Removal of Organic Dyes, Polymers and Surfactants Using Carbonaceous Materials Derived from Walnut Shells.

A series of new granular carbonaceous adsorbents was prepared via single-stage physical and chemical activation of walnut shells. Their suitability for removing various types of organic pollutants (represented by dyes, surfactants and water-soluble polymers) from the liquid phase was assessed. The activation of the precursor was carried out with CO2 and H3PO4 using conventional heating. Activated biocarbons were characterized in terms of chemical composition, acidic-basic nature of the surface, textural and electrokinetic properties as well as thermal stability. Depending on the type of activating agent used during the activation procedure, the obtained biocarbons differed in terms of specific surface area (from 401 to 1361 m2/g) and the type of porous structure produced (microporosity contribution in the range of 45-75%). Adsorption tests proved that the effectiveness of removing organic pollutants from the liquid phase depended to a large extent on the type of prepared adsorbent as well as the chemical nature and the molecular size of the adsorbate used. The chemically activated sample showed greater removal efficiency in relation to all tested pollutants. Its maximum adsorption capacity for methylene blue, poly(acrylic acid), poly(ethylene glycol) and Triton X-100 reached the levels of 247.1, 680.9, 38.5 and 61.8 mg/g, respectively.

CMC: Cancer miRNA Census - a list of cancer-related miRNA genes.

A growing body of evidence indicates an important role of miRNAs in cancer; however, there is no definitive, convenient-to-use list of cancer-related miRNAs or miRNA genes that may serve as a reference for analyses of miRNAs in cancer. To this end, we created a list of 165 cancer-related miRNA genes called the Cancer miRNA Census (CMC). The list is based on a score, built on various types of functional and genetic evidence for the role of particular miRNAs in cancer, e.g. miRNA-cancer associations reported in databases, associations of miRNAs with cancer hallmarks, or signals of positive selection of genetic alterations in cancer. The presence of well-recognized cancer-related miRNA genes, such as MIR21, MIR155, MIR15A, MIR17 or MIRLET7s, at the top of the CMC ranking directly confirms the accuracy and robustness of the list. Additionally, to verify and indicate the reliability of CMC, we performed a validation of criteria used to build CMC, comparison of CMC with various cancer data (publications and databases), and enrichment analyses of biological pathways and processes such as Gene Ontology or DisGeNET. All validation steps showed a strong association of CMC with cancer/cancer-related processes confirming its usefulness as a reference list of miRNA genes associated with cancer.

Impact of Sclerotinia sclerotiorum Infection on Lettuce (Lactuca sativa L.) Survival and Phenolics Content-A Case Study in a Horticulture Farm in Poland.

Sclerotinia sclerotiorum is a cause of a prevalent and destructive disease that attacks many horticultural food crops, such as lettuce. This soil-borne necrotrophic fungal pathogen causes significant economic losses in worldwide lettuce production annually. Furthermore, current methods utilized for management and combatting the disease, such as biocontrol, are insufficient. In this study, three cultivars of lettuce (one Crispy and two Leafy cultivars of red and green lettuce) were grown in central Poland (Lodz Voivodeship), a widely known Polish horticultural region. In the summer and early autumn, lettuce cultivars were grown in control and S. sclerotiorum-infected fields. The lettuce cultivars (Templin, Lollo Rossa, and Lollo Bionda) differed phenotypically and in terms of the survival of the fungal infection. The Crispy iceberg Templin was the most susceptible to S. sclerotiorum infection compared to the other cultivars during both vegetation seasons. The total content of phenolic compounds, flavonoids, and anthocyanins varied among cultivars and fluctuated during infection. Moreover, phenolic content was affected by vegetation season with alterable environmental factors such as air temperature, humidity, soil temperature, and pH. The most increased levels of phenolics, both flavonoids and anthocyanins in infected plants, were observed in the Leafy red Lollo Rossa cultivar in both crops. However, the highest survival/resistance to the fungus was noticed for Lollo Rossa in the summer crop and Lollo Bionda in the autumn crop.

Consequences of genetic variants in miRNA genes.

