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PubMed2026

Metabolic engineering of Candida yeasts for biotechnological applications.

Candida yeasts represent a versatile yet underexploited platform for industrial biotechnology. These yeasts utilize a remarkably broad range of carbon sources, particularly for hydrophobic carbon sources, coupled with robust growth and diverse biosynthetic capacities, making them promising hosts for sustainable production of chemicals, fuels, and proteins. Despite these advantages, industrial deployment of Candida species has been hindered by concerns regarding opportunistic pathogenicity and the historical lack of efficient genetic manipulation tools, leading to a substantial gap between metabolic potential and practical utilization. Recent advances in functional genomics, genome editing, and systems metabolic engineering are rapidly overcoming these barriers, enabling more precise and efficient strain development. In this review, we systematically summarize recent progress in the metabolic engineering of Candida species as microbial cell factories, with particular emphasis on expanding genetic toolkits, utilizting renewable and non-conventional carbon sources, and biosynthesizing high-value compounds. In addition, we propose a biosafety-oriented classification framework to support their safe industrial deployment. Finally, we discuss current challenges and emerging opportunities, emphasizing that the synergy of synthetic biology and artificial intelligence-driven design holds the key to unlocking the biotechnological potential of Candida yeasts.

Candida↗
PubMed1995

Editing efficiency of hepatitis delta virus RNA is related to the course of infection in woodchucks.

Based on evidence in vitro which shows that the small form of hepatitis delta virus (HDV) antigen (S-HDAg) initiates virus replication, whereas the long form (L-HDAg) encoded by the edited L-genome inhibits replication, we first put forward the hypothesis that HDV RNA editing efficiency, i.e. the intracellular L/S-genome ratio, could be a determining factor on the course of the infection. In order to analyse the precise sequence of events after infection, woodchuck carriers of woodchuck hepatitis virus (WHV) were superinfected with HDV and sequential changes in HDV RNA editing efficiency were analysed in relation to the duration of viraemia. Our findings show that: (1) in both transiently and persistently viraemic woodchucks, the percentage of L-genome is higher at the early stage of onset of the disease than at the late stage; (2) at the early stage of onset the percentage of L-genome is higher in cases with transient viraemia than in those with persistent viraemia; (3) a relatively greater decrease in L-genome is seen later in transiently viraemic animals than in those that remain persistently viraemic. In view of the above findings in vivo and other supporting evidence in vitro, we propose a hypothesis for the pathogenesis of HDV. This hypothesis predicts the outcome of acute infection and we suggest a gene therapeutic approach for this disease based on the intracellular accumulation (or increase) of the L-genome.

Animals↗
PubMed2026

Base editing reveals an essential role for NANOG in human embryogenesis.

Understanding how the first cell lineages in human development are specified and maintained has fundamental importance and clinical implications for regenerative medicine, infertility and pregnancy loss. Although mouse models have provided valuable insights into transcription factors regulating early development, translating these findings to human embryos has been limited by ethical, technical and biological constraints. Functional studies of transcription factors in human embryos have been hindered by nuclease-based genome editing approaches that induce genotoxicity1-3. Here, to overcome this, we applied ABE8e adenine base editing4,5 to precisely target an exon splice donor site, resulting in a splicing defect and functional knockout of the developmental regulator NANOG in human embryos. This approach did not trigger genotoxicity and showed limited off-target editing. Loss of NANOG disrupts pluripotent epiblast specification and instead cells differentiate towards a primitive endoderm (yolk sac) or trophectoderm (placental) transcriptional programme. Retention of primitive endoderm differentiation in NANOG-edited human embryos reveals a functional compensation that is distinct from mouse, underscoring the importance of directly investigating human development. Our findings demonstrate an essential role for NANOG in human pluripotency and epiblast specification and highlight the utility of base editing for functional interrogation of human development.

Journal Article↗
PubMed1985

A digital sound editor.

A series of digital computer programs which facilitate the production and control of acoustic stimuli for hearing assessment and research are described. The package, which is available for PDP 11 computers under RT-11, allows sounds to be digitized, adjusted for amplitude and/or dc offset, edited while in digital form, and output to file or tape. The waveform editor package includes facilities to edit sounds in time--with some sections removed or added with temporal precision of 0.1 msec or better. Two or more sounds may also be combined for stereo or monaural (sound-on-sound) output, or two may be concatenated. Together, the programs permit a wide range of manipulations useful in preparing sound stimuli for use in hearing experiments or in clinical audiometry.

