A novel bioprospecting strategy via 13C-based high-throughput probing of active methylotrophs inhabiting oil reservoir surface soil.

Methane-oxidizing bacteria (MOB) have long been considered as a microbial indicator for oil and gas prospecting. However, due to the phylogenetically narrow breath of ecophysiologically distinct MOB, classic culture-dependent approaches could not discriminate MOB population at fine resolution, and accurately reflect the abundance of active MOB in the soil above oil and gas reservoirs. Here, we presented a novel microbial anomaly detection (MAD) strategy to quantitatively identify specific indicator methylotrophs in the surface soils for bioprospecting oil and gas reservoirs by using a combination of 13C-DNA stable isotope probing (SIP), high-throughput sequencing (HTS), quantitative PCR (qPCR) and geostatistical analysis. The Chunguang oilfield of the Junggar Basin was selected as a model system in western China, and type I methanotrophic Methylobacter was most active in the topsoil above the productive oil wells, while type II methanotrophic Methylosinus predominated in the dry well soils, exhibiting clear differences between non- and oil reservoir soils. Similar results were observed by quantification of Methylobacter pmoA genes as a specific bioindicator for the prediction of unknown reservoirs by grid sampling. A microbial anomaly distribution map based on geostatistical analysis further showed that the anomalous zones were highly consistent with petroleum, geological and seismic data, and validated by subsequent drilling. Over seven years, a total of 24 wells have been designed and drilled into the targeted anomaly, and the success rate via the MAD prospecting strategy was 83 %. Our results suggested that molecular techniques are powerful tools for oil and gas prospecting. This study indicates that the exploration efficiency could be significantly improved by integrating multi-disciplinary information in geophysics and geomicrobiology while reducing the drilling risk to a greater extent.

Salt tolerance evaluation and mini-core collection development in Miscanthus sacchariflorus and M. lutarioriparius.

Marginal lands, such as those with saline soils, have potential as alternative resources for cultivating dedicated biomass crops used in the production of renewable energy and chemicals. Optimum utilization of marginal lands can not only alleviate the competition for arable land use with primary food crops, but also contribute to bioenergy products and soil improvement. Miscanthus sacchariflorus and M. lutarioriparius are prominent perennial plants suitable for sustainable bioenergy production in saline soils. However, their responses to salt stress remain largely unexplored. In this study, we utilized 318 genotypes of M. sacchariflorus and M. lutarioriparius to assess their salt tolerance levels under 150 mM NaCl using 14 traits, and subsequently established a mini-core elite collection for salt tolerance. Our results revealed substantial variation in salt tolerance among the evaluated genotypes. Salt-tolerant genotypes exhibited significantly lower Na+ content, and K+ content was positively correlated with Na+ content. Interestingly, a few genotypes with higher Na+ levels in shoots showed improved shoot growth characteristics. This observation suggests that M. sacchariflorus and M. lutarioriparius adapt to salt stress by regulating ion homeostasis, primarily through enhanced K+ uptake, shoot Na+ exclusion, and Na+ sequestration in shoot vacuoles. To evaluate salt tolerance comprehensively, we developed an assessment value (D value) based on the membership function values of the 14 traits. We identified three highly salt-tolerant, 50 salt-tolerant, 127 moderately salt-tolerant, 117 salt-sensitive, and 21 highly salt-sensitive genotypes at the seedling stage by employing the D value. A mathematical evaluation model for salt tolerance was established for M. sacchariflorus and M. lutarioriparius at the seedling stage. Notably, the mini-core collection containing 64 genotypes developed using the Core Hunter algorithm effectively represented the overall variability of the entire collection. This mini-core collection serves as a valuable gene pool for future in-depth investigations of salt tolerance mechanisms in Miscanthus.

Identifying Active Rather than Total Methanotrophs Inhabiting Surface Soil Is Essential for the Microbial Prospection of Gas Reservoirs.

