Stratifying ICIs-responsive tumor microenvironment in HCC: from parsing out immune-hypoxic crosstalk to clinically applicable MRI-radiomics models.

BACKGROUND: We aimed to redefine Immune checkpoint inhibitors (ICIs)-responsive "hot" TME and develop a corresponding stratification model to maximize ICIs-efficacy in Hepatocellular Carcinoma (HCC). METHODS: Hypoxic scores were designed, and the relevance to immunotherapy responses were validated in pan-cancers through single cell analysis. Multi-omics analysis using the hypoxic scores and immune infiltrate abundance was performed to redefine the ICIs-responsive TME subtype in HCC patients from TCGA (n = 363) and HCCDB database (n = 228). The immune hypoxic stress index (IHSI) was constructed to stratify the ICIs-responsive TME subtype, with exploring biological mechanism in vitro and in vivo. MRI-radiomics models were built for clinical applicability. RESULTS: The hypoxic scores were lower in the dominant cell-subclusters of responders in pan-cancers. The higher immune infiltrate-normoxic (HIN) subtype was redefined as the ICIs-responsive TME. Stratification of the HIN subtype using IHSI effectively identified ICIs-responders in Melanoma (n = 122) and urological cancer (n = 22). TRAF3IP3, the constituent gene of IHSI, was implicated in ICIs-relevant "immune-hypoxic" crosstalk by stimulating MAVS/IFN-I pathway under normoxic condition. MRI-radiomics models assessing TRAF3IP3 with HIF1A expression (AUC > 0.80) screened ICIs-Responders in HCC cohort (n = 75). CONCLUSION: The hypoxic-immune stratification redefined ICIs-responsive TME and provided MRI-Radiomics models for initial ICIs-responders screening, with IHSI facilitating further identification.

TMEM106A transcriptionally regulated by promoter methylation is involved in invasion and metastasis of hepatocellular carcinoma.

Expression of transmembrane protein 106A (TMEM106A) has been reported to be dysregulated in several types of cancers. However, the role of TMEM106A in hepatocellular carcinoma (HCC) is still unknown. In the present study, we demonstrate that TMEM106A is markedly downregulated in HCC compared with normal liver tissue. In particular, tumor-specific DNA methylation of TMEM106A is frequently observed in tumor tissues from HCC patients. Immunohistochemistry and pyrosequencing reveal a significant relationship between TMEM106A methylation and downregulation of protein expression. Receiver operating characteristic (ROC) curve analysis reveals that methylation of TMEM106A in tumor samples is different from that in non-malignant adjacent tissues of HCC patients. Moreover, HCC patients with TMEM106A hypermethylation have a poor clinical prognosis. 5-Aza-2'-deoxycytidin treatment of hypermethylated TMEM106A in highly metastatic HCC cells increases the expression of TMEM106A. Functional assays reveal that overexpression of TMEM106A significantly suppresses the malignant behavior of HCC cells in vitro and decreases tumorigenicity and lung metastasis in vivo. Mechanistically, TMEM106A inhibits epithelial mesenchymal transition (EMT) of HCC cells through inactivation of the Erk1/2/Slug signaling pathway. In conclusion, our findings demonstrate that TMEM106A is an inhibitor of HCC EMT and metastasis, and TMEM106A is often transcriptionally downregulated by promoter methylation, which results in reduced levels of TMEM106A protein and predicts poor survival outcomes for HCC patients.

Water consumption and biodiversity: Responses to global emergency events.

