Synonymous mutations in representative yeast genes are mostly strongly non-neutral.

Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral1-5. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar-albeit nonidentical-between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.

Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast.

In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to Matia-González et al. (2021, 2015).

Identification of Lung and Blood Microbiota Implicated in COVID-19 Prognosis.

The implications of the microbiome on Coronavirus disease 2019 (COVID-19) prognosis has not been thoroughly studied. In this study we aimed to characterize the lung and blood microbiome and their implication on COVID-19 prognosis through analysis of peripheral blood mononuclear cell (PBMC) samples, lung biopsy samples, and bronchoalveolar lavage fluid (BALF) samples. In all three tissue types, we found panels of microbes differentially abundant between COVID-19 and normal samples correlated to immune dysregulation and upregulation of inflammatory pathways, including key cytokine pathways such as interleukin (IL)-2, 3, 5-10 and 23 signaling pathways and downregulation of anti-inflammatory pathways including IL-4 signaling. In the PBMC samples, six microbes were correlated with worse COVID-19 severity, and one microbe was correlated with improved COVID-19 severity. Collectively, our findings contribute to the understanding of the human microbiome and suggest interplay between our identified microbes and key inflammatory pathways which may be leveraged in the development of immune therapies for treating COVID-19 patients.

Trans-kingdom RNAs and their fates in recipient cells: advances, utilization, and perspectives.

The phenomenon and potential mechanisms of trans-kingdom RNA silencing (or RNA interference, RNAi) are among the most exciting topics in science today. Based on trans-kingdom RNAi, host-induced gene silencing (HIGS) has been widely applied to create crops with resistance to various pests and pathogens, overcoming the limitations of resistant cultivars. However, a lack of transformation technology in many crops limits the application of HIGS. Here, we describe the various fates of trans-kingdom RNAs in recipient organisms. Based on the assumption that small RNAs can be transferred between the host and its microbiome or among microbiome members, we propose a possible alternative strategy for plant protection against pathogens without the need for crop genetic modification.

Diversity of fungal isolates from fungus-growing termite Macrotermes barneyi and characterization of bioactive compound from Xylaria escharoidea.

Owing to their potential applications, as well as their structural diversity, the discovery of novel secondary metabolites from insect-associated fungi has been of interest to researchers in recent years. The aim of this study was therefore to estimate the diversity of fungi associated with fungus-growing termites and bioprospecting these for potential secondary metabolites. In total, 18 fungal species were isolated and described from the gut and comb of Macrotermes barneyi based on 18S ribosomal DNA gene sequence analysis. Antimicrobial activity assays were carried out on all the known fungi, and nine isolates were recorded as active against pathogenic fungi. Xylaria escharoidea, the best performing isolate, was grown at laboratory scale and 4,8-dihydroxy-3,4-dihydronaphthalen-1(2H) was isolated and characterized. The minimum inhibitory concentration of this isolated compound against tested pathogenic organisms was found to be 6.25 µg. In addition, molecular docking studies have revealed that 4,8-dihydroxy-3,4-dihydronaphthalen-1(2H) is a prominent antibacterial agent with a marked interaction with key residues on protein A (agrAC ) that regulates the accessory gene. The findings of this study support the drug discovery of antimicrobial properties in insect-associated fungi, which may lead to novel secondary metabolites.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Transboundary Pathogenic microRNA Analysis Framework for Crop Fungi Driven by Biological Big Data and Artificial Intelligence Model.

Plant fungal diseases have been affecting the world's agricultural production and economic levels for a long time, such as rice blast, gray tomato mold, potato late blight etc. Recent studies have shown that fungal pathogens transmit microRNA as an effector to host plants for infection. However, bioassay-based verification analysis is time-consuming and challenging, and it is difficult to analyze from a global perspective. With the accumulation of fungal and plant-related data, data analysis methods can be used to analyze pathogenic fungal microRNA further. Based on the microRNA expression data of fungal pathogens infecting plants before and after, this paper discusses the selection strategy of sample data, the extraction strategy of pathogenic fungal microRNA, the prediction strategy of a fungal pathogenic microRNA target gene, the bicluster-based fungal pathogenic microRNA functional analysis strategy and experimental verification methods. A general analysis pipeline based on machine learning and bicluster-based function module was proposed for plant-fungal pathogenic microRNA.The pipeline proposed in this paper is applied to the infection process of Magnaporthe oryzae and the infection process of potato late blight. It has been verified to prove the feasibility of the pipeline. It can be extended to other relevant crop pathogen research, providing a new idea for fungal research on plant diseases. It can be used as a reference for understanding the interaction between fungi and plants.

Reconstruction and functional annotation of Ascosphaera apis full-length transcriptome utilizing PacBio long reads combined with Illumina short reads.

