Heterochromatin repeat organization at an individual level: Rex1BD and the 14-3-3 protein coordinate to shape the epigenetic landscape within heterochromatin repeats.

In eukaryotic cells, heterochromatin is typically composed of tandem DNA repeats and plays crucial roles in gene expression and genome stability. It has been reported that silencing at individual units within tandem heterochromatin repeats exhibits a position-dependent variation. However, how the heterochromatin is organized at an individual repeat level remains poorly understood. Using a novel genetic approach, our recent study identified a conserved protein Rex1BD required for position-dependent silencing within heterochromatin repeats. We further revealed that Rex1BD interacts with the 14-3-3 protein to regulate heterochromatin silencing by linking RNAi and HDAC pathways. In this review, we discuss how Rex1BD and the 14-3-3 protein coordinate to modulate heterochromatin organization at the individual repeat level, and comment on the biological significance of the position-dependent effect in heterochromatin repeats. We also identify the knowledge gaps that still need to be unveiled in the field.

Rex1BD and the 14-3-3 protein control heterochromatin organization at tandem repeats by linking RNAi and HDAC.

Tandem DNA repeats are often organized into heterochromatin that is crucial for genome organization and stability. Recent studies revealed that individual repeats within tandem DNA repeats can behave very differently. How DNA repeats are assembled into distinct heterochromatin structures remains poorly understood. Here, we developed a genome-wide genetic screen using a reporter gene at different units in a repeat array. This screen led to identification of a conserved protein Rex1BD required for heterochromatin silencing. Our structural analysis revealed that Rex1BD forms a four-helix bundle structure with a distinct charged electrostatic surface. Mechanistically, Rex1BD facilitates the recruitment of Clr6 histone deacetylase (HDAC) by interacting with histones. Interestingly, Rex1BD also interacts with the 14-3-3 protein Rad25, which is responsible for recruiting the RITS (RNA-induced transcriptional silencing) complex to DNA repeats. Our results suggest that coordinated action of Rex1BD and Rad25 mediates formation of distinct heterochromatin structure at DNA repeats via linking RNAi and HDAC pathways.

Exportation of Monkeypox Virus From the African Continent.

BACKGROUND: The largest West African monkeypox outbreak began September 2017, in Nigeria. Four individuals traveling from Nigeria to the United Kingdom (n = 2), Israel (n = 1), and Singapore (n = 1) became the first human monkeypox cases exported from Africa, and a related nosocomial transmission event in the United Kingdom became the first confirmed human-to-human monkeypox transmission event outside of Africa. METHODS: Epidemiological and molecular data for exported and Nigerian cases were analyzed jointly to better understand the exportations in the temporal and geographic context of the outbreak. RESULTS: Isolates from all travelers and a Bayelsa case shared a most recent common ancestor and traveled to Bayelsa, Delta, or Rivers states. Genetic variation for this cluster was lower than would be expected from a random sampling of genomes from this outbreak, but data did not support direct links between travelers. CONCLUSIONS: Monophyly of exportation cases and the Bayelsa sample, along with the intermediate levels of genetic variation, suggest a small pool of related isolates is the likely source for the exported infections. This may be the result of the level of genetic variation present in monkeypox isolates circulating within the contiguous region of Bayelsa, Delta, and Rivers states, or another more restricted, yet unidentified source pool.

The Role of Clt1-Regulated Xylan Metabolism in Melanin and Toxin Formation for the Pathogenicity of Curvularia lunata in Maize.

We previously reported that the BTB (brica-brac, tramtrack, and broad) domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata, that were regulated by Clt1. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan, which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production-deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Isolation and Characterization of Akhmeta Virus from Wild-Caught Rodents (Apodemus spp.) in Georgia.

In 2013, a novel orthopoxvirus was detected in skin lesions of two cattle herders from the Kakheti region of Georgia (country); this virus was named Akhmeta virus. Subsequent investigation of these cases revealed that small mammals in the area had serological evidence of orthopoxvirus infections, suggesting their involvement in the maintenance of these viruses in nature. In October 2015, we began a longitudinal study assessing the natural history of orthopoxviruses in Georgia. As part of this effort, we trapped small mammals near Akhmeta (n = 176) and Gudauri (n = 110). Here, we describe the isolation and molecular characterization of Akhmeta virus from lesion material and pooled heart and lung samples collected from five wood mice (Apodemus uralensis and Apodemus flavicollis) in these two locations. The genomes of Akhmeta virus obtained from rodents group into 2 clades: one clade represented by viruses isolated from A. uralensis samples, and one clade represented by viruses isolated from A. flavicollis samples. These genomes also display several presumptive recombination events for which gene truncation and identity have been examined.IMPORTANCE Akhmeta virus is a unique Orthopoxvirus that was described in 2013 from the country of Georgia. This paper presents the first isolation of this virus from small mammal (Rodentia; Apodemus spp.) samples and the molecular characterization of those isolates. The identification of the virus in small mammals is an essential component to understanding the natural history of this virus and its transmission to human populations and could guide public health interventions in Georgia. Akhmeta virus genomes harbor evidence suggestive of recombination with a variety of other orthopoxviruses; this has implications for the evolution of orthopoxviruses, their ability to infect mammalian hosts, and their ability to adapt to novel host species.

