Growth Phenotype Research Articles

The RNA helicase Dbp10 coordinates assembly factor association with PTC maturation during ribosome biogenesis.

During ribosome biogenesis a plethora of assembly factors and essential enzymes drive the unidirectional maturation of nascent pre-ribosomal subunits. The DEAD-box RNA helicase Dbp10 is suggested to restructure pre-ribosomal rRNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Here, we show that point mutations within conserved catalytic helicase-core motifs of Dbp10 yield a dominant-lethal growth phenotype. Such dbp10 mutants, which stably associate with pre-60S intermediates, impair pre-60S biogenesis at a nucleolar stage prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3. Furthermore, the binding of the GTPase Nug1 to particles isolated directly via mutant Dbp10 bait proteins is specifically inhibited. The N-terminal domain of Nug1 interacts with Dbp10 and the methyltransferase Spb1, whose pre-60S incorporation is also reduced in absence of functional Dbp10 resulting in decreased methylation of 25S rRNA nucleotide G2922. Our data suggest that Dbp10's helicase activity generates the necessary framework for assembly factor docking thereby permitting PTC rRNA methylation and the progression of pre-60S maturation.

Exogenous Serotonin (5-HT) Promotes Mesocotyl and Coleoptile Elongation in Maize Seedlings under Deep-Seeding Stress through Enhancing Auxin Accumulation and Inhibiting Lignin Formation

Serotonin (5-HT), an indoleamine compound, has been known to mediate many physiological responses of plants under environmental stress. The deep-seeding (≥20 cm) of maize seeds is an important cultivation strategy to ensure seedling emergence and survival under drought stress. However, the role of 5-HT in maize deep-seeding tolerance remains unexplored. Understanding the mechanisms and evaluating the optimal concentration of 5-HT in alleviating deep-seeding stress could benefit maize production. In this study, two maize inbred lines were treated with or without 5-HT at both sowing depths of 20 cm and 3 cm, respectively. The effects of different concentrations of 5-HT on the growth phenotypes, physiological metabolism, and gene expression of two maize inbred lines were examined at the sowing depths of 20 cm and 3 cm. Compared to the normal seedling depth of 3 cm, the elongation of the mesocotyl (average elongation 3.70 cm) and coleoptile (average elongation 0.58 cm), secretion of indole-3-acetic acid (IAA; average increased 3.73 and 0.63 ng g−1 FW), and hydrogen peroxide (H2O2; average increased 1.95 and 0.63 μM g−1 FW) in the mesocotyl and coleoptile were increased under 20 cm stress, with a concomitant decrease in lignin synthesis (average decreased 0.48 and 0.53 A280 g−1). Under 20 cm deep-seeding stress, the addition of 5-HT activated the expression of multiple genes of IAA biosynthesis and signal transduction, including Zm00001d049601, Zm00001d039346, Zm00001d026530, and Zm00001d049659, and it also stimulated IAA production in both the mesocotyl and coleoptile of maize seedlings. On the contrary, 5-HT suppressed the expression of genes for lignin biosynthesis (Zm00001d016471, Zm00001d005998, Zm00001d032152, and Zm00001d053554) and retarded the accumulation of H2O2 and lignin, resulting in the elongation of the mesocotyl and coleoptile of maize seedlings. A comprehensive evaluation analysis showed that the optimum concentration of 5-HT in relieving deep-seeding stress was 2.5 mg/L for both inbred lines, and 5-HT therefore could improve the seedling emergence rate and alleviate deep-seeding stress in maize seedlings. These findings could provide a novel strategy for improving maize deep-seeding tolerance, thus enhancing yield potential under drought and water stress.

The divalent metal ion exporter IhpABC is required to maintain iron homeostasis under low to moderate environmental iron conditions in the bacterium Bradyrhizobium japonicum.

