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PubMed2025

Refined and benchmarked homemade media for cost-effective, weekend-free human pluripotent stem cell culture.

BACKGROUND: Cost-effective, practical, and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However, the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover, we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly, the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. METHODS: We formulated B8 with commercially available, growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry, and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability, genome editing efficiency, single-cell cloning, and differentiation in both monolayer and organoids. Finally, we validated key findings using male (H1) and female (H9) human embryonic stem cells. RESULTS: hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast, cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time, B8+ led to gene expression changes indicative of marked lineage priming, reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state, consistent with intermittent media starvation. CONCLUSIONS: Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8, integrating some features of B8 while resembling cE8, emerges as a robust and practical option with limited compromises. B8+, though advantageous in some contexts, warrants caution due to lineage priming effects that may impact differentiation outcomes.

hiPSC; pluripotency; culture media; thermostable F↗

PubMed2004

The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase that edits acetyl primer units.

Type II polyketide synthases (PKSs) synthesize polyfunctional aromatic polyketides through iterative condensations of malonyl extender units. The biosynthesis of most aromatic polyketides is initiated through an acetate unit derived from decarboxylation of malonyl-acyl carrier protein (ACP). Modification of this primer unit represents a powerful method of generating novel polyketides. We have demonstrated that recombination of the initiation module from the R1128 PKS with heterologous elongation modules afforded regioselectively modified polyketides containing alternative primer units. With the exception of the role of the acyltransferase homologue ZhuC, the catalytic cycle of the initiation module has been well explored. ZhuC, along with the ketosynthase III homologue ZhuH and the ACP(p) ZhuG, is essential for the in vivo biosynthesis of aromatic polyketides derived from non-acetate primer units. Here we have studied the role of ZhuC using PKS proteins reconstituted in vitro. We show that the tetracenomycin (tcm) minimal PKS can be directly primed with non-acetate acyl groups. In the presence of approximately 10 microM hexanoyl-ZhuG or approximately 100 microM hexanoyl-CoA, the tcm minimal PKS synthesized hexanoyl-primed analogues of octaketides SEK4 and SEK4b, as well as acetate-primed decaketides SEK15 and SEK15b at comparable levels. Addition of ZhuC abolished synthesis of the acetate-primed decaketides, resulting in exclusive synthesis of the hexanoyl-primed octaketides. In the absence of alternative acyl donors, ZhuC severely retarded the activity of the tcm minimal PKS. The editing capabilities of ZhuC were directly revealed by demonstrating that ZhuC has 100 times greater specificity for acetyl- and propionyl-ACP as compared to hexanoyl- and octanoyl-ACP. Thus, by purging the acetate primer units that otherwise dominate polyketide chain initiation, ZhuC (and presumably its homologues in other PKSs such as the doxorubicin and frenolicin PKSs) allows alternative primer units to be utilized by the elongation module in vivo. The abilities of other alkylacyl primer units to prime the tcm minimal PKS were also investigated in this report.

Acylation↗

PubMed1996

The prevalence of mental disorders in rural office practice.

OBJECTIVE: To determine the prevalence of mental disorders in rural primary care office practice. DESIGN: Patient interview; chart review. SETTING: Two rural primary care office practices. PATIENTS: Three hundred-fifty scheduled or walk-in patients age 18 years or older. MEASUREMENTS: Medical Outcomes Study 36-Item Short-Form Health Survey (MOS SF-36), the Primary Care Evaluation of Mental Disorders (PRIME-MD), physical health using Greenfield's index of coexistent disease (ICED), and health care utilization using the number of office visits and total office and laboratory charges six months before until six months after the interview. RESULTS: Of these patients 34% met criteria for one or more of the 18 mental disorders evaluated by the PRIME-MD; 19% met criteria for specific disorders according to criteria from the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (DSM-III-R). Mood disorders were most common (21.7%), followed by anxiety disorders (12.3%), somatoform disorders (11.1%), probable alcohol abuse or dependence (6.0%), and eating disorders (2.0%). By logistic regression, there was an association of age, sex, race (black), and education with lower prevalence of various mental disorder categories. Even after adjustment for demographic variables and physical health (ICED score), those with PRIME-MD diagnoses had significantly lower function as measured by the eight MOS SF-36 scales and higher utilization of office services (p < .001). CONCLUSIONS: The prevalence of mental disorders in rural primary care office practice is as high as in urban office practice.

Adult↗

PubMed1998

Impaired mismatch extension by a herpes simplex DNA polymerase mutant with an editing nuclease defect.

