Elife. 2024 Feb 22;12:RP89267. doi: 10.7554/eLife.89267.

ABSTRACT

Extinction is a specific example of learning where a previously reinforced stimulus or response is no longer reinforced, and the previously learned behaviour is no longer necessary and must be modified. Current theories suggest extinction is not the erasure of the original learning but involves new learning that acts to suppress the original behaviour. Evidence for this can be found when the original behaviour recovers following the passage of time (spontaneous recovery) or reintroduction of the reinforcement (i.e. reinstatement). Recent studies have shown that pharmacological manipulation of noradrenaline (NA) or its receptors can influence appetitive extinction; however, the role and source of endogenous NA in these effects are unknown. Here, we examined the role of the locus coeruleus (LC) in appetitive extinction. Specifically, we tested whether optogenetic stimulation of LC neurons during extinction of a food-seeking behaviour would enhance extinction evidenced by reduced spontaneous recovery in future tests. LC stimulation during extinction trials did not change the rate of extinction but did serve to reduce subsequent spontaneous recovery, suggesting that stimulation of the LC can augment reward-related extinction. Optogenetic inhibition of the LC during extinction trials reduced responding during the trials where it was applied, but no long-lasting changes in the retention of extinction were observed. Since not all LC cells expressed halorhodopsin, it is possible that more complete LC inhibition or pathway-specific targeting would be more effective at suppressing extinction learning. These results provide further insight into the neural basis of appetitive extinction, and in particular the role of the LC. A deeper understanding of the physiological bases of extinction can aid development of more effective extinction-based therapies.

PMID:38386378 | DOI:10.7554/eLife.89267

Adv Sci (Weinh). 2024 Feb 22:e2304501. doi: 10.1002/advs.202304501. Online ahead of print.

ABSTRACT

CD8+ T cells are critical for host antitumor responses, whereas persistent antigenic stimulation and excessive inflammatory signals lead to T cell dysfunction or exhaustion. Increasing early memory T cells can improve T cell persistence and empower T cell-mediated tumor eradication, especially for adoptive cancer immunotherapy. Here, it is reported that tumor-associated monocytes (TAMos) are highly correlated with the accumulation of CD8+ memory T cells in human cancers. Further analysis identifies that TAMos selectively reprogram CD8+ T cells into T central memory-like (TCM -like) cells with enhanced recall responses. L-NMMA, a pan nitric oxide synthase inhibitor, can mitigate TAMo-mediated inhibition of T cell proliferation without affecting TCM -like cell generation. Moreover, the modified T cells by TAMo exposure and L-NMMA treatment exhibit long-term persistence and elicit superior antitumor effects in vivo. Mechanistically, the transmembrane protein CD300LG is involved in TAMo-mediated TCM -like cell polarization in a cell-cell contact-dependent manner. Thus, the terminally differentiated TAMo subset (CD300LGhigh ACElow ) mainly contributes to TCM -like cell development. Taken together, these findings establish the significance of TAMos in boosting T-cell antitumor immunity.

PMID:38386350 | DOI:10.1002/advs.202304501

Plant Dis. 2024 Feb 22. doi: 10.1094/PDIS-12-23-2711-PDN. Online ahead of print.

