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Candidatus Patescibacteria, also known as candidate phyla radiation (CPR), including the class-level uncultured clade JAEDAM01 (formerly a subclass of Gracilibacteria/GN02/BD1-5), are ubiquitous in activated sludge. However, their characteristics and relationships with other organisms are largely unknown. They are believed to be episymbiotic, endosymbiotic or predatory. Despite our understanding of their limited metabolic capacity, their precise roles remain elusive due to the difficulty in cultivating and identifying them. In previous research, we successfully recovered high-quality metagenome-assembled genomes (MAGs), including a member of JAEDAM01 from activated sludge flocs. In this study, we designed new probes to visualize the targeted JAEDAM01-associated MAG HHAS10 and identified its host using fluorescence in situ hybridization (FISH). The FISH observations revealed that JAEDAM01 HHAS10-like cells were located within dense clusters of Zoogloea, and the fluorescence brightness of zoogloeal cells decreased in the vicinity of the CPR cells. The Zoogloea MAGs possessed genes related to extracellular polymeric substance biosynthesis, floc formation and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The JAEDAM01 MAG HHAS10 possessed genes associated with type IV pili, competence protein EC and PHA degradation, suggesting a Zoogloea-dependent lifestyle in activated sludge flocs. These findings indicate a new symbiotic relationship between JAEDAM01 and Zoogloea., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Several plant-associated microbes synthesize the auxinic plant growth regulator phenylacetic acid (PAA) in culture; however, the role of PAA in plant-pathogen interactions is not well understood. In this study, we investigated the role of PAA during interactions between the phytopathogenic bacterium Pseudomonas syringae strain Pto DC3000 ( Pto DC3000) and the model plant host, Arabidopsis thaliana . Previous work demonstrated that indole-3-acetaldehyde dehydrogenase A (AldA) of Pto DC3000 converts indole-3-acetaldehyde (IAAld) to the auxin indole-3-acetic acid (IAA). Here, we further demonstrated the biochemical versatility of AldA by conducting substrate screening and steady-state kinetic analyses, and showed that AldA can use both IAAld and phenylacetaldehyde as substrates to produce IAA and PAA, respectively. Quantification of auxin in infected plant tissue showed that AldA-dependent synthesis of either IAA or PAA by Pto DC3000 does not contribute significantly to the increase in auxin levels in infected A. thaliana leaves. Using available arogenate dehydratase ( adt ) mutant lines of A. thaliana compromised for PAA synthesis, we observed that a reduction in PAA-Asp and PAA-Glu is correlated with elevated levels of IAA and increased susceptibility. These results provide evidence that PAA/IAA homeostasis in A. thaliana influences the outcome of plant-microbial interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lee, Harper, Lee, Qi, Clay, Aoi, Jez, Kasahara, Blodgett and Kunkel.)

Loss-of-function mutations in methyl-CpG binding protein 2 ( MECP2 ) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in ∼1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Here, using a human MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2 -induced phenotypes. Our approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2 . We discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, we found that MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, our study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.

Acetogens are among the key microorganisms involved in the bioproduction of commodity chemicals from diverse carbon resources, such as biomass and waste gas. Thermophilic acetogens are particularly attractive because fermentation at higher temperatures offers multiple advantages. However, the main target product is acetic acid. Therefore, it is necessary to reshape metabolism using genetic engineering to produce the desired chemicals with varied carbon lengths. Although such metabolic engineering has been hampered by the difficulty involved in genetic modification, a model thermophilic acetogen, M. thermoacetica ATCC 39073, is the case with a few successful cases of C2 and C3 compound production, other than acetate. This brief report attempts to expand the product spectrum to include C4 compounds by using strain Y72 of Moorella thermoacetica . Strain Y72 is a strain related to the type strain ATCC 39073 and has been reported to have a less stringent restriction-modification system, which could alleviate the cumbersome transformation process. A simplified procedure successfully introduced a key enzyme for acetoin (a C4 chemical) production, and the resulting strains produced acetoin from sugars and gaseous substrates. The culture profile revealed varied acetoin yields depending on the type of substrate and culture conditions, implying the need for further engineering in the future. Thus, the use of a user-friendly chassis could benefit the genetic engineering of M. thermoacetica ., Competing Interests: This work has been included in a patent application by Hiroshima University and AIST., (Copyright © 2024 Kato, Fujii, Kato, Wada, Watanabe, Nakamichi, Aoi, Morita, Murakami and Nakashimada.)