Bacterial iron export mitigates high iron stress, but a role for it under lower iron conditions has not been established. MbfA is the high iron stress exporter in Bradyrhizobium japonicum. Here, we identify the ihpABC genes in a selection for secondary site mutations that suppress the poor growth phenotype of feoAB mutants defective in iron acquisition. IhpABC belongs to the RND tripartite efflux pump family. High iron conditions that derepress the mbfA gene partially rescued the growth of an ihpC mutant but reverted the feoB ihpC mutant to the feoB growth phenotype. The ihpA mutant grown under low iron conditions accumulated higher levels of iron compared to the wild type, and it displayed aberrant iron-responsive gene expression. The mbfA mutant was more sensitive than the wild type to H2 O2 , but the ihpA mutant was not sensitive. The ihpA mutant accumulated more Zn, Co and Cd than was found in the wild type, and growth of the mutant was more sensitive to inhibition by ZnCl2 , CoCl2 and CdCl2 . The findings suggest that IhpABC is a divalent metal ion exporter that helps maintain iron homeostasis under low to moderate environmental iron levels. Thus, iron export is not limited to managing high iron stress.

Comparison of the arsenic protective effects of four nanomaterials on pakchoi in an alkaline soil

Accurately applying engineered nanoparticles (NPs) in farmland stress management is important for sustainable agriculture and food safety. We investigated the protective effects of four engineered NPs (SiO2, CeO2, ZnO, and S) on pakchoi under arsenic (As) stress using pot experiments. The results showed that CeO2, SiO2, and S NPs resulted in biomass reduction, while ZnO NPs (100 and 500 mg kg−1) significantly increased shoot height. Although 500 mg kg−1 S NPs rapidly dissolved to release SO42−, reducing soil pH and pore water As content and further reducing shoot As content by 21.6 %, the growth phenotype was inferior to that obtained with 100 mg kg−1 ZnO NPs, probably due to acid damage. The addition of 100 mg kg−1 ZnO NPs not only significantly reduced the total As content in pakchoi by 23.9 % compared to the As-alone treatment but also enhanced plant antioxidative activity by increasing superoxide dismutase (SOD) and peroxidase (POD) activities and decreasing malondialdehyde (MDA) content. ZnO NPs in soil might inhibit As uptake by roots by increasing the dissolved organic carbon (DOC) by 19.12 %. According to the DLVO theory, ZnO NPs were the most effective in preventing As in pore water from entering plant roots due to their smaller hydrated particle size. Redundancy analysis (RDA) further confirmed that DOC and SO42− were the primary factors controlling plant As uptake under the ZnO NP and S NP treatments, respectively. These findings provide an important basis for the safer and more sustainable application of NP-conjugated agrochemicals.

Genome-wide characterization of the xyloglucan endotransglucosylase/hydrolase family genes and their response to plant hormone in sugar beet

Xyloglucan endotransglucosylase/hydrolases (XTHs) play a crucial role in plant growth and development. However, their functional response to phytohormone in sugar beet still remains obscure. In this study, we identified 30 putative BvXTH genes in the sugar beet genome. Phylogenetic and evolutionary relationship analysis revealed that they were clustered into three groups and have gone through eight tandem duplication events under purifying selection. Gene structure and motif composition analysis demonstrated that they were highly conserved and all contained one conserved glycoside hydrolase family 16 domain (Glyco_hydro_16) and one xyloglucan endotransglycosylase C-terminus (XET_C) domain. Transcriptional expression analysis exhibited that all BvXTHs were ubiquitously expressed in leaves, root hairs and tuberous roots, and most of them were up-regulated by brassinolide (BR), jasmonic acid (JA), abscisic acid (ABA) and gibberellic acid (GA3). Further mutant complementary experiment demonstrated that expression of BvXTH17 rescued the retarded growth phenotype of xth22, an Arabidopsis knock out mutant of AtXTH22. The findings in our work provide fundamental information on the structure and evolutionary relationship of the XTH family genes in sugar beet, and reveal the potential function of BvXTH17 in plant growth and hormone response.