ABSTRACT

In March 2021, unusual plant stuning, collar, and wet root rot of lettuce (Lactuca sativa L.) during the rosette stage was observed in two commercial fields in Serbia (44°58'N, 20°32'E; 44°45'N, 20°43'E). Disease incidence in the fields (≈ 0.9 ha each) was approximately 15 and 20%, respectively. Initial above-ground symptoms were yellowing and wilting of leaves, while below-ground symptoms were collar, wet root rot, and lesions becoming necrotic. Eventually, whole plants wilted, collapsed, and died. A total of 35 symptomatic plants were collected from the fields, and diseased tissues were cut into small pieces, surface sterilized, and plated on potato dextrose agar (PDA). Isolation resulted in 20 morphologically uniform monoconidial isolates. The isolates formed white to creamy colonies, gradually becoming salmon pink, slimy, or moist in appearance, with sparse aerial mycelia. Numerous hyphal coils with conidiophores and hyaline, smooth-surfaced, ellipsoid to ovoid, septate or aseptate conidia were formed (4.5 to 10.1×1.2 to 3.7 μm (n = 100)). To confirm the species identity, the internal transcribed spacer (ITS) region and the D1/D2 region of a selected representative isolate 13-3-c were amplified and sequenced by using primer pairs ITS1/ITS4 (White et al. 1990) and N1/N2 (O'Donnell and Gray 1995), respectively. The sequences were deposited in GenBank (ITS: OR880564 and D1/D2: OR880567). Sequence analysis revealed 100% nucleotide identity with P. cucumerina isolates from different countries deposited in the NCBI GenBank, including isolate MH860704 (Vu et al. 2019) (ITS region) and isolate KY662256 (Su et al. 2017) (D1/D2 region). Neighbor-joining analysis was conducted based on the combined ITS and D1/D2 regions, and the tree was constructed with the substitution models (1,000 bootstrap). The combined phylogeny confirmed that the sequences shared a common clade with P. cucumerina. Hence, morphological, microscopic, and molecular characterization confirmed the pathogen as P. cucumerina (Palm et al., 1995; Carlucci et al., 2012). In a pathogenicity assay, 10 isolates were tested. Five 30-day-old lettuce plants (cv. Majska Kraljica) per isolate were root-dipped in the conidial suspensions (1×105 conidia/ml). The 10 inoculated plants were transplanted into 1 L pots containing sterile substrate (Floragard, Germany). Plants treated with sterile distilled water were used as controls. Plants were maintained in a greenhouse at 25 to 28°C under a 12-hour photoperiod (Cai et al., 2021). Four weeks after inoculation, stunting, chlorosis, and wilting of plants were observed, while collars and roots exhibited typical decaying symptoms. No symptoms were observed on the control plants. The pathogen was reisolated from symptomatic tissue as previously described. Koch's postulates were completed by confirming the identity of reisolates based on morphological features. To our knowledge, this is the first report of P. cucumerina on lettuce or any other crop in Serbia. P. cucumerina is already known as a pathogen of lettuce and other hosts grown in many countries worldwide, as well as in some European countries (Belgium, England, Italy, the Netherlands, and Switzerland) (Zhang et al. 2019). This emerging pathogen may cause significant economic losses in lettuce production in Serbia and in the entire Balkan region. Our results may help to develop effective management strategies based on accurate and timely identification and regular pathogen monitoring.

PMID:38386304 | DOI:10.1094/PDIS-12-23-2711-PDN

Synth Syst Biotechnol. 2024 Feb 8;9(2):217-222. doi: 10.1016/j.synbio.2024.01.011. eCollection 2024 Jun.

ABSTRACT

The protein inverse folding problem, designing amino acid sequences that fold into desired protein structures, is a critical challenge in biological sciences. Despite numerous data-driven and knowledge-driven methods, there remains a need for a user-friendly toolkit that effectively integrates these approaches for in-silico protein design. In this paper, we present DIProT, an interactive protein design toolkit. DIProT leverages a non-autoregressive deep generative model to solve the inverse folding problem, combined with a protein structure prediction model. This integration allows users to incorporate prior knowledge into the design process, evaluate designs in silico, and form a virtual design loop with human feedback. Our inverse folding model demonstrates competitive performance in terms of effectiveness and efficiency on TS50 and CATH4.2 datasets, with promising sequence recovery and inference time. Case studies further illustrate how DIProT can facilitate user-guided protein design.

PMID:38385151 | PMC:PMC10876589 | DOI:10.1016/j.synbio.2024.01.011

Front Cell Dev Biol. 2024 Feb 7;12:1284988. doi: 10.3389/fcell.2024.1284988. eCollection 2024.

ABSTRACT

Ca2+ is a key secondary messenger that modulates sperm motility by tuning flagellar movement in various species. The sperm-specific Ca2+ channel, CatSper, is a primary Ca2+ gate that is essential for male fertility in mammals. CatSper-mediated Ca2+ signaling enables sperm to develop hyperactivated motility and fertilize the eggs in the female tract. Therefore, altered CatSper function compromises the entry of Ca2+ into the sperm, followed by impairing hyperactivation and male fertility. However, methods to evaluate the function of the CatSper channel are limited to patch clamping and functional imaging using Ca2+ dye. Previous studies have revealed that various parameters for sperm motility are highly correlated with intracellular Ca2+ levels in mouse. Here, I cover a step-by-step protocol to analyze the change in Ca2+-mediated sperm motility by using computer-assisted semen analysis (CASA) to evaluate the functional normality of the CatSper channel in sperm. This approach analyzes sperm motility parameters during intracellular Ca2+ chelation followed by in vitro capacitation to recover intracellular Ca2+ via the activated CatSper channel. Thus, this Ca2+-handling method is handy and could be broadly applied in reproductive biology labs and clinics that have CASA equipment to examine the functional normality of the CatSper channel.