MicroRNAs (miRNAs) are small non-coding RNAs that posttranscriptionally regulate the expression of most genes. They are involved in regulating many physiological processes, and aberrations in the levels of different miRNAs play an important role in the development of many diseases, including autoimmune diseases, neuropsychiatric diseases, and cancers. Although miRNAs are being intensively studied and levels of many miRNAs are either specifically increased or decreased in particular diseases, very little is known about the genetic variations of miRNA genes and their impact on the functioning of miRNA genes and human diseases. To shed more light on the potential effects of genetic variants in miRNA genes, we review here representative examples of SNPs, mutations linked to Mendelian diseases, and cancer somatic mutations located in miRNA genes and discuss their potential effects on the expression of miRNA genes, i.e., the structure and processing of miRNA precursors, the levels of generated miRNAs, miRNA target recognition/silencing, and impact on human diseases.

Mutations in the miR-142 gene are not common in myeloproliferative neoplasms.

Recent data indicate that MIR142 is the most frequently mutated miRNA gene and one of the most frequently mutated noncoding elements in all cancers, with mutations occurring predominantly in blood cancers, especially diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. Functional analyses show that the MIR142 alterations have profound consequences for lympho- and myelopoiesis. Furthermore, one of the targets downregulated by miR-142-5p is CD274, which encodes PD-L1 that is elevated in many cancer types, including myeloproliferative neoplasms (MPNs). To extend knowledge about the occurrence of MIR142 mutations, we sequenced the gene in a large panel of MPNs [~ 700 samples, including polycythemia vera, essential thrombocythemia, primary myelofibrosis (PMF), and chronic myeloid leukemia], neoplasm types in which such mutations have never been tested, and in panels of acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL). We identified 3 mutations (one in a PMF sample and two others in one CLL sample), indicating that MIR142 mutations are rare in MPNs. In summary, mutations in MIR142 are rare in MPNs; however, in specific subtypes, such as PMF, their frequency may be comparable to that observed in CLL or AML.

miRMut: Annotation of mutations in miRNA genes from human whole-exome or whole-genome sequencing.

Here, we present the miRMut protocol to annotate mutations found in miRNA genes based on whole-exome sequencing (WES) or whole-genome sequencing (WGS) results. The pipeline assigns mutation characteristics, including miRNA gene IDs (miRBase and MirGeneDB), mutation localization within the miRNA precursor structure, potential RNA-binding motif disruption, the ascription of mutation according to Human Genome Variation Society (HGVS) nomenclature, and miRNA gene characteristics, such as miRNA gene confidence and miRNA arm balance. The pipeline includes creating tabular and graphical summaries. For complete details on the use and execution of this protocol, please refer to Urbanek-Trzeciak et al. (2020).

Variant Identification in BARD1, PRDM9, RCC1, and RECQL in Patients with Ovarian Cancer by Targeted Next-generation Sequencing of DNA Pools.

Several ovarian cancer susceptibility genes have been discovered, but more are likely to exist. In this study, we aimed to analyze knowledge-based selected genes, that is, BARD1, PRDM9, RCC1, and RECQL, in which pathogenic germline variants have been reported in patients with breast and/or ovarian cancer. As deep sequencing of DNA samples remains costly, targeted next-generation sequencing of DNA pools was utilized to screen the exons of BARD1, PRDM9, RCC1, and RECQL in approximately 400 Polish ovarian cancer cases. A total of 25 pools of 16 samples (including several duplicated samples with known variants) were sequenced on the NovaSeq6000 and analyzed with SureCall (Agilent) application. The set of variants was filtrated to exclude spurious variants, and, subsequently, the identified rare genetic variants were validated using Sanger sequencing. No pathogenic mutation was found within the analyzed cohort of patients with ovarian cancer. Validation genotyping of filtered rare silent and missense variants revealed that the majority of them were true alterations, especially those with a higher mutation quality value. The high concordance (R2 = 0.95) of population allele frequency for 44 common SNPs in the European control population (gnomAD) and our experiment confirmed the reliability of pooled sequencing. Mutations in BARD1, PRDM9, RCC1, and RECQL do not contribute substantially to the risk of ovarian cancer. Pooled DNA sequencing is a cost-effective and reliable method for the initial screening of candidate genes; however, it still requires validation of identified rare variants. PREVENTION RELEVANCE: BARD1, PRDM9, RCC1, and RECQL are not high/moderate-risk ovarian cancer susceptibility genes. Pooled sequencing is a reliable and cost-effective method to detect rare variants in candidate genes.

