Reverse Libraries Research Articles

Etravirine Prevents West Nile Virus and Chikungunya Virus Infection Both In Vitro and In Vivo by Inhibiting Viral Replication.

Diseases transmitted by arthropod-borne viruses such as West Nile virus (WNV) and chikungunya virus (CHIKV) pose threat to global public health. Unfortunately, to date, there is no available approved drug for severe symptoms caused by both viruses. It has been reported that reverse transcriptase inhibitors can effectively inhibit RNA polymerase activity of RNA viruses. We screened the anti-WNV activity of the FDA-approved reverse transcriptase inhibitor library and found that 4 out of 27 compounds showed significant antiviral activity. Among the candidates, etravirine markedly inhibited WNV infection in both Huh 7 and SH-SY5Y cells. Further assays revealed that etravirine inhibited the infection of multiple arboviruses, including yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and CHIKV. A deeper study at the phase of action showed that the drug works primarily during the viral replication process. This was supported by the strong interaction potential between etravirine and the RNA-dependent RNA polymerase (RdRp) of WNV and alphaviruses, as evaluated using molecular docking. In vivo, etravirine significantly rescued mice from WNV infection-induced weight loss, severe neurological symptoms, and death, as well as reduced the viral load and inflammatory cytokines in target tissues. Etravirine showed antiviral effects in both arthrophlogosis and lethal mouse models of CHIKV infection. This study revealed that etravirine is an effective anti-WNV and CHIKV arbovirus agent both in vitro and in vivo due to the inhibition of viral replication, providing promising candidates for clinical application.

Plant Promoters: Their Identification, Characterization, and Role in Gene Regulation.

One of the strategies to overcome diseases or abiotic stress in crops is the use of improved varieties. Genetic improvement could be accomplished through different methods, including conventional breeding, induced mutation, genetic transformation, or gene editing. The gene function and regulated expression through promoters are necessary for transgenic crops to improve specific traits. The variety of promoter sequences has increased in the generation of genetically modified crops because they could lead to the expression of the gene responsible for the improved trait in a specific manner. Therefore, the characterization of the promoter activity is necessary for the generation of biotechnological crops. That is why several analyses have focused on identifying and isolating promoters using techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR), genetic libraries, cloning, and sequencing. Promoter analysis involves the plant genetic transformation method, a potent tool for determining the promoter activity and function of genes in plants, contributing to understanding gene regulation and plant development. Furthermore, the study of promoters that play a fundamental role in gene regulation is highly relevant. The study of regulation and development in transgenic organisms has made it possible to understand the benefits of directing gene expression in a temporal, spatial, and even controlled manner, confirming the great diversity of promoters discovered and developed. Therefore, promoters are a crucial tool in biotechnological processes to ensure the correct expression of a gene. This review highlights various types of promoters and their functionality in the generation of genetically modified crops.

Synthesis Strategy of Reversible Circuits on DNA Computers

DNA computers and quantum computers are gaining attention as alternatives to classical digital computers. DNA is a biological material that can be reprogrammed to perform computing functions. Quantum computing performs reversible computations by nature based on the laws of quantum mechanics. In this paper, DNA computing and reversible computing are combined to propose novel theoretical methods to implement reversible gates and circuits in DNA computers based on strand displacement reactions, since the advantages of reversible logic gates can be exploited to improve the capabilities and functionalities of DNA computers. This paper also proposes a novel universal reversible gate library (URGL) for synthesizing n-bit reversible circuits using DNA to reduce the average length and cost of the constructed circuits when compared with previous methods. Each n-bit URGL contains building blocks to generate all possible permutations of a symmetric group of degree n. Our proposed group (URGL) in the paper is a permutation group. The proposed implementation methods will improve the efficiency of DNA computer computations as the results of DNA implementations are better in terms of quantum cost, DNA cost, and circuit length.

Identification of Haemaphysalis longicornis Genes Differentially Expressed in Response to Babesia microti Infection.

