Laser Ablation Ionization Mass Spectrometry: A Space Prototype System for In Situ Sulphur Isotope Fractionation Analysis on Planetary Surfaces

The signatures of element isotope fractionation can be used for the indirect identification of extant or extinct life on planetary surfaces or their moons. Element isotope fractionation signatures are very robust against the harsh environmental conditions, such as temperature or irradiation, which typically prevail on solar system bodies. Sulphur is a key element for life as we know it and bacteria exist, such as sulphur reducing bacteria, that can metabolize sulphur resulting in isotope fractionations of up to −70‰ δ34S. Geochemical processes are observed to fractionate up to values of −20‰ δ34S hence, fractionation exceeding that value might be highly indicative for the presence of life. However, the detection of sulphur element isotope fractionation in situ, under the assumption that life has existed or still does exist, is extremely challenging. To date, no instrument developed for space application showed the necessary detection sensitivity or measurement methodology for such an identification. In this contribution, we report a simple measurement protocol for the accurate detection of sulphur fractionation δ34S using our prototype laser ablation ionization mass spectrometer system designed for in situ space exploration missions. The protocol was elaborated based on measurements of five sulphur containing species that were sampled at different Mars analogue field sites, including two cave systems in Romania and the Río Tinto river environment in Spain. Optimising the laser pulse energy of our laser ablation ionization mass spectrometer (LIMS) allowed the identification of a peak-like trend of the 34S/32S ratio, where the maximum, compared to internal standards, allowed to derive isotope fractionation with an estimated δ34S accuracy of ∼2‰. This accuracy is sufficiently precise to differentiate between abiotic and biotic signatures, of which the latter, induced by, e.g., sulphate-reducing microorganism, may fractionate sulphur isotopes by more than −70‰ δ34S. Our miniature LIMS system, including the discussed measurement protocol, is simple and can be applied for life detection on extra-terrestrial surfaces, e.g., Mars or the icy moons like Europa.

Fractionation Analysis of Iron in Coastal Rivers to Yantai Sishili Bay with a Bismuth Microrods-Based Electrochemical Sensor

As an essential metal micronutrient, Fe plays an important role in the marine biogeochemical cycling process, and the bioavailability of Fe has a direct relationship with its fractions in water. The fractionation analysis of iron in main coastal rivers to Yantai Sishili Bay was achieved with an electrochemical sensor based on bismuth microrods (BiMRs). The sensor was characterized by scanning electron microscope and electrochemical methods, and the reliability of the sensor was verified by the determination of the standard samples. Different fractions of iron in coastal river waters, including total iron (TFe), total dissolved iron (TDFe) and particulate iron (PFe), have been determined by combining simple sample pretreatments and cathodic stripping voltammetry with the BiMRs-based sensor. The average concentrations of TFe in Guangdang River, Xin’an River and Yuniao River were 4.02, 3.66 and 4.42 μmol L−1, respectively. The main fractionation of iron in three rivers was PFe, which accounts for 84.46%, 87.56% and 92.34%, respectively. Furthermore, the relationships between iron concentration and tidal action, salinity, dissolved oxygen and other factors were also investigated.

Manganese fractionation analysis in specific soil and sediment samples

Manganese has been determined in soil- and sediment samples taken from selected regions with high manganese concentrations anthropogenic and/or geogenic. The total content of manganese in chosen sediment- and soil samples has been determined applying FAAS after microwave digestion and the manganese fractions after sequential extraction procedures using galvanostatic stripping chronopotentiometry. The highest content of manganese has been determined in sediment from Hôrka (6243.6 ± 56.2 mg kg-1), while the lowest value has been obtained in the sediment from Kráľová (278.6 ± 3.9 mg kg-1). Using a modified Tessier’s procedure it was found, that manganese in sediments from Kráľová is associated mainly with the carbonate fraction (>50%), while in sediments from Lozorno and Hôrka it is associated primarily with the Mn and Fe oxide fraction (80% and 42% respectively).

