Phylogenetic diversity of functional genes in deep-sea cold seeps: a novel perspective on metagenomics.

BACKGROUND: Leakages of cold, methane-rich fluids from subsurface reservoirs to the sea floor are termed cold seeps. Recent exploration of the deep sea has shed new light on the microbial communities in cold seeps. However, conventional metagenomic methods largely rely on reference databases and neglect the phylogeny of functional genes. RESULTS: In this study, we developed the REMIRGE program to retrieve the full-length functional genes from shotgun metagenomic reads and fully explored the phylogenetic diversity in cold seep sediments. The abundance and diversity of functional genes involved in the methane, sulfur, and nitrogen cycles differed in the non-seep site and five cold seep sites. In one Haima cold seep site, the divergence of functional groups was observed at the centimeter scale of sediment depths, with the surface layer potentially acting as a reservoir of microbial species and functions. Additionally, positive correlations were found between specific gene sequence clusters of relevant genes, indicating coupling occurred within specific functional groups. CONCLUSION: REMIRGE revealed divergent phylogenetic diversity of functional groups and functional pathway preferences in a deep-sea cold seep at finer scales, which could not be detected by conventional methods. Our work highlights that phylogenetic information is conducive to more comprehensive functional profiles, and REMIRGE has the potential to uncover more new insights from shotgun metagenomic data. Video Abstract.

Long-term influence of chloroxylenol on anaerobic microbial community: Performance, microbial interaction, and antibiotic resistance gene behaviors.

The use of antibacterial and disinfection products is increasing in recent years. Para-chloro-meta-xylenol (PCMX), a widely used antimicrobial agent, has been detected in various environments. Herein, the impacts of PCMX with long-term exposure on anaerobic sequencing batch reactors were investigated. The high concentration (50 mg/L, GH group) PCMX severely inhibited the nutrient removal process, and the low concentration group (0.5 mg/L, GL group) slightly affected the removal efficiency which was recovered after 120 days of adaptation compared to the control group (0 mg/L, GC group). Cell viability tests indicated that PCMX inactivated the microbes. A significant reduction in bacterial α-diversity was observed in the GH but not the GL group. The microbial communities were shifted upon PCMX exposure, among which Olsenella, Novosphingobium, and Saccharibacteria genera incertae Sedis became the predominant genera in the GH groups. Network analyses showed that PCMX significantly reduced the complexity and interactions of the microbial communities, consistent with the negative impacts on bioreactor performance. Real-time PCR analysis indicated that PCMX affected the behavior of antibiotic resistance genes (ARGs), and the relationship between ARGs and bacterial genera gradually became complicated after long-term exposure. Most detected ARGs decreased on Day 60 but increased on Day 120 especially in the GL group, implying the potential risk of environment-relevant concentration of PCMX in the ecosystems. This study provides new insights into the understanding of the impacts and risks of PCMX on wastewater treatment processes.

A Multi-Layer Intrusion Detection System for SOME/IP-Based In-Vehicle Network.

The automotive Ethernet is gradually replacing the traditional controller area network (CAN) as the backbone network of the vehicle. As an essential protocol to solve service-based communication, Scalable service-Oriented MiddlewarE over IP (SOME/IP) is expected to be applied to an in-vehicle network (IVN). The increasing number of external attack interfaces and the protocol's vulnerability makes SOME/IP in-vehicle networks vulnerable to intrusion. This paper proposes a multi-layer intrusion detection system (IDS) architecture, including rule-based and artificial intelligence (AI)-based modules. The rule-based module is used to detect the SOME/IP header, SOME/IP-SD message, message interval, and communication process. The AI-based module acts on the payload. We propose a SOME/IP dataset establishment method to evaluate the performance of the proposed multi-layer IDS. Experiments are carried out on a Jetson Xavier NX, showing that the accuracy of AI-based detection reached 99.7761% and that of rule-based detection was 100%. The average detection time per packet is 0.3958 ms with graphics processing unit (GPU) acceleration and 0.6669 ms with only a central processing unit (CPU). After vehicle-level real-time analyses, the proposed IDS can be deployed for distributed or select critical advanced driving assistance system (ADAS) traffic for detection in a centralized layout.