PMID:38385023 | PMC:PMC10879342 | DOI:10.3389/fcell.2024.1284988

iScience. 2024 Feb 5;27(3):109133. doi: 10.1016/j.isci.2024.109133. eCollection 2024 Mar 15.

ABSTRACT

Despite adjuvant chemotherapy and endocrine therapy in luminal breast cancer (LBC), relapses are common. Addressing this, we aim to develop a prognostic model to refine adjuvant therapy strategies, particularly for patients at high recurrence risk. Notably, obesity profoundly affects the tumor microenvironment (TME) of LBC. However, it is unclear whether obesity-related biological features can effectively screen high-risk patients. Utilizing weighted gene coexpression network analysis (WGCNA) on RNA sequencing (RNAseq) data, we identified seven obese LBC genes (OLGs) closely associated with patient prognosis. Subsequently, we developed a luminal obesity-gene clinical prognostic index (LOG-CPI), combining a 7-gene signature, TNM staging, and age. Its predictive efficacy was confirmed across validation datasets and a clinical cohort (5-year accuracy = 0.828, 0.760, 0.751, and 0.792, respectively). LOG-CPI emerges as a promising predictor for clinical prognosis and treatment response, helping distinguish molecular and immunological features in LBC patients and guiding clinical practice by identifying varying prognoses.

PMID:38384850 | PMC:PMC10879711 | DOI:10.1016/j.isci.2024.109133

iScience. 2024 Jan 29;27(3):109037. doi: 10.1016/j.isci.2024.109037. eCollection 2024 Mar 15.

ABSTRACT

Changes in glycosylation patterns have been associated with malignant transformation and clinical outcomes in several cancer types, prompting ongoing research into the mechanisms involved and potential clinical applications. In this study, we performed an extensive transcriptomic analysis of glycosylation-related genes and pathways, using publicly available bulk and single cell transcriptomic datasets from tumor samples and cancer cell lines. We identified genes and pathways strongly associated with different tumor types, which may represent novel diagnostic biomarkers. By using single cell RNA-seq data, we characterized the contribution of different cell types to the overall tumor glycosylation. Transcriptomic analysis of cancer cell lines revealed that they present a simplified landscape of genes compared to tissue. Lastly, we describe the association of different genes and pathways with the clinical outcome of patients. These results can serve as a resource for future research aimed to unravel the role of the glyco-code in cancer.

PMID:38384845 | PMC:PMC10879703 | DOI:10.1016/j.isci.2024.109037

Biodes Res. 2024 Feb 21;6:0025. doi: 10.34133/bdr.0025. eCollection 2024.

ABSTRACT

The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components. Previously, extracytoplasmic function sigma factors (ECFs), a diverse group of alternative sigma factors with distinct promoter specificities, were shown to have great potential as context-independent regulators, but so far, they have only been used in a few model species. Here, we show that the alphaproteobacterium Sinorhizobium meliloti, which has been proposed as a plant-associated bacterial chassis for synthetic biology, has a similar phylogenetic ECF acceptance range as the gammaproteobacterium Escherichia coli. A common set of orthogonal ECF-based regulators that can be used in both bacterial hosts was identified and used to create 2-step delay circuits. The genetic circuits were implemented in single copy in E. coli by chromosomal integration using an established method that utilizes bacteriophage integrases. In S. meliloti, we demonstrated the usability of single-copy pABC plasmids as equivalent carriers of the synthetic circuits. The circuits were either implemented on a single pABC or modularly distributed on 3 such plasmids. In addition, we provide a toolbox containing pABC plasmids compatible with the Golden Gate (MoClo) cloning standard and a library of basic parts that enable the construction of ECF-based circuits in S. meliloti and in E. coli. This work contributes to building a context-independent and species-overarching ECF-based toolbox for synthetic biology applications.

PMID:38384496 | PMC:PMC10880074 | DOI:10.34133/bdr.0025

Eur J Clin Microbiol Infect Dis. 2024 Feb 22. doi: 10.1007/s10096-024-04777-z. Online ahead of print.