Computers↗
PubMed2002

[The meaning of the sixth edition of "Organon der Heilkunst"].

Like any artist, the paractitioner of the healing art should exactly know his tools, i.e. his medicines and the directions for their application. In addition, he should be able to rely on their genuineness. Regarding the tool "Organon", considerable uncertainty has been expressed by some physicians about the authenticity of Hahnemann's instructions for preparing and applying Q-potencies, as described in the sixth edition of the Organon of Medicine published by Richard Haehl in 1921. Since 1992, however, the first text-critical edition of the sixth edition of the Organonis available. Unlike Haehl's edition this one is exclusively based on Hahnemann's original manuscript and precisely transcribing all its handwriting, vouches for its authenticity. Hence there is no reason left to ignore Hahnemanns instructions concerning Q-potencies. This paper presents the history of the reception of Q-potencies as well as their prerequisites and evolution up to Hahnemann's final modifications of his earlier directions. As it turns out, these late instructions of Hahnemann do not mean complete change of all his previous opinions. Rather they are the logical completion of a course followed by him for ten years already. Q-potencies were Hahnemann's solution of the following therapeutic dilemma: on the one side physicians are inclined to repeat the dose of a high potency as often as possible in order to accelerate the process of healing; on the other side they should refrain from repeating the dose to avoid violent aggravations of the state of the patient.

Books↗
PubMed2026

Precision Medicine in Transfusion-Dependent and Non-Transfusion-Dependent β-Thalassemia: Toward Personalized Diagnosis and Therapy.

β-thalassemia comprises a clinically heterogeneous group of disorders in which anemia severity, transfusion exposure, iron loading, and organ complications vary widely among individuals. This structured narrative review summarizes practical applications of precision medicine in transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT), with explicit attention to which strategies apply to each clinical category. Literature indexed in PubMed and Scopus from 2000 to 2025 was reviewed using terms related to thalassemia, precision medicine, magnetic resonance imaging (MRI), chelation tailoring, next-generation sequencing (NGS), fetal hemoglobin (HbF) modifiers, luspatercept, mitapivat, hepcidin, gene therapy, gene editing, and artificial intelligence (AI). Evidence was synthesized descriptively because interventions, outcomes, and populations were heterogeneous, and no pooled meta-analysis was performed. In TDT, precision care is centered on individualized transfusion planning, extended red-cell antigen matching, MRI-guided cardiac and hepatic iron monitoring, organ-directed chelation intensification, and selection of disease-modifying or curative approaches. In NTDT, precision care emphasizes accurate phenotype classification, MRI liver iron concentration, because serum ferritin may underestimate iron burden, selective chelation, surveillance for NTDT-specific complications, and individualized use of agents that improve anemia. Personalized chelation should include deferiprone, either alone or in combination, when cardiac iron is increased. Comprehensive molecular diagnosis should include HBB together with HBA1 and HBA2 assessment, while secondary and tertiary modifiers help explain phenotypic variability and complication risk. Hepcidin and growth differentiation factor 15 (GDF-15) are discussed as investigational biomarkers; transferrin saturation is not recommended for routine iron-overload assessment in thalassemia. AI currently has its strongest role in screening and diagnosis, whereas risk-stratification models remain exploratory. Equitable implementation requires standardized TDT/NTDT pathways, regional MRI and genomics access, longitudinal registries, and multidisciplinary interpretation.

Humans↗
PubMed2025

Cellular Inflammation-Induced Cleavage of Phosphorothioate DNA Locker Activates CRISPR/Cas9 Regulator for Gene Editing.

The CRISPR technology is a highly promising strategy for developing a versatile toolbox to engineer genetic circuits. However, achieving precise and specific control over the activity of the CRISPR/Cas9 system in response to intracellular processes remains a challenging endeavor. In this study, we present a cellular inflammation-induced activation of an engineered CRISPR/Cas9 regulator for gene regulation. A phosphorothioate (PS)-modified DNA sequence, referred as the "locker," is employed to deactivate single guide RNA (sgRNA), whose locker sequence complements the spacer region of sgRNA. In the presence of myeloperoxidase during cellular inflammation, a halogenation process is triggered, leading to the generation of HClO, specifically cleaving the PS site of locker and activating CRISPR/Cas9 for gene editing. The target GFP gene has been successfully edited, downregulating the GFP protein expression in HeLa cells. This study provides valuable insights into the CRISPR-based gene regulation through specific endogenous processes.