Mechanistic studies on the N-heterocyclic carbene-catalyzed reaction of isatin-derived enals with hydrazones.

The possible reaction mechanism and origin of stereoselectivity of the NHC-catalyzed annulation reaction between isatin-derived enals and hydrazones were theoretically studied by using density functional theory (DFT). According to the computational results, the Michael addition process was identified to be the stereoselectivity determining step and led to the experimentally observed S-configured product predominantly. The distortion-interaction analysis showed that the electrostatic interaction between two interactive fragments controls the stereoselectivity. Moreover, the types of interactions were further verified by non-covalent interaction analysis, in which the ππ, C-HF and LPπ interactions involved in the favorable transition state are the key for determining the stereoselectivity.

Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

Novel Orthopoxvirus Infection in an Alaska Resident.

BACKGROUND.: Human infection by orthopoxviruses is being reported with increasing frequency, attributed in part to the cessation of smallpox vaccination and concomitant waning of population-level immunity. In July 2015, a female resident of interior Alaska presented to an urgent care clinic with a dermal lesion consistent with poxvirus infection. Laboratory testing of a virus isolated from the lesion confirmed infection by an Orthopoxvirus. METHODS.: The virus isolate was characterized by using electron microscopy and nucleic acid sequencing. An epidemiologic investigation that included patient interviews, contact tracing, and serum testing, as well as environmental and small-mammal sampling, was conducted to identify the infection source and possible additional cases. RESULTS.: Neither signs of active infection nor evidence of recent prior infection were observed in any of the 4 patient contacts identified. The patient's infection source was not definitively identified. Potential routes of exposure included imported fomites from Azerbaijan via the patient's cohabiting partner or wild small mammals in or around the patient's residence. Phylogenetic analyses demonstrated that the virus represents a distinct and previously undescribed genetic lineage of Orthopoxvirus, which is most closely related to the Old World orthopoxviruses. CONCLUSIONS.: Investigation findings point to infection of the patient after exposure in or near Fairbanks. This conclusion raises questions about the geographic origins (Old World vs North American) of the genus Orthopoxvirus. Clinicians should remain vigilant for signs of poxvirus infection and alert public health officials when cases are suspected.

Detection and Molecular Characterization of Zoonotic Poxviruses Circulating in the Amazon Region of Colombia, 2014.

During 2014, cutaneous lesions were reported in dairy cattle and farmworkers in the Amazon Region of western Colombia. Samples from 6 patients were analyzed by serologic and PCR testing, and results demonstrated the presence of vaccinia virus and pseudocowpox virus. These findings highlight the need for increased poxvirus surveillance in Colombia.

Two novel poxviruses with unusual genome rearrangements: NY_014 and Murmansk.

The genome sequence and annotation of two novel poxviruses, NY_014 and Murmansk, are presented. Despite being isolated on different continents and from different hosts, the viruses are relatively similar, albeit distinct species. The closest known relative of the novel viruses is Yoka poxvirus. Five novel genes were found in these genomes, two of which were MHC class I homologs. Although the core of these genomes was well conserved, the terminal regions showed significant variability with large deletions and surprising evidence of recombination with orthopoxviruses.

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat.

The genome of Eptesipoxvirus (EPTV) is the first poxvirus genome isolated from a microbat. The 176,688 nt sequence, which is believed to encompass the complete coding region of the virus, is 67% A+T and is predicted to encode 191 genes. 11 of these genes have no counterpart in GenBank and are therefore unique to EPTV. The presence of a distantly related ortholog of Vaccinia virus F5L in EPTV uncovered a link with fragmented F5L orthologs in Molluscum contagiosum virus/squirrelpox and clade II viruses. Consistent with the unique position of EPTV approximately mid-point between the orthopoxviruses and the clade II viruses, EPTV has 11 genes that are specific to the orthopoxviruses and 13 genes that are typical, if not exclusive, to the clade II poxviruses. This mosaic nature of EPTV blurs the distinction between the old description of the orthopoxvirus and clade II groups. Genome annotation and characterization failed to find any common virulence genes shared with the other poxvirus isolated from bat (pteropoxvirus); however, EPTV encodes 3 genes that may have been transferred to or from deerpox and squirrelpox viruses; 2 of these, a putative endothelin-like protein and a MHC class I-like protein are likely to have immunomodulatory roles.

