transition through T ≤ 20 K seen in the simulations that accompanies dramatic lattice expansion and configurational energy change with temperature in the adlayer. Moreover, the transition is unique in that it appears to be continuous and understandable on the basis of very simple dynamical arguments. The results suggest that such behavior could be present and influential in a wide range of molecules whose dynamical (time averaged) shapes are very close to their static symmetry, in contrast to surface systems with lower symmetry constituents and sharp rotational transitions. The transition is also observed in annealed, percolating adlayers and its dependence on lattice topology is discussed. C 60 Fullerenes have been of experimental and theoretical interest since their discovery almost thirty years ago. Their elastic properties and high degree of symmetry coupled with the interesting behavior of endohedral fullerenes make them ideal systems for study and innovation. When placed on a surface such as graphite or graphene, the interactions of the C 60 adlayer with the graphite 1-7 result in a wide range of epitaxial characteristics and phase transitions dependent upon the area density of the system, which have been studied theoretically 3-7 and experimentally. 7-17 Fullerene layers on graphitic structures 18-21 as well as fullerenes in nanomechanical systems 22-24 have also been studied computationally. Experimentally, the C 60 /gr system presents a single layer lacking strong orientational epitaxy over a wide temperature range 5-17 and a second layer which exhibits dendritic growth. 10 At all temperatures investigated so far the molecules themselves behave essentially as free rotors. Mainly because most of the interesting physics takes place for C 60 /gr systems at room temperature and much higher, the low-temperature behavior of these systems has not been explored. Computational studies * Author to whom correspondence should be addressed.have provided a wealth of insight into the behavior of C 60 /gr systems-complete with a prediction about structural ordering 21 -but the preponderance of work has been done with spherically averaged potentials which are known not to address the low-temperature behavior of the system. 4 18 In addition the all-atom work that has been published deals with the high temperature regime or specific dynamics related to nanoscale bearings and nanodevices. [22][23][24] The purpose of this Letter is to report results of ultra-lowtemperature (T ≤ 20 K) all atom Molecular Dynamics simulations of fullerene patches on graphite, which predict anomalous behavior of the adlayer as the system evolves through a hindered rotational transition. Because the static symmetry of the adsorbate molecules is so close to their dynamic symmetry, the rotational transition couples with dramatic configu...
N,N,N’,N’-Tetramethyl-formamidinium chloride (2a) reacts with elemental sodium in various solvents to give N,N,N’,N’,N’’,N’’-hexamethyl-guanidinium chloride (4a). The reaction of 2a with potassium affords N,N,N’,N’,N’’,N’’,N’’’,N’’’-octamethyl-oxamidinium dichloride (3a). From the reaction of 2a with magnesium in different solvents in general result mixtures of the salts 4a, 3a and N,N,N’,N’-tetramethyl-formamidinium chloride (10a). The composition of these mixtures depends on the solvent and the reaction temperature. Similar results are obtained, when a zinc/copper couple is used instead of magnesium. Very likely from 2a and magnesium or zinc, respectively, organometallic intermediates 11, 12 are formed which could be trapped by aromatic aldehydes and phenylisocyanate. The salt 2a can be reductively coupled by a low-valent titanium reagent to give the oxamidinium salt 3a.
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