Profile of Basal Cell Carcinoma Mutations and Copy Number Alterations - Focus on Gene-Associated Noncoding Variants.

Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5'UTRs, 3'UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, ~50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3'UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5'UTR, and CHCHD2 frequently showed mutations in the 5'UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified a frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general.

What Is New in the Field of Industrial Wastes Conversion into Polyhydroxyalkanoates by Bacteria?

The rising global consumption and industrialization has resulted in increased food processing demand. Food industry generates a tremendous amount of waste which causes serious environmental issues. These problems have forced us to create strategies that will help to reduce the volume of waste and the contamination to the environment. Waste from food industries has great potential as substrates for value-added bioproducts. Among them, polyhydroxyalkanaotes (PHAs) have received considerable attention in recent years due to their comparable characteristics to common plastics. These biodegradable polyesters are produced by microorganisms during fermentation processes utilizing various carbon sources. Scale-up of PHA production is limited due to the cost of the carbon source metabolized by the microorganisms. Therefore, there is a growing need for the development of novel microbial processes using inexpensive carbon sources. Such substrates could be waste generated by the food industry and food service. The use of industrial waste streams for PHAs biosynthesis could transform PHA production into cheaper and more environmentally friendly bioprocess. This review collates in detail recent developments in the biosynthesis of various types of PHAs produced using waste derived from agrofood industries. Challenges associated with this production bioprocess were described, and new ways to overcome them were proposed.

A pan-cancer atlas of somatic mutations in miRNA biogenesis genes.

It is a well-known and intensively studied phenomenon that the levels of many miRNAs are differentiated in cancer. miRNA biogenesis and functional expression are complex processes orchestrated by many proteins cumulatively called miRNA biogenesis proteins. To characterize cancer somatic mutations in the miRNA biogenesis genes and investigate their potential impact on the levels of miRNAs, we analyzed whole-exome sequencing datasets of over 10 000 cancer/normal sample pairs deposited within the TCGA repository. We identified and characterized over 3600 somatic mutations in 29 miRNA biogenesis genes and showed that some of the genes are overmutated in specific cancers and/or have recurrent hotspot mutations (e.g. SMAD4 in PAAD, COAD and READ; DICER1 in UCEC; PRKRA in OV and LIN28B in SKCM). We identified a list of miRNAs whose level is affected by particular types of mutations in either SMAD4, SMAD2 or DICER1 and showed that hotspot mutations in the RNase domains in DICER1 not only decrease the level of 5p-miRNAs but also increase the level of 3p-miRNAs, including many well-known cancer-related miRNAs. We also showed an association of the mutations with patient survival. Eventually, we created an atlas/compendium of miRNA biogenesis alterations providing a useful resource for different aspects of biomedical research.

Pan-cancer analysis of somatic mutations in miRNA genes.

BACKGROUND: miRNAs are considered important players in oncogenesis, serving either as oncomiRs or suppressormiRs. Although the accumulation of somatic alterations is an intrinsic aspect of cancer development and many important cancer-driving mutations have been identified in protein-coding genes, the area of functional somatic mutations in miRNA genes is heavily understudied. METHODS: Here, based on the analysis of large genomic datasets, mostly the whole-exome sequencing of over 10,000 cancer/normal sample pairs deposited within the TCGA repository, we undertook an analysis of somatic mutations in miRNA genes. FINDINGS: We identified and characterized over 10,000 somatic mutations and showed that some of the miRNA genes are overmutated in Pan-Cancer and/or specific cancers. Nonrandom occurrence of the identified mutations was confirmed by a strong association of overmutated miRNA genes with KEGG pathways, most of which were related to specific cancer types or cancer-related processes. Additionally, we showed that mutations in some of the overmutated genes correlate with miRNA expression, cancer staging, and patient survival. INTERPRETATION: Our study is the first comprehensive Pan-Cancer study of cancer somatic mutations in miRNA genes. It may help to understand the consequences of mutations in miRNA genes and the identification of miRNA functional mutations. The results may also be the first step (form the basis and provide the resources) in the development of computational and/or statistical approaches/tools dedicated to the identification of cancer-driver miRNA genes. FUNDING: This work was supported by research grants from the Polish National Science Centre 2016/22/A/NZ2/00184 and 2015/17/N/NZ3/03629.

