西藏林芝真蚋亚属三新种（双翅目：蚋科）

本文记述西藏林芝真蚋亚属Ｅｕｓｉｍｕｌｉｕｍ三种：凸端真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｃｏｎｃａｖｕｓｔｙｌｕｍｓｐ．ｎｏｖ．、林芝真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｌｉｎｇｚｉｅｎｓｅｓｐ．ｎｏｖ．、裂缘真蚋Ｓｉｍｕｌｉｕｍ（Ｅｕｓｉｍｕｌｉｕｍ）ｓｃｈｉｚｏｌｏｍｕｎｓｐ．ｎｏｖ 。     

Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis

Cellulose is inherently resistant to breakdown, and the native crystalline structure (cellulose I) of cellulose is considered to be one of the major factors limiting its potential in terms of cost-competitive lignocellulosic biofuel production. Here we report the impact of ionic liquid pretreatment on the cellulose crystalline structure in different feedstocks, including microcrystalline cellulose (Avicel), switchgrass (Panicum virgatum), pine ( Pinus radiata ), and eucalyptus ( Eucalyptus globulus ), and its influence on cellulose hydrolysis kinetics of the resultant biomass. These feedstocks were pretreated using 1-ethyl-3-methyl imidazolium acetate ([C2mim][OAc]) at 120 and 160 °C for 1, 3, 6, and 12 h. The influence of the pretreatment conditions on the cellulose crystalline structure was analyzed by X-ray diffraction (XRD). On a larger length scale, the impact of ionic liquid pretreatment on the surface roughness of the biomass was determined by small-angle neutron scattering (SANS). Pretreatment resulted in a loss of native cellulose crystalline structure. However, the transformation processes were distinctly different for Avicel and for the biomass samples. For Avicel, a transformation to cellulose II occurred for all processing conditions. For the biomass samples, the data suggest that pretreatment for most conditions resulted in an expanded cellulose I lattice. For switchgrass, first evidence of cellulose II only occurred after 12 h of pretreatment at 120 °C. For eucalyptus, first evidence of cellulose II required more intense pretreatment (3 h at 160 °C). For pine, no clear evidence of cellulose II content was detected for the most intense pretreatment conditions of this study (12 h at 160 °C). Interestingly, the rate of enzymatic hydrolysis of Avicel was slightly lower for pretreatment at 160 °C compared with pretreatment at 120 °C. For the biomass samples, the hydrolysis rate was much greater for pretreatment at 160 °C compared with pretreatment at 120 °C. The result for Avicel can be explained by more complete conversion to cellulose II upon precipitation after pretreatment at 160 °C. By comparison, the result for the biomass samples suggests that another factor, likely lignin-carbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity.     

Intramolecular folding in human ILPR fragment with three C-rich repeats

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br(2) footprinting experiments on a mixture of the ILPR-I3 and a 5'-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures.     

MarcoPolo: a method to discover differentially expressed genes in single-cell RNA-seq data without depending on prior clustering

The standard analysis pipeline for single-cell RNA-seq data consists of sequential steps initiated by clustering the cells. An innate limitation of this pipeline is that an imperfect clustering result can irreversibly affect the succeeding steps. For example, there can be cell types not well distinguished by clustering because they largely share the global structure, such as the anterior primitive streak and mid primitive streak cells. If one searches differentially expressed genes (DEGs) solely based on clustering, marker genes for distinguishing these types will be missed. Moreover, clustering depends on many parameters and can often be subjective to manual decisions. To overcome these limitations, we propose MarcoPolo, a method that identifies informative DEGs independently of prior clustering. MarcoPolo sorts out genes by evaluating if the distributions are bimodal, if similar expression patterns are observed in other genes, and if the expressing cells are proximal in a low-dimensional space. Using real datasets with FACS-purified cell labels, we demonstrate that MarcoPolo recovers marker genes better than competing methods. Notably, MarcoPolo finds key genes that can distinguish cell types that are not distinguishable by the standard clustering. MarcoPolo is built in a convenient software package that provides analysis results in an HTML file.     

Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPR_n=4). Mobilities of ILPR_n=4 in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPR_n=4 most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPR_n=2). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPR_n=4 at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex–quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPR_n=4 that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.     

Temperature control methods in a laser tweezers system

Two methods of temperature control of a dual-beam optical-tweezers system are compared. In the first method, we used a 975 nm infrared laser to raise the temperature 5.6 degrees C/100 mW in a nonheating (830 nm) optical trap. The temperature increment logarithmically decreases toward the periphery of the heating beam, causing a fluid convection of 8 mum/s inside a 180 microm thick microchamber. In the second method, heating or cooling fluid was pumped through copper jackets that were placed on the water immersion objectives on both sides of the microchamber to control its temperature from 4.5 degrees C to 68 degrees C. The temperature controlled by the second method was both stable and homogeneous, inducing little fluid convection that would disturb single-molecule applications. An analysis of the power spectrum of the thermal force on a trapped bead showed no detectable vibration due to the liquid circulation. In both methods, force was measured directly by sensors of the momentum flux of light, independent of environmental disturbances including refractive index changes that vary with temperature. The utility of the second method was demonstrated in single-molecule experiments by measuring the mechanical stretch of a 41 kbp lambda double-stranded DNA at temperatures ranging from 8.4 degrees C to 45.6 degrees C.     

Bti的溶血特性及其在Bti制剂毒力检测中的应用

实验利用Bti(苏云金杆菌以色列亚种）对兔红细胞（RBC）的溶血特性,以A541特征光吸收对Bti毒蛋白溶液体积或其对数作回归分析。结果表明：二者具有良好的直线关系。相关系数r≥0．95。方差分析表明：不同梯度的Bti每蛋白溶液处理之间差异极显著,处理内各重复之间差异不显著,同一天内的重复检测没有显著差异。本又给出了Bti制剂毒力检测的参考程度,并就该程度应用于Bti毒力快速检测作了较为详尽的探讨。     

A negative regulatory role for auxin in sulphate deficiency response in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Sulphate is a major macronutrient required for the synthesis of the sulphur (S)-containing amino acid cysteine and thus is critical for cellular metabolism, growth and development and response to various abiotic and biotic stresses. A recent genome-wide expression study suggested that several auxin-inducible genes were up-regulated by S deficiency in Arabidopsis. Here, we examined the relationship between auxin signaling and S deficiency. Investigation of DR5::GUS expression patterns indicates that auxin accumulation and/or response is suppressed by S deficiency. Consistently, S deficiency resulted in the suppression of lateral root development, but the axr1-3 mutant was insensitive to this response. Furthermore, the activation of the promoter for the putative thioglucosidase gene (At2g44460) by S deficiency was suppressed by auxin, cytokinin and abscisic acid (ABA). Interestingly, the activation of At2g44460 by S deficiency is regulated by the availability of carbon and nitrogen nutrients in a tissue-specific manner. These results demonstrate that auxin plays a negative role in signaling to S deficiency. Given that activation of the genes encoding the sulphate transporter SULTR1;2 and 5′-adenylylsulphate reductase APR2 are suppressed by cytokinin only, we hypothesize that while cytokinin may play an important role in general S deficiency response, auxin might be only involved in a subset of S deficiency responses such as the release of thiol groups from the S storage sources. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.