Scales Bridging in the Model of Growth of Animals, a Holistic Slant

Scales bridging problem has been studied in mathematics and the natural sciences for decades. In this study we concentrate on a model of growth of animals. The model of growth of pigs was built in two different techniques continuum, and non-local. The both techniques model the same process and use the same variables. However, the outcomes that one can acquire from them are different. In this study, in this model, the problem how to bridge scales between genotype, and phenotypes of ontogenetic growth remain intact; it has not been considered. We narrowed the gap instead and discussed scales bridging problem between biochemistry of digestion, and the growth of animals. This range of scales the model potentially can bridge. In this study we discuss what does it mean bridge a scale. How to identify the scale to bridge. What means one has, to perform it. In this research non-local modelling technique was used. It was shown that this technique has sufficient power to make known emergent events in animals’ growth. The use of the non-local modelling technique to bridge the scale was discussed.    

CRISPR-Based Functional Transcriptomics Defines the Tumor-Dependency and Molecular Determinants of Long Noncoding RNAs in Multiple Myeloma

Long noncoding RNAs (lncRNAs) constitute most elements in the human transcriptome, yet we lack a comprehensive understanding of their role in tumor cells. Here, in multiple myeloma (MM), we exploited the RNA-targeting activity of the CRISPR-Cas13d endonuclease to develop a functional transcriptomics screening platform that enabled not only the transcriptome-wide identification of tumor-promoting-lncRNAs (tp-lncRNAs) in MM cells - outperforming any prior attempt - but also provided mechanistic information about their sub-cellular site-of-function, the isoform-conferring activity, and their downstream molecular pathways. Using RNA-seq data from CD138+ plasma cells from 319 clinically annotated, homogeneously treated, newly diagnosed MM patients, we first identified 5,304 lncRNAs that were expressed in ≥10% of patients, and then designed and generated a pooled lentiviral library with 9 sgRNAs per identified lncRNA, ~500 non-targeting sgRNAs as negative controls, and 9 sgRNAs targeting known protein-coding oncogenes in MM, such as IRF4, as positive controls. We infected this library at low MOI (<0.3) in five MM cell lines (AMO1, H929, KMS11, OPM2, R8226), each expressing a Cas13d-GFP fusion protein. After 3 weeks, we tested for sgRNAs that were relatively depleted or enriched in the MM cell population using deep sequencing and the MAGeCK robust rank aggregation algorithm. From this, we identified ~600 tp-lncRNAs upon which MM cell lines exhibit significant growth dependency, making up ~12% of the identified lncRNA transcriptome, significantly larger than the previous estimate of 3-5%. The tp-lncRNAs overlapped across MM cell lines: 17% were observed in all cell lines tested, 26% in four cell lines, 34% in at least three cell lines, and 53% in at least two cell lines. The dependency score of many tp-lncRNAs was on a par or even superior to the dependency score of the positive control IRF4 and, as expected, the top-scoring tp-lncRNAs included RROL, MALAT1, and NEAT1. We used a small library of 3,500 gRNAs to validate the dependency on 314 tp-lncRNAs and further validated the dependency on 12 tp-lncRNAs by individually targeting them using antisense oligonucleotides or CRISPR-Cas13d. These studies were followed by downstream transcriptomic analyses using either qRT-PCR or RNA-seq, which validated the growth phenotypes and informed on function. Interestingly, when comparing CRISPR-Cas13d to the current state-of-the-art CRISPR interference screens, we found that the CRISPR-Cas13d platform was superior, with a 4- to 30-fold increase in identifying tp-lncRNAs among 913 lncRNAs of intergenic origin in the same cell lines, and with the additional ability to identify tp-lncRNAs generated at gene loci that are antisense to protein-coding genes. We further investigated the sub-cellular localization and site-of-function of the tp-lncRNAs by using a Cas13d that accumulates either in the cytosol or in the nucleus as well as sub-cellular RNA-seq and single molecule RNA FISH. This way, while confirming that most tp-lncRNAs were functional and enriched in the cell nuclei, we also identified 20 hits providing dependency in the cytosol, including the small nucleolar RNA host genes 6 and 10. We observed that the lncRNA transcriptome investigated in this study contained several transcript variants produced from the same lncRNA genes, which differ in their sequences and likely in their structures and functions. Importantly, our screening platform was able to precisely identify tp-lncRNAs isoform(s) conferring dependencies even among many isoforms, a result that was corroborated by RNA-seq and/or qRT-PCR analysis after individually targeting the tp-lncRNA isoforms with Cas13d. Finally, from our screens, we identified and validated the MM dependency on the novel lncRNA Plasma Cell Specific RNA 1 ( PLASP-1), which is one of the most abundant lncRNAs in MM and normal plasma cells (median TPM>25), whereas it has significantly lower expression or is absent in 54 normal tissues and cell types. PLASP-1 is an un-spliced transcript that directly interacts with spliceosome proteins, as established in living cells. We are currently investigating its functional impact on the splicing machinery and the results will be presented. Overall, this novel platform comprehensively defines tumor dependency on lncRNAs in MM and provides information to guide further mechanistic investigation.