To further our understanding of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) E17 guideline and promote effective implementation, a public workshop was held in Japan by regulatory agency and industry representatives. In this workshop, important concepts explained in the ICH E17 guideline, such as intrinsic/extrinsic ethnic factors that influence treatment effects ("effect modifiers") and the holistic evaluation of consistency of treatment effect were actively discussed through case studies. The importance of holistic evaluation of consistency was recognized, and it was concluded that the evaluation and relevant discussion should be shared with regulatory authorities, sponsors, and broader stakeholders., (© 2024 The Authors. Clinical Pharmacology & Therapeutics © 2024 American Society for Clinical Pharmacology and Therapeutics.)

Plants adopt optimal tolerance strategies depending on the intensity and duration of stress. Retaining water is a priority under short-term drought conditions, whereas maintaining growth and reproduction processes takes precedence over survival under conditions of prolonged drought. However, the mechanism underlying changes in the stress response depending on the degree of drought is unclear. Here, we report that SNF1-related protein kinase 2 (SnRK2) substrate 1 (SNS1) is involved in this growth regulation under conditions of drought stress. SNS1 is phosphorylated and stabilized by SnRK2 protein kinases reflecting drought conditions. It contributes to the maintenance of growth and promotion of flowering as drought escape by repressing stress-responsive genes and inducing FLOWERING LOCUS T (FT) expression, respectively. SNS1 interacts with the histone methylation reader proteins MORF-related gene 1 (MRG1) and MRG2, and the SNS1-MRG1/2 module cooperatively regulates abscisic acid response. Taken together, these observations suggest that the phosphorylation and accumulation of SNS1 in plants reflect the intensity and duration of stress and can serve as a molecular scale for maintaining growth and adopting optimal drought tolerance strategies under stress conditions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Isopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO 2 -emission petrochemical methods that are not sustainable. Therefore, alternative low-CO 2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method., Results: Moorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H 2 resulted in higher IPA production at the specific rate of 0.03 h -1 . The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh., Conclusions: The successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens., (© 2024. The Author(s).)

Cancer genome sequencing consortiums have recently catalogued an abundance of somatic mutations, across a wide range of human cancers, in the chromatin-modifying enzymes that regulate gene expression. Defining the molecular mechanisms underlying the potentially oncogenic functions of these epigenetic mutations could serve as the basis for precision medicine approaches to cancer therapy. MLL4 encoded by the KMT2D gene highly mutated in a large number of human cancers, is a key histone lysine monomethyltransferase within the Complex of Proteins Associated with Set1 (COMPASS) family that regulates gene expression through enhancer function, potentially functioning as a tumor suppressor. We report that the KMT2D mutations which cause MLL4 protein truncation also alter MLL4's subcellular localization, resulting in loss-of-function in the nucleus and gain-of-function in the cytoplasm. We demonstrate that isogenic correction of KMT2D truncation mutation rescues the aberrant localization phenotype and restores multiple regulatory functions of MLL4, including COMPASS integrity/stabilization, histone H3K4 mono-methylation, enhancer activation, and therefore transcriptional regulation. Moreover, isogenic correction diminishes the sensitivity of KMT2D -mutated cancer cells to targeted metabolic inhibition. Using immunohistochemistry, we identified that cytoplasmic MLL4 is unique to the tissue of bladder cancer patients with KMT2D truncation mutations. Using a preclinical carcinogen model of bladder cancer in mouse, we demonstrate that truncated cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D -mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome., Competing Interests: Competing interests statement:J.J.M.: Consultant: Merck, AstraZeneca, Incyte, Janssen, BMS, UroGen, Prokarium, Imvax, Pfizer, Seagen/Astellas, Compensation for talks/educational courses: AUA, OncLive, Olympus, UroToday, Clinical Trials: SWOG, Genentech, Merck, AstraZeneca, Incyte.