Haemaphysalis longicornis is a tick and a vector of various pathogens, including the human pathogenetic Babesia microti. The objective of this study was to identify female H. longicornis genes differentially expressed in response to infection with B. microti Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to Babesia infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including ola1, crt and Vg2. Functional analysis of up-regulated genes in fed or Babesia-infected ticks by RNA interference showed that knockdown of crt and Vg2 in infected ticks and knockdown of ola1 in uninfected ticks accelerated engorgement. In contrast, Vg1 knockdown in infected ticks had delayed engorgement. Knockdown of crt and Vg1 in infected ticks decreased engorged female weight. Vg2 knockdown reduced B. microti infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of H. longicornis genes in blood feeding and B. microti infection.

Screening of Differentially Expressed Microsporidia Genes from Nosema ceranae Infected Honey Bees by Suppression Subtractive Hybridization.

The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

Regulation of vitellogenin gene expression under the negative modulator, gonad-inhibiting hormone in Penaeus monodon

Vitellogenesis is a principal process during ovarian maturation in crustaceans. This process is negatively regulated by gonad-inhibiting hormone (GIH), a neuronal peptide hormone from eyestalks. However, the detailed mechanism through which GIH regulates Vg expression is still ambiguous. In this study, suppression subtractive hybridization (SSH) under specific GIH-knockdown condition was utilized to determine the expression of genes in the ovary that may act downstream of GIH to control vitellogenin synthesis in Penaeus monodon. The total of 102 and 82 positive clones of up-regulated and down-regulated genes in GIH- knockdown shrimp were identified from the forward and reverse SSH libraries, respectively. Determination of the expression profiles of these reproduction-related genes during ovarian development revealed that the expression of calreticulin (CALR) was significantly reduced in vitellogenic ovary suggesting its role in vitellogenesis. Suppression of CALR by specific dsRNA showed elevated vitellogenin (Vg) transcript level in the ovary at day 7 post-dsRNA injection. Since CALR can bind to steroid hormone receptors and prevents the binding of the receptor to its responsive element to regulate gene expression, it is possible that CALR is an inhibitory mediator of vitellogenin synthesis via steroidal pathway. Our results posted a possible novel pathway of GIH signaling that might interfere the steroid signaling cascade to mediate Vg synthesis in the shrimp.

An Improved Design of n-Bit Universal Reversible Gate Library

Reversible logic has been considered as an important solution to the power dissipation problem in the existing electronic devices. Many universal reversible libraries that include more than one type of gates have been proposed in the literature. This paper proposes a novel reversible n-bit gate that is proved to be universal for synthesizing reversible circuits. Reducing the reversible circuit synthesis problem to permutation group allows Schreier-Sims Algorithm for the strong generating set-finding problem to be used in the synthesize of reversible circuits using the proposed gate. A novel optimization rules will be proposed to further optimize the synthesized circuits in terms of the number of gates, the quantum cost and the utilization of library to achieve better results than that shown in the literature.

Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization.

In our previous study, we obtained a large number of differentially expressed genes (DEGs) between second-generation merozoites (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix using RNA sequencing (RNA-seq). Here, we report two subtractive cDNA libraries for MZ2 (forward library) and MZ3 (reverse library) that were constructed using suppression subtractive hybridization (SSH). PCR amplification revealed that the MZ2 and MZ3 libraries contained approximately 96.7% and 95% recombinant clones, respectively, and the length of the inserted fragments ranged from 0.5 to 1.5kb. A total of 106 and 111 unique sequences were obtained from the MZ2 and MZ3 libraries, respectively, and were assembled into 13 specific consensus sequences (contigs or genes) (5 from MZ2 and 8 from MZ3). The qRT-PCR results revealed that 11 out of 13 genes were differentially expressed between MZ-2 and MZ-3. Of 13 genes, 11 genes were found in both SSH and our RNA-seq data and displayed a similar expression trend between SSH and RNA-seq data, and the remaining 2 genes have not been reported in both E. necatrix genome and our RNA-seq data. Among the 11 genes, the expression trends of 8 genes were highly consistent between SSH and our RNA-seq data. These DEGs may provide specialized functions related to the life-cycle transitions of Eimeria species.