Long Non-Coding RNA LINC01410 Promoted Tumor Progression via the ErbB Signaling Pathway by Targeting STAT5 in Gallbladder Cancer

ObjectivesLong non-coding RNAs (lncRNAs) have been recently emerging as crucial molecules in multiple human cancers. However, their expression patterns, roles as well as the underlying mechanisms in gallbladder cancer (GBC) remain largely unclear.Materials and MethodsThe expression of lncRNAs in GBC was downloaded from GEO database. Quantitative real-time polymerase chain reaction (qRT-PCR) and RNA in situ hybridization (ISH) were used to detect the expression of lncRNAs in GBC tissues. The full-sequence of LINC01410 was determined by RACE assay. Subcellular distribution of LINC01410 was examined by nuclear/cytoplasmic RNA fractionation analysis. Loss- and gain-of-function experiments were conducted to explore the biological functions of LINC01410 in vitro and in vivo. RNA pull-down, RNA immune-precipitation (RIP), and Western blot assay were conducted to investigate the mechanisms underlying the biological function of LINC01410 in GBC.ResultsLINC01410 was significantly upregulated in the GBC tissues compared to adjacent non-tumor tissues. High LINC01410 expression was significantly associated with poor prognosis of GBC patients. We identified LINC01410 to be 2,877 bp in length and mainly localized in the cytoplasm of GBC cells. Overexpression of LINC01410 promoted GBC cell proliferation, migration, and invasion in vitro and GBC progression in vivo, whereas LINC01410 downregulation rescued these effects in vitro. From RNA pull-down and RIP assay, we identified that STAT5 was a critical downstream target of LINC01410. Furthermore, ErbB signaling pathway was involved in the malignant phenotypes of GBC mediated by LINC01410.ConclusionsOur results suggested that LINC01410 was an important lncRNA that promoted GBC progression via targeting STAT5 and activating ErbB signaling pathway.

LA-ICP-MS on hardened cement paste: laser-material interaction, signal formation and optimization of laser fluence

In almost all applications of concrete components, both the transport of substances such as chlorides, sulphates, acids, CO2, etc. through the pore structure into the concrete and the resulting local chemical and physical processes have a negative effect on the lifetime of the structure. Most data are actually obtained from layer-by-layer mechanical sampling of, for instance, bore dust, followed by chemical analysis. Several groups have previously demonstrated the enormous potential of LA-ICP-MS for monitoring these multi element processes both qualitatively and quantitatively and with high spatial resolution. However, there has been no fundamental investigation of laser-material interaction, aerosol particle formation, fractionation analysis or the effect of cement-specific parameters such as the water to cement (w/c) ratio on signal intensity. This paper presents the ablation mechanisms of a frequently used 213 nm quintupled Nd:YAG ns laser operating on the HCP (hardened cement paste) multi-phase system in comparison with amorphous and well-characterized NIST 612 glass. It includes energy-signal considerations, crater evaluations after multiple shots using different energy densities and aerosol structures captured on filters. The investigation determined a linear energy to signal behavior in a range of 2–6 J/cm2, while the ablation mechanism is different to common mechanisms obtained for glass or brass. The aerosol captured on the filter material displays cotton-like structures as well as defined spherical particles, which is comparable to observations made with NIST glass aerosols.

LncRNA HOXC-AS3 Promotes Epithelial-to-mesenchymal Transition through Decreasing the Expression of FBXL17 in Cervical Squamous Cell Carcinoma

Background:The expression and biological roles of a novel lncRNA HOXC-AS3 in cervical squamous cell carcinoma (CESC) are largely unknown.Methods:LncRNA HOXC-AS3 was identified from The Cancer Genome Atlas (TCGA) database. Real time quantitative PCR (qRT-PCR) was used to evaluate the expression of HOXC-AS3 in 68 pairs of CESC tumor tissues and adjacent normal tissues. Kaplan-Meier survival analysis was used to assess the significance of HOXC-AS3 in predicting overall survival (OS). Correlations between HOXC-AS3 expression and clinicopathological parameters were assessed using Chi-square test. The biological functions of HOXC-AS3 in CESC were investigated using loss-of and gain-of function assays, which including cell proliferation, migration and invasion. Furthermore, the underlying mechanism was revealed using bioinformatics prediction, qRT-PCR and western blot.Results:HOXC-AS3 expression was significantly up-regulated in CESC tumor tissues. Up-regulated HOXC-AS3 expression was correlated with advanced stage and poor OS. Over-expression of HOXC-AS3 promoted tumor cell proliferation, migration and invasion. Conversely, HOXC-AS3 knockdown significantly inhibited these effects. Subcellular fractionation analysis revealed that HOXC-AS3 was mainly localized in cell nuclei. Further mechanistic analyses demonstrated that HOXC-AS3 down-regulated the expression of FBXL17. The promotive effects of HOXC-AS3 on cell proliferation, migration and invasion could be repressed by FBXL17. Moreover, HOXC-AS3 activated the EMT process to promote the progression of CESC. Conclusion:In conclusion, our study indicated that HOXC-AS3 acted as an oncogenic lncRNA in CESC through downregulating FBXL17 expression and activating EMT process.

