Molecular systematics of citrus-associated Alternaria species

The causal agents of Alternaria brown spot of tangerines and tangerine hybrids, Alternaria leaf spot of rough lemon and Alternaria black rot of citrus historically have been referred to as Alternaria citri or A. alternata. Ten species of Alternaria recently were described among a set of isolates from leaf lesions on rough lemon (Citrus jambhiri) and tangelo (C. paradisi × C. reticulata), and none of these isolates was considered representative of A. alternata or A. citri. To test the hypothesis that these newly described morphological species are congruent with phylogenetic species, selected Alternaria brown spot and leaf spot isolates, citrus black rot isolates (post-harvest pathogens), isolates associated with healthy citrus tissue and reference species of Alternaria from noncitrus hosts were scored for sequence variation at five genomic regions and used to estimate phylogenies. These data included 432 bp from the 5' end of the mitochondrial ribosomal large subunit (mtLSU), 365 bp from the 5' end of the beta-tubulin gene, 464 bp of an endopolygalacturonase gene (endoPG) and 559 and 571 bp, respectively, of two anonymous genomic regions (OPA1-3 and OPA2-1). The mtLSU and beta-tubulin phylogenies clearly differentiated A. limicola, a large-spored species causing leaf spot of Mexican lime, from the small-spored isolates associated with citrus but were insufficiently variable to resolve evolutionary relationships among the small-spored isolates from citrus and other hosts. Sequence analysis of translation elongation factor alpha, calmodulin, actin, chitin synthase and 1, 3, 8-trihydroxynaphthalene reductase genes similarly failed to uncover significant variation among the small-spored isolates. Phylogenies estimated independently from endoPG, OPA1-3 and OPA2-1 data were congruent, and analysis of the combined data from these regions revealed nine clades, eight of which contained small-spored, citrus-associated isolates. Lineages inferred from analysis of the combined dataset were in general agreement with described morphospecies, however, three clades contained more than one morphological species and one morphospecies (A. citrimacularis) was polyphyletic. Citrus black rot isolates also were found to be members of more than a single lineage. The number of morphospecies associated with citrus exceeded that which could be supported under a phylogenetic species concept, and isolates in only five of nine phylogenetic lineages consistently were correlated with a specific host, disease or ecological niche on citrus. We advocate collapsing all small-spored, citrus-associated isolates of Alternaria into a single phylogenetic species, A. alternata.     

Soil Properties and their Spatial Pattern in a Degraded Sandy Grassland under Post-grazing Restoration, Inner Mongolia, Northern China

In this study, we use classical and geostatistical methods to identify characteristics of some selected soil properties including soil particle size distribution, soil organic carbon, total nitrogen, pH and electrical conductivity and their spatial variation in a 5-year recovery degraded sandy grassland after two different grazing intensity disturbance: post-heavy-grazing restoration grassland (HGR) and post-moderately grazing restoration grassland (MGR), respectively, in Horqin steppe, Inner Mongolia, northern China. The objective was to examine effect of grazing intensity on spatial heterogeneity of soil properties. One hundred soil samples were taken from the soil layer 0–15 cm in depth of a grid of 10 m×10 m under each treatment. The results showed that soil fine fractions (very fine sand, 0.1–0.05 mm and silt + clay, <0.05 mm), soil organic carbon and total nitrogen concentrations were significant lower and their coefficients of variation significant higher under the HGR than under the MGR. Geostatistical analysis of soil heterogeneity revealed that soil particle size fractions, organic carbon and total nitrogen showed different degree of spatial dependence with exponential or spherical semivariograms on the scale measured under HGR and MGR. The spatial structured variance account for a large proportion of the sample variance in HGR plot ranging from 88% to 97% for soil particle fractions, organic C and total N, however, except for organic C (88.8%), the structured variance only account for 50% of the sample variance for soil particle fractions and total N in the MGR plot. The ranges of spatial autocorrelation for coarse-fine sand, very fine sand, silt + clay, organic C and total N were 13.7 m, 15.8 m, 15.2 m, 22.2 m and 21.9 m in HGR plot, respectively, and was smaller than in MGR plot with the corresponding distance of 350 m, 144.6 m, 45.7 m, 27.3 m and 30.3 m, respectively. This suggested that overgrazing resulted in an increase in soil heterogeneity. Soil organic C and total N were associated closely with soil particle fractions, and the kriging-interpolated maps showed that the spatial distribution of soil organic C and total N corresponded to the distribution patterns of soil particle fractions, indicating that high degree of spatial heterogeneity in soil properties was linked to the distribution of vegetative and bare sand patches. The results suggested that the degree of soil heterogeneity at field scale can be used as an index for indicating the extent of grassland desertification. Also, the changes in soil heterogeneity may in turn influence vegetative succession and restoration process of degraded sandy grassland ecosystem.     