Perfect Concurrent Fault Detection in CMOS Logic Circuits Using Parity Preservative Reversible Gates

Reversible logic has 100% fault observability meaning that a fault in any circuit node propagates to the output stage. In other words, reversible circuits are latent-fault-free. Our motivation is to incorporate this unique feature of reversible logic to design CMOS circuits having perfect or 100% Concurrent Error Detection (CED) capability. For this purpose, we propose a new fault preservative reversible gate library called Even Target - Mixed Polarity Multiple Control Toffoli (ET-MPMCT). By using ET-MPMCT, we ensure that the evenness/oddness of applied 1’s at input, is preserved at all levels of a circuit including output level unless there is a faulty node. A single fault always destroys the parity of input at the output. Our design strategy has two steps for a given function: 1) implement the function with our proposed reversible ET-MPMCT gate library; and 2) apply reversible-to-CMOS gate conversion. For the first step, we propose two approaches. For our first approach in step 1, we first need to have a reversible form of the given function if it is irreversible. Then, synthesize the reversible function using Mixed Polarity Multiple Control Toffoli (MPMCT) gate library by conventional reversible logic synthesis techniques. Finally, the synthesized circuit is converted to ET-MPMCT constructed circuit which is fault preservative. Our second approach, is an ESOP - Exclusive Sum of Products - based synthesis approach modified for our proposed fault preservative ET-MPMCT gate library. It does work with both irreversible and reversible functions. We synthesize our circuits with both approaches in step 1 and choose the circuit with lower number of reversible gates to be fed to step 2 of our design strategy. In second step of our design strategy, we convert our fault preservative reversible circuits into their CMOS counterparts. The performance of our designs is compared with other CED schemes in the literature in terms of area, power consumption, delay and detection rate. Simulations are done with Cadence Genus tool using TSMC 40nm technology. Clearly, results are in favor of our proposed techniques.

Comprehensive comparative compositional study of the vapour phase of cigarette mainstream tobacco smoke and tobacco heating product aerosol

A simple direct sample collection/dilution and introduction method was developed using quartz wool and Tenax/sulficarb sorbents for thermal desorption and comprehensive two-dimensional gas chromatography (TD-GC × GC) analyses of volatile organic compounds from vapour phase (VP) fractions of aerosol produced by tobacco heating products (THP1.0) and 3R4F mainstream tobacco smoke (MTS). Analyses were carried out using flame ionisation detection (FID) for semi-quantification and both low and high resolution time-of-flight mass spectrometry (LR/HR-TOFMS) for qualitative comparison and peak assignment. Qualitative analysis was carried out by combining identification data based on linear retention indices (LRIs) with a match window of ±10 index units, mass spectral forward and reverse library searches (from LR and HRTOFMS spectra) with a match factor threshold of >700 (both forward and reverse), and accurate mass values of ± 3 ppm for increased confidence in peak identification. Using this comprehensive approach of data mining, a total of 79 out of 85 compounds and a total of 198 out of 202 compounds were identified in THP1.0 aerosol and in 3R4F MTS, respectively. Among the identified analytes, a set of 35 compounds was found in both VP sample types. Semi-quantitative analyses were carried out using a chemical class-based external calibration method. Acyclic, alicyclic, aromatic hydrocarbons and ketones appeared to be prominent in 3R4F MTS VP, whereas larger amounts of aldehydes, ketones, heterocyclic hydrocarbons and esters were present in THP1.0 aerosol VP. The results demontsrate the capability and versatility of the method for the characterization and comparison of complex aerosol samples and highlighted the relative chemical simplicity of THP1.0 aerosol in comparison to MTS.