Humans↗
PubMed2024

Global quantification of off-target activity by base editors.

Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.

Gene Editing↗
PubMed1994

Trypanosoma brucei mitochondria contain RNA helicase activity.

Mitochondrial gene expression in kinetoplastid organisms such as Trypanosoma, Leishmania and Crithidia requires a posttranscriptional RNA processing event known as kRNA editing. During editing, uridine nucleotides get inserted and deleted into pre-mRNAs directed by small, metabolically stable RNAs, termed guide RNAs. Although the precise mechanism of the reaction is not understood, the accepted working model describes the formation of extended anti-parallel RNA helices between gRNA molecules with pre- and partially edited mRNAs as intermediates. These duplex structures must be separated to ensure the sequential action of multiple gRNAs in a 3' to 5' polarity on the mRNA molecule. In spite of this fact, no unwinding activity has heretofore been identified in kinetoplastid mitochondria. We report the characterisation of a RNA helicase activity within Trypanosoma brucei mitochondrial extracts. The activity unwinds 25- and 48 bp, tailed RNA duplex structures but fails to separate DNA strands. It can be destroyed by heat denaturation as well as by proteinase K treatment. The activity requires magnesium cations and acts in a NTP/dNTP dependent manner. Hydrolysis of a nucleoside triphosphate is required rather than mere NTP binding as deduced from a comparison of unwinding in the presence of ATP and AMP-PCP. RNA duplexes mimicking presumed kRNA editing intermediates are substrates of the unwinding activity and therefore, we address the possible involvement of a RNA helicase activity during kRNA editing.

Adenosine Triphosphate↗
PubMed2004

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

Animals↗
PubMed2025

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Schizosaccharomyces↗
PubMed2026

Epigenetic and Transcriptional Regulatory Networks Underlying Psoriasis Pathogenesis.

Psoriasis is a chronic, immune-mediated dermatologic disorder characterized by the hyperproliferation of keratinocytes and dysregulated immune signaling. Although genome-wide association studies have identified susceptibility loci, the multifactorial nature of the disease underlines the importance of nongenetic regulatory mechanisms. Among these epigenetic modifications are those that critically link genetic predisposition with environmental stimuli. This review offers an in-depth overview of the current insights into the role of epigenetic regulation in the pathophysiology of psoriasis. Key mechanisms, including aberrant DNA methylation, histone post-translational modifications (eg, H3K27ac, H3K4me3), and dysregulated noncoding RNAs, are discussed in the context of inflammatory signaling and immune cell function. This review also explores how environmental factors such as UV radiation and air pollution induce the epigenetic reprogramming that perpetuates the proinflammatory state. Furthermore, it highlights the translational potential of targeting epigenetic regulators and epigenome-editing technologies, including clustered regularly interspaced short palindromic repeats (CRISPR) fusion systems, as precision therapeutic strategies. In parallel, advances in single-cell epigenomics, spatial transcriptomics, and the profiling of circulating biomarkers offer novel diagnostic tools. Despite advances, challenges persist, including the limited predictive value of preclinical models and variable epigenetic profiles. Positioning epigenetics as the bridge between genetic risk, environmental triggers, and therapeutic advances, this review presents a framework for precision medicine in psoriasis.

Humans↗
PubMed1989

Image analysis of restriction enzyme fingerprint autoradiograms.

A genome mapping system has been developed that reads and assembles data from clones analysed by restriction enzyme fragmentation and polyacrylamide gel electrophoresis. Input data for the system can be most effectively obtained by the use of a scanning densitometer and image-processing package, such as that described in this article. The image-processing procedure involves preliminary location of bands, cooperative tracking of lanes by correlation of adjacent bands, a precise densitometric pass, alignment of the marker bands with the standard, optional interactive editing, and normalization of the accepted bands.

Animals↗
PubMed1992

Computer methods for locating kinetoplastid cryptogenes.