The D368A mutation within the 3'-5'-exonuclease domain of the herpes simplex type 1 DNA polymerase inactivates this nuclease and severely interferes with virus viability. Compared with the wild type enzyme, the D368A mutant exhibits substantially elevated rates of incorrect nucleotide incorporation, as measured in a LacZ reversion assay. This high rate occurs in the presence of high levels of dNTPs, a condition that forces the enzyme to extend mismatched primers. Hence, the mutant fails to correct many misincorporations that are removed in the wild type. In addition, the mutant shows a much reduced ability to replicate DNA templates primed with a 3'-mismatch as compared with wild type. This extension defect also appears more severe than observed for replicases which naturally lack editing nucleases. Based on these findings, we suggest that the inability of the D368A herpes simplex mutant polymerase to replicate beyond a mismatched base pair severely inhibits viral replication.

Amino Acid Sequence↗

PubMed2026

Precision Engineering of Evolution-Resilient Rice against Bacterial Blight.

The persistent conflict between rice and Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight, exemplifies a dynamic genetic arms race in agriculture. The cyclical deployment and erosion of major resistance (R) genes highlight the high adaptive potential of Xoo and the need for strategies that are durable rather than absolute. This review synthesizes a paradigm shift from reactive, single R-gene deployment toward proactive engineering of evolution-resilient resistance. We explore the molecular-genetic basis of Xoo adaptability, including TAL effector diversification, non-TAL virulence functions, genome variation, and immune suppression mechanisms. In response, we propose a framework for durable disease management with three connected components: precision disarmament through editing of susceptibility-gene effector-binding elements and executor/decoy designs; smart induction through targeted delivery and immune priming; and ecological fortification through protective microbiomes. We also discuss the limits, trade-offs, and field-validation requirements of these approaches. Integrating frontier technologies with evolutionary genetics, predictive genomics, and pathogen population dynamics can help develop rice varieties and deployment systems that are more difficult for Xoo populations to overcome.

CRISPR↗

PubMedDate not supplied

Changes in the biochemical oxygen demand procedure in the 21st edition of Standard Methods for the Examination of Water and Wastewater.

The dilution biochemical oxygen demand (BOD) test has widespread application for design and operation of wastewater treatment processes, evaluating the quality of natural waters, and assessing the effect of wastewater discharges on these waters. While standardization of the BOD-measuring method has become of prime importance in maintaining dependable data acquisition, changes are made as needed in response to questions raised by analysts and to accommodate new applications. The purpose of this article is to describe changes that have been incorporated in the 20th and 21st editions of Standard Methods for the Examination of Water and Wastewater (APHA et al., 1998 and 2004). These changes include changes in text format to clarify the procedural steps, allowance for use of bottle sizes ranging from 60 mL or larger, improvements in quality-control procedures, and improvements in the method of calculating BOD. Other changes include allowance for the use of allylthiourea for nitrification inhibition and broadening the source of seed that can be used for inoculation of BOD samples.

Biodegradation, Environmental↗

PubMed2004

Methods for analysis of mitochondrial tRNA editing in Acanthamoeba castellanii.

We present here methodology for assaying 5'-terminal editing of mitochondrial tRNAs in the amoeboid protist Acanthamoeba castellanii. This type of editing involves replacement of one or more nucleotides within the first three positions at the 5' end of a tRNA substrate. The assay procedure involves RNA ligase-mediated joining of the 5' and 3' ends of a tRNA, use of the resulting circularized tRNA as template for cDNA synthesis primed by tRNA-specific primers over a region that encompasses the ligated 5' and 3' halves of the acceptor stem, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of double-stranded cDNA product. This approach has the advantage that it simultaneously reveals potential editing events on the 5' and 3' side of an acceptor stem, as well as serves to identify mature tRNAs (characterized by having a 3'-CCAOH motif) and partially processed intermediates.

Acanthamoeba↗

PubMed1999

Role of APC in the selection of immunodominant T cell epitopes.

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.

Amino Acid Sequence↗

PubMed1983

Scene perception: a failure to find a benefit from prior expectancy or familiarity.

In our everyday world, we typically have an expectancy as to the kinds of scenes that we will see from one glance to the next. Also, many of the scenes that we do see are familiar in the sense that they have been experienced before. Do these factors influence the perception of a scene? In three experiments, priming subjects with a verbal descriptor of a scene was not found to improve reliably the perception of that scene as assessed by the speed and accuracy of detecting an incongruity between an object and its setting (Experiments 1 and 2) or a specified target object (Experiment 3). Also, in attempting to perceive these scenes, subjects could not capitalize on the residue from prior exposures of a scene's background, even though those backgrounds had been processed to the point where semantic information had been extracted from them. Although these results are inconsistent with recent speculations on the role of frames in scene perception, recent experiments on the perception of a scene from a single fixation, and film-editing practice with "flash cuts." The implications of these results are that the mechanisms for perceiving and interpreting nondegraded real-world scenes are so quick and efficient that conditions can readily be found in which priming and prior exposures of substantial portions of scenes are not helpful for perceiving and judging certain aspects of those scenes.

