Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging.

In the realm of biological research, the invention of super-resolution microscopy (SRM) has enabled the visualization of ultrafine sub-cellular structures and their functions in live cells at the nano-scale level, beyond the diffraction limit, which has opened up a new window for advanced biomedical studies to unravel the complex unknown details of physiological disorders at the sub-cellular level with unprecedented resolution and clarity. However, most of the SRM techniques are highly reliant on the personalized special photophysical features of the fluorophores. In recent times, there has been an unprecedented surge in the development of robust new fluorophore systems with personalized features for various super-resolution imaging techniques. To date, xanthene, cyanine, oxazine and BODIPY cores have been authoritatively utilized as the basic fluorophore units in most of the small-molecule-based organic fluorescent probe designing strategies for SRM owing to their excellent photophysical characteristics and easy synthetic acquiescence. Since the future of next-generation SRM studies will be decided by the availability of advanced fluorescent probes and these four fluorescent building blocks will play an important role in progressive new fluorophore design, there is an urgent need to review the recent advancements in designing fluorophores for different SRM methods based on these fluorescent dye cores. This review article not only includes a comprehensive discussion about the recent developments in designing fluorescent probes for various SRM techniques based on these four important fluorophore building blocks with special emphasis on their effective integration into live cell super-resolution bio-imaging applications but also critically evaluates the background of each of the fluorescent dye cores to highlight their merits and demerits towards developing newer fluorescent probes for SRM.

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and four disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases, with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild-type full-length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic Caenorhabditis elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress, which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

Whole exome sequencing identifies two novel variants in PHEX and DMP1 in Malaysian children with hypophosphatemic rickets.

BACKGROUND: Hypophosphatemic rickets (HR) is a genetic disease of phosphate wasting that is characterized by defective bone mineralization. The most common cause of the disease is mutations in the phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) gene. The aims of this study were to identify the gene variants responsible for HR in three cases of Malaysian origin from three independent families and to describe their clinical, biochemical, and radiological features. METHODS: Whole exome sequencing (WES) was performed on all patients and their parents, followed by Sanger sequencing validation. Bioinformatics tools were used to provide supporting evidence for pathogenicity of variants. To confirm that a mutation is de novo, paternity test was carried out. High resolution melting curve analysis was performed to assess the allele frequency in normal controls for mutations that were found in the patients. RESULTS: The patients showed typical characteristics of HR including lower limb deformity, hypophosphatemia, and elevated alkaline phosphatase. WES revealed two variants in the PHEX gene and one variant in the dentin matrix protein 1 (DMP1) gene. Two of the three variants were novel, including c.1946_1954del (p.Gly649_Arg651del) in PHEX and c.54 + 1G > A in DMP1. Our data suggests that the novel p.Gly649_Arg651del variant is likely pathogenic for HR disease. CONCLUSIONS: This study extends the variant spectrum of the PHEX and DMP1 genes. Our findings indicate that WES is an advantageous approach for diagnosis of genetic diseases which are heterogeneous.

Gut-disc axis: A cause of intervertebral disc degeneration and low back pain?

PURPOSE: Low back pain (LBP), a widely prevalent and costly disease around the world, is mainly caused by intervertebral disc (IVD) degeneration (IDD). Although numerous factors may trigger this degenerative process, microbiome dysbiosis has recently been implicated as one of the likely causes. However, the exact relationship between the microbiome and IDD is not well understood. This review summarizes the potential mechanisms and discusses microbiome dysbiosis's possible influence on IDD and LBP. METHODS: Prospective literature review. RESULTS: Alterations in microbiome composition and host responses to the microbiota causing pathological bone development and involution, led to the concept of gut-bone marrow axis and gut-bone axis. Moreover, the concept of the gut-disc axis was also proposed to explain the microbiome's role in IDD and LBP. According to the existing evidence, the microbiome could be an important factor for inducing and aggravating IDD through changing or regulating the outside and inside microenvironment of the IVD. Three potential mechanisms by which the gut microbiota can induce IVD and cause LBP are: (1) translocation of the bacteria across the gut epithelial barrier and into the IVD, (2) regulation of the mucosal and systemic immune system, and (3) regulation of nutrient absorption and metabolites formation at the gut epithelium and its diffusion into the IVD. Furthermore, to investigate whether IVD is initiated by pathogenic bacteria and establish the correlation between the presence of certain microbial groups with the disease in question, microbiome diversity analysis based on16S rRNA data can be used to characterise stool/blood microbiota from IVD patients. CONCLUSION: Future studies on microbiome, fungi and viruses in IDD is necessary to revolutionize our thinking about their possible role in the development of IVD diseases. Furthermore, we believe that inflammation inhibition and interruption of amplification of cascade reaction in IVD by targeting the gut and IVD microbiome is worthwhile for the treatment of IDD and LBP. LEVEL OF EVIDENCE I: Diagnostic: individual cross-sectional studies with the consistently applied reference standard and blinding.

