Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.     

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.     

Detection, evaluation and minimization of nonenzymatic deamidation in proteomic sample preparation

Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the (13)C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4-9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37 °C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deamidation: -Q-V-, -Q-L- and -Q-G- and, to a lesser extent, -Q-A- and -Q-E-.     

Effects of clonal integration,nutrients and cadmium on growth of the aquatic macrophyte Pistia stratiotes

许多湿地同时遭受养分和有毒金属的污染,其植被多数为克隆植物。我们假设,富养化和克隆整合可以通过增加植物的生长来提高对有毒金属污染的植物修复能力,即使在毒性胁迫环境下也是如此。为验证此假说,我们将常见、广布的匍匐茎漂浮植物大薸(Pistia stratiotes L.)的单个分株种植在两种不同的养分条件下和两种镉污染处理(无镉或含0.6 mg L⁻¹ 的镉)中42天。通过保持或切断母株与其后代分株之间的连接来维持或阻止克隆整合,并通过保留或移除切断后的后代分株来控制克隆内竞争的有无。由于高养分条件下克隆后代分株的数量增加了一倍,因此镉处理下大薸的生物量在高养分条件下几乎是在低养分条件下的两倍。切断匍匐茎连接对整个克隆(母株和后代分株的总和)的生物量没有影响。切断连接后去除后代分株对镉污染下母株的生物量没有显著的影响,但却显著增加了无镉污染下母株的生物量。这些研究结果支持富养化可以提高水生植物对有毒金属污染的修复能力这一假说,但并不支持克隆整合有利于植物修复的假说。因此,水生植物(如大薸)可能有助于对同时遭受有毒金属和养分污染的湿地进行修复,但克隆片段化对植物的这种修复能力可能没有显著影响。     

Multiclonal tussocks in the grass Achnatherum splendens (Trinius) Nevskia (Poaceae)

Clonal plant species often form genetically diverse populations, even when sexual reproduction in a population is rarely observed. Here we test whether the spatially discrete clusters of plants (tussocks of graminoids) formed within populations of some clonal species can likewise be multiclonal. We sampled leaves of ramets (shoots) within 20 tussocks of the grass Achnatherum splendens in the Otindag Sandland in Inner Mongolia, China, and genotyped the ramets using standard molecular protocols. The 20 tussocks were allocated to three classes: (i) small, circular, (ii) large, circular and (iii) large, irregular. Most tussocks (80%) were multiclonal and some contained at least eight different clones. Irregularly shaped tussocks contained twice as many clones as circular tussocks; neither size nor cover within a tussock affected number of clones per tussock, and the smaller clones in a tussock showed no tendency to occur on the edge or near the center of a tussock. These patterns seem more consistent with formation of multiclonal tussocks by coalescence than by colonization. Therefore, individual tussocks, especially large, irregular ones, cannot a priori be treated as genetic individuals without assessing their genetic information in, e.g., population demography, genetics and evolution studies.     

The relative advantages of plasticity and fixity in different environments: when is it good for a plant to adjust?

Plant populations and species differ greatly in phenotypic plasticity. This could be because plasticity is advantageous under some conditions and disadvantageous or not advantageous under others. We distinguish adaptive from injurious and neutral plasticity and discuss when selection should favor adaptive plasticity over genetic differentiation or lack of phenotypic variation. It seems reasonable to hypothesize that selection is likely to favor plasticity when an environmental factor varies on the same spatial scale as the plant response unit, when the plant can respond to an environmental factor faster than the level of the factor changes, and when environmental variation is highly but not completely predictable. Phenotypic plasticity might also tend to be more advantageous when mean resource availability is high rather than low, when a response can occur late in development rather than early, and when a response is reversible rather than irreversible. There is substantial evidence for the hypothesis that predictability favors plasticity. However, available evidence does not support the hypothesis that high mean resource availability necessarily favors plasticity. Testing hypotheses about when it is good for a plant to adjust is central to understanding the diversity of plasticity in plants.     

Effects of clonal integration on plant plasticity in Fragaria chiloensis

The ability of clonal plants to transport substances between ramets located in different microsites also allows them to modify the plastic responses of individual ramets to local environmental conditions. By equalising concentrations of substances between ramets, physiological integration might decrease responses to local conditions. However, integration has also been observed to increase plasticity and induce novel plastic responses in ramets. To ask how integration modifies plant plasticity in the clonal herb, Fragaria chiloensis, ramets were given either low light and high nitrogen or high light and low nitrogen, simulating a pattern of resource patchiness in their native habitat. Ramets in contrasting light/nitrogen treatments were either connected or single. Effects of light/nitrogen and connection were measured at three levels of morphological organisation, the organ, the ramet, and the clonal fragment. Connection between ramets reduced or had no effect on plastic responses in leaf size at the level of the plant organ. This suggested that integration dampened certain plastic responses. Connection induced a new plastic response at the level of the clonal fragment, an increase in allocation to vegetative reproduction in patches of low light and high nitrogen. It is concluded that clonal integration can have different effects on plant plasticity at different levels of plant organisation. It appears that, at least in this species, integration can increase plasticity at the level of the clonal fragment and concentrate vegetative reproduction in particular microsite types.     

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis

The role of human prostatic acid phosphatase (PAcP, {"type":"entrez-protein","attrs":{"text":"P15309","term_id":"130730"}}P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed ^PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate ^PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.