The biological role of the repetitive DNA sequences in the human genome remains an outstanding question. Recent long-read human genome assemblies have allowed us to identify a function for one of these repetitive regions. We have uncovered a tandem array of conserved primate-specific retrogenes encoding the protein Elongin A3 (ELOA3), a homolog of the RNA polymerase II (RNAPII) elongation factor Elongin A (ELOA). Our genomic analysis shows that the ELOA3 gene cluster is conserved among primates and the number of ELOA3 gene repeats is variable in the human population and across primate species. Moreover, the gene cluster has undergone concerted evolution and homogenization within primates. Our biochemical studies show that ELOA3 functions as a promoter-associated RNAPII pause-release elongation factor with distinct biochemical and functional features from its ancestral homolog, ELOA. We propose that the ELOA3 gene cluster has evolved to fulfil a transcriptional regulatory function unique to the primate lineage that can be targeted to regulate cellular hyperproliferation.

The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Although the percentage of multi-regional clinical trials (MRCTs) submitted for drug approval in Japan increased significantly since the 2007 publication of the regulatory guideline, "Basic principles on global clinical trials", strategic collaborations between Asian countries will be important to promote MRCTs in accordance with the ICH E17 guideline published in 2017. In this study, characteristics of MRCTs reviewed for drug approval in Japan, especially those with participation by South-East Asia and East Asia, were investigated to explore opportunities for collaborations on global drug development in Asia. More than 90% of reviewed trials were conducted as global MRCTs. In addition to Japan, South-East Asia has participated in various types of MRCTs in terms of total numbers of subjects and countries. However, South-East Asia participation was lower in large-size MRCTs (total sample size ≥ 1000) than in middle- (500 ≤ total sample size < 1000) and small-size MRCTs (total sample size < 500). Furthermore, similar clinical trials for the same indications to the MRCTs without South-East Asia were rarely conducted separately in South-East Asia. Participation of other Asian countries did not affect the percentage of Japanese subjects enrolled in an MRCT, but did significantly increase the percentage of participating Asian subjects. These results suggest that additional opportunities for collaboration on MRCTs may be possible between Japan and other Asian countries, especially more collaborations with South-East Asia in the large-size MRCTs. More data of Asian populations from MRCTs will be useful for exploring an important ethnic factor affecting drug response, and will provide a sound scientific basis in considering the application of the pooled data concept in Asia, as described in the ICH E17 guideline., (© 2023. The Author(s), under exclusive licence to The Drug Information Association, Inc.)

During Arabidopsis seed coat development, copious amounts of mucilage polysaccharides are produced in the epidermal cells. When hydrated on imbibition, these polysaccharides expand and are released to encapsulate the seed as a two-layered hydrogel. Polysaccharides are synthesized from UDP-sugars by glycosyltransferases (GTs) and several GTs, with differing activities, have been identified that contribute to mucilage polysaccharide synthesis. How these GTs orchestrate production of the complex polysaccharides found in mucilage remains to be determined. In this study, we generated a range of multiple GT mutants using either CRISPR/Cas9 targeted mutation or genetic crosses of existing T-DNA insertion mutants. Four traits for mucilage amounts or macromolecular properties were examined for four replicate seed lots from 31 different GT mutant combinations. This data provides a valuable resource for future genetic, biochemical, structural, and functional studies of the roles and properties of polysaccharides present in Arabidopsis mucilage and the relative contributions of different GTs to mucilage production., (© 2023. Springer Nature Limited.)