Spatial distribution patterns across multiple microbial taxonomic groups.

Even in the vertical dimension, soil bacterial communities are spatially distributed in a distance-decay relationship (DDR). However, whether this pattern is universal among all soil microbial taxonomic groups, and how body size influences this distribution, remains elusive. Our study consisted of obtaining 140 soil samples from two adjacent ecosystems in the Yellow River Delta (YRD), both nontidal and tidal, and measuring the DDR between topsoil and subsoil for bacteria, archaea, fungi and protists (rhizaria). Our results showed that the entire community generally fitted the DDR patterns (P < 0.001), this was also true at the kingdom level (P < 0.001, with the exception of the fungal community), and for most individual phyla (47/75) in both ecosystems and with soil depth. Meanwhile, these results presented a general trend that the community turnover rate of nontidal soils was higher than tidal soils (P < 0.05), and that the rate of topsoil was also higher than that of subsoil (P < 0.05). Additionally, microbial spatial turnover rates displayed a negative relationship with body sizes in nontidal topsoil (R2 = 0.29, P = 0.009), suggesting that the smaller the body size of microorganisms, the stronger the spatial limitation was in this environment. However, in tidal soils, the body size effect was negligible, probably owing to the water's fluidity. Moreover, community assembly was judged to be deterministic, and heterogeneous selection played a dominant role in the different environments. Specifically, the spatial distance was much more influential, while the soil salinity in these ecosystems was the major environmental factor in selecting the distributions of microbial communities. Overall, this study revealed that microbial community compositions at different taxonomic levels followed relatively consistent distribution patterns and mechanisms in this coastal area.

Environmental DNA metabarcoding reveals the influence of human activities on microeukaryotic plankton along the Chinese coastline.

Microeukaryotic plankton, with its extremely diverse taxa, is a key component in both the marine food web and biogeochemical cycling. Coastal seas, which are home to the numerous microeukaryotic plankton that underpin the functions of these aquatic ecosystems, are often impacted by human activities. However, understanding the biogeographical patterns of diversity and community structure of microeukaryotic plankton and the role that major shaping factors play at the continent scale is still a challenge in coastal ecology. Here, the biogeographic patterns of biodiversity, community structure, and co-occurrence patterns were investigated by environmental DNA (eDNA) based approaches. Unlike most eDNA studies, we combined several methods (in silico PCR, mock and environmental communities) to systematically evaluate the specificity and coverage of primers to overcome the limitation of marker selection on biodiversity recovery. The 1380F/1510R primer set showed the best performance for the amplification of coastal plankton with the highest coverage, sensitivity, and resolution. We showed a unimodal pattern for planktonic alpha diversity with latitude (P < 0.001), and nutrient-related factors (NO3N, NO2N, and NH4N) were the leading predictors for spatial patterning. Significant regional biogeographic patterns and potential drivers for planktonic communities were found across coastal regions. All communities generally fitted the regional distance-decay relationship (DDR) model with the strongest spatial turnover rate was found in the Yalujiang (YLJ) estuary (P < 0.001). The environmental factors, especially inorganic nitrogen and heavy metals (HMs), had the greatest impact on planktonic community similarity in the Beibu Bay (BB) and East China Sea (ECS). Furthermore, we observed spatial plankton co-occurrence patterns, and the networked topology and structure were strongly driven by potential anthropogenic activity factors (nutrients and HMs). Overall, our study provided a systematic approach for metabarcode primer selection in eDNA-based biodiversity monitoring and revealed that the spatial pattern of the microeukaryotic plankton community was mainly controlled by regional human activity-related factors.

The Stability of Phyto-Zooplanktonic Networks Varied with Zooplanktonic Sizes in Chinese Coastal Ecosystem.