Identification and characterization of sex related genes in Actinidia arguta by suppression subtractive hybridization

Abstract Actinidia arguta, with sweet flavour, high nutritional value and health promoting role, is a typical dioecious plant. But dioecy represents an important constraint in kiwifruit breeding programmes and also requires identification of male and female genotypes before planting an orchard. Therefore, understanding molecular basis of sex determination in A. arguta is important for molecular identification of seedling gender at early stage. PCR-based suppressive subtractive hybridization (SSH) was performed to screen genes related to sex determination. A total of 533 and 508 positive clones, from forward and reverse libraries respectively, were selected for sequencing. The clean ESTs were assembled into 309 (forwad) and 369 (reverse) unigenes, and 228 and 299 of them hit known homologous sequences of the NCBI nr database. Based on Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, four unigenes that might be involved in sex determination were selected for validation by qRT-PCR. The expression of PME was very strong in male flower at late development stage, with 72 times higher than that of female flower. Reversely, male flower exhibited extremely low expression level of ACO, MAN1 and MYC2 at late development stage as compared with female flower. The variable expression patterns of these genes between male and female flowers suggested their importance in controlling sex expression. These results advanced our understanding of the molecular mechanism of sex determination in A. arguta.

Reverse and Random Decoy Methods for False Discovery Rate Estimation in High Mass Accuracy Peptide Spectral Library Searches.

Spectral library searching (SLS) is an attractive alternative to sequence database searching (SDS) for peptide identification due to its speed, sensitivity, and ability to include any selected mass spectra. While decoy methods for SLS have been developed for low mass accuracy peptide spectral libraries, it is not clear that they are optimal or directly applicable to high mass accuracy spectra. Therefore, we report the development and validation of methods for high mass accuracy decoy libraries. Two types of decoy libraries were found to be suitable for this purpose. The first, referred to as Reverse, constructs spectra by reversing a library's peptide sequences except for the C-terminal residue. The second, termed Random, randomly replaces all non-C-terminal residues and either retains the original C-terminal residue or replaces it based on the amino-acid frequency of the library's C-terminus. In both cases the m/z values of fragment ions are shifted accordingly. Determination of FDR is performed in a manner equivalent to SDS, concatenating a library with its decoy prior to a search. The utility of Reverse and Random libraries for target-decoy SLS in estimating false-positives and FDRs was demonstrated using spectra derived from a recently published synthetic human proteome project (Zolg, D. P.; et al. Nat. Methods 2017, 14, 259-262). For data sets from two large-scale label-free and iTRAQ experiments, these decoy building methods yielded highly similar score thresholds and spectral identifications at 1% FDR. The results were also found to be equivalent to those of using the decoy-free PeptideProphet algorithm. Using these new methods for FDR estimation, MSPepSearch, which is freely available search software, led to 18% more identifications at 1% FDR and 23% more at 0.1% FDR when compared with other widely used SDS engines coupled to postprocessing approaches such as Percolator. An application of these methods for FDR estimation for the recently reported "hybrid" library search (Burke, M. C.; et al. J. Proteome Res. 2017, 16, 1924-1935) method is also made. The application of decoy methods for high mass accuracy SLS permits the merging of these results with those of SDS, thereby increasing the assignment of more peptides, leading to deeper proteome coverage.

WSSV-responsive gene expression under the influence of PmVRP15 suppression

The viral responsive protein 15 from black tiger shrimp Penaeus monodon (PmVRP15), is highly up-regulated and produced in the hemocytes of shrimp with white spot syndrome virus (WSSV) infection. To investigate the differential expression of genes from P. monodon hemocytes that are involved in WSSV infection under the influence of PmVRP15 expression, suppression subtractive hybridization (SSH) of PmVRP15-silenced shrimp infected with WSSV was performed. The 189 cDNA clones of the forward library were generated by subtracting the cDNAs from WSSV-infected and PmVRP15 knockdown shrimp with cDNAs from WSSV-infected and GFP knockdown shrimp. For the opposite subtraction, the 176 cDNA clones in the reverse library was an alternative set of genes in WSSV-infected shrimp hemocytes in the presence of PmVRP15 expression. The abundant genes in forward SSH library had a defense/homeostasis of 26%, energy/metabolism of 23% and in the reverse SSH library a hypothetical protein with unknown function was found (30%). The differential expressed immune-related genes from each library were selected for expression analysis using qRT-PCR. All selected genes from the forward library showed high up-regulation in the WSSV-challenged PmVRP15 knockdown group as expected. Interestingly, PmHHAP, a hemocyte homeostasis associated protein, and granulin-like protein, a conserved growth factor, are extremely up-regulated in the absence of PmVRP15 expression in WSSV-infected shrimp. Only transcript level of transglutaminase II, that functions in regulating hematopoietic tissue differentiation and inhibits mature hemocyte production in shrimp, was obviously down-regulated as observed from SSH results. Taken together, our results suggest that PmVRP15 might have a function relevant to hemocyte homeostasis during WSSV infection.