Polyhydroxyalkanoates Synthesized by Aeromonas Species: Trends and Challenges.

The negative effects of petrochemical-derived plastics on the global environment and depletion of global fossil fuel supplies have paved the way for exploring new technologies for the production of bioplastics. Polyhydroxyalkanoates (PHAs) are considered an alternative for synthetic polymers because of their biodegradability, biocompatibility, and non-toxicity. Many bacteria have been reported to have the ability to synthesize PHAs. Among them, the Aeromonas species seem to be ideal hosts for the industrial production of these biopolyesters due to their robust growth, simple growth requirements, their ability for the synthesis of homopolymers, co-polymers, and terpolymers with unique material properties. Some Aeromonas strains were able to produce PHAs in satisfactory amounts from simple carbon sources. Efforts have been made to use genetically modified Aeromonas strains for enhanced PHAs and to obtain bacteria with modified compositions and improved properties. This review discusses the current state of knowledge of polyhydroxyalkanoates synthesized by Aeromonas species, with a special focus on their potential, challenges, and progress in PHA synthesis.

Somatic Mutations in miRNA Genes in Lung Cancer-Potential Functional Consequences of Non-Coding Sequence Variants.

A growing body of evidence indicates that miRNAs may either drive or suppress oncogenesis. However, little is known about somatic mutations in miRNA genes. To determine the frequency and potential consequences of miRNA gene mutations, we analyzed whole exome sequencing datasets of 569 lung adenocarcinoma (LUAD) and 597 lung squamous cell carcinoma (LUSC) samples generated in The Cancer Genome Atlas (TCGA) project. Altogether, we identified 1091 somatic sequence variants affecting 522 different miRNA genes and showed that half of all cancers had at least one such somatic variant/mutation. These sequence variants occurred in most crucial parts of miRNA precursors, including mature miRNA and seed sequences. Due to our findings, we hypothesize that seed mutations may affect miRNA:target interactions, drastically changing the pool of predicted targets. Mutations may also affect miRNA biogenesis by changing the structure of miRNA precursors, DROSHA and DICER cleavage sites, and regulatory sequence/structure motifs. We identified 10 significantly overmutated hotspot miRNA genes, including the miR-379 gene in LUAD enriched in mutations in the mature miRNA and regulatory sequences. The occurrence of mutations in the hotspot miRNA genes was also shown experimentally. We present a comprehensive analysis of somatic variants in miRNA genes and show that some of these genes are mutational hotspots, suggesting their potential role in cancer.

siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer.

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3' arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5' end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3' overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3' overhangs.

Sequence features of Drosha and Dicer cleavage sites affect the complexity of isomiRs.

The deep-sequencing of small RNAs has revealed that different numbers and proportions of miRNA variants called isomiRs are formed from single miRNA genes and that this effect is attributable mainly to imprecise cleavage by Drosha and Dicer. Factors that influence the degree of cleavage precision of Drosha and Dicer are under investigation, and their identification may improve our understanding of the mechanisms by which cells modulate the regulatory potential of miRNAs. In this study, we focused on the sequences and structural determinants of Drosha and Dicer cleavage sites, which may explain the generation of homogeneous miRNAs (in which a single isomiR strongly predominates) as well as the generation of heterogeneous miRNAs. Using deep-sequencing data for small RNAs, we demonstrate that the generation of homogeneous miRNAs requires more sequence constraints at the cleavage sites than the formation of heterogeneous miRNAs. Additionally, our results indicate that specific Drosha cleavage sites have more sequence determinants in miRNA precursors than specific cleavage sites for Dicer and that secondary structural motifs in the miRNA precursors influence the precision of Dicer cleavage. Together, we present the sequence and structural features of Drosha and Dicer cleavage sites that influence the heterogeneity of the released miRNAs.