The linkages between phytoplankton and zooplankton are crucial for the stability of complex food webs and the flow of energy within the marine ecosystem. Despite body size exhibiting multiple effects on the planktonic community assembly and the dispersal scale, its role in determining the stability of phyto-zooplanktonic co-occurrence patterns remains unclear. Here, we focused on more than 13,000 kilometers of the Chinese coast to study the diatom-dominated plankton ecosystem and to report the significant negative effects of zooplanktonic body sizes on the topological properties of phyto-zooplanktonic networks (PZNs) by using more than 500 species from 251 samples taken along the coastline. PZNs tended to be more complex and stable when phytoplankton associated with smaller zooplankton. Particularly, the subnetworks of dominant phytoplankton displayed differences with different zooplanktonic body sizes. The zooplankton with larger and smaller body sizes tended to interact with dinoflagellates and diatoms, respectively. Additionally, abiotic factors (i.e., water temperature, pH, salinity, and metal concentrations) displayed significant effects on PZNs via the shifting of zooplanktonic composition, and the zooplanktonic body sizes altered the network modules' associations with different environmental factors. Our study elucidated the general relationship between zooplanktonic body sizes and the stability of PZNs, which provides new insights into marine food webs. IMPORTANCE Body size is a key life trait of aquatic plankton that affects organisms' metabolic rates and ecological functions; however, its specific effects on interactions between phytoplankton and zooplankton are poorly understood. We collected planktonic species and their body size data along more than 13,000 kilometers of coastline to explore the role of zooplanktonic body size in maintaining the stability of phyto-zooplanktonic networks (PZNs). We found that zooplankton play a more important role in maintaining PZN stability than do phytoplankton as well as that the PZN would be more complex and stable with smaller zooplankton. Furthermore, this work revealed that body size significantly determined the relationships between environmental factors and network structure. Overall, these findings lay a general relationship between zooplanktonic body sizes and the stability of PZNs, which helps us further explore the micro food web of coastal ecosystems.

CAMTA1 gene affects the ischemia-reperfusion injury by regulating CCND1.

Epigenetic modulations lead to changes in gene expression, including DNA methylation, histone modifications, and noncoding RNAs. In recent years, epigenetic modifications have been related to the pathogenesis of different types of cancer, cardiovascular disease, and other diseases. Emerging evidence indicates that DNA methylation could be associated with ischemic stroke (IS) and plays a role in pathological progression, but the underlying mechanism has not yet been fully understood. In this study, we used human methylation 850K BeadChip to analyze the differences in gene methylation status in the peripheral blood samples from two groups (3 IS patients vs. 3 healthy controls). According to their bioinformatics profiling, we found 278 genes with significantly different methylation levels. Seven genes with the most significant methylation modifications were validated in two expanded groups (100 IS patients vs. 100 healthy controls). The CAMTA1 gene had significantly different methylation changes in patients compared to the controls. To understand the CAMTA1 function in stroke, we generated CAMTA1 knockout in SH-SY5Y cells. RNA seq results in CAMTA1 knockout cells revealed the pathways and gene set enrichments involved in cellular proliferation and cell cycle. Furthermore, a series of experiments demonstrated that in the oxygen-glucose deprivation/re-oxygenation (OGD/R) model system, the expression of cyclin D1, an essential regulator of cell cycle progression, was increased in SH-SY5Y CAMTA1 KO cells. Increasing evidence demonstrated that ischemic stress could inappropriately raise cyclin D1 levels in mature neurons. However, the molecular signals leading to an increased cyclin D1 level are unclear. Our findings demonstrate for the first time that the CAMTA1 gene could regulate cyclin D1 expression and implicate their role in strokes.

Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of ischaemic stroke.