Antioxidant Effects of Resveratrol in Intervertebral Disk.

Intervertebral disk (IVD) degeneration (IVDD) can cause various spinal degenerative diseases. Cumulative evidence has indicated that IVDD can result from inflammation, apoptosis, autophagy, biomechanical changes and other factors. Currently, lack of conservative treatment for degenerative spinal diseases leads to an urgent demand for clinically applicable medication to ameliorate the progression of IVDD. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a polyphenol compound extracted from red wine or grapes, has shown protective effects on IVD, alleviating the progression of IVDD. Resveratrol has been demonstrated as a scavenger of free radicals both in vivo and in vitro. The antioxidant effects of resveratrol are likely attributed to its regulation on mitochondrial dysfunction or the elimination of reactive oxygen species. This review will summarize the mechanisms of the reactive oxygen species production and elaborate the mechanisms of resveratrol in retarding IVDD progression, providing a comprehensive understanding of the antioxidant effects of resveratrol in IVD.

Single-Cell Transcriptome Profiling Reveals Multicellular Ecosystem of Nucleus Pulposus during Degeneration Progression.

Although degeneration of the nucleus pulposus (NP) is a major contributor to intervertebral disc degeneration (IVDD) and low back pain, the underlying molecular complexity and cellular heterogeneity remain poorly understood. Here, a comprehensive single-cell resolution transcript landscape of human NP is reported. Six novel human NP cells (NPCs) populations are identified by their distinct molecular signatures. The potential functional differences among NPC subpopulations are analyzed. Predictive transcripts, transcriptional factors, and signal pathways with respect to degeneration grades are explored. It is reported that fibroNPCs is the subpopulation for end-stage degeneration. CD90+NPCs are observed to be progenitor cells in degenerative NP tissues. NP-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including granulocytic myeloid-derived suppressor cells (G-MDSCs). Integrin αM (CD11b) and oxidized low density lipoprotein receptor 1 (OLR1) as surface markers of NP-derived G-MDSCs are uncovered. The G-MDSCs are found to be enriched in mildly degenerated (grade II and III) NP tissues compared to severely degenerated (grade IV and V) NP tissues. Their immunosuppressive function and alleviation effects on NPCs' matrix degradation are revealed in vitro. Collectively, this study reveals the NPC-type complexity and phenotypic characteristics in NP, thereby providing new insights and clues for IVDD treatment.

A non-genetic, cell cycle-dependent mechanism of platinum resistance in lung adenocarcinoma.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

YB-1 Knockdown Inhibits the Proliferation of Mesothelioma Cells through Multiple Mechanisms.

Y-box binding protein-1 (YB-1) is a multifunctional oncoprotein that has been shown to regulate proliferation, invasion and metastasis in a variety of cancer types. We previously demonstrated that YB-1 is overexpressed in mesothelioma cells and its knockdown significantly reduces tumour cell proliferation, migration, and invasion. However, the mechanisms driving these effects are unclear. Here, we utilised an unbiased RNA-seq approach to characterise the changes to gene expression caused by loss of YB-1 knockdown in three mesothelioma cell lines (MSTO-211H, VMC23 and REN cells). Bioinformatic analysis showed that YB-1 knockdown regulated 150 common genes that were enriched for regulators of mitosis, integrins and extracellular matrix organisation. However, each cell line also displayed unique gene expression signatures, that were differentially enriched for cell death or cell cycle control. Interestingly, deregulation of STAT3 and p53-pathways were a key differential between each cell line. Using flow cytometry, apoptosis assays and single-cell time-lapse imaging, we confirmed that MSTO-211H, VMC23 and REN cells underwent either increased cell death, G1 arrest or aberrant mitotic division, respectively. In conclusion, this data indicates that YB-1 knockdown affects a core set of genes in mesothelioma cells. Loss of YB-1 causes a cascade of events that leads to reduced mesothelioma proliferation, dependent on the underlying functionality of the STAT3/p53-pathways and the genetic landscape of the cell.