Given that it was a once-in-a-century emergency event, the confinement measures related to the coronavirus disease 2019 (COVID-19) pandemic caused diverse disruptions and changes in life and work patterns. These changes significantly affected water consumption both during and after the pandemic, with direct and indirect consequences on biodiversity. However, there has been a lack of holistic evaluation of these responses. Here, we propose a novel framework to study the impacts of this unique global emergency event by embedding an environmentally extended supply-constrained global multi-regional input-output model (MRIO) into the drivers-pressure-state-impact-response (DPSIR) framework. This framework allowed us to develop scenarios related to COVID-19 confinement measures to quantify country-sector-specific changes in freshwater consumption and the associated changes in biodiversity for the period of 2020-2025. The results suggest progressively diminishing impacts due to the implementation of COVID-19 vaccines and the socio-economic system's self-adjustment to the new normal. In 2020, the confinement measures were estimated to decrease global water consumption by about 5.7% on average across all scenarios when compared with the baseline level with no confinement measures. Further, such a decrease is estimated to lead to a reduction of around 5% in the related pressure on biodiversity. Given the interdependencies and interactions across global supply chains, even those countries and sectors that were not directly affected by the COVID-19 shocks experienced significant impacts: Our results indicate that the supply chain propagations contributed to 79% of the total estimated decrease in water consumption and 84% of the reduction in biodiversity loss on average. Our study demonstrates that the MRIO-enhanced DSPIR framework can help quantify resource pressures and the resultant environmental impacts across supply chains when facing a global emergency event. Further, we recommend the development of more locally based water conservation measures-to mitigate the effects of trade disruptions-and the explicit inclusion of water resources in post-pandemic recovery schemes. In addition, innovations that help conserve natural resources are essential for maintaining environmental gains in the post-pandemic world.

[Quantitative model for patchouli alcohol in Pogostemon cablin by near-infrared spectroscopy].

OBJECTIVE: To establish a near-infrared spectroscopy quantitative model for rapid determination of the patchouli alcohol content in Pogostemon cablin. METHOD: The gas chromatography was adopted for determining the content of patchouli alcohol content in 102 batches of P. cablin samples. Their near-infrared spectrograms were collected and preprocessed by standard normal variate and the first derivative of Savitsky-Golay. The quantitative model of patchouli alcohol content was established by the partial least squares regression analysis. RESULT: According to the correction model established in this study, the root-mean-square error of calibration, the root-mean-square error of prediction and the root-mean-square error of cross-validation of the calibration model for Patchouli alcohol were 0.991 10, 0.012 9, 0.012 8 and 0.033 15, respectively. CONCLUSION: The near-infrared spectroscopy quantitative model established in this study is stable, accurate and reliable for the rapid determination of the content of patchouli alcohol in P. cablin.

Construction and Validation of an Epigenetic Regulator Signature as A Novel Biomarker For Prognosis, Immunotherapy, And Chemotherapy In Hepatocellular Carcinoma.

Background: Epigenetic modification regulates various aspects of cancer biology, from tumor growth and invasion to immune microenvironment modulation. Whether epigenetic regulators (EGRs) can decide tumor malignant degree and risk of immune evasion in liver hepatocellular carcinoma (LIHC) remains unclear. Method: An EGR signature called "EGRscore" was constructed based on bulk RNA-seq data of EGR in hepatocellular carcinoma (HCC). The correlation between EGRscore and overall survival (OS) was validated in HCC cohorts and other tumor cohorts. Mutation profiles, copy number alterations (CNAs), enriched pathways, and response to immunotherapy and chemotherapy were compared between EGRscore-high and EGRscore-low patients. Results: We found that EGRscore was associated with OS in HCC as well as several tumors including glioma, uveal melanoma (UVM), and kidney tumors. A mechanism study demonstrated that the distinct mutation profile of TP53 was present in EGRscore-high and EGRscore-low patients. Meanwhile, EGRscore-low patients were characterized with immune cells that promote killing tumors. Furthermore, EGRscore was associated with genes regulating drug resistance in HCC. Finally, we indicated that EGRscore-low patients had higher response rates to immunotherapy and targeted therapy. Conclusions: EGRscore could be used to distinguish OS, tumor progression, mutation pattern, and immune microenvironment. The present study contributes to improving hepatocellular carcinoma patient prognosis and predicting response to immunotherapy.

An Immune-Related Gene Signature Predicting Prognosis and Immunotherapy Response in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is inflammation-associated cancer with high incidence and poor prognosis. In the last decade, immunotherapy has become an important strategy for managing HCC. OBJECTIVE: This study aimed to establish an immune-related gene signature for predicting prognosis and immunotherapy response in HCC. METHODS: We identified immune-related differentially expressed genes (IRDEGs) based on The Cancer Genome Atlas (TCGA) database and the Immunology Database and Analysis Portal (ImmPort) database. The weighted gene co-expression network analysis (WGCNA) and Cox proportional hazard model were utilized to determine hub immune-related genes (IRGs). The TIDE tool and R package pRRophetic were used to assess the correlation between the immune-related gene signature and the clinical responses to immunotherapy and chemotherapy. RESULTS: By using WGCNA combined with Cox proportional hazard model, PRC1, TOP2A, TPX2, and ANLN were identified as hub IRGs. The prognostic value of the newly developed gene signature (IRGPI) was demonstrated in both the TCGA database and the Gene Expression Omnibus (GEO) database. The TIDE tool showed that the high- and low-IRGPI groups presented significantly different tumor immune microenvironment and immunotherapy responses. Furthermore, the high-IRGPI group also had significantly lower chemoresistance to cisplatin than the low-IRGPI group. CONCLUSION: The IRGPI is a tool for predicting prognosis as well as responsiveness to immunotherapy and chemotherapy in HCC.