A facultative anaerobic, Gram-strain-negative, rod-shaped bacterium (strain NBU2970 T ) was isolated by using modified ichip in situ cultivation from a marine sediment sample collected from Meishan Island in the East China Sea. Strain NBU2970 T grew optimally at 37 °C, with a NaCl concentration of 2.0 % (w/v) and at pH 7.0. The 16S rRNA gene sequence analyses revealed that strain NBU2970 T represents a novel species with the genus Muricauda , sharing highest sequence identities with Muricauda beolgyonensis BB-My12 T (96.1 %), Muricauda alvinocaridis SCR12 T (96.0 %), Muricauda taeanensis 105 T (96.0 %) and Muricauda ruestringensis B1 T (95.6 %). Phylogenetic analyses also indicated that strain NBU2970 T clustered with the genus Muricauda and was closely related to M. beolgyonensis BB-My12 T and M. ruestringensis B1 T . The draft genome sequence of strain NBU2970 T was composed of six contigs with a size of 3.2 Mbp, containing 3045 protein-coding genes and 38 RNA genes. The DNA G+C content was 43.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU2970 T and related species of the genus Muricauda were well below the threshold limit for prokaryotic species delineation. The major cellular fatty acids were iso-C 15 : 0 , iso-C 15 : 1 G and iso-C 17 : 0 3-OH. The only respiratory quinone was MK-6. The major polar lipid was phosphatidylethanolamine. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU2970 T is considered to be a representative of a novel species in the genus Muricauda , for which the name Muricauda meishanensis sp. nov. is proposed. The type strain is NBU2970 T (=KCTC 82915 T =MCCC 1K06394 T ).

Acetogens grow autotrophically and use hydrogen (H 2 ) as the energy source to fix carbon dioxide (CO 2 ). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H 2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H 2 and CO 2 . We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H 2 and CO 2 ., (Copyright © 2023 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia , and Verrucomicrobiota . Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Seo, Jung, Epstein, Zhang, Owen, Baba, Yamamoto, Harada, Nakashimada, Kato, Aoi and He.)

The polymerase-associated factor 1 complex (PAF1C) is a key, post-initiation transcriptional regulator of both promoter-proximal pausing and productive elongation catalyzed by RNA Pol II and is also involved in transcriptional repression of viral gene expression during human immunodeficiency virus-1 (HIV-1) latency. Using a molecular docking-based compound screen in silico and global sequencing-based candidate evaluation in vivo, we identified a first-in-class, small-molecule inhibitor of PAF1C (iPAF1C) that disrupts PAF1 chromatin occupancy and induces global release of promoter-proximal paused RNA Pol II into gene bodies. Transcriptomic analysis revealed that iPAF1C treatment mimics acute PAF1 subunit depletion and impairs RNA Pol II pausing at heat shock-down-regulated genes. Furthermore, iPAF1C enhances the activity of diverse HIV-1 latency reversal agents both in cell line latency models and in primary cells from persons living with HIV-1. In sum, this study demonstrates that efficient disruption of PAF1C by a first-in-class, small-molecule inhibitor may have therapeutic potential for improving current HIV-1 latency reversal strategies.

Microbial electrosynthesis system (MES) is a promising method that can use carbon dioxide, which is a greenhouse gas, to produce methane which acts as an energy source, without using organic substances. However, this bioelectrical reduction reaction can proceed at a certain high applied voltage when coupled with water oxidation in the anode coated with metallic catalyst. When coupled with the oxidation of HS - to SO 4 2- , methane production is thermodynamically more feasible, thus implying its production at a considerably lower applied voltage. In this study, we demonstrated the possibility of electrotrophic methane production coupled with HS - oxidation in a cost-effective bioanode chamber in the MES without organic substrates at a low applied voltage of 0.2 V. In addition, microbial community analyses of biomass enriched in the bioanode and biocathode were used to reveal the most probable pathway for methane production from HS - oxidation. In the bioanode, electroautotrophic SO 4 2- production accompanied with electron donation to the electrode is performed mainly by the following two steps: first, incomplete sulfide oxidation to sulfur cycle intermediates (SCI) is performed; then the produced SCI are disproportionated to HS - and SO 4 2- . In the biocathode, methane is produced mainly via H 2 and acetate by electron-accepting syntrophic bacteria, homoacetogens, and acetoclastic archaea. Here, a new eco-friendly MES with biological H 2 S removal is established., (Copyright © 2022. Published by Elsevier B.V.)

It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.