RNA imaging in living cells - methods and applications.

Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications.

RAN translation and frameshifting as translational challenges at simple repeats of human neurodegenerative disorders.

Repeat-associated disorders caused by expansions of short sequences have been classified as coding and noncoding and are thought to be caused by protein gain-of-function and RNA gain-of-function mechanisms, respectively. The boundary between such classifications has recently been blurred by the discovery of repeat-associated non-AUG (RAN) translation reported in spinocerebellar ataxia type 8, myotonic dystrophy type 1, fragile X tremor/ataxia syndrome and C9ORF72 amyotrophic lateral sclerosis and frontotemporal dementia. This noncanonical translation requires no AUG start codon and can initiate in multiple frames of CAG, CGG and GGGGCC repeats of the sense and antisense strands of disease-relevant transcripts. RNA structures formed by the repeats have been suggested as possible triggers; however, the precise mechanism of the translation initiation remains elusive. Templates containing expansions of microsatellites have also been shown to challenge translation elongation, as frameshifting has been recognized across CAG repeats in spinocerebellar ataxia type 3 and Huntington's disease. Determining the critical requirements for RAN translation and frameshifting is essential to decipher the mechanisms that govern these processes. The contribution of unusual translation products to pathogenesis needs to be better understood. In this review, we present current knowledge regarding RAN translation and frameshifting and discuss the proposed mechanisms of translational challenges imposed by simple repeat expansions.

Self-duplexing CUG repeats selectively inhibit mutant huntingtin expression.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by the expansion of the CAG repeat in the translated sequence of the HTT gene. This expansion generates a mutant huntingtin protein that contains an abnormally elongated polyglutamine tract, which, together with mutant transcript, causes cellular dysfunction. Currently, there is no curative treatment available to patients suffering from HD; however, the selective inhibition of the mutant allele expression is a promising therapeutic option. In this study, we developed a new class of CAG repeat-targeting silencing reagents that consist of self-duplexing CUG repeats. Self-duplex formation was induced through one or several U-base substitutions. A number of self-duplexing guide-strand-only short interfering RNAs have been tested through transfection into cells derived from HD patients, showing distinct activity profiles. The best reagents were highly discriminatory between the normal and mutant HTT alleles (allele selectivity) and the HTT transcript and other transcripts containing shorter CAG repeats (gene selectivity). We also demonstrated that the self-duplexing CUG repeat short interfering RNAs use the RNA interference pathway to elicit silencing, and repeat-targeting reagents showed similar activity and selectivity when expressed from short hairpin RNA vectors to achieve more durable silencing effects.

Triplet repeats in transcripts: structural insights into RNA toxicity.

Tandem repeats of various trinucleotide motifs are frequent entities in transcripts, and RNA structures formed by these sequences depend on the motif type and number of reiterations. The functions performed by normal triplet repeats in transcripts are poorly understood, but abnormally expanded repeats of certain types trigger pathogenesis in several human genetic disorders known as the triplet repeat expansion diseases (TREDs). The diseases caused by expanded non-coding CUG and CGG repeats in transcripts include myotonic dystrophy type 1 and fragile X-associated tremor ataxia syndrome. Another group of disorders in which transcripts containing translated CAG repeats play an auxiliary role in pathogenesis include Huntington's disease and several spinocerebellar ataxias. In this review, we gathered existing knowledge regarding the structural features of triplet repeats in transcripts and discussed this in the context of various pathogenic mechanisms assigned to toxic RNA repeats. These mechanisms include aberrant alternative splicing, the inhibition of nuclear transport and export, induction of the innate immune response, alteration of a microRNA biogenesis pathway and abnormal activation of an RNA interference pathway. We also provide ideas for future investigations to reveal further mechanisms of pathogenesis directly triggered by mutant RNA repeats in TREDs.