Characterization of the Key Aroma Compounds in Chinese Syrah Wine by Gas Chromatography-Olfactometry-Mass Spectrometry and Aroma Reconstitution Studies.

The key aroma compounds and the organoleptic quality of two Chinese Syrah wines from the Yunnan Shangri-La region and Ningxia Helan mountain region were characterized. The most important eighty aroma-active compounds were identified by Gas Chromatography-Olfactometry. In both Syrah samples, ethyl 2-methylpropanoate, ethyl 3-methylbutanoate, 3-methylbutyl acetate, 2- and 3-methyl-1-butanol, ethyl hexanoate, ethyl octanoate, 2-phenethyl acetate, methional, 3-methylbutanoic acid, hexanoic acid, octanoic acid, ß-damascenone, guaiacol, 2-phenylethanol, trans-whiskylactone, 4-ethylguaiacol, eugenol, 4-ethylphenol, and sotolon were detected to have the highest odor intensities. In the chemical analysis, 72 compounds were quantitated by Stir Bar Sorptive Extraction combined with Gas Chromatography Mass Spectrometry. Based on the Odor Activity Value (OAV), the aromas were reconstituted by combining aroma compounds in the synthetic wine, and sensory descriptive analysis was used to verify the chemical data. Fatty acid ethyl esters, acetate esters, and ß-damascenone were found with higher OAVs in the more fruity-smelling sample of Helan Mountain rather than Shangri-La.

The platelet-activating factor acetylhydrolase gene derived from Trichoderma harzianum induces maize resistance to Curvularia lunata through the jasmonic acid signaling pathway.

Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen.

Genome sequence and virulence variation-related transcriptome profiles of Curvularia lunata, an important maize pathogenic fungus.

BACKGROUND: Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China. Genome sequencing of the pathogen will provide important information for globally understanding its virulence mechanism. RESULTS: We report the genome sequences of a highly virulent C. lunata strain. Phylogenomic analysis indicates that C. lunata was evolved from Bipolaris maydis (Cochliobolus heterostrophus). The highly virulent strain has a high potential to evolve into other pathogenic stains based on analyses on transposases and repeat-induced point mutations. C. lunata has a smaller proportion of secreted proteins as well as B. maydis than entomopathogenic fungi. C. lunata and B. maydis have a similar proportion of protein-encoding genes highly homologous to experimentally proven pathogenic genes from pathogen-host interaction database. However, relative to B. maydis, C. lunata possesses not only many expanded protein families including MFS transporters, G-protein coupled receptors, protein kinases and proteases for transport, signal transduction or degradation, but also many contracted families including cytochrome P450, lipases, glycoside hydrolases and polyketide synthases for detoxification, hydrolysis or secondary metabolites biosynthesis, which are expected to be crucial for the fungal survival in varied stress environments. Comparative transcriptome analysis between a lowly virulent C. lunata strain and its virulence-increased variant induced by resistant host selection reveals that the virulence increase of the pathogen is related to pathways of toxin and melanin biosynthesis in stress environments, and that the two pathways probably have some overlaps. CONCLUSIONS: The data will facilitate a full revelation of pathogenic mechanism and a better understanding of virulence differentiation of C. lunata.

Insertional mutagenesis and cloning of the gene required for the biosynthesis of the non-host-specific toxin in Cochliobolus lunatus that causes maize leaf spot.

The maize pathotype Cochliobolus lunatus causes Curvularia leaf spot by producing a non-host-specific toxin known as methyl 5-(hydroxymethyl) furan-2-carboxylate (M5HF2C). However, related research that explores the genes that control the production of this toxin is rare. In the current work, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed to tag the gene required for the biosynthesis of the toxin. Of the 3,000 ATMT transformants recovered, 4 showed a significant decline in pathogenicity on maize leaves; 1 transformant, T806, produced no detectable M5HF2C. Genomic DNA that flanks the integrated plasmid was recovered from one of the mutants. A cosmid clone of the wild-type strain was isolated using the recovered DNA as a probe. The results of the structural and functional analyses of the region corresponding to the tagged site were then used as a basis to successfully clone one gene, called Clt-1. Bioinformatics analysis showed that the gene coded a BTB domain-containing protein that comprises 745 amino acids. Southern analysis revealed that the gene was localized in the genome as a single copy. The essential roles of Clt-1 in both toxin production and pathogenicity were confirmed by gene disruption experiments. In summary, the novel gene Clt-1 is closely associated with toxin production and pathogen virulence in leaves of susceptible varieties.