CD300f epitopes are specific targets for acute myeloid leukemia with monocytic differentiation.

Antibody-based therapy in acute myeloid leukemia (AML) has been marred by significant hematologic toxicity due to targeting of both hematopoietic stem and progenitor cells (HSPCs). Achieving greater success with therapeutic antibodies requires careful characterization of the potential target molecules on AML. One potential target is CD300f, which is an immunoregulatory molecule expressed predominantly on myeloid lineage cells. To confirm the value of CD300f as a leukemic target, we showed that CD300f antibodies bind to AML from 85% of patient samples. While one CD300f monoclonal antibody (mAb) reportedly did not bind healthy hematopoietic stem cells, transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. Importantly for antibody targeting, the extracellular region of CD300f can be present with or without the exon 4-encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f-specific antibodies. Furthermore, binding of one mAb, DCR-2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP-D2. Detailed analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that lacked exon 4 compared to AML with monocytic differentiation. Analysis of a small cohort of AML cells revealed that the UP-D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. This provides the opportunity to develop an antibody-based strategy to target AMLs with monocytic differentiation but not healthy CD34+ HSPCs. This would be a major step forward in developing effective anti-AML therapeutic antibodies with reduced hematologic toxicity.

Variation of soil bacterial communities along a chronosequence of Eucalyptus plantation.

Eucalyptus is harvested for wood and fiber production in many tropical and sub-tropical habitats globally. Plantation has been controversial because of its influence on the surrounding environment, however, the influence of massive Eucalyptus planting on soil microbial communities is unclear. Here we applied high-throughput sequencing of the 16S rRNA gene to assess the microbial community composition and diversity of planting chronosequences, involving two, five and ten years of Eucalyptus plantation, comparing to that of secondary-forest in South China. We found that significant changes in the composition of soil bacteria occurred when the forests were converted from secondary-forest to Eucalyptus. The bacterial community structure was clearly distinct from control and five year samples after Eucalyptus was grown for 2 and 10 years, highlighting the influence of this plantation on local soil microbial communities. These groupings indicated a cycle of impact (2 and 10 year plantations) and low impact (5-year plantations) in this chronosequence of Eucalyptus plantation. Community patterns were underpinned by shifts in soil properties such as pH and phosphorus concentration. Concurrently, key soil taxonomic groups such as Actinobacteria showed abundance shifts, increasing in impacted plantations and decreasing in low impacted samples. Shifts in taxonomy were reflected in a shift in metabolic potential, including pathways for nutrient cycles such as carbon fixation, which changed in abundance over time following Eucalyptus plantation. Combined these results confirm that Eucalyptus plantation can change the community structure and diversity of soil microorganisms with strong implications for land-management and maintaining the health of these ecosystems.

Metagenomic Functional Potential Predicts Degradation Rates of a Model Organophosphorus Xenobiotic in Pesticide Contaminated Soils.

Chemical contamination of natural and agricultural habitats is an increasing global problem and a major threat to sustainability and human health. Organophosphorus (OP) compounds are one major class of contaminant and can undergo microbial degradation, however, no studies have applied system-wide ecogenomic tools to investigate OP degradation or use metagenomics to understand the underlying mechanisms of biodegradation in situ and predict degradation potential. Thus, there is a lack of knowledge regarding the functional genes and genomic potential underpinning degradation and community responses to contamination. Here we address this knowledge gap by performing shotgun sequencing of community DNA from agricultural soils with a history of pesticide usage and profiling shifts in functional genes and microbial taxa abundance. Our results showed two distinct groups of soils defined by differing functional and taxonomic profiles. Degradation assays suggested that these groups corresponded to the organophosphorus degradation potential of soils, with the fastest degrading community being defined by increases in transport and nutrient cycling pathways and enzymes potentially involved in phosphorus metabolism. This was against a backdrop of taxonomic community shifts potentially related to contamination adaptation and reflecting the legacy of exposure. Overall our results highlight the value of using holistic system-wide metagenomic approaches as a tool to predict microbial degradation in the context of the ecology of contaminated habitats.

Soil aggregation and associated microbial communities modify the impact of agricultural management on carbon content.