Methane-oxidizing bacteria (MOB) have long been recognized as an important bioindicator for oil and gas exploration. However, due to their physiological and ecological diversity, the distribution of MOB in different habitats varies widely, making it challenging to authentically reflect the abundance of active MOB in the soil above oil and gas reservoirs using conventional methods. Here, we selected the Puguang gas field of the Sichuan Basin in Southwest China as a model system to study the ecological characteristics of methanotrophs using culture-independent molecular techniques. Initially, by comparing the abundance of the pmoA genes determined by quantitative PCR (qPCR), no significant difference was found between gas well and non-gas well soils, indicating that the abundance of total MOB may not necessarily reflect the distribution of the underlying gas reservoirs. 13C-DNA stable isotope probing (DNA-SIP) in combination with high-throughput sequencing (HTS) furthermore revealed that type II methanotrophic Methylocystis was the absolutely predominant active MOB in the non-gas-field soils, whereas the niche vacated by Methylocystis was gradually filled with type I RPC-2 (rice paddy cluster-2) and Methylosarcina in the surface soils of gas reservoirs after geoscale acclimation to trace- and continuous-methane supply. The sum of the relative abundance of RPC-2 and Methylosarcina was then used as specific biotic index (BI) in the Puguang gas field. A microbial anomaly distribution map based on the BI values showed that the anomalous zones were highly consistent with geological and geophysical data, and known drilling results. Therefore, the active but not total methanotrophs successfully reflected the microseepage intensity of the underlying active hydrocarbon system, and can be used as an essential quantitative index to determine the existence and distribution of reservoirs. Our results suggest that molecular microbial techniques are powerful tools for oil and gas prospecting.

Variation and characterization of prometryn in oysters (Crassostrea gigas) after seawater exposure.

Prometryn (PRO) is frequently detected in shellfish of international trade among triazine herbicides because of its wide application in agriculture and aquaculture worldwide. Nevertheless, the variations of PRO remain unclear in aquatic organisms, which affect the accuracy of their food safety risk assessment. In the present study, the tissue-specific accumulation, biotransformation, and potential metabolic pathway of PRO were reported in oyster species Crassostrea gigas for the first time. The experiments were conducted through semi-static seawater exposure with low and high concentrations of PRO (at nominal concentrations of 10 and 100 µg/L) via daily renewal over 22 days, followed by 16 days of depuration in clean seawater. The characterization of prometryn in oysters was then evaluated through the bioaccumulation behavior, elimination pathway and metabolic transformation, comparing with other organisms. The digestive gland and gonad were found to be the main target organs during uptake. In addition, the highest bioconcentration factor of 67.4 ± 4.1 was observed when exposed to low concentration. The level of PRO in oyster tissues rapidly decreased within 1 day during depuration, with an elimination rate of >90 % for the gill. Moreover, four metabolites of PRO were identified in oyster samples of exposed groups, including HP, DDIHP, DIP, and DIHP, in which HP was the major metabolite. Considering the mass percentage of hydroxylated metabolites higher than 90 % in oyster samples, PRO poses a larger threat to aquatic organisms than rat. Finally, the biotransformation pathway of PRO in C. gigas was proposed, the major metabolic process of which was hydroxylation along with N-dealkylation. Meanwhile, the newly discovered biotransformation of PRO in oyster indicates the importance of monitoring environmental levels of PRO in cultured shellfish, to prevent possible ecotoxicological effects as well as to ensure the safety of aquatic products.

Multi-omics prediction of oat agronomic and seed nutritional traits across environments and in distantly related populations.

KEY MESSAGE: Integration of multi-omics data improved prediction accuracies of oat agronomic and seed nutritional traits in multi-environment trials and distantly related populations in addition to the single-environment prediction. Multi-omics prediction has been shown to be superior to genomic prediction with genome-wide DNA-based genetic markers (G) for predicting phenotypes. However, most of the existing studies were based on historical datasets from one environment; therefore, they were unable to evaluate the efficiency of multi-omics prediction in multi-environment trials and distantly related populations. To fill those gaps, we designed a systematic experiment to collect omics data and evaluate 17 traits in two oat breeding populations planted in single and multiple environments. In the single-environment trial, transcriptomic BLUP (T), metabolomic BLUP (M), G + T, G + M, and G + T + M models showed greater prediction accuracy than GBLUP for 5, 10, 11, 17, and 17 traits, respectively, and metabolites generally performed better than transcripts when combined with SNPs. In the multi-environment trial, multi-trait models with omics data outperformed both counterpart multi-trait GBLUP models and single-environment omics models, and the highest prediction accuracy was achieved when modeling genetic covariance as an unstructured covariance model. We also demonstrated that omics data can be used to prioritize loci from one population with omics data to improve genomic prediction in a distantly related population using a two-kernel linear model that accommodated both likely casual loci with large-effect and loci that explain little or no phenotypic variance. We propose that the two-kernel linear model is superior to most genomic prediction models that assume each variant is equally likely to affect the trait and can be used to improve prediction accuracy for any trait with prior knowledge of genetic architecture.