ABSTRACT

PURPOSE: Several model studies suggested the implementation of latent tuberculosis infection (LTBI) testing and treatment could greatly reduce the incidence of tuberculosis (TB) and achieve the 2035 target of the "End TB" Strategy in China. The present study aimed to evaluate the cost-effectiveness of LTBI testing and TB preventive treatment among key population (≥ 50 years old) susceptible to TB at community level in China.

METHODS: A Markov model was developed to investigate the cost-effectiveness of LTBI testing using interferon gamma release assay (IGRA) and subsequent treatment with 6-month daily isoniazid regimen (6H) (as a standard regimen for comparison) or 6-week twice-weekly rifapentine and isoniazid regimen (6-week H2P2) in a cohort of 10,000 adults with an average initial age of 50 years.

RESULTS: In the base-case analysis, LTBI testing and treatment with 6H was dominated (i.e., more expensive with a lower quality-adjusted life year (QALY)) by LTBI testing and treatment with 6-week H2P2. LTBI testing and treatment with 6-week H2P2 was more effective than no intervention at a cost of $20,943.81 per QALY gained, which was below the willingness-to-pay (WTP) threshold of $24,211.84 per QALY gained in China. The one-way sensitivity analysis showed the change of LTBI prevalence was the parameter that most influenced the results of the incremental cost-effectiveness ratios (ICERs).

CONCLUSION: As estimated by a Markov model, LTBI testing and treatment with 6-week H2P2 was cost-saving compared with LTBI testing and treatment with 6H, and it was considered to be a cost-effective option for TB control in rural China.

PMID:38383889 | DOI:10.1007/s10096-024-04777-z

Nature. 2024 Feb 21. doi: 10.1038/s41586-024-07067-y. Online ahead of print.

ABSTRACT

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.

PMID:38383780 | DOI:10.1038/s41586-024-07067-y

Microbiome. 2024 Feb 22;12(1):31. doi: 10.1186/s40168-024-01758-4.

ABSTRACT

BACKGROUND: People living with HIV (PLWH), even when viral replication is controlled through antiretroviral therapy (ART), experience persistent inflammation. This inflammation is partly attributed to intestinal microbial dysbiosis and translocation, which may lead to non-AIDS-related aging-associated comorbidities. The extent to which living with HIV - influenced by the infection itself, ART usage, sexual orientation, or other associated factors - affects the biological age of the intestines is unclear. Furthermore, the role of microbial dysbiosis and translocation in the biological aging of PLWH remains to be elucidated. To investigate these uncertainties, we used a systems biology approach, analyzing colon and ileal biopsies, blood samples, and stool specimens from PLWH on ART and people living without HIV (PLWoH) as controls.

RESULTS: PLWH exhibit accelerated biological aging in the colon, ileum, and blood, as measured by various epigenetic aging clocks, compared to PLWoH. Investigating the relationship between microbial translocation and biological aging, PLWH had decreased levels of tight junction proteins in the intestines, along with increased microbial translocation. This intestinal permeability correlated with faster biological aging and increased inflammation. When investigating the relationship between microbial dysbiosis and biological aging, the intestines of PLWH had higher abundance of specific pro-inflammatory bacteria, such as Catenibacterium and Prevotella. These bacteria correlated with accelerated biological aging. Conversely, the intestines of PLWH had lower abundance of bacteria known for producing the anti-inflammatory short-chain fatty acids, such as Subdoligranulum and Erysipelotrichaceae, and these bacteria were associated with slower biological aging. Correlation networks revealed significant links between specific microbial genera in the colon and ileum (but not in feces), increased aging, a rise in pro-inflammatory microbe-related metabolites (e.g., those in the tryptophan metabolism pathway), and a decrease in anti-inflammatory metabolites like hippuric acid.

CONCLUSIONS: We identified specific microbial compositions and microbiota-related metabolic pathways that are intertwined with intestinal and systemic biological aging. This microbial signature of biological aging is likely reflecting various factors including the HIV infection itself, ART usage, sexual orientation, and other aspects associated with living with HIV. A deeper understanding of the mechanisms underlying these connections could offer potential strategies to mitigate accelerated aging and its associated health complications. Video Abstract.

PMID:38383483 | DOI:10.1186/s40168-024-01758-4

BMC Genom Data. 2024 Feb 21;25(1):21. doi: 10.1186/s12863-024-01207-z.

ABSTRACT

BACKGROUND: GGC and GCC short tandem repeats (STRs) are of various evolutionary, biological, and pathological implications. However, the fundamental two-repeats (dyads) of these STRs are widely unexplored.