STAT5B germline heterozygous variant presenting with lymphadenopathy, atopy, and short stature

Background: Signal Transducers and Activators of Transcription (STAT) proteins are fundamental for multiple cellular processes, including immunity. STAT5B serves as a signal transducer downstream of cytokine and growth factor-mediated activation. Aberrations in STAT5B are associated with the development of atopy, immunodeficiencies, autoimmunity, cancers, hematological disease, growth disorders, and lung disease. Biallelic STAT5B variants are associated with loss-of-function of the gene, and cause a recessive disorder characterized by growth hormone insensitivity and immune dysregulation. Clinically significant heterozygous variants result in either dominant negative or gain-of-function effects; these latter variants are rare and their impact on the immune system is less clearly delineated than those responsible for the recessive form of the condition. Aim: We describe the presentation, immune workup, and genetic findings in a pediatric patient with a novel STAT5B heterozygous variant. Methods: A thorough retrospective review of the patient’s chart was performed. Results: A four-year-old male presented with history of lymphadenopathy, eczema, asthma, food allergy, short stature, and recurrent upper and lower respiratory tract infections. Clinical trio whole exome sequencing identified a novel heterozygous variant (c.1A>G; p. Met1?) in STAT5B. His mother harbours the same heterozygous variant and has a history remarkable for atopy. Immune investigations on the child revealed persistent elevated IgE. The patient’s variant targets the STAT5B N-terminal domain, and represents the first case with a heterozygous variant affecting this region. Conclusion: We present a novel STAT5B variant associated with a dominantly-inherited growth and immune phenotype. This is the first report of a heterozygous variant affecting the N-terminal domain in association with a clinical phenotype, expanding the genotypic landscape of this disorder. Further investigations and follow up responses to growth hormone replacement are needed to better delineate the functional effect of the variant identified in this family. Statement of Novelty: We report a novel germline heterozygous STAT5B variant in a paediatric patient with lymphadenopathy, atopy, and short stature.

A dual-subcellular localized β-glucosidase confers pathogen and insect resistance without a yield penalty in maize.

Maize is one of the most important crops for food, cattle feed and energy production. However, maize is frequently attacked by various pathogens and pests, which pose a significant threat to maize yield and quality. Identification of quantitative trait loci and genes for resistance to pests will provide the basis for resistance breeding in maize. Here, a β-glucosidase ZmBGLU17 was identified as a resistance gene against Pythium aphanidermatum, one of the causal agents of corn stalk rot, by genome-wide association analysis. Genetic analysis showed that both structural variations at the promoter and a single nucleotide polymorphism at the fifth intron distinguish the two ZmBGLU17 alleles. The causative polymorphism near the GT-AG splice site activates cryptic alternative splicing and intron retention of ZmBGLU17 mRNA, leading to the downregulation of functional ZmBGLU17 transcripts. ZmBGLU17 localizes in both the extracellular matrix and vacuole and contribute to the accumulation of two defence metabolites lignin and DIMBOA. Silencing of ZmBGLU17 reduces maize resistance against P.aphanidermatum, while overexpression significantly enhances resistance of maize against both the oomycete pathogen P. aphanidermatum and the Asian corn borer Ostrinia furnacalis. Notably, ZmBGLU17 overexpression lines exhibited normal growth and yield phenotype in the field. Taken together, our findings reveal that the apoplastic and vacuolar localized ZmBGLU17 confers resistance to both pathogens and insect pests in maize without a yield penalty, by fine-tuning the accumulation of lignin and DIMBOA.