LncRNA USP2-AS1 Promotes Hepatocellular Carcinoma Growth by Enhancing YBX1-Mediated HIF1α Protein Translation Under Hypoxia.

Recently, the role of lncRNAs in tumorigenesis and development has received increasing attention, but the mechanism underlying lncRNAs-mediated tumor growth in the hypoxic microenvironment of solid tumors remains obscure. Using RNA sequencing, 25 hypoxia-related lncRNAs were found to be upregulated in HCC, of which lncRNA USP2-AS1 were significantly increased under hypoxia. We further confirmed that USP2-AS1 was significantly upregulated in liver cancer using FISH assay and that USP2-AS1 was associated with advanced liver cancer and increased tumor size. Furthermore, overexpression of USP2-AS1 under hypoxia dramatically increased HCC proliferation and clone formation, whereas the opposite results were observed after USP2-AS1 knockdown. We also found that overexpression of USP2-AS1 increased migration and invasion of HCC cells, while USP2-AS1 knockdown led to the opposite effect. In addition, USP2-AS1 knockdown can increase the efficacy of lenvatinib in our mice tumor xenograft model. Our findings also suggest that USP2-AS1 could increase the protein level of HIF1α by enhancing YBX1 protein binding to HIF1α mRNA under hypoxia and the therapeutic effect of lenvatinib can be enhanced by combination with HIF1α inhibitors in liver cancer.

GPR174 knockdown enhances blood flow recovery in hindlimb ischemia mice model by upregulating AREG expression.

Regulatory T cells (Tregs) are critically involved in neovascularization, an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174), which is a regulator of Treg function and development, in neovascularization remains elusive. Here, we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs, thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically, GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via Gαs/cAMP/PKA signal pathway activation. Collectively, these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases.

MTDH antisense oligonucleotides reshape the immunosuppressive tumor microenvironment to sensitize Hepatocellular Carcinoma to immune checkpoint blockade therapy.

Immune checkpoint blockade (ICB) therapy is an important treatment option for individuals with cancer, but it has certain limitations. Identifying a better target that can overcome tumor immune escape and stimulate T cell activity is critical. This research aimed to delve into the molecular mechanism underlying the immunoregulatory function of metadherin (MTDH), which is a novel and potential therapeutic target in hepatocellular cancer (HCC). A small interfering RNA library was screened using the luciferase reporter assay and PD-L1 promoter. The Cancer Genome Atlas database and HCC tissues were used to investigate the relationship between MTDH and PD-L1. The association between MTDH and ß-catenin/lymphoid enhancer binding factor (LEF-1) was discovered by co-immunoprecipitation. The chromatin immunoprecipitation assay was used to investigate the interaction of MTDH with the PD-L1 promoter when LEF-1 expression was silenced. Locked nucleic acid antisense oligonucleotides (ASOs) were used to inhibit MTDH. We utilized in vitro co-cultures and in vivo syngeneic tumor development experiments to confirm the effectiveness of MTDH ASO combined with PD-1 monoclonal antibody (mAb). MTDH was demonstrated to be a PD-L1 modulator. MTDH increased PD-L1 expression and upregulated PD-L1 transcriptional activity through ß-catenin/LEF-1 signaling. More importantly, MTDH ASO improved the anti-PD-1 response and increased cytotoxic T-cell infiltration in PD-1 mAb-treated malignancies. MTDH effectively predicts the therapeutic efficacy of ICB therapy. Our results imply that combining MTDH ASO with PD-1 mAb could be a promising therapeutic strategy for HCC. In addition, MTDH is a potential novel biomarker for predicting the effectiveness of immune checkpoint inhibitor treatment.

Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma.