Soil carbon (C) stabilisation is known to depend in part on its distribution in structural aggregates, and upon soil microbial activity within the aggregates. However, the mechanisms and relative contributions of different microbial groups to C turnover in different aggregates under various management practices remain unclear. The aim of this study was to determine the role of soil aggregation and their associated microbial communities in driving the responses of soil organic matter (SOM) to multiple management practices. Our results demonstrate that higher amounts of C inputs coupled with greater soil aggregation in residue retention management practices has positive effects on soil C content. Our results provide evidence that different aggregate size classes support distinct microbial habitats which supports the colonisation of different microbial communities. Most importantly our results indicate that the effects of management practices on soil C is modulated by soil aggregate sizes and their associated microbial community and are more pronounced in macro-aggregate compared with micro-aggregate sizes. Based on our findings we recommend that differential response of management practices and microbial control on the C turnover in macro-aggregates and micro-aggregate should be explicitly considered when accounting for management impacts on soil C turnover.

The pangenome of hexaploid bread wheat.

There is an increasing understanding that variation in gene presence-absence plays an important role in the heritability of agronomic traits; however, there have been relatively few studies on variation in gene presence-absence in crop species. Hexaploid wheat is one of the most important food crops in the world and intensive breeding has reduced the genetic diversity of elite cultivars. Major efforts have produced draft genome assemblies for the cultivar Chinese Spring, but it is unknown how well this represents the genome diversity found in current modern elite cultivars. In this study we build an improved reference for Chinese Spring and explore gene diversity across 18 wheat cultivars. We predict a pangenome size of 140 500 ± 102 genes, a core genome of 81 070 ± 1631 genes and an average of 128 656 genes in each cultivar. Functional annotation of the variable gene set suggests that it is enriched for genes that may be associated with important agronomic traits. In addition to variation in gene presence, more than 36 million intervarietal single nucleotide polymorphisms were identified across the pangenome. This study of the wheat pangenome provides insight into genome diversity in elite wheat as a basis for genomics-based improvement of this important crop. A wheat pangenome, GBrowse, is available at http://appliedbioinformatics.com.au/cgi-bin/gb2/gbrowse/WheatPan/, and data are available to download from http://wheatgenome.info/wheat_genome_databases.php.

WheatGenome.info: A Resource for Wheat Genomics Resource.

An integrated database with a variety of Web-based systems named WheatGenome.info hosting wheat genome and genomic data has been developed to support wheat research and crop improvement. The resource includes multiple Web-based applications, which are implemented as a variety of Web-based systems. These include a GBrowse2-based wheat genome viewer with BLAST search portal, TAGdb for searching wheat second generation genome sequence data, wheat autoSNPdb, links to wheat genetic maps using CMap and CMap3D, and a wheat genome Wiki to allow interaction between diverse wheat genome sequencing activities. This portal provides links to a variety of wheat genome resources hosted at other research organizations. This integrated database aims to accelerate wheat genome research and is freely accessible via the web interface at http://www.wheatgenome.info/ .

Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat.

Despite being a major international crop, our understanding of the wheat genome is relatively poor due to its large size and complexity. To gain a greater understanding of wheat genome diversity, we have identified single nucleotide polymorphisms between 16 Australian bread wheat varieties. Whole-genome shotgun Illumina paired read sequence data were mapped to the draft assemblies of chromosomes 7A, 7B and 7D to identify more than 4 million intervarietal SNPs. SNP density varied between the three genomes, with much greater density observed on the A and B genomes than the D genome. This variation may be a result of substantial gene flow from the tetraploid Triticum turgidum, which possesses A and B genomes, during early co-cultivation of tetraploid and hexaploid wheat. In addition, we examined SNP density variation along the chromosome syntenic builds and identified genes in low-density regions which may have been selected during domestication and breeding. This study highlights the impact of evolution and breeding on the bread wheat genome and provides a substantial resource for trait association and crop improvement. All SNP data are publically available on a generic genome browser GBrowse at www.wheatgenome.info.

Molecular marker databases.

The detection and analysis of genetic variation plays an important role in plant breeding and this role is increasing with the continued development of genome sequencing technologies. Molecular genetic markers are important tools to characterize genetic variation and assist with genomic breeding. Processing and storing the growing abundance of molecular marker data being produced requires the development of specific bioinformatics tools and advanced databases. Molecular marker databases range from species specific through to organism wide and often host a variety of additional related genetic, genomic, or phenotypic information. In this chapter, we will present some of the features of plant molecular genetic marker databases, highlight the various types of marker resources, and predict the potential future direction of crop marker databases.