PTEN regulates RPA1 and protects DNA replication forks

Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.     

拟南芥根毛衰老死亡过程的PCD检测

根毛是植物体吸收养分的重要器官, 自然条件下根毛的寿命很短, 仅能存活2–3周, 随即脱落死亡。以模式植物拟南芥(Arabidopsis thaliana)根毛为材料, 对根毛死亡的细胞学特征进行了报道。结果发现, 根毛衰老死亡后细胞内的原生质体发生了收缩, 并在胞质中观察到凝集物的出现; 通过原位末端标记(TUNEL)检测, 发现幼根上的根毛细胞核DNA发生了片段化。上述结果表明, 拟南芥根毛的衰老死亡很可能是植物体自主调控的程序性细胞死亡(PCD)。另外, 当根毛衰老死亡后,细胞核大多会迁移到靠近根毛基部的位置, 且正常的长管状根毛发生旋转扭曲。     

Characterization of eight polymorphic microsatellite loci for the giant grouper (Epinephelus lanceolatus Bloch)

Eight polymorphic microsatellite loci were isolated and characterized using a small insert genomic DNA library for the giant grouper (Epinephelus lanceolatus Bloch, 1790), a commercially valuable marine fish in tropical waters. They showed polymorphism information content ranging from 0.177 to 0.775, allele numbers ranging from two to 10, effective allele numbers ranging from 1.227 to 5.012, and observed and expected heterozygosities from 0.2 to 0.733 and from 0.185 to 0.801, respectively, which we anticipate will be useful for population genetic studies of the giant grouper.     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Human Organoids as a Promising Platform for Fighting COVID-19

The coronavirus disease 2019 (COVID-19) global pandemic evoked by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a major public health problem with significant morbidity and mortality. Understanding the pathogenesis and molecular mechanisms underlying this novel virus is crucial for both fundamental research and clinical trials in order to devise effective therapies and vaccination regimens. Basic research on SARS-CoV-2 largely depends on ex vivo models that allow viral invasion and replication. Organoid models are now emerging as a valuable tool to investigate viral biology and disease progression, serving as an efficient platform to investigate potential therapies for COVID-19. Here, we summarize various human stem cell-derived organoid types employed in SARS-CoV-2 studies. We highlight key findings from these models, including cell tropisms and molecular mechanisms in viral infection. We also describe their use in identifying potential therapeutic agents against SARS-CoV-2. As more and more advanced organoids emerge, they will facilitate the understanding of disease pathogenesis for drug development in this dreaded pandemic.     

An obligate intermediate along the slow folding pathway of a group II intron ribozyme

Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135. We find that structural collapse and native folding of Domain 1 precede assembly of the entire ribozyme, indicating that D1 contains an on-pathway intermediate to folding of the D135 ribozyme. Subsequent docking of Domains 3 and 5, for which D1 provides a preorganized scaffold, appears to be very fast and independent of one another. In contrast to other RNAs, the D135 ribozyme undergoes slow tertiary collapse to a compacted state, with a rate constant that is also limited by the formation D1. These findings provide a new paradigm for RNA folding and they underscore the diversity of RNA biophysical behaviors.