BACKGROUND & AIMS: Cancer stemness and immune evasion are closely associated and play critical roles in tumor development and resistance to immunotherapy. However, little is known about the underlying molecular mechanisms that coordinate this association. METHODS: The expressions of heterogeneous nuclear ribonucleoprotein M (HNRNPM) in 240 hepatocellular carcinoma (HCC) samples, public databases, and liver development databases were analyzed. Chromatin immunoprecipitation assays were performed to explore the associations between stem-cell transcription factors and HNRNPM. HNRNPM-regulated alternative splicing (AS) and its binding motif were identified by RNA-seq and RIP-seq. HNRNPM-specific antisense oligonucleotides were developed to explore potential therapeutic targets in HCC. CD8+ T cells that were co-cultured with tumor cells were sorted by flow cytometry assays. RESULTS: We identified an elevated oncofetal splicing factor in HCC, HNRNPM, that unifies and regulates the positive association between cancer stemness and immune evasion. HNRNPM knockdown abolished HCC tumorigenesis and diminished cancer stem cell properties in vitro and in vivo. Mechanistically, HNRNPM regulated the AS of MBD2 by binding its flanking introns, whose isoforms played opposing roles. Although MBD2a and MBD2c competitively bound to CpG islands in the FZD3 promoter, MBD2a preferentially increased FZD3 expression and then activated the WNT/ß-catenin pathway. Interestingly, FZD3 and ß-catenin further provided additional regulation by targeting OCT4 and SOX2. We found that HNRNPM inhibition significantly promoted CD8+ T cell activation and that HNRNPM- antisense oligonucleotides effectively inhibited WNT/ß-catenin to enhance anti-programmed cell death protein-1 immunotherapy by promoting CD8+ T cell infiltration. CONCLUSIONS: HNRNPM has a tumor-intrinsic function in generating an immunosuppressive HCC environment through an AS-dependent mechanism and demonstrates proof of the concept of targeting HNRNPM in tailoring HCC immunotherapeutic approaches.

Identification and validation of a new gene signature predicting prognosis of hepatocellular carcinoma patients by network analysis of stemness indices.

Background: Stem cells play an important role in hepatocellular carcinoma (HCC). However, their precise effect on HCC tumorigenesis and progression remains unclear. The present study aimed to characterize stem cell-related gene expression in HCC.Methods: The mRNA expression-based stemness index (mRNAsi) was used to analyze the clinical characteristics and prognosis of HCC patients. The weighted gene co-expression network analysis (WGCNA) was used to construct a gene co-expression network of 374 HCC patients. Finally, six genes were used to construct the prognosis signature.Results: HCC patients had a higher mRNAsi score than healthy people, suggesting poor prognosis. Two gene modules highly related to mRNAsi were identified. Multivariate Cox analysis was carried out to establish a Cox proportional risk regression model. The risk score for each patient was the sum of the product of each gene expression and its coefficient. Survival analysis suggested that the low-risk group had a significantly better prognosis.Conclusions: The established six-gene signature was able to predict patient prognosis accurately. This new signature should be verified in prospective studies in order to determine patient prognosis in clinical decision-making.

Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1.

BACKGROUND: Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. METHODS: We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. RESULTS: SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. CONCLUSIONS: SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

Profiles of alternative splicing landscape in breast cancer and their clinical significance: an integrative analysis based on large-sequencing data.

BACKGROUND: Alternative splicing (AS) is closely correlated with the initiation and progression of carcinoma. The systematic analysis of its biological and clinical significance in breast cancer (BRCA) is, however, lacking. METHODS: Clinical data and RNA-seq were obtained from the TCGA dataset and differentially expressed AS (DEAS) events between tumor and paired normal BRCA tissues were identified. Enrichment analysis was then used to reveal the potential biological functions of DEAS events. We performed protein-protein interaction (PPI) analysis of DEAS events by using STRING and the correlation network between splicing factors (SFs) and AS events was constructed. The LASSO Cox model, Kaplan-Meier and log-rank tests were used to construct and evaluate DEAS-related risk signature, and the association between DEAS events and clinicopathological features were then analyzed. RESULTS: After strict filtering, 35,367 AS events and 973 DEAS events were detected. DEAS corresponding genes were significantly enriched in pivotal pathways including cell adhesion, cytoskeleton organization, and extracellular matrix organization. A total of 103 DEAS events were correlated with disease free survival. The DEAS-related risk signature stratified BRCA patients into two groups and the area under curve (AUC) was 0.754. Moreover, patients in the high-risk group had enriched basel-like subtype, advanced clinical stages, proliferation, and metastasis potency. CONCLUSIONS: Collectively, the profile of DEAS landscape in BRCA revealed the potential biological function and prognostic value of DEAS events.

Splicing factors: Insights into their regulatory network in alternative splicing in cancer.

More than 95% of all human genes are alternatively spliced after transcription, which enriches the diversity of proteins and regulates transcript and/or protein levels. The splicing isoforms produced from the same gene can manifest distinctly, even exerting opposite effects. Mounting evidence indicates that the alternative splicing (AS) mechanism is ubiquitous in various cancers and drives the generation and maintenance of various hallmarks of cancer, such as enhanced proliferation, inhibited apoptosis, invasion and metastasis, and angiogenesis. Splicing factors (SFs) play pivotal roles in the recognition of splice sites and the assembly of spliceosomes during AS. In this review, we mainly discuss the similarities and differences of SF domains, the details of SF function in AS, the effect of SF-driven pathological AS on different hallmarks of cancer, and the main drivers of SF expression level and subcellular localization. In addition, we briefly introduce the application prospects of targeted therapeutic strategies, including small-molecule inhibitors, siRNAs and splice-switching oligonucleotides (SSOs), from three perspectives (drivers, SFs and pathological AS). Finally, we share our insights into the potential direction of research on SF-centric AS-related regulatory networks.

Establishment and validation of a novel autophagy-related gene signature for patients with breast cancer.

BACKGROUND: There exists considerable evidence conforming that autophagy may play an important role in the biological process of breast cancer. This study aimed to construct and evaluate a novel autophagy-related gene signature as a potential prognostic factor and therapeutic target in breast cancer patients based on high-throughput sequencing datasets. MATERIALS & METHODS: Autophagy-related genes obtained from the Human Autophagy Database and high-sequencing data obtained from The Cancer Genome Atlas (TCGA) were analyzed to identify differential expressed genes (DEGs) between tumor and normal tissues. Then GO and KEGG analysis were performed to explore potential biological and pathological functions of DEGs. Autophagy-related prognostic genes were identified by univariate COX regression analysis. Subsequently stepwise model selection using the Alkaike information criterion (AIC) and multivariate COX regression model was performed to construct autophagy-related gene signature. Then patients were divided into high- and low-risk groups based on the risk score identified by the autophagy-related gene signature. Multivariate COX regression model and stratification analysis were used to specify the prognostic value of this gene signature in whole cohort and various subgroups. T-test and ANOVA analysis were used to compare the expression differences of continuous variables (5 prognostic genes and risk score) in binary and multiple category groups respectively. Kaplan-Meier analysis, log-rank tests and the area under receiver operating characteristic (ROC) curve (AUC) were conducted to validate the accuracy and precise of the autophagy-related gene signature based on GSE20685 and GSE21653 datasets. RESULTS: We profiled autophagy-related DEGs in normal and breast tumor tissues. GO and KEGG analysis indicated that autophagy-related DEGs might participate in breast cancer occurrence, development and drug resistance. Then we identified five autophagy-related genes (EIF4EBP1, ATG4A, BAG1, MAP1LC3A and SERPINA1) that had significantly prognostic values for breast cancer. Autophagy-related gene signature was constructed and patients were divided into high- and low- risk groups based on their risk score. Patients in the high-risk group tended to have shorter overall survival (OS) and relapse-free survival (RFS) times than those in the low-risk group (OS: HR = 1.620, 95%CIs: 1.345-1.950; P < 0.001; RFS: HR = 1.487, 95%CIs: 1.248-1.771, P < 0.001). Autophagy-related gene signature had significant prognostic value in stratified subgroups especially in advanced breast cancer subgroups (T3-4; N2-3; stage III-IV). Its prognostic value was further confirmed in two GEO validation datasets (GSE20685: P = 6.795e-03; GSE21653: P = 1.383e-03). Finally, association analysis between clinicopathological factors and gene signature showed the risk score was higher in patients with ER/PR negative, higher clinical stage or T stage (P < 0.01). CONCLUSION: We established and confirmed a novel autophagy-related gene signature for patients with breast cancer that had independent survival prognostic value especially in advanced breast cancer subgroups. Our research might promote the molecular mechanism study of autophagy-related genes in breast cancer.

Suitability of various DNA extraction methods for a traditional Chinese paocai system.

Traditional paocai brine (PB), which is continuously propagated by back-slopping and contains various species of lactic acid bacteria (LAB), is critical for the flavor of paocai. Culture-independent approaches are commonly used to investigate the microbial communities of fermented food. To evaluate the influence of different DNA (DNA) extraction methods on estimates of bacterial community profiles from 4 PBs, the lysis efficiency, DNA yield, purity and denaturing gradient gel electrophoresis (DGGE) profiles of V3 region of a 16S ribosomal ribonucleic acid gene were acquired. The cell lysis pattern of SDS + beads and Lysing matrix E+ beads (methods 3 and 4) showed higher cell lysis efficiency than SDS and SDS + Lysozyme (methods 1 and 2) in all PBs. SDS + beads obtained the largest DNA yield of the 4 methods. Moreover, methods 3 and 4 resulted in higher H' values and generated more global bacteria profiles than other methods. Overall, our results demonstrate that the properties of PB significantly affect the efficiency of DNA extraction methods. Methods 3 and 4 were both suitable for DNA extraction from PB. Method 3 is more economic, simple and rapid than method 4 for large-scale studies of the bacterial profiles of PB.

An integrated laboratory method to measure and verify directional hydraulic conductivity in fine-to-medium sandy sediments.

The constant-head permeameter test (CHPT) is widely used in sandy samples as a standard method in the laboratory to investigate hydraulic conductivity (K). However, it neither can be used to consistently determine directional hydraulic conductivity (DHC) nor guarantee the comparability of measured K values of samples with different sizes. Therefore, this paper proposes an integrated laboratory method, called modified CHPT (MCHPT), for the efficient determination and verification of consistent DHC values in fine-to-medium sandy sediments, based on a new methodological framework. A precise and standardized procedure for preparing the experimental setup of MCHPT was conducted, based on the integrated experimental setup of CHPT and tracer tests. Moreover, a formula was yielded for the time-optimized sample saturation control. In comparison with grain size-based methods, the validity of consistent Kh and Kv values determined by MCHPT was convincing.

[Characteristics of macrozoobenthic assemblages and their relationship with aquatic environmental factors in streams of Lake Dianchi watershed in the wet season].

The characteristics of macrozoobenthic assemblages and their relationship with aquatic environmental factors in the 29 streams of the Lake Dianchi (LD) watershed in the wet season were studied. Biosurveys of macrozoobenthic assemblages during July and August 2009, as well as 17 mensal aquatic physicochemical parameters from September 2008 to August 2009 were carried out at 29 sampling sites. The objectives were to demonstrate the characteristics of macrozoobenthic assemblages in streams of the LD watershed in the wet season, to identify the key aquatic environmental factors affecting these assemblages, and to investigate the relationship of spatial patterns between the Shannon-Weaver diversity index of the assemblages and the assessment of aquatic environmental quality. Results identified 3 divisions, 7 families, and 8 genera of macrozoobenthos, including 4 families and 5 genera of Annelida, 2 families and 2 genera of Mollusca, and 1 family and 1 genus of Arthropoda. Among these, Limnodrilus (Annelida) , a tolerant species, was the dominant genus. TN, NH4(+)-N, TP, and DO were the key aquatic environmental factors affecting the macrozoobenthic assemblages, respectively 2.03-32.00, 0.34-26.66, 0.09-3.20, and 0.10-6.80 mg/L. The spatial patterns of the Shannon-Weaver diversity index of macrozoobenthic assemblages and the assessments of aquatic environmental quality were both greatest in the streams flowing into the east LD (Luolonghe, Laoyuhe, and Nanchonghe rivers), followed by those flowing into the south LD (Yunihe, Laochaihe, Baiyuhe, Cixianghe, Dongdahe, Zhonghe and Guchenghe rivers), and least in those flowing into the north LD (Wangjiaduiqu, Xinyunlianghe, Laoyunlianghe, Wulonghe, Daguanhe, Xibahe, Chuanfanghe, Cailianhe, Jinjiahe, Panlongjiang, Daqinghe, Haihe, Liujiabaoxianghe, Xiaoqinghe, Wujiabaoxianghe, Xiabahe, Laobaoxianghe, Xinbaoxianghe, and Maliaohe rivers).