Semicarbazide Accumulation, Distribution and Chemical Forms in Scallop (Chlamys farreri) after Seawater Exposure.

Semicarbazide is a newly recognized marine pollutant and has the potential to threaten marine shellfish, the ecological equilibrium and human health. In this study, we examined the accumulation, distribution, and chemical forms of semicarbazide in scallop tissues after exposure to 10, 100, and 1000 µg/L for 30 d at 10 °C. We found a positive correlation between semicarbazide residues in the scallops and the exposure concentration (p < 0.01). Semicarbazide existed primarily in free form in all tissues while bound semicarbazide ranged from 12.1 to 32.7% and was tissue-dependent. The time for semicarbazide to reach steady-state enrichment was 25 days and the highest levels were found in the disgestive gland, followed by gills while levels in gonads and mantle were similar and were lowest in adductor muscle. The bioconcentration factor (BCF) of semicarbazide at low exposure concentrations was higher than that at high exposure concentrations. These results indicated that the scallop can uptake semicarbazide from seawater and this affects the quality and safety of these types of products when used as a food source.

Beta1-integrin/Hedgehog-Gli1 signaling pathway fuels the diameter-dependent osteoblast differentiation on different TiO2 nanotubes: The optimal-diameter nanotubes for osteoblast differentiation.

Micro/nanotextured topographies (MNTs) can modulate cell-biomaterial interactions mostly by their controllable geometrics. Among them, TiO2 nanotubes, regarded as having a highly controllable nanoscale geometry, have been extensively investigated and applied and significantly affect diameter-dependent cell biological behaviors. In this study, we used five typical MNTs decorated with TiO2 nanotubes with diameters of 30, 50, 70, 100 and 120 nm to explore the optimal nanotube diameter for improving the biofunctional properties and to more deeply understand the underlying mechanisms by which these MNTs affect osteogenic differentiation by revealing the effect of beta1-integrin/Hedgehog-Gli1 signaling on this process. The MNTs affected MG63 osteoblast-like cell spreading, osteogenic gene expression (BMP-2, Runx2 and ALP), mineralization and ALP activity in a diameter-dependent pattern, and the optimal TiO2 nanotube diameter of 70 nm provided the best microenvironment for osteogenic differentiation as well as beta1-integrin/Hedgehog-Gli1 signaling activation. This enhanced osteogenic differentiation by the optimal-diameter TiO2 nanotubes of 70 nm was attenuated via suppression of the beta1-integrin/ Hedgehog-Gli1 signaling, which indicated a significant role of this pathway in mediating the diameter-dependent osteogenic differentiation promotional effect of MNTs with different TiO2 nanotube diameters. These results might provide deeper insights into the signal transduction mechanisms by which different nanoscale geometries influence cellular functions for biomaterial modification and biofunctionalization.

Human Ccr4 and Caf1 Deadenylases Regulate Proliferation and Tumorigenicity of Human Gastric Cancer Cells via Modulating Cell Cycle Progression.

Cancer cells generally have reprogrammed gene expression profiles to meet the requirements of survival, continuous division, and metastasis. An interesting question is whether the cancer cells will be affected by interfering their global RNA metabolism. In this research, we found that human Ccr4a/b (hCcr4a/b) and Caf1a/b (hCaf1a/b) deadenylases, the catalytic components of the Ccr4-Not complex, were dysregulated in several types of cancers including stomach adenocarcinoma. The impacts of the four deadenylases on cancer cell growth were studied by the establishment of four stable MKN28 cell lines with the knockdown of hCcr4a/b or hCaf1a/b or transient knockdown in several cell lines. Depletion of hCcr4a/b or hCaf1a/b significantly inhibited cell proliferation and tumorigenicity. Mechanistic studies indicated that the cells were arrested at the G2/M phase by knocking down hCaf1a, while arrested at the G0/G1 phase by depleting hCaf1b or hCcr4a/b. The four enzymes did not affect the levels of CDKs and cyclins but modulated the levels of CDK-cyclin inhibitors. We identified that hCcr4a/b, but not hCaf1a/b, targeted the p21 mRNA in the MKN28 cells. Furthermore, depletion of any one of the four deadenylases dramatically impaired processing-body formation in the MKN28 and HEK-293T cells. Our results highlight that perturbating global RNA metabolism may severely affect cancer cell proliferation, which provides a potential novel strategy for cancer treatment.