Cognition↗

PubMed2025

Cell-type-specific response to silicon treatment in soybean leaves revealed by single-nucleus RNA sequencing and targeted gene editing.

Mineral nutrient uptake and deposition profoundly influence plant development, stress resilience, and productivity. Silicon (Si), though classified as a non-essential element, significantly influences a plant's physiology, particularly in fortifying defense responses and mitigating stress. While the genetic and molecular mechanisms of Si uptake and transport are well studied in monocots, particularly rice, their role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. We identified distinct cellular populations, including a unique Si-induced or Si-associated cell cluster within vascular cells, suggesting a specialized mechanism of Si distribution. Si treatment notably induced the expression of defense-related genes, with a pronounced enrichment in vascular cells, underscoring their pivotal role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in phytoalexin biosynthesis, salicylic acid, and immune receptor signaling, suggesting transcriptional priming of genes involved in defense responses. Further investigation of Si transporters revealed precise expression of an Si efflux gene in epidermal cells in response to Si treatment. We also validated the role of efflux Si transporters using a Xenopus oocyte assay and CRISPR/Cas9 genome editing of composite soybean plant roots. This study provides critical insights into the biotic stress regulatory networks influenced by Si treatment in soybean leaves at the single-cell level, thus laying the foundation for enhancing stress tolerance through optimized mineral nutrient uptake.

Glycine max↗

PubMed2004

Bangkok 2004. In Thailand, drug users have to fight for their rights.

Injection drug users have been overlooked in the Thai government's response to HIV/AIDS, but not in its war on drugs. They experience constant police harassment and ineffective services, and face stigma and discrimination in society and in the health-care setting. Paisan Suwannawong, of the Thai Drug Users' Network, was scheduled to speak at the Opening Ceremonies of the XV International AIDS Conference in Bangkok on 11 July 2004. But he was placed last on a very long agenda that included Thai Prime Minister Thaksin Shinawatra, United Nations Secretary-General Kofi Annan, and Miss Universe. By the time he spoke, all the dignitaries and almost all the delegates had left the hall. In this edited version of his address, Paisan provides a personal testimonial about what it is like to be a drug user living with HIV/AIDS in Thailand, outlines the problems with the government's response, and describes other obstacles to delivering treatment and other services to drug users.

Human Rights↗

PubMedDate not supplied

Detection and treatment of depressive syndromes in a rural island clinic.

BACKGROUND: Major depression is a serious and often persistent problem for 5 to 10 percent of patients in primary care. The detection and treatment of depression can be a particular challenge in rural settings. This study describes patterns of care for depression at a rural primary care clinic on an island off the coast of Washington State. METHODS: For a period of 2 months, 226 primary care patients were evaluated for depression using a two-stage screening method. Structured chart abstraction was conducted for patients meeting criteria for major depression or dysthymia. RESULTS: Ten percent of the study sample met Diagnostic and Statistical Manual of Medical Disorders, fourth edition, criteria for major depression, 4 percent met criteria for dysthymia, and 8 percent met criteria for major depression and dysthymia. Of the patients who had a diagnosis of major depression or dysthymia by the PRIME-MD, approximately 50 percent had their condition detected by their primary care provider, and approximately 50 percent received guideline level antidepressant treatment. Less than 40 percent of patients whose depression was treated with antidepressants made more than two visits during the 8 weeks after the initial prescription. CONCLUSIONS: These findings are consistent with those from other primary care settings and suggest that there are many opportunities for improving the quality of care for depressed patients in rural primary care settings.

Adult↗

PubMed2026

Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.

Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.

epidermolysis bullosa (EB)↗

PubMed2026

Humanizing acidic mammalian chitinase variants establish lung immune conditioning and control environmentally driven inflammation and fibrosis.

Chitin, a widespread environmental particle constituent, triggers lung inflammation but is degraded by chitinases. In humans, single-nucleotide polymorphisms (SNPs) in CHIA (acidic mammalian chitinase; AMCase) are associated with lung disease, suggesting that chitinase variants influence responses to airborne particles. Here, we edit the mouse Chia1 locus to generate humanized (hChia) mice harboring common human SNPs. Compared with controls expressing disease-protective SNPs, hChia mice lack robust chitinase activity and fail to degrade natural chitin substrates. Lung-resident lymphocytes and macrophages are spontaneously primed and sensitive to inflammatory triggering by environmental chitin. Immune cell infiltration correlates with airway chitin following challenge, and hChia mice exhibit exacerbated inflammatory and fibrotic lung disease. In humans with acute respiratory failure, alveolar hemorrhage coincides with environmentally derived chitin particles that are susceptible to chitinase degradation, attenuating inflammatory cell responses. Thus, environmental chitin and chitinase activity are crucial determinants of lung immune conditioning with potential therapeutic applications.