Mitochondria serve as axonal shuttle for Cox7c mRNA through mechanism that involves its mitochondrial targeting signal

Localized protein synthesis plays a key role in spatiotemporal regulation of the cellular proteome. Neurons, which extend axons over long distances, heavily depend on this process. However, the mechanisms by which axonal mRNAs are transported to protein target sites are not fully understood. Here, we describe a novel role for mitochondria in shuttling a nuclear encoded mRNA along axons. Fractionation analysis and smFISH revealed that the mRNA encoding Cox7c protein is preferentially associated with mitochondria from a neuronal cell line and from primary motor neuron axons. Live cell imaging of MS2-tagged Cox7c or Cryab control mRNA in primary motor neurons further confirmed the preferential colocalization of Cox7c mRNA with mitochondria. More importantly, Cox7c demonstrated substantial co-transport with mitochondria along axons. Intriguingly, the coding region, rather than the 3UTR, was found to be the key domain for the co-transport. Furthermore, we show that puromycin treatment as well as hindering the synthesis of the mitochondrial targeting signal (MTS) reduced the co-localization. Overall, our results reveal a novel mRNA transport mode which exploits mitochondria as a shuttle and translation of the MTS as a recognition feature. Thus, mitochondria may play a role in spatial regulation of the axonal transcriptome and self-sustain their own proteome at distant neuronal sites.

Disruption of Dense Granular Protein 2 (GRA2) Decreases the Virulence of Neospora caninum

Neospora caninum causes abortions in cattle and nervous system dysfunction in dogs. Dense granular proteins (GRAs) play important roles in virulence; however, studies on NcGRA functions are limited. In the present study, multiple methods, including site-directed mutagenesis; CRISPR/Cas9 gene editing; Western blotting; quantitative polymerase chain reaction; confocal microscopy; plaque, invasion, egress, and replication assays; animal assays of survival rate and parasite burden; and hematoxylin–eosin staining, were used to characterize the NcGRA2 protein, construct an NcGRA2 gene disruption (ΔNcGRA2) strain, and explore its virulence in vivo and vitro. The results showed that NcGRA2 shared 31.31% homology with TgGRA2 and was colocalized with NcGRA6 at the posterior end of tachyzoites and the intravacuolar network of parasitophorous vacuoles (PVs). Cell fractionation analysis showed that NcGRA2 behaved as a transmembrane and membrane-coupled protein. The ΔNcGRA2 strain was constructed by coelectroporation of the NcGRA2-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA2) and DHFR-TS DNA donor and verified at the protein, genome, and transcriptional levels and by immunofluorescence localization analysis. The in vitro virulence results showed that the ΔNcGRA2 strain displayed smaller plaques, similar invasion and egress abilities, and slower intracellular growth. The in vivo virulence results showed a prolonged survival time, lower parasite burden, and mild histopathological changes. Overall, the present study indicates that NcGRA2, as a dense granular protein, forms the intravacuolar network structure of PVs and weakens N. caninum virulence by slowing proliferation. These data highlight the roles of NcGRA2 and provide a foundation for research on other protein functions in N. caninum.

Efficient Homogeneous TEMPO-mediated Oxidation of Cellulose in Lithium Bromide Hydrates

An appropriate cellulose-dissolving solvent is critical for the homogeneous oxidation of cellulose using TEMPO (2, 2, 6, 6-tetramethylpiperidine-1-oxyl)-mediated system. Herein, TEMPO/NaClO/NaClO2 system in lithium bromide hydrates (LBHs) was developed for the homogeneous selective-oxidation of cellulose, which was two-stage protocol involving cellulose dissolution and homogeneous oxidation. Specifically, cellulose was firstly dissolved in LiBr⋅3.5H2O and offered the optimal pH (5.6) for the subsequent TEMPO/NaClO/NaClO2 oxidation without precipitation of the cellulose chains. Effect of reaction conditions on cellulose oxidation was investigated. The results showed that high degree of oxidation (DO) and evenly distributed carboxyl could be achieved. The particle size gradually decreased with DO, and oxidized cellulose with high water solubility and amorphous structure could be obtained. Furthermore, FT-IR, 13C NMR and fractionation analysis verified that cellulose was successfully converted and the carboxyl uniformly distributed onto the cellulose chains. This TEMPO-mediated system using LBHs as solvent presented an efficient method on the homogeneous selective oxidation of primary hydroxyl in cellulose.