“A picture is worth a thousand words”: Reverse image lookup and digital library assessment

This brief communication builds on the application of content‐based image retrieval (CBIR) and reverse image lookup (RIL), a graduated form of CBIR, as assessment tools for digital library image reuse. It combines literature on the definition, history, usefulness, and limitations of RIL and includes a brief analysis of the 4 published digital library image reuse assessment case studies. In its conclusion, the communication paper proposes that RIL offers benefits for digital library managers in the assessment of their collections.

2D wavelet transform data compression with error level guarantee for Z-map models

AbstractMost of workpiece shapes in NC milling simulations are in Z-map representations that require a very large amount of data to precisely hold a high resolution model. An irreversible compression algorithm for Z-map models using a two-dimensional Haar wavelet transform is proposed to resolve this tight memory situation for an ordinary PC. A shape model is first transformed by using Haar wavelet to build a wavelet synopsis tree while the maximum errors caused by virtually truncating high-frequency components are simultaneously calculated. The total amount of the shape data can be reduced by truncating particular sections of the wavelet components that satisfy the error threshold given by the user. Our algorithm guarantees that any error due to its irreversible compression processes is smaller than the specified level measured against the original model. A series of experiments were conducted using an Apple iMac with a 3.2 GHz CPU and 8 GB of memory. The experiments were performed with 16 sample shape models on 512×512 to 8192×8192 grids to evaluate the compression efficiency of the proposed method. Experimental results confirmed that our compression algorithm requires approximately 20–30 ms for 512×512 models and 7 s for 8192×8192 models under a maximum error level of 10× 10−6 m (a typical criteria for NC milling simulations). The compressed binaries outputted by the proposed method are generally 25–35% smaller than the baseline results by gzip, one of common reversible compression libraries, while these two methods require almost the same level of computational costs.Highlights Discarding diagonal components in WT significantly reduces data amount. The proposed method outperforms a reversible method by 25–35% in size reduction. Most of computational time is consumed by the reversible compression step. The proposed method compresses 5122 models in 20 ms, 81922 models in 7 s.

Identification of heavy metal pollutant tolerance-associated genes in Avicennia marina (Forsk.) by suppression subtractive hybridization

The halophytic Avicennia marina (Forsk.) is one of the pioneer mangroves along the south coast of China. It is an appropriate material for understanding molecular mechanisms of heavy metal tolerance in mangrove plants. A forward and a reverse cDNA library was constructed by PCR-based suppressive subtractive hybridization (SSH) to isolate these tolerance-associated genes from A. marina leaves. A total of 99 ESTs obtained from the forward and reverse libraries showed significant differential expressions. Twenty-nine genes selected by SSH were studied by real-time PCR in order to analyze their expression level. Most of these genes' expression increased in leaves under Cd stress, which suggests that these genes contribute to the heavy metal tolerance in A. marina. The diversity of these genes indicated that heavy metal stress resulted in a complex response in mangrove plants. This could prove a useful approach for further exploring the molecular mechanisms behind such heavy metal tolerance.

Deep Sequencing of Suppression Subtractive Hybridisation Drought and Recovery Libraries of the Non-model Crop Trifolium repens L.