The mtDNA copy number can affect the function of mitochondria and play an important role in the development of diseases. However, there are few studies on the mechanism of mtDNA copy number variation and its effects in IS. The specific mechanism of mtDNA copy number variation is still unclear. In this study, mtDNA copy number of 101 IS patients and 101 normal controls were detected by qRT-PCR, the effect of D-loop variation on mtDNA copy number of IS patients was explored. Then, a TFAM gene KD-OE PC12 cell model was constructed to explore the effect of mtDNA copy number variation on mitochondrial function. The results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). The relative expression of TFAM gene mRNA in the cells of the OGD/R treatment group was significantly lower than that of the control group (p < 0.05). In addition, after TFAM gene knockdown and over-expression plasmids were transfected into HEK 293T cells, mtDNA copy number and ATP production level of Sh-TFAM transfection group was significantly decreased (p < 0.05), while mtDNA copy number and ATP production level of OE-TFAM transfected group were significantly higher than that of blank control group and OE-ctrl negative control group (p < 0.01). Our study demonstrated that mitochondrial D-loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, thus affecting the mitochondrial metabolism and function of nerve cells, participating in the pathological damage mechanism of IS.

Fungal dynamics and potential functions during anaerobic digestion of food waste.

Fungi could play an important role during anaerobic digestion (AD), but have received less attention than prokaryotes. Here, AD bioreactors of food waste were performed to explore fungal succession and their potential ecological and engineering value. We found that similar patterns in fungal biomass and diversity, decreasing from the initial time point (Day 0) to the lowest value within 3-6 days and then started to rise and stabilized between 9 and 42 days. Throughout the entire AD process, variations in fungal community composition were observed and dominant fungal taxa have the potential ability to degrade complex organic matter and alleviate fatty acid and ammonia accumulation. Furthermore, we found that deterministic processes gradually dominated fungal assembly succession (up to 84.85% at the final stage), suggesting changing environmental status responsible for fungal community dynamics and specifically, fungal community structure, diversity and biomass were regulated by different environmental variables or the same variables with opposite effects. AD bioreactors could directionally select specific fungal taxa over time, but some highly abundant fungi could not be mapped to any fungal species with defined function in the reference database, so function prediction relying on PICRUSt2 may underestimate fungal function in AD systems. Collectively, our study confirmed fungi have important ecological and engineering values in AD systems.

The large-scale spatial patterns of ecological networks between phytoplankton and zooplankton in coastal marine ecosystems.

Although autotrophic phytoplankton and heterotrophic zooplankton both play important roles in the food web of marine ecosystem, their comprehensive interactions and spatial patterns at continental scale remain poorly studied. Here, we collected 251 seawater samples along 13,000 km of Chinese coastline, and microscopically investigated the latitudinal gradients of planktonic diversities. In total, 307 phytoplanktonic and 311 zooplanktonic species were visually identified. Using the newly developed Inter-Domain Ecological Networks (IDENs) approach, the phytoplankton-zooplankton interaction networks were constructed. We found that the phyto-zooplankton network structure was varied across three regions, more complex and numerous connections along the southern coast than in the north. In addition, some particular associations between zooplanktonic and phytoplanktonic groups were found to be localized in specific regions. Furthermore, the seawater temperature and salinity were the major driving force for shaping planktonic interaction networks. These results provide a deeper understanding of planktonic biogeography and phytoplankton-zooplankton interaction patterns.

Genetic variations in ABCA1/G1 associated with plasma lipid levels and risk of ischemic stroke.