Enhancement of biodiesel yield and characteristics through in-situ solvo-thermal co-transesterification of wet microalgae with spent coffee grounds.

This study evaluated in-situ co-transesterification of wet spent coffee ground (SCG)/microalgae mixture for enhanced biodiesel production. SCG and microalgae showed lipid contents of 16.0 and 23.6 wt%, respectively. A total of 27 transesterification runs were performed using wet SCG:algae (1:1, w/w) at different temperatures, times, and solvent ratios. Box-Behnken quadratic model suggested 198 °C, 6 mL solvent g-1 biomass, and reaction time of 132 min as the optimum conditions for maximum biodiesel yield. At different SCG/microalgae blend ratios, pure microalgae showed the highest biodiesel yield of 20.15 wt%. Increase of SCG ratio resulted in significant reduction in the biodiesel yield, reaching the lowest value of 11.2 wt% using pure SCG. On the other hand, SCG showed better biodiesel characteristics than microalgae regarding iodine value, cetane number, and oxidation stability. The present results confirmed that SCG-algae blend results in dual effect of enhancing biodiesel yield and quality, comparing to the individual transesterification.

New Pathways of Mutational Change in SARS-CoV-2 Proteomes Involve Regions of Intrinsic Disorder Important for Virus Replication and Release.

The massive worldwide spread of the SARS-CoV-2 virus is fueling the COVID-19 pandemic. Since the first whole-genome sequence was published in January 2020, a growing database of tens of thousands of viral genomes has been constructed. This offers opportunities to study pathways of molecular change in the expanding viral population that can help identify molecular culprits of virulence and virus spread. Here we investigate the genomic accumulation of mutations at various time points of the early pandemic to identify changes in mutationally highly active genomic regions that are occurring worldwide. We used the Wuhan NC_045512.2 sequence as a reference and sampled 15 342 indexed sequences from GISAID, translating them into proteins and grouping them by month of deposition. The per-position amino acid frequencies and Shannon entropies of the coding sequences were calculated for each month, and a map of intrinsic disorder regions and binding sites was generated. The analysis revealed dominant variants, most of which were located in loop regions and on the surface of the proteins. Mutation entropy decreased between March and April of 2020 after steady increases at several sites, including the D614G mutation site of the spike (S) protein that was previously found associated with higher case fatality rates and at sites of the NSP12 polymerase and the NSP13 helicase proteins. Notable expanding mutations include R203K and G204R of the nucleocapsid (N) protein inter-domain linker region and G251V of the viroporin encoded by ORF3a between March and April. The regions spanning these mutations exhibited significant intrinsic disorder, which was enhanced and decreased by the N-protein and viroporin 3a protein mutations, respectively. These results predict an ongoing mutational shift from the spike and replication complex to other regions, especially to encoded molecules known to represent major ß-interferon antagonists. The study provides valuable information for therapeutics and vaccine design, as well as insight into mutation tendencies that could facilitate preventive control.

The hierarchical micro-/nanotextured topographies promote the proliferation and angiogenesis-related genes expression in human umbilical vein endothelial cells by initiation of Hedgehog-Gli1 signaling.

The hierarchical microtextured/nanotextured topographies have been recognized to have better tissue integration properties, but the underlying mechanisms are only partially understood. Hedgehog signaling plays a pivotal role in developmental and homeostatic angiogenesis. We suppose that the Hedgehog-Gli1 signaling may play a significant role in the response of endothelial cells to microtextured/nanotextured topographies (MNTs). To confirm this hypothesis, we produced the MNTs decorated with TiO2 nanotubes of two different diameters (25 and 70 nm), and the proliferation, apoptosis, angiogenesis-related genes expression and Hedgehog signaling activity of human umbilical vein endothelial cells (HUVECs) grown onto these MNTs were measured. Our results showed that the MNTs induced significantly high expression of Sonic Hedgehog (SHH), Smoothened (SMO) and GLI1 in the HUVECs as well as high activation of Hedgehog-Gli1 signaling, compared to the smooth surface. The HUVECs grown on the MNTs showed significantly high levels of adhesion, proliferation and expression of angiogenesis-related genes, including angiopoietin-1 (ANG-1), vascular endothelial growth factor (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (ENOS); these enhancements were attenuated by siRNA-mediated depletion of SMO, which indicated a significant role of Hedgehog-Gli1 signaling in mediating the enhanced effect of the MNTs on the angiogenic potential of HUVECs. This study may contribute to the modification of biomaterial surfaces for better tissue integration and clinical performance.