Sulfinamide Formation from the Reaction of Bacillithiol and Nitroxyl.

Bacillithiol (BSH) replaces glutathione (GSH) as the most prominent low-molecular-weight thiol in many low G + C gram-positive bacteria. BSH plays roles in metal binding, protein/enzyme regulation, detoxification, redox buffering, and bacterial virulence. Given the small amounts of BSH isolated from natural sources and relatively lengthy chemical syntheses, the reactions of BSH with pertinent reactive oxygen, nitrogen, and sulfur species remain largely unexplored. We prepared BSH and exposed it to nitroxyl (HNO), a reactive nitrogen species that influences bacterial sulfur metabolism. The profile of this reaction was distinct from HNO oxidation of GSH, which yielded mixtures of disulfide and sulfinamide. The reaction of BSH and HNO (generated from Angeli's salt) gives only sulfinamide products, including a newly proposed cyclic sulfinamide. Treatment of a glucosamine-cysteine conjugate, which lacks the malic acid group, with HNO forms disulfide, implicating the malic acid group in sulfinamide formation. This finding supports a mechanism involving the formation of an N-hydroxysulfenamide intermediate that dehydrates to a sulfenium ion that can be trapped by water or internally trapped by an amide nitrogen to give the cyclic sulfinamide. The biological relevance of BSH reactivity toward HNO is provided through in vivo experiments demonstrating that Bacillus subtilis exposed to HNO shows a growth phenotype, and a strain unable to produce BSH shows hypersensitivity toward HNO in minimal medium cultures. Thiol analysis of HNO-exposed cultures shows an overall decrease in reduced BSH levels, which is not accompanied by increased levels of BSSB, supporting a model involving the formation of an oxidized sulfinamide derivative, identified in vivo by high-pressure liquid chromatography/mass spectrometry. Collectively, these findings reveal the unique chemistry and biology of HNO with BSH in bacteria that produce this biothiol.

Detecting radio- and chemoresistant cells in 3D cancer co-cultures using chromatin biomarkers

The heterogenous treatment response of tumor cells limits the effectiveness of cancer therapy. While this heterogeneity has been linked to cell-to-cell variability within the complex tumor microenvironment, a quantitative biomarker that identifies and characterizes treatment-resistant cell populations is still missing. Herein, we use chromatin organization as a cost-efficient readout of the cells’ states to identify subpopulations that exhibit distinct responses to radiotherapy. To this end, we developed a 3D co-culture model of cancer spheroids and patient-derived fibroblasts treated with radiotherapy. Using the model we identified treatment-resistant cells that bypassed DNA damage checkpoints and exhibited an aggressive growth phenotype. Importantly, these cells featured more condensed chromatin which primed them for treatment evasion, as inhibiting chromatin condensation and DNA damage repair mechanisms improved the efficacy of not only radio- but also chemotherapy. Collectively, our work shows the potential of using chromatin organization to cost-effectively study the heterogeneous treatment susceptibility of cells and guide therapeutic design.

Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas.

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter. Using fluorescent protein tagging, we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth, especially in high light, reduced ATP level, strong ascorbate accumulation, and diminished non-photochemical quenching in high light. On the other hand, total cellular phosphorous content was unaffected, and the phenotype of the Crpht4-7 mutants could not be alleviated by ample Pi supply. CrPHT4-7-overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters substantially recovered its slow growth phenotype, demonstrating that CrPHT4-7 transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any substantial ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in C. reinhardtii.

Integrated genomic and functional analyses of human skin-associated Staphylococcus reveal extensive inter- and intra-species diversity.