BACKGROUND: ATP binding cassette transporters ABCA1 and ABCG1 play a crucial role in cholesterol efflux and reverse cholesterol transport (RCT), thereby rendering ischemic stroke (IS) susceptibility. Variants of ABCA1/G1 have been implicated in etiology of IS. This study aimed to investigate the association between single-nucleotide polymorphisms (SNPs) of ABCA1/G1 with plasma lipid variability and the risk of IS in Chinese Han Population. METHODS: Totally 249 IS patients and 226 healthy controls were enrolled and 10 SNPs of ABCA1/G1 were screened for genotyping by kompetitive allele-specific polymerase chain reaction (KASP) and validated by sanger sequencing. The logistic regression analysis was performed to identify risk alleles of IS and appropriate genetic model. The genetic risk scores (GRS) and predicted risks for all individuals was computed. Based on different plasma lipid levels, we applied stratified analyses for subgroups. Linkage disequilibrium (LD) test was used to explore different functional haplotype combinations. Association between specific allele or genotype of the SNPs of ABCA1/G1 and plasma lipid or lipoproteins levels were also investigated. RESULTS: Besides total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), significant differences of clinical data were observed between IS and control group. The rare GG genotype frequencies of rs4149338 on ABCA1 was higher in IS patients than those in controls (11.4%, 4.6%, respectively, P = 0.037). Frequencies of rs57137919 on ABCG1 for rare AA genotype was lower in IS group than those in control group (4.6%, 13.3%, respectively, P = 0.030). GRS showed ability to discriminate IS patients and controls (AUC = 0.633, P < 0.001). Haplotype A-A (rs4149339-rs4149338) was correlated with reduced risk of IS (P = 0.023). Association analysis showed that subjects with rare AA genotype of rs57137919 had the lowest LDL-C levels while rare GG genotype of rs4149338 had lower TC level than those with AA genotype. The mRNA expression of ABCG1 was higher in IS patients, especially in the patients with frequent GG genotype of rs57137919, and was positively correlated with higher ABCG1 expression level and plasma LDL-C level. CONCLUSIONS: Polymorphisms of ABCA1/G1 associated with varieties of plasma lipid levels and risk of IS.

Fungi and cercozoa regulate methane-associated prokaryotes in wetland methane emissions.

Wetlands are natural sources of methane (CH4) emissions, providing the largest contribution to the atmospheric CH4 pool. Changes in the ecohydrological environment of coastal salt marshes, especially the surface inundation level, cause instability in the CH4 emission levels of coastal ecosystems. Although soil methane-associated microorganisms play key roles in both CH4 generation and metabolism, how other microorganisms regulate methane emission and their responses to inundation has not been investigated. Here, we studied the responses of prokaryotic, fungal and cercozoan communities following 5 years of inundation treatments in a wetland experimental site, and molecular ecological networks analysis (MENs) was constructed to characterize the interdomain relationship. The result showed that the degree of inundation significantly altered the CH4 emissions, and the abundance of the pmoA gene for methanotrophs shifted more significantly than the mcrA gene for methanogens, and they both showed significant positive correlations to methane flux. Additionally, we found inundation significantly altered the diversity of the prokaryotic and fungal communities, as well as the composition of key species in interactions within prokaryotic, fungal, and cercozoan communities. Mantel tests indicated that the structure of the three communities showed significant correlations to methane emissions (p < 0.05), suggesting that all three microbial communities directly or indirectly contributed to the methane emissions of this ecosystem. Correspondingly, the interdomain networks among microbial communities revealed that methane-associated prokaryotic and cercozoan OTUs were all keystone taxa. Methane-associated OTUs were more likely to interact in pairs and correlated negatively with the fungal and cercozoan communities. In addition, the modules significantly positively correlated with methane flux were affected by environmental stress (i.e., pH) and soil nutrients (i.e., total nitrogen, total phosphorus and organic matter), suggesting that these factors tend to positively regulate methane flux by regulating microbial relationships under inundation. Our findings demonstrated that the inundation altered microbial communities in coastal wetlands, and the fungal and cercozoan communities played vital roles in regulating methane emission through microbial interactions with the methane-associated community.

Epigenetic regulation mechanism of DNA methylation and miRNAs on the expression of the ALOX5AP gene in patients with ischemic stroke.