Development of bioreactor scale-down model using orthogonal projections to latent structures method and CO2 supplementation.

Scale-down model qualification is an important step for developing a large-scale cell culture process to enhance process understanding and support process characterization studies. Traditionally, only harvest data are used to show consistency between small-scale and large-scale bioreactor performance, allowing attributes that are dynamic over the cell culture period to be overlooked. A novel statistical method, orthogonal projections to latent structures (OPLS) analysis, can be utilized to compare time-course cell culture data across scales. Here we describe an example where OPLS is used to identify gaps between small-scale and large-scale bioreactor performances. In this case, differences in the partial pressure of carbon dioxide (pCO2) and lactate profiles were observed between small- and large-scale bioreactors, which were linked to differences in the product-quality attributes fragments and galactosylation. An improved small-scale model was developed, leading to improved consistency in the process performance and product qualities across scales and qualification of the scale-down model for regulatory submissions. This new statistical approach can provide valuable insights into process understanding and process scale-up.

The SUN-family protein Sad1 mediates heterochromatin spatial organization through interaction with histone H2A-H2B.

Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1HBM and H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1's functionality.

Bubble-Guidance Breaking Gas Shield for Highly Efficient Overall Water Splitting.

Overall water splitting is a promising technology for sustainable hydrogen production, but the primary challenge is removing bubbles from the electrode surface quickly to increase hydrogen production. Inspired by the directional fluid transport properties of natural biological surfaces like Nepenthes peristome and Morpho butterfly's wings, here a strategy is demonstrated to achieve highly efficient overall water splitting by a bubble-guidance electrode, that is, an anisotropic groove-micro/nanostructured porous electrode (GMPE). Gradient groove micro/nanostructures on the GMPE serve as high-speed bubble transmission channels and exhibit superior bubble-guidance capabilities. The synergistic effect of the asymmetric Laplace pressure generated between microscale porous structure and groove patterns and the buoyancy along the groove patterns pushes the produced bubbles directionally to spread, transport, and detach from the electrode surface in time. Moreover, the low adhesive nanosheet arrays are beneficial to reduce bubble size and increase bubble release frequency, which cooperatively improve mass transfer with the microscale structure. Notably, GMPE outperforms planar-micro/nanostructured porous electrode (PMPE) in hydrogen/oxygen evolution reactions, with GMPE||GMPE showing better water splitting performance than commercially available RuO2||20 wt.% Pt/C. This work improves electrodes for better mass transfer and kinetics in electrochemical reactions at solid-liquid-gas interfaces, offering insight for designing and preparing gas-involved photoelectrochemical electrodes.

Recent insights into mechanisms preventing ectopic centromere formation.

The centromere is a specialized chromosomal structure essential for chromosome segregation. Centromere dysfunction leads to chromosome segregation errors and genome instability. In most eukaryotes, centromere identity is specified epigenetically by CENP-A, a centromere-specific histone H3 variant. CENP-A replaces histone H3 in centromeres, and nucleates the assembly of the kinetochore complex. Mislocalization of CENP-A to non-centromeric regions causes ectopic assembly of CENP-A chromatin, which has a devastating impact on chromosome segregation and has been linked to a variety of human cancers. How non-centromeric regions are protected from CENP-A misincorporation in normal cells is largely unexplored. Here, we review the most recent advances on the mechanisms underlying the prevention of ectopic centromere formation, and discuss the implications in human disease.

Trichoderma biodiversity in major ecological systems of China.

An investigation of Trichoderma biodiversity involving a large-scale environmental gradient was conducted to understand the Trichoderma distribution in China. A total of 3,999 isolates were isolated from forestry, grassland, wetland and agriculture ecosystems, and 50 species were identified based on morphological characteristics and sequence analysis of genetic markers. Trichoderma harzianum showed the largest proportion of isolates and the most extensive distribution. Hypocrea semiorbis, T. epimyces, T. konilangbra, T. piluliferum, T. pleurotum, T. pubescens, T. strictipilis, T. hunua, T. oblongisporum and an unidentified species, Trichoderma sp. MA 3642, were first reported in China. Most Trichoderma species were distributed in Jilin and Heilongjiang Provinces in northeast China and the fewest were distributed in Qinghai Province. Based on the division of ecological and geographic factors, forestry ecosystems and low-altitude regions have the greatest species biodiversity of Trichoderma.