White clover is a short-lived perennial whose persistence is greatly affected by abiotic stresses, particularly drought. The aim of this work was to characterize its molecular response to water deficit and recovery following re-hydration to identify targets for the breeding of tolerant varieties. We created a white clover reference transcriptome of 16,193 contigs by deep sequencing (mean base coverage 387x) four Suppression Subtractive Hybridization (SSH) libraries (a forward and a reverse library for each treatment) constructed from young leaf tissue of white clover at the onset of the response to drought and recovery. Reads from individual libraries were then mapped to the reference transcriptome and processed comparing expression level data. The pipeline generated four robust sets of transcripts induced and repressed in the leaves of plants subjected to water deficit stress (6,937 and 3,142, respectively) and following re-hydration (6,695 and 4,897, respectively). Semi-quantitative polymerase chain reaction was used to verify the expression pattern of 16 genes. The differentially expressed transcripts were functionally annotated and mapped to biological processes and pathways. In agreement with similar studies in other crops, the majority of transcripts up-regulated in response to drought belonged to metabolic processes, such as amino acid, carbohydrate, and lipid metabolism, while transcripts involved in photosynthesis, such as components of the photosystem and the biosynthesis of photosynthetic pigments, were up-regulated during recovery. The data also highlighted the role of raffinose family oligosaccharides (RFOs) and the possible delayed response of the flavonoid pathways in the initial response of white clover to water withdrawal. The work presented in this paper is to our knowledge the first large scale molecular analysis of the white clover response to drought stress and re-hydration. The data generated provide a valuable genomic resource for marker discovery and ultimately for the improvement of white clover.

New Design of Universal Reversible Gate Library

We present new algorithms to synthesize exact universal reversible gate library for various types of gates and costs. We use the powerful algebraic software GAP for implementation and examination of our algorithms and the reversible logic synthesis problems have been reduced to group theory problems. It is shown that minimization of arbitrary cost functions of gates and orders of magnitude are faster than its previously counterparts for reversible logic synthesis. Experimental results show that a significant improvement over the previously proposed synthesis algorithm is obtained compared with the existing approaches to reversible logic synthesis.

Majority-layered T hybridization using quantum-dot cellular automata

AbstractThe atomistic quantum-dot cellular automata (QCA) based implementations of the reversible circuits have got tremendous exposures in the last few days, due to “room-temperature workability” of the QCA. The researchers are in serious need of a methodology that can realize the area-efficient QCA counterparts of reversible benchmark circuits. In this work, a novel methodology named majority-layered T hybridization is proposed to synthesize the reversible circuits using QCA. Firstly the reversible library consisting of CNTS Gates have been generated to validate the usability of the proposed methodology. Then, an elementary QCA module of 3×3 Toffoli Gate have been proposed and extended in the realization of 4×4, 5×5 and 6×6 Toffoli Gates (multi-control Toffoli Gates). The proper mathematical modelling of the several QCA design metrics like effective area, delay and O-cost has been established. The QCA counterpart of 3×3 Toffoli Gate reports 18.61% less effective area and 8.33% less O-cost compared to th...

Suppressive subtractive hybridization reveals different gene expression between high and low virulence strains of Cladosporium cladosporioides

Cladosporium cladosporioides is a ubiquitous fungus, causing infections in plants, humans, and animals. Suppression subtractive hybridization (SSH) and quantitative real-time PCR (qRT-PCR) were used in this study to identify differences in gene expression between two C. cladosporioides strains, the highly virulent Z20 strain and the lowly virulent Zt strain. A total of 61 unigenes from the forward library and 42 from the reverse library were identified. Gene ontology (GO) analysis showed that these genes were involved in various biological processes, cellular components and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the unigenes in the forward library corresponded to 5 different pathways and the reverse library unigenes were involved in 3 different pathways. The qRT-PCR results indicated that expressions of APL1, GUD1, CSE1, SPBC3E7.04c and MFS were significantly different between Z20 and Zt strains, while genes encoding the senescence-associated proteins, pse1, nup107, mip1, pex2, icl1 and α/β hydrolase exhibited no significant differences between the two strains. In addition, we found that 5 unigenes encoding mip1, chk1, icl1, α/β hydrolase and β-glucosidase may be associated with pathogenicity. One unigene (MFS) may be related to the resistance to 14 α-demethylase inhibitor fungicides, and 5 unigenes (PEX2, NUP107, PSE1, APL1, and SPBC3E7.04c) may be related to either low spore yield or earlier aging of the Zt strain. Our study may help better understand the molecular mechanism of C. cladosporioides infection, and therefore improve the treatment and prevention of C. cladosporioides induced diseases.