Identifying genetic diversity of avirulence genes in Leptosphaeria maculans using whole genome sequencing.

Next generation sequencing technology allows rapid re-sequencing of individuals, as well as the discovery of single nucleotide polymorphisms (SNPs), for genomic diversity and evolutionary analyses. By sequencing two isolates of the fungal plant pathogen Leptosphaeria maculans, the causal agent of blackleg disease in Brassica crops, we have generated a resource of over 76 million sequence reads aligned to the reference genome. We identified over 21,000 SNPs with an overall SNP frequency of one SNP every 2,065 bp. Sequence validation of a selection of these SNPs in additional isolates collected throughout Australia indicates a high degree of polymorphism in the Australian population. In preliminary phylogenetic analysis, isolates from Western Australia clustered together and those collected from Brassica juncea stubble were identical. These SNPs provide a novel marker resource to study the genetic diversity of this pathogen. We demonstrate that re-sequencing provides a method of validating previously characterised SNPs and analysing differences in important genes, such as the disease related avirulence genes of L. maculans. Understanding the genetic characteristics of this devastating pathogen is vital in developing long-term solutions to managing blackleg disease in Brassica crops.

Dispersion and domestication shaped the genome of bread wheat.

Despite the international significance of wheat, its large and complex genome hinders genome sequencing efforts. To assess the impact of selection on this genome, we have assembled genomic regions representing genes for chromosomes 7A, 7B and 7D. We demonstrate that the dispersion of wheat to new environments has shaped the modern wheat genome. Most genes are conserved between the three homoeologous chromosomes. We found differential gene loss that supports current theories on the evolution of wheat, with greater loss observed in the A and B genomes compared with the D. Analysis of intervarietal polymorphisms identified fewer polymorphisms in the D genome, supporting the hypothesis of early gene flow between the tetraploid and hexaploid. The enrichment for genes on the D genome that confer environmental adaptation may be associated with dispersion following wheat domestication. Our results demonstrate the value of applying next-generation sequencing technologies to assemble gene-rich regions of complex genomes and investigate polyploid genome evolution. We anticipate the genome-wide application of this reduced-complexity syntenic assembly approach will accelerate crop improvement efforts not only in wheat, but also in other polyploid crops of significance.

Single nucleotide polymorphism discovery from wheat next-generation sequence data.

Single nucleotide polymorphisms (SNPs) are the most abundant type of molecular genetic marker and can be used for producing high-resolution genetic maps, marker-trait association studies and marker-assisted breeding. Large polyploid genomes such as wheat present a challenge for SNP discovery because of the potential presence of multiple homoeologs for each gene. AutoSNPdb has been successfully applied to identify SNPs from Sanger sequence data for several species, including barley, rice and Brassica, but the volume of data required to accurately call SNPs in the complex genome of wheat has prevented its application to this important crop. DNA sequencing technology has been revolutionized by the introduction of next-generation sequencing, and it is now possible to generate several million sequence reads in a timely and cost-effective manner. We have produced wheat transcriptome sequence data using 454 sequencing technology and applied this for SNP discovery using a modified autoSNPdb method, which integrates SNP and gene annotation information with a graphical viewer. A total of 4,694,141 sequence reads from three bread wheat varieties were assembled to identify a total of 38 928 candidate SNPs. Each SNP is within an assembly complete with annotation, enabling the selection of polymorphism within genes of interest.

Next-generation sequencing applications for wheat crop improvement.

Bread wheat (Triticum aestivum; Poaceae) is a crop plant of great importance. It provides nearly 20% of the world's daily food supply measured by calorie intake, similar to that provided by rice. The yield of wheat has doubled over the last 40 years due to a combination of advanced agronomic practice and improved germplasm through selective breeding. More recently, yield growth has been less dramatic, and a significant improvement in wheat production will be required if demand from the growing human population is to be met. Next-generation sequencing (NGS) technologies are revolutionizing biology and can be applied to address critical issues in plant biology. Technologies can produce draft sequences of genomes with a significant reduction to the cost and timeframe of traditional technologies. In addition, NGS technologies can be used to assess gene structure and expression, and importantly, to identify heritable genome variation underlying important agronomic traits. This review provides an overview of the wheat genome and NGS technologies, details some of the problems in applying NGS technology to wheat, and describes how NGS technologies are starting to impact wheat crop improvement.