Ascosphaera apis is a widespread fungal pathogen of honeybee larvae that results in chalkbrood disease, leading to heavy losses for the beekeeping industry in China and many other countries. This work was aimed at generating a full-length transcriptome of A. apis using PacBio single-molecule real-time (SMRT) sequencing. Here, more than 23.97 Gb of clean reads was generated from long-read sequencing of A. apis mycelia, including 464,043 circular consensus sequences (CCS) and 394,142 full-length non-chimeric (FLNC) reads. In total, we identified 174,095 high-confidence transcripts covering 5141 known genes with an average length of 2728 bp. We also discovered 2405 genic loci and 11,623 isoforms that have not been annotated yet within the current reference genome. Additionally, 16,049, 10,682, 4520 and 7253 of the discovered transcripts have annotations in the Non-redundant protein (Nr), Clusters of Eukaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, 1205 long non-coding RNAs (lncRNAs) were identified, which have less exons, shorter exon and intron lengths, shorter transcript lengths, lower GC percent, lower expression levels, and fewer alternative splicing (AS) evens, compared with protein-coding transcripts. A total of 253 members from 17 transcription factor (TF) families were identified from our transcript datasets. Finally, the expression of A. apis isoforms was validated using a molecular approach. Overall, this is the first report of a full-length transcriptome of entomogenous fungi including A. apis. Our data offer a comprehensive set of reference transcripts and hence contributes to improving the genome annotation and transcriptomic study of A. apis.

A novel dimorphic microsporidian Ameson iseebi sp. nov. infecting muscle of the Japanese spiny lobster, Panulirus japonicus, in western Japan.

Japanese spiny lobsters (Panulirus japonicus) exhibiting white opaque abdominal muscle were found in Mie and Wakayama prefectures, in mid-Western Japan. Microscopically, two types of microsporidian spores, ovoid and rod-shaped, were observed infecting the muscle. Histologically, both types of spore were detected inside myofibers of the abdomen, appendages, and cardiac muscles and were often both observed in a single myofiber simultaneously. Transmission electron microscopy revealed that ovoid spores have villous projections on the surface, and that ovoid and rod-shaped spores have a polar filament with 12 coils and 6 to 8 coils respectively. Merogonic and sporogonic stages were observed around ovoid spores, but rarely around rod-shaped spores. The small subunit ribosomal DNA sequences obtained from both spore types were identical to each other, indicating that this microsporidian exhibits a clear spore dimorphism. Phylogenetic analysis based on the rDNA sequences indicates that this microsporidian is part of a clade consisting of the genera Ameson and Nadelspora, with the most closely related species being A. herrnkindi found in the Caribbean spiny lobster P. argus. Based on ultrastructural features, molecular phylogenetic data, host type and geographical differences among known species in these genera, the species found in whitened abdominal muscles of the Japanese spiny lobster is described as Ameson iseebi sp. nov.

Quantitation of RNA by a fluorometric method using the SYTO RNASelect stain.

Quantitation of even trace amounts of RNA has biological significance. However, existing methods of RNA estimation are not capable of eliminating the interference of other impurities, especially DNA. In this study, we developed a rapid and sensitive method for fluorometric estimation of RNA using an RNA-specific dye, SYTO RNASelect. A good linear correlation between the fluorescence intensity and RNA concentration was observed using this method. The maximal fluorescence intensity of DNA was only 2.9% of the fluorescence intensity of 40 µg/mL RNA, demonstrating the high RNA specificity of the SYTO RNASelect method.

MIST: a Multilocus Identification System for Trichoderma.

Due to the rapid expansion in microbial taxonomy, precise identification of common industrially and agriculturally relevant fungi such as Trichoderma species is challenging. In this study, we introduce the online multilocus identification system (MIST) for automated detection of 349 Trichoderma species based on a set of three DNA barcodes. MIST is based on the reference databases of validated sequences of three commonly used phylogenetic markers collected from public databases. The databases consist of 414 complete sequences of the nuclear rRNA internal transcribed spacers (ITS) 1 and 2, 583 sequence fragments of the gene encoding translation elongation factor 1-alpha (tef1), and 534 sequence fragments of the gene encoding RNA polymerase subunit 2 (rpb2). Through MIST, information from different DNA barcodes can be combined and the identification of Trichoderma species can be achieved based on the integrated parametric sequence similarity search (blastn) performed in the manner of a decision tree classifier. In the verification process, MIST provided correct identification for 44 Trichoderma species based on DNA barcodes consisting of tef1 and rpb2 markers. Thus, MIST can be used to obtain an automated species identification as well as to retrieve sequences required for manual identification by means of phylogenetic analysis.IMPORTANCE The genus Trichoderma is important to humankind, with a wide range of applications in industry, agriculture, and bioremediation. Thus, quick and accurate identification of Trichoderma species is paramount, since it is usually the first step in Trichoderma-based research. However, it frequently becomes a limitation, especially for researchers who lack taxonomic knowledge of fungi. Moreover, as the number of Trichoderma-based studies has increased, a growing number of unidentified sequences have been stored in public databases, which has made the species identification more ambiguous. In this study, we provide an easy-to-use tool, MIST, for automated species identification, a list of Trichoderma species, and corresponding sequences of reference DNA barcodes. Therefore, this study will facilitate the research on the biodiversity and applications of the genus Trichoderma.