RESULTS: On a genome-wide scale, we mapped (GGC)2 and (GCC)2 dyads in human, and found monumental colonies (distance between each dyad < 500 bp) of extraordinary density, and in some instances periodicity. The largest (GCC)2 and (GGC)2 colonies were intergenic, homogeneous, and human-specific, consisting of 219 (GCC)2 on chromosome 2 (probability < 1.545E-219) and 70 (GGC)2 on chromosome 9 (probability = 1.809E-148). We also found that several colonies were shared in other great apes, and directionally increased in density and complexity in human, such as a colony of 99 (GCC)2 on chromosome 20, that specifically expanded in great apes, and reached maximum complexity in human (probability 1.545E-220). Numerous other colonies of evolutionary relevance in human were detected in other largely overlooked regions of the genome, such as chromosome Y and pseudogenes. Several of the genes containing or nearest to those colonies were divergently expressed in human.

CONCLUSION: In conclusion, (GCC)2 and (GGC)2 form unprecedented genomic colonies that coincide with the evolution of human and other great apes. The extent of the genomic rearrangements leading to those colonies support overlooked recombination hotspots, shared across great apes. The identified colonies deserve to be studied in mechanistic, evolutionary, and functional platforms.

PMID:38383300 | DOI:10.1186/s12863-024-01207-z

Bioinformatics. 2024 Feb 21:btae104. doi: 10.1093/bioinformatics/btae104. Online ahead of print.

ABSTRACT

MOTIVATION: Creating knowledge bases and ontologies is a time consuming task that relies on manual curation. AI/NLP approaches can assist expert curators in populating these knowledge bases, but current approaches rely on extensive training data, and are not able to populate arbitrarily complex nested knowledge schemas.

RESULTS: Here we present Structured Prompt Interrogation and Recursive Extraction of Semantics (SPIRES), a Knowledge Extraction approach that relies on the ability of Large Language Models (LLMs) to perform zero-shot learning (ZSL) and general-purpose query answering from flexible prompts and return information conforming to a specified schema. Given a detailed, user-defined knowledge schema and an input text, SPIRES recursively performs prompt interrogation against an LLM to obtain a set of responses matching the provided schema. SPIRES uses existing ontologies and vocabularies to provide identifiers for matched elements. We present examples of applying SPIRES in different domains, including extraction of food recipes, multi-species cellular signaling pathways, disease treatments, multi-step drug mechanisms, and chemical to disease relationships. Current SPIRES accuracy is comparable to the mid-range of existing Relation Extraction (RE) methods, but greatly surpasses an LLM's native capability of grounding entities with unique identifiers. SPIRES has the advantage of easy customization, flexibility, and, crucially, the ability to perform new tasks in the absence of any new training data. This method supports a general strategy of leveraging the language interpreting capabilities of LLMs to assemble knowledge bases, assisting manual knowledge curation and acquisition while supporting validation with publicly-available databases and ontologies external to the LLM.

AVAILABILITY: SPIRES is available as part of the open source OntoGPT package: https://github.com/monarch-initiative/ontogpt.

PMID:38383067 | DOI:10.1093/bioinformatics/btae104

Biochem Biophys Res Commun. 2024 Feb 15;703:149659. doi: 10.1016/j.bbrc.2024.149659. Online ahead of print.

ABSTRACT

Utilizing RNA sequence (RNA-Seq) splice junction data from a cohort of 1841 B-cell acute lymphoblastic leukemia (B-ALL) patients we define transcriptionally distinct isoforms of ARID5B, a risk-associated gene identified in genome wide association studies (GWAS), which associate with disease survival. Short (S) and long (L) ARID5B transcripts, which differ in an encoded BAH-like chromatin interaction domain, show remarkable correlation to the isoform splicing pattern. Testing of the ARID5B proximal promoter of the S & L isoforms indicated that both are functionally independent in luciferase reporter assays. Increased short isoform expression is associated with decreased event-free and overall survival. The abundance of short and long transcripts strongly correlates to B-ALL prognostic stratification, where B-ALL subtypes with poor outcomes express a higher proportion of the S-isoform. These data demonstrate that the analysis of independent promoters and alternative splicing events are essential for improved risk stratification and a more complete understanding of disease pathology.

PMID:38382358 | DOI:10.1016/j.bbrc.2024.149659

Talanta. 2024 Feb 17;272:125790. doi: 10.1016/j.talanta.2024.125790. Online ahead of print.