AMCase↗

PubMed2004

Genesis of the strand-biased signature in somatic hypermutation of rearranged immunoglobulin variable genes.

The history and current development of the reverse transcriptase model of somatic hypermutation (RT-model) is reviewed with particular reference to the genesis of strand-biased mutation signatures in rearranged immunoglobulin variable genes (V(D)J). The recent disagreement in the field as to whether strand bias really exists or not has been critically analysed and the confusion traced to the putative presence, in some mutated V(D)J sequence collections, of polymerase chain reaction (PCR)-recombinant artefacts. Recent analysis of somatic hypermutation in xeroderma pigmentosum variant patients, by the group of PJ Gearhart and others, has established that the Y-family translesion DNA repair enzyme, DNA polymerase eta (eta), is responsible for the striking A-T targeted strand-bias mutation signature seen in all mouse and human collections of somatically mutated V(D)J sequences. This evidence, together with our own recent demonstration that human DNA polymerase eta is a reverse transcriptase, leads to the conclusion that the strand-biased A-T mutation signature is caused either by: (i) error-prone DNA-dependent DNA repair synthesis by pol-eta of single-strand nicks preferentially in the non-transcribed strand; and/or (ii) by error-prone cDNA synthesis of the transcribed strand by pol-eta using the pre-mRNA as the copying template, primed by the nicked transcribed DNA strand, followed by replacement of the original transcribed strand by cDNA. Analysis of the total mutation pattern also suggests that the major transitions observed in SHM (A-->G, C-->T and G-->A) can be explained by known RNA editing mechanisms active on pre-mRNA which are then written into cDNA during synthesis of the transcribed strand by error-prone cellular reverse transcriptases such as pol-eta.

Animals↗

PubMed2002

Prime-sight and blindsight.

Listening to subject's commentaries can be a useful spur to novel scientific departures, as in studies of blindsight. Recently further testing (after a gap of 17 years) has been possible with subject DB, who was a blindsight patient (the first) tested intensively over a period of 10 years and who was the subject of the book, (Blindsight OUP, 1986, 2nd edition, 1998). Essentially his original capacity is the same or somewhat more sensitive. Some further types of discriminations have now been tested that were not possible in the original study. But a new feature was discovered, namely that he describes conscious after-images of a wide range of inducing stimuli of which he is unconscious. The properties of the after-images are briefly described, such as their occurrence following unseen colour patches, luminance changes, shape, spatial frequency, their long durations, showing inter-ocular transfer, as well as their conforming to Emmert's Law. Various approaches are discussed towards finding the source of their generation, such as the use of after-images of colour mixes of separate inputs to the two eyes ("cortical yellow"). They are also discussed in terms of after-images in normal subjects that are generated by imagination. The discovery offers a rare potential opportunity to compare the brain activity associated with both the conscious and unconscious attributes of the precisely the same stimuli in the same locus in the same subject.

Adult↗

PubMed2002

Natural killer cells and dendritic cells: rendezvous in abused tissues.

Natural killer (NK) cells and dendritic cells (DCs) are two types of specialized cell of the innate immune system, the reciprocal interaction of which results in a potent, activating cross-talk. For example, DCs can prime resting NK cells, which, in turn, after activation, might induce DC maturation. However, NK cells negatively regulate the function of DCs also by killing immature DCs in peripheral tissues. Moreover, a subset of NK cells, after migration to secondary lymphoid tissues, might have a role in the editing of mature DCs based on the selective killing of mature DCs that do not express optimal surface densities of MHC class I molecules. So, cognate interactions between NK cells and DCs provide a coordinated mechanism that is involved not only in the regulation of innate immunity, but also in the promotion of appropriate downstream adaptive responses for defence against pathogens.

Animals↗

PubMed2026

Ultra-sensitive profiling of CRISPR-Cas off-target effects with Tracking-seq2.

Accurate detection of off-target activity in primary human cells is crucial for ensuring the safety of gene therapies, yet existing methods often lack sufficient sensitivity. To address this limitation, we develop Tracking-seq2, an advanced technology that integrates exogenous 5' → 3' exonuclease treatment and non-homologous end joining (NHEJ) pathway inhibitors