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

Similarities and spatial variations of bacterial and fungal communities in field rice planthopper (Hemiptera: Delphacidae) populations.

Rice planthoppers are notorious plant sap-feeding pests which cause serious damage. While several microbes in rice planthoppers have been broadly characterized, the abundance and diversity of bacteria and fungi in field planthoppers are largely unknown. This study investigated the bacterial and fungal community compositions of Chinese wild rice planthoppers Laodelphax striatellus and Sogatella furcifera using parallel 16S rRNA gene amplicon and internal transcribed space region sequencing. The bacteria varied significantly between the species and were partitioned significantly by sex, tissues and host environments in each species. The majority of bacteria were affiliated with the genera Wolbachia, Cardinium, Rickettsia and Pantoea. The abundance of Wolbachia was negatively correlated with that of Cardinium in both planthopper species. Compared with bacteria, the abundance and diversity of fungi did not differ between sexes but both were enriched in the gut. The bacterial community as a whole showed no significant correlation with the fungal community. The majority of fungi were related to Sarocladium, Alternaria, Malassezia, Aspergillus and Curvularia. A phylogenetic analysis revealed that these fungi were closely related to botanic symbionts or pathogens. Our results provide novel insights into the bacteria and fungi of rice planthoppers.

A new lineage of crayfish-infecting Microsporidia: The Cambaraspora floridanus n. gen. n. sp. (Glugeida: Glugeidae) complex from Floridian freshwaters (USA).

Crayfish are a vital ecological asset in their native range but can be highly damaging as invasive species. Knowledge of their diseases, including high levels of research on Aphanomyces astaci (crayfish plague), show that disease plays a vital role during crayfish invasions. Microsporidian diseases in crayfish are less studied but are thought to have important links to crayfish health and invasion dynamics. In this study we provide a systematic description of a novel microsporidian parasite from the Floridian crayfish, Procambarus paeninsulanus, with additional genetic identification from related Microsporidia from Procambarus fallax, Cambarellus shufeldtii and Cambarellus blacki. This novel microsporidium from P. paeninsulanus is described in a new genus, Cambaraspora, and species, Cambaraspora floridanus, and represents a novel member of the Clade V Microsporidia within the Glugeidae. The parasite develops in the muscle tissue of P. paeninsulanus, within a sporophorous vesicle, and produces a spore with 19-21 turns of the polar filament measuring 6.136 ± 0.84 µm in length and 2.12 ± 0.23 µm in width. The muscle-infecting nature of the parasite suggests that it is horizontally transmitted. Genetic data for the 18S of the parasite from all hosts confirms its assignment to Clade V and reveal it to be a relative of multiple fish-infecting parasites. It shows closest genetic relationship to Glugea plecoglossi, but branches alongside multiple microsporidia from fish, crustaceans and eDNA isolates. The information presented here suggests that this novel parasite may have the potential to infect piscine hosts and is a likely mortality driver in the P. paeninsulanus population. Its potential as a control agent or wildlife disease invasion threat is explored, as well as the placement of this novel microsporidium within the Glugeidae.

Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera.

Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

A simple procedure for detecting Dekkera bruxellensis in wine environment by RNA-FISH using a novel probe.

Dekkera bruxellensis, considered the major microbial contaminant in wine production, produces 4-ethylphenol, a cause of unpleasant odors. Thus, identification of this yeast before wine spoilage is crucial. Although challenging, it could be achieved using a simple technique: RNA-FISH. To reach it is necessary to design probes that allow specific detection/identification of D. bruxellensis among the wine microorganisms and in the wine environment and, if possible, using low formamide concentrations. Therefore, this study was focused on: a) designing a DNA-FISH probe to identify D. bruxellensis that matches these requirements and b) determining the applicability of the RNA-FISH procedure after the end of the alcoholic fermentation and in wine. A novel DNA-FISH D. bruxellensis probe with good performance and specificity was designed. The application of this probe using an in-suspension RNA-FISH protocol (applying only 5% of formamide) allowed the early detection/identification of D. bruxellensis at low cell densities (5 × 102 cell/mL). This was possible by flow cytometry independently of the growth stage of the target cells, both at the end of the alcoholic fermentation and in wine even in the presence of high S. cerevisiae cell densities. Thus, this study aims to contribute to facilitate the identification of D. bruxellensis before wine spoilage occurs, preventing economic losses to the wine industry.