5-lipoxygenase-activating protein (FLAP), encoded by the arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene, can adjust the biogenesis of proinflammatory leukotrienes to increase the adhesion and permeability of the vascular internal wall. Moreover, it participates in the process of atherosclerosis and is closely associated with ischemic stroke (IS). Accumulating evidence has shown that the expression levels of the ALOX5AP gene are upregulated in patients with IS. However, the mechanism of ALOX5AP action in IS remain elusive. The present study hypothesized that epigenetic regulation, including DNA methylation and microRNA (miR/miRNA) regulation, affects the expression levels of the ALOX5AP gene. Therefore, 200 patients with a first diagnosis of acute IS and 200 healthy control subjects were enrolled in the present study. Initially, the mRNA expression levels of the ALOX5AP gene were examined by reverse transcription-quantitative PCR. It was found that the mRNA levels of ALOX5AP gene in the IS group were significantly higher compared with controls (P<0.05). Subsequently, the methylation status of 17 CpG sites located in the promoter region of ALOX5AP was assessed by MethyTarget sequencing. However, the levels of methylation exhibited no significant differences between IS and control groups (P>0.05). Moreover, the expression levels of miR-335 and miR-495 were examined as two potential miRNAs targeting the ALOX5AP gene. The expression levels of miR-335 and miR-495 in the IS group were significantly lower compared with the control group (P<0.05). Finally, the luciferase assay results indicated that the luciferase activity of the experimental group following co-transfection of miRNA mimic and wild-type reporter gene plasmid was significantly lower compared with the other experimental groups (P<0.05), suggesting that miR-335 and miR-495 could specifically bind to the 3'-untranslated region of the ALOX5AP gene, thereby downregulating its expression. The present study provided preliminary evidence demonstrating that epigenetic regulation affects the expression of the ALOX5AP gene in patients with IS.

[Microorganisms in the typical anaerobic digestion system of organic solid wastes: a review].

The facultative anaerobic and strict anaerobic microorganisms enriched and acclimated during the anaerobic digestion process are crucial for the efficiency of the anaerobic digestion system. Most of the problems encountered during running anaerobic digestion processes could be effectively improved via stimulation of microbial metabolic activity. Benefited from the rapid development of microbiome techniques, deeper insights into the microbial diversity in anaerobic digestion systems, e.g. the microbe-microbe interactions and microbe-environment interactions, have been gained. A complex and intricate metabolic network exists in the anaerobic digestion system of solid organic wastes. However, little is known about these interactions and the underlying mechanisms. This review briefly summarized the representative interactions between microbial communities during anaerobic digestion process discovered to date. In addition, typical issues encountered during the anaerobic digestion of solid organic wastes and how microbes can tackle and alleviate these issues were discussed. Finally, future priorities on microbiome research were proposed based on present contribution of microbiome analysis in anaerobic digestion system.

Warming reshaped the microbial hierarchical interactions.

Global warming may alter microbially mediated ecosystem functions through reshaping of microbial diversity and modified microbial interactions. Here, we examined the effects of 5-year experimental warming on different microbial hierarchical groups in a coastal nontidal soil ecosystem, including prokaryotes (i.e., bacteria and archaea), fungi, and Cercozoa, which is a widespread phylum of protists. Warming significantly altered the diversity and structure of prokaryotic and fungal communities in soil and additionally decreased the complexity of the prokaryotic network and fragmented the cercozoan network. By using the Inter-Domain Ecological Network approach, the cross-trophic interactions among prokaryotes, fungi, and Cercozoa were further investigated. Under warming, cercozoan-prokaryotic and fungal-prokaryotic bipartite networks were simplified, whereas the cercozoan-fungal network became slightly more complex. Despite simplification of the fungal-prokaryotic network, the strengthened synergistic interactions between saprotrophic fungi and certain prokaryotic groups, such as the Bacteroidetes, retained these phyla within the network under warming. In addition, the interactions within the fungal community were quite stable under warming conditions, which stabilized the interactions between fungi and prokaryotes or protists. Additionally, we found the microbial hierarchical interactions were affected by environmental stress (i.e., salinity and pH) and soil nutrients. Interestingly, the relevant microbial groups could respond to different soil properties under ambient conditions, whereas under warming these two groups tended to respond to similar soil properties, suggesting network hub species responded to certain environmental changes related to warming, and then transferred this response to their partners through trophic interactions. Finally, warming strengthened the network modules' negative association with soil organic matters through some fungal hub species, which might trigger soil carbon loss in this ecosystem. Our study provides new insights into the response and feedback of microbial hierarchical interactions under warming scenario.