RNA editing in the mitochondria of kinetoplastid protoza involves the insertion and/or deletion of precise numbers of uridine residues at precise locations in the numbers of uridine residues at precise locations in the transcribed RNA of certain genes. These genes are known as cryptogenes. In this paper we study computational algorithms to search for unknown cryptogenes and for the associated templates for insertion of uridines, gRNA sequences. The pairwise similarity search algorithm of Smith and Waterman (1) is modified to study this problem. The algorithm searches for unknown gRNAs given the cryptogene sequence. The method is tested on 4 known cryptogenes from L.tarentolae which are known to have 7 associated gRNAs. The statistical distribution of the longest gRNA when comparing random sequences is derived. Finally we develop an algorithm to search for cryptogenes using amino acid sequences from related proteins.

Algorithms↗
PubMed2025

Tdrd15 is dispensable for male fertility and spermatogenesis in the golden hamster.

Tudor domain-containing proteins (TDRDs) constitute an evolutionarily conserved protein family and are critical for germline development and piRNA pathway regulation, with established roles in male fertility. While multiple TDRD family members have been functionally linked to spermatogenic impairment, the precise biological role of TDRD15 remains to be elucidated. We used CRISPR/Cas9-mediated gene editing to generate Tdrd15 knockout (KO) golden hamsters (Mesocricetus auratus), a model necessitated by the absence of a functional Tdrd15 ortholog in the mouse genome, to investigate its function in male reproduction. Phylogenetic analysis demonstrated that TDRD15 is strongly conserved among eutherian mammals, with testis-restricted expression patterns in hamsters. Despite the successful induction of frameshift mutations and significant transcriptional knockdown, Tdrd15 KO males maintained normal fertility parameters, including unaltered testicular architecture, spermatogenic progression (confirmed by periodic acidic-Schiff (PAS) staining and immunohistochemistry), and sperm quality metrics determined using a computer-assisted analysis. Quantitative polymerase chain reaction (qPCR) revealed compensatory overexpression of paralogous Tdrd genes in KO testes, implying functional redundancy within this protein family. This study provides the first experimental evidence that TDRD15 is dispensable for male fertility in golden hamsters under physiological conditions, thereby challenging the prevailing assumptions of its obligatory function in spermiogenesis. Altogether, these findings support a more targeted allocation of research efforts within the field of male reproductive biology.

Animals↗
PubMed1999

Kinetoplastid RNA editing does not require the terminal 3' hydroxyl of guide RNA, but modifications to the guide RNA terminus can inhibit in vitro U insertion.

During RNA editing in kinetoplastid parasites, trans-acting guide RNAs (gRNAs) direct the insertion and deletion of U residues at precise sites in mitochondrial pre-mRNAs. We show here that some modifications to the 3' terminal ribose of gRNA inhibit its ability to direct in vitro U insertion. However, we found that gRNAs lacking this moiety in some circumstances support in vitro editing. Thus, the 3' OH is not required. Inhibition resulting from gRNA modification can be overcome by increasing the gRNA-pre-mRNA base-pairing potential upstream of the editing site, suggesting an importance for this interaction to productive processing.

Animals↗
PubMed2026

Advances in organoids for personalized medicine: from technological development to clinical application.

Organoids, three-dimensional cell culture models derived from patient tissues or stem cells, have emerged as a cutting-edge technology in personalized medicine, owing to their remarkable ability to closely recapitulate in vivo tissue architecture and function. This review provides a comprehensive overview of the technological evolution and construction methodologies of organoids, highlighting their significant applications in oncology, genetic disorders, infectious diseases, and drug screening. This review examines how organoids enable precision medicine by preserving genomic fidelity, predicting drug sensitivity, and creating disease models via gene editing. Despite these advances, organoid technology faces several technical challenges that impede its full clinical translation. Addressing these obstacles is critical for realizing the potential of organoids in individualized therapeutic strategies. This article aims to delineate current progress and future directions in organoid research, furnishing a theoretical foundation and guiding future investigations towards enhancing personalized treatment paradigms.

disease modeling↗
PubMed2006

Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination.

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNA(Leu). The editing domain of LeuRS deacylates the mischarged Ile-tRNA(Leu) and Met-tRNA(Leu). We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 A, 2.4 A, and 3.2 A resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

Amino Acid Sequence↗