Comparative genome-scale analysis of Pichia pastoris variants informs selection of an optimal base strain.

Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y-11430 (CBS7435), GS115, X-33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y-11430 exhibited the highest activity of genes involved in methanol utilization, up to two-fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X-33 displayed a six-fold higher transformation efficiency and up to 1.2-fold higher titers than Y-11430. X-33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X-33 into Y-11430 resulted in an optimized base strain that provided up to four-fold higher transformation efficiency and three-fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts.

A formal redefinition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) and reassignment of species based on molecular phylogenetics.

The microsporidian genera Nosema and Vairimorpha comprise a clade described from insects. Currently the genus Nosema is defined as having a dimorphic life cycle characterized by diplokaryotic stages and diplosporoblastic sporogony with two functionally and morphologically distinct spore types ("early" or "primary" and "environmental"). The Vairimorpha life cycle, in addition to a Nosema-type diplokaryotic sporogony, includes an octosporoblastic sporogony producing eight uninucleate spores (octospores) within a sporophorous vesicle. Molecular phylogeny, however, has clearly demonstrated that the genera Nosema and Vairimorpha, characterized by the absence or presence of uninucleate octospores, respectively, represent two polyphyletic taxa, and that octosporogony is turned on and off frequently within taxa, depending on environmental factors such as host species and rearing temperature. In addition, recent studies have shown that both branches of the Vairimorpha-Nosema clade contain species that are uninucleate throughout their life cycle. The SSU rRNA gene sequence data reveal two distinct clades, those closely related to Vairimorpha necatrix, the type species for the genus Vairimorpha, and those closely related to Nosema bombycis, the type species for the genus Nosema. Here, we redefine the two genera, giving priority to molecular character states over those observed at the developmental, structural or ultrastructural levels and present a list of revised species designations. Using this approach, a series of species are renamed (combination novum) and members of two genera, Rugispora and Oligosporidium, are reassigned to Vairimorpha because of their phylogenetic position. Moreover, the family Nosematidae is redefined and includes the genera Nosema and Vairimorpha comprising a monophyletic lineage of Microsporidia.

Ultrastructural and molecular characterization of Nosema alticae sp. nov. (Microsporidia: Nosematidae), pathogen of the flea beetle, Altica hampei Allard, 1867 (Coleoptera: Chrysomelidae).

In this study, the first microsporidian pathogen from Altica hampei (Coleoptera: Chrysomelidae) is described based on light microscopy, ultrastructural characteristics and comparative 16S SSU rDNA analysis. All developmental stages of the microsporidium are diplokaryotic and in direct contact with the host cell cytoplasm. Giemsa-stained mature spores are oval in shape and measured 3.82 ± 0.35 µm in length and 2.54 ± 0.27 µm in width. The polar filament of the binucleate spores is isofilar with 12-14 coils. Coils are 140.28 ± 4.88 nm (135.59-147.06; n = 36) in diameter and consist of six concentric layers of different electron density and thickness. The spores have a relatively thick (161.72 ± 29.19 nm) trilaminar spore wall. Morphological, ultrastructural and molecular features indicate that the described microsporidium belongs to the genus Nosema and is named Nosema alticae sp. nov.

A comprehensive comparison and analysis of computational predictors for RNA N6-methyladenosine sites of Saccharomyces cerevisiae.

N6-methyladenosine (m6A) modification, as one of the commonest post-transcription modifications in RNAs, has been reported to be highly related to many biological processes. Over the past decade, several tools for m6A sites prediction of Saccharomyces cerevisiae have been developed and are freely available online. However, the quality of predictions by these tools is difficult to quantify and compare. In this study, an independent dataset M6Atest6540 was compiled to systematically evaluate nine publicly available m6A prediction tools for S. cerevisiae. The experimental results indicate that RAM-ESVM achieved the best performance on M6Atest6540; however, most models performed substantially worse than their performances reported in the original papers. The benchmark dataset Met2614, which was used as the training dataset for the nine methods, were further analyzed by using a position bias index. The results demonstrated the significantly different bias of dataset Met2614 compared with the RNA segments around m6A sites recorded in RMBase. Moreover, newMet2614 was collected by randomly selecting RNA segments from non-redundant data recorded in RMBase, and three different kinds of features were extracted. The performances of the models built on Met2614 and newMet2614 with the features were compared, which shows the better generalization of models built on newMet2614. Our results also indicate the position-specific propensity-based features outperform other features, although they are also easily over-fitted on a biased dataset.