Fenton-like reaction driving the degradation and uptake of multi-walled carbon nanotubes mediated by bacterium.

Carbon nanotubes (CNTs) have been widely studied because of their potential applications. The increasing applications of CNTs and less known of their environmental fates rise concerns about their safety. In this study, the biotransformation of multi-walled carbon nanotubes (MWCNTs) by Labrys sp. WJW was investigated. Within 16 days, qPCR analysis showed that cell numbers increased 4.92 ± 0.36 folds using 100 mg/L MWCNTs as the sole carbon source. The biotransformation of MWCNTs, which led to morphology and functional group change, was evidenced by transmission electron microscopy and X-ray photoelectron spectroscopy analyses. Raman spectra illustrated that more defects and disordered carbon appeared on MWCNTs during incubation. The underlying biotransformation mechanism of MWCNTs through an extracellular bacterial Fenton-like reaction was demonstrated. In this bacteria-mediated reaction, the OH production was induced by reduction of H2O2 involved a continuous cycle of Fe(II)/Fe(III). Bacterial biotransformation of MWCNTs will provide new insights into the understanding of CNTs bioremediation processes.

Sampling cores and sequencing depths affected the measurement of microbial diversity in soil quadrats.

Due to the massive quantity and broad phylogeny, an accurate measurement of microbial diversity is highly challenging in soil ecosystems. Initially, the deviation caused by sampling should be adequately considered. Here, we attempted to uncover the effect of different sampling strategies on α diversity measurement of soil prokaryotes. Four 1 m2 sampling quadrats in a typical grassland were thoroughly surveyed through deep 16S rRNA gene sequencing (over 11 million reads per quadrat) with numerous replicates (33 soil sampling cores with total 141 replicates per quadrat). We found the difference in diversity was relatively small when pooling soil cores before and after DNA extraction and sequencing, but they were both superior to a non-pooling strategy. Pooling a small number of soil cores (i.e., 5 or 9) combined with several technical replicates is sufficient to estimate diversities for soil prokaryotes, and there is great flexibility in pooling original samples or data at different experimental steps. Additionally, the distribution of local α diversity varies with sampling core number, sequencing depth, and abundance distribution of the community, especially for high orders of Hill diversity index (i.e., Shannon entropy and inverse Simpson index). For each grassland soil quadrat (1 m2), retaining 100,000 reads after taxonomic clustering might be a realistic option, as these number of reads can efficiently cover the majority of common species in this area. Our findings provide important guidance for soil sampling strategy, and the general results can serve as a basis for further studies.

Association Study Between Genetic Variation in Whole Mitochondrial Genome and Ischemic Stroke.

Mitochondrial DNA (mtDNA) affects the mitochondrial function, which is potentially related to susceptibility to ischemic stroke (IS). However, study on IS genetics by whole mitochondrial genome sequencing has not been extensively explored. Therefore, a two-stage study was designed to explore the relationship between the whole mitochondrial genome variants and IS. In the first stage, whole mitochondrial genomes of 52 IS patients and 55 controls were sequenced by next-generation sequencing. Fifty-three mtDNA mutation sites which may be related to the pathogenesis of IS were discovered. Nine unreported mtDNA variation sites were found for the first time. In the second larger Chinese cohort, we confirmed that m.T195C and m.T12338C in the mitochondrial D-loop region were the protective factors of IS, especially m.T195C and m.C311T in the LAA subtype. In conclusion, our study provided population genetic information and a reference for IS-relevant research, with wide applications in diagnosis, therapeutic treatments and prediction of IS.