Human skin is stably colonized by a distinct microbiota that functions together with epidermal cells to maintain a protective physical barrier. Staphylococcus, a prominent genus of the skin microbiota, participates in colonization resistance, tissue repair, and host immune regulation in strain-specific manners. To unlock the potential of engineering skin microbial communities, we aim to characterize the diversity of this genus within the context of the skin environment. We reanalyzed an extant 16S rRNA amplicon dataset obtained from distinct body sites of healthy volunteers, providing a detailed biogeographic depiction of staphylococcal species that colonize our skin. S. epidermidis, S. capitis, and S. hominis were the most abundant staphylococcal species present in all volunteers and were detected at all body sites. Pan-genome analysis of isolates from these three species revealed that the genus-core was dominated by central metabolism genes. Species-restricted-core genes encoded known host colonization functions. The majority (~68%) of genes were detected only in a fraction of isolate genomes, underscoring the immense strain-specific gene diversity. Conspecific genomes grouped into phylogenetic clades, exhibiting body site preference. Each clade was enriched for distinct gene sets that are potentially involved in site tropism. Finally, we conducted gene expression studies of select isolates showing variable growth phenotypes in skin-like medium. In vitro expression revealed extensive intra- and inter-species gene expression variation, substantially expanding the functional diversification within each species. Our study provides an important resource for future ecological and translational studies to examine the role of shared and strain-specific staphylococcal genes within the skin environment.

BIOM-29. FIBRONECTIN TYPE III DOMAIN CONTAINING 3B AS PROGNOSTIC BIOMARKER AND REGULATORY TARGET FOR MALIGNANT BRAIN TUMOR

Abstract Malignant brain tumor (MBT) is an aggressive tumor with high drug-resistance and recurrence rates. Using ‘Oncopression’ big data analysis, we found that a fibronectin-related protein, fibronectin type III domain containing 3B (FNDC3B), was specifically overexpressed in brain tumor patients and conducted a pilot and mechanism study on the role of FNDC3B in MBT. We evaluated the functional significance of FNDC3B in MBT using FNDC3B specific siRNA and performing cell viability assay, real-time qPCR, Western blot, 3D-invasion assay, and wound healing assay. Furthermore, using proteome profiler human phospho-kinase arrays, we investigated the correlated signaling which activated while FNDC3B-mediated MBT cell progression. We found that down-regulation of FNDC3B decreased tumor cell proliferation, migratory and invasiveness properties as well as stemness phenotype which are hallmarks of cancer progression. Furthermore, we demonstrate that PI3K/AKT signaling pathways were involved mainly in FNDC3B-related tumor growth and mesenchymal phenotypes. Although follow-up studies are necessary, the results of this study suggest that FNDC3B is worth investigating as a novel alternative diagnostic and therapeutic target candidate for MBT.

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.

Genome instability footprint under rapamycin and hydroxyurea treatments.

The mutational processes dictating the accumulation of mutations in genomes are shaped by genetic background, environment and their interactions. Accurate quantification of mutation rates and spectra under drugs has important implications in disease treatment. Here, we used whole-genome sequencing and time-resolved growth phenotyping of yeast mutation accumulation lines to give a detailed view of the mutagenic effects of rapamycin and hydroxyurea on the genome and cell growth. Mutation rates depended on the genetic backgrounds but were only marginally affected by rapamycin. As a remarkable exception, rapamycin treatment was associated with frequent chromosome XII amplifications, which compensated for rapamycin induced rDNA repeat contraction on this chromosome and served to maintain rDNA content homeostasis and fitness. In hydroxyurea, a wide range of mutation rates were elevated regardless of the genetic backgrounds, with a particularly high occurrence of aneuploidy that associated with dramatic fitness loss. Hydroxyurea also induced a high T-to-G and low C-to-A transversion rate that reversed the common G/C-to-A/T bias in yeast and gave rise to a broad range of structural variants, including mtDNA deletions. The hydroxyurea mutation footprint was consistent with the activation of error-prone DNA polymerase activities and non-homologues end joining repair pathways. Taken together, our study provides an in-depth view of mutation rates and signatures in rapamycin and hydroxyurea and their impact on cell fitness, which brings insights for assessing their chronic effects on genome integrity.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.