ABSTRACT

The investigation of exosomes holds substantial importance in the field of disease diagnosis and prognosis, where in the rapid and low-loss isolation of exosomes emerges as a crucial step owing to their exceedingly low concentration in bodily fluids. Herein, bifunctional immunoaffinity magnetic nanoparticles (BI-NPs) were successfully constructed by binding Ti4+ and antibody to magnetic nanoparticles through host-guest interaction. Exosomes in the cell culture supernatant could be effectively captured by the BI-NPs and then gently eluted with α-CD and 10% ammonia solution. Following the elution process, the content of captured exosome protein was determined to be up to 97 μg/mL. A total of 2822 protein groups were identified in the exosomes isolated by BI-NPs, 1060 protein groups were derived from exosomes. At the same time, the mass of exosome protein obtained by BI-NPs was more than 2 times that of UC isolation, and the above results indicated that BI-NPs had high purity enrichment performance. This is attributed to the fact that BI-NPs combine the dual affinity of Ti4+ and antibody to achieve efficient enrichment of exosomes, in addition to the mild elution property of BI-NPs due to the presence of the host-guest system. BI-NPs offer a novel approach for the isolation of exosomes, in order to further promote the application of exosomes in the required fields.

PMID:38382302 | DOI:10.1016/j.talanta.2024.125790

Plant Cell. 2024 Feb 21:koae054. doi: 10.1093/plcell/koae054. Online ahead of print.

ABSTRACT

The phytohormone auxin is at times called the master regulator of plant processes and has been shown to be a central player in embryo development, the establishment of the polar axis, early aspects of seedling growth, as well as growth and organ formation during later stages of plant development. The Plant Cell has been key, since the inception of the journal, to developing an understanding of auxin biology. Auxin regulated plant growth control is accomplished by both changes in the levels of active hormones and the sensitivity of plant tissues to these concentration changes. In this historical review, we chart auxin research as it has progressed in key areas and highlighting the role The Plant Cell played in these scientific developments. We focus on understanding auxin-responsive genes, transcription factors, reporter constructs, perception and signal transduction processes. Auxin metabolism is discussed from the development of tryptophan auxotrophic mutants, the molecular biology of conjugate formation and hydrolysis, indole-3-butyric acid metabolism and transport, and key steps in indole-3-acetic acid biosynthesis, catabolism and transport. This progress leads to an expectation of a more comprehensive understanding of the systems biology of auxin and the spatial and temporal regulation of cellular growth and development.

PMID:38382088 | DOI:10.1093/plcell/koae054

Nucleic Acids Res. 2024 Feb 21:gkae120. doi: 10.1093/nar/gkae120. Online ahead of print.

ABSTRACT

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA emerging during library preparation and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package.

PMID:38381903 | DOI:10.1093/nar/gkae120

Sci Adv. 2024 Feb 23;10(8):eadl1258. doi: 10.1126/sciadv.adl1258. Epub 2024 Feb 21.

ABSTRACT

Adrenal Cushing's syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing's driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAcW196G is unexpectedly distinct from other described Cushing's variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653-cubic angstrom cleft between the catalytic core of PKAcW196G and type II regulatory subunits (RII), but only a 395-cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAcW196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing's syndrome.

PMID:38381834 | DOI:10.1126/sciadv.adl1258

Microb Biotechnol. 2024 Feb;17(2):e14418. doi: 10.1111/1751-7915.14418.

ABSTRACT

CRISPR-Cas systems evolved in prokaryotes to implement a powerful antiviral immune response as a result of sequence-specific targeting by ribonucleoproteins. One of such systems consists of an RNA-guided RNA endonuclease, known as CRISPR-Cas13. In very recent years, this system is being repurposed in different ways in order to decipher and engineer gene expression programmes. Here, we discuss the functional versatility of the CRISPR-Cas13 system, which includes the ability for RNA silencing, RNA editing, RNA tracking, nucleic acid detection and translation regulation. This functional palette makes the CRISPR-Cas13 system a relevant tool in the broad field of systems and synthetic biology.

PMID:38381083 | DOI:10.1111/1751-7915.14418

Elife. 2024 Feb 21;12:RP88793. doi: 10.7554/eLife.88793.

ABSTRACT

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for Escherichia coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzyme's activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycle's electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

PMID:38381041 | DOI:10.7554/eLife.88793