Two-stage clustering (TSC): a pipeline for selecting operational taxonomic units for the high-throughput sequencing of PCR amplicons

Clustering 16S/18S rRNA amplicon sequences into operational taxonomic units (OTUs) is a critical step for the bioinformatic analysis of microbial diversity. Here, we report a pipeline for selecting OTUs with a relatively low computational demand and a high degree of accuracy. This pipeline is referred to as two-stage clustering (TSC) because it divides tags into two groups according to their abundance and clusters them sequentially. The more abundant group is clustered using a hierarchical algorithm similar to that in ESPRIT, which has a high degree of accuracy but is computationally costly for large datasets. The rarer group, which includes the majority of tags, is then heuristically clustered to improve efficiency. To further improve the computational efficiency and accuracy, two preclustering steps are implemented. To maintain clustering accuracy, all tags are grouped into an OTU depending on their pairwise Needleman-Wunsch distance. This method not only improved the computational efficiency but also mitigated the spurious OTU estimation from 'noise' sequences. In addition, OTUs clustered using TSC showed comparable or improved performance in beta-diversity comparisons compared to existing OTU selection methods. This study suggests that the distribution of sequencing datasets is a useful property for improving the computational efficiency and increasing the clustering accuracy of the high-throughput sequencing of PCR amplicons. The software and user guide are freely available at http://hwzhoulab.smu.edu.cn/paperdata/.     

Palmitate contributes to insulin resistance through downregulation of the Src-mediated phosphorylation of Akt in C2C12 myotubes

The mechanisms of free fatty acid (FFA)-induced peripheral insulin resistance remain elusive. This study aimed to investigate the effect of palmitate, a saturated fatty acid, on glucose metabolism in C2C12 myotubes, and to explore the underlying mechanisms. In it, palmitate decreased insulin-stimulated glucose uptake and consumption in a dose-dependent manner, and it reduced the insulin-stimulated phosphorylation of Akt at Thr308 and Ser473, but had no effect on the protein expression of PI3K-p85 or the activity of PI3K. Additionally, it inhibited the insulin-stimulated phosphorylation of Src at Tyr416, causing a reduction in the Src-mediated phosphorylation of Akt. Inhibition of Src by PP2 resulted in decreases in insulin-stimulated glucose uptake and phosphorylation of Src at Tyr416 and Akt at Thr308 and Ser473. The findings indicate that palmitate contributes to insulin resistance by inhibiting the Src-mediated phosphorylation of Akt in C2C12 myotubes, and this provides insight into the molecular mechanisms of FFA-induced insulin resistance.     

Yidiyin, a Chinese herbal decoction, improves erectile dysfunction in diabetic patients and rats through the NO-cGMP pathway

The nitric-oxide (NO)-cyclic-guanosine-monophosphate (cGMP) pathway plays a key role in penile erection. Erectile dysfunction (ED) is a complication in male diabetic patients that impacts their quality of 1ife. Recently, Yidiyin, a Chinese herbal decoction, is used to treat diabetic ED, but convincing evidence is lacking, and the potential mechanisms remain uncertain. In the study, diabetic ED patients had low scores on international index of erectile function-5 (IIEF-5), and administration of Yidiyin and hypoglycemic drugs for 16 weeks ameliorated patients' scores on IIEF-5 more than the hypoglycemic drug alone. Moreover, streptozotocin-induced diabetes severely impaired rats' erectile function and the activity of the NO-cGMP pathway in the corpora cavernosum, and treatment with Yidiyin for 4 weeks obviously increased the rats' erectile function, remarkably enhanced the activity of nitric oxide synthase (NOS), and elevated the contents of NO and cGMP. Our findings indicate that Yidiyin improves diabetic ED probably by enhancing the NO-cGMP pathway.     

摄食声对草鱼幼鱼的诱集作用

以草鱼(Ctenopharyngodon idellus)幼鱼为实验对象, 进行了声音播放实验, 旨在探究草鱼幼鱼对水下录制的草鱼摄食浮萍声音(简称摄食声)的行为反应。以不播放声音的草鱼鱼群作为对照, 探讨了4 种单频音(500、1000、2000和3000 Hz)和摄食声对草鱼游泳行为和在水槽内的分布的影响。结果表明: 在播放单频音时, 3min内草鱼的趋音游泳速度和逗留时间与对照组无显著性差异(P>0.05); 在播放摄食声时, 3min内草鱼的趋音游泳速度和逗留时间显著高于4种单频音组和对照组(P<0.05); 在播放单频音时, 20min内草鱼的平均游泳速度、水槽内的分布和趋音率与对照组无显著性差异(P>0.05); 在播放摄食声时, 20min内草鱼的平均游泳速度和趋音率都显著高于4种单频音组和对照组(P<0.05)。草鱼摄食声对草鱼幼鱼有诱集作用, 为声音诱鱼技术研究提供了科学依据。     

贵州部分洞穴裸灶螽的鉴定及其遗传多样性分析

贵州是中国具有丰富喀斯特洞穴生态系统的省份之一,裸灶螽作为洞穴中的优势物种又为生态环境指示种,其遗传多样性能够反应洞穴生物多样性.为评估洞穴开发对洞穴生物多样性的影响,本研究通过形态鉴定和分子辅助鉴定的方式对来自贵州省毕节、黔西南、铜仁和黔南4个地区8个洞穴97号裸灶螽标本进行鉴定,并对其遗传多样性进行分析.结果共鉴定...     

外源激素与温度对花楸树种子萌发的影响

三种外源激素和萌发温度对花楸树种子萌发影响的研究结果表明,25℃（16h光／8h暗,10d）-5℃（暗110d）变温可极显著提高种子发芽率和种子发芽势,但极显著延迟发芽初始时间。200mg·L^-16-BA溶液中吸胀2d后经历25℃·5℃变温可使种子发芽初始时间最短（33d）,发芽率（62．25％）和发芽势（57．88％）最高。GA3或ABA与温度的结合处理对种子萌发的促进作用小于6-BA与温度的综合处理。     

Phosphorus deficiency-induced reduction in root hydraulic conductivity in Medicago falcata is associated with ethylene production

Plants grown in phosphorus-deficient solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lpr) and enhanced ethylene production. To uncover the relationship between the reduction in Lpr and increase in ethylene production, we investigated effect of phosphorus (P) deficiency on ethylene production and Lpr in legume plants of Medicago falcata L. There was an increase in ethylene production and a reduction of Lpr of M. falcata roots when M. falcata seedlings grown in P sufficient solutions (0.5 mM H₂PO₄^2?) were transferred to P-deficient solutions (5 μM H₂PO₄^2?). Antagonists of ethylene biosynthesis, CoCl₂ and aminoethoxyvinylglycine (AVG), abolished the P deficiency-induced ethylene production. Root hydraulic conductivity of M. falcata seedlings grown in P-sufficient solutions was insensitive to CoCl₂ and AVG, while the two chemicals enhanced Lpr for those grown in P-deficient solutions, suggesting that P deficiency-induced decrease in Lpr can be reversed by inhibiting ethylene production. Ethylene precursor 1-amino cyclopropane-1-carboxylic acid (ACC) and ethylene donor ethephon had greater inhibitory effect on Lpr of P-sufficient seedlings than that of P-deficient seedlings. Root hydraulic conductivity of P-sufficient seedlings was more sensitive to HgCl₂ than that of P-deficient seedlings. Taken together, these findings suggest that ethylene induced by P deficiency may play an important role in modulation of root hydraulic conductivity by affecting aquaporins in plants.     

Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB,c-Jun N-terminal kinase,and p38 mitogen-activated protein kinase

Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V–fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.     

Different nitrogen saturation thresholds for above-, below-, and total net primary productivity in a temperate steppe

Identifying the thresholds for the positive responses of total net primary productivity (NPP) to nitrogen (N) enrichment is an essential prerequisite for predicting the benefits of N deposition on ecosystem carbon sequestration. However, the responses of below-ground NPP (BNPP) to N enrichment are unknown in many ecosystems, which limits our ability to understand the carbon cycling under the scenario of increasing N availability. We examined the changes in above-ground NPP (ANPP), BNPP, and NPP of a temperate meadow steppe across a wide-ranging N addition gradient (0, 2, 5, 10, 20, and 50 g N m⁻² year⁻¹) during 5 years. Both ANPP and NPP increased nonlinearly with N addition rates. The N saturation threshold for ANPP (T_A) and NPP (T_N) was at the rate of 13.11 and 6.70 g N m⁻² year⁻¹, respectively. BNPP decreased with increasing N addition when N addition rates ˃5 g N m⁻² year⁻¹, resulting in much lower T_N than T_A. Soil N enrichment played a key role in driving the negative impacts of high N addition rates on BNPP, and consequently on the earlier occurrence of N saturation threshold for NPP. Our results highlight the negative effects of soil N enrichment on NPP in natural grasslands super-saturated with N. Furthermore, by considering ANPP and BNPP simultaneously, our results indicate that previous findings from above-ground might have over-estimated the positive effects of N deposition on primary productivity.     

The sequencing bias relaxed characteristics of Hi-C derived data and implications for chromatin 3D modeling

The 3D chromatin structure modeling by chromatin interactions derived from Hi-C experiments is significantly challenged by the intrinsic sequencing biases in these experiments. Conventional modeling methods only focus on the bias among different chromatin regions within the same experiment but neglect the bias arising from different experimental sequencing depth. We now show that the regional interaction bias is tightly coupled with the sequencing depth, and we further identify a chromatin structure parameter as the inherent characteristics of Hi-C derived data for chromatin regions. Then we present an approach for chromatin structure prediction capable of relaxing both kinds of sequencing biases by using this identified parameter. This method is validated by intra and inter cell-line comparisons among various chromatin regions for four human cell-lines (K562, GM12878, IMR90 and H1hESC), which shows that the openness of chromatin region is well correlated with chromatin function. This method has been executed by an automatic pipeline (AutoChrom3D) and thus can be conveniently used.