Adaptive laboratory evolution of Rhodococcus rhodochrous DSM6263 for chlorophenol degradation under hypersaline condition

BackgroundNormally, a salt amount greater than 3.5% (w/v) is defined as hypersaline. Large amounts of hypersaline wastewater containing organic pollutants need to be treated before it can be discharged into the environment. The most critical aspect of the biological treatment of saline wastewater is the inhibitory/toxic effect exerted on bacterial metabolism by high salt concentrations. Although efforts have been dedicated to improving the performance through the use of salt-tolerant or halophilic bacteria, the diversities of the strains and the range of substrate spectrum remain limited, especially in chlorophenol wastewater treatment.ResultsIn this study, a salt-tolerant chlorophenol-degrading strain was generated from Rhodococcus rhodochrous DSM6263, an original aniline degrader, by adaptive laboratory evolution. The evolved strain R. rhodochrous CP-8 could tolerant 8% NaCl with 4-chlorophenol degradation capacity. The synonymous mutation in phosphodiesterase of strain CP-8 may retard the hydrolysis of cyclic adenosine monophosphate (cAMP), which is a key factor reported in the osmoregulation. The experimentally verified up-regulation of intracellular cAMP level in the evolved strain CP-8 contributes to the improvement of growth phenotype under high osmotic condition. Additionally, a point mutant of the catechol 1,2-dioxygenase, CatAN211S, was revealed to show the 1.9-fold increment on activity, which the mechanism was well explained by molecular docking analysis.ConclusionsThis study developed one chlorophenol-degrading strain with extraordinary capacity of salt tolerance, which showed great application potential in hypersaline chlorophenol wastewater treatment. The synonymous mutation in phosphodiesterase resulted in the change of intracellular cAMP concentration and then increase the osmotic tolerance in the evolved strain. The catechol 1,2-dioxygenase mutant with improved activity also facilitated chlorophenol removal since it is the key enzyme in the degradation pathway.

Optimal phenotypic adaptation in fluctuating environments

Phenotypic adaptation is a universal feature of biological systems navigating highly variable environments. Recent empirical data support the role of memory-driven decision making in cellular systems navigating uncertain future nutrient landscapes, wherein a distinct growth phenotype emerges in fluctuating conditions. We develop a simple stochastic mathematical model to describe memory-driven cellular adaptation required for systems to optimally navigate such uncertainty. In this framework, adaptive populations traverse dynamic environments by inferring future variation from a memory of prior states, and memory capacity imposes a fundamental trade-off between the speed and accuracy of adaptation to new fluctuating environments. Our results suggest that the observed growth reductions that occur in fluctuating environments are a direct consequence of optimal decision making and result from bet hedging and occasional phenotypic-environmental mismatch. We anticipate that this modeling framework will be useful for studying the role of memory in phenotypic adaptation, including in the design of temporally varying therapies against adaptive systems.

UHPLC/MS-Based Untargeted Metabolomics Reveals Metabolic Characteristics of Clinical Strain of Mycoplasma bovis.

Mycoplasma bovis is a global concern for the cattle industry owing to its high rates of infection and resulting morbidity, but its pathogenesis remains poorly understood. Metabolic pathways and characteristics of M. bovis clinical strain were elucidated by comparing the differential expression of metabolites between M. bovis clinical strain NX114 and M. bovis international reference strain PG45. Metabolites of M. bovis in the logarithmic stage were analyzed based on the non-targeted metabolomic technology of ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). We found 596 metabolites with variable expression, of which, 190 had substantial differences. Differential metabolite analysis of M. bovis NX114 showed organic acids and their derivatives, nucleosides, and nucleotide analogs as important components. We found O-Phospho-L-serine (SEP) as a potential signature metabolite and indicator of pathogenicity. The difference in nucleic acid metabolites reflects the difference in growth phenotypes between both strains of M. bovis. According to KEGG enrichment analysis, the ABC transporter synthesis route had the most differential metabolites of the first 15 differential enrichment pathways. This study reflects the species-specific differences between two strains of M. bovis and further enriches our understanding of its metabolism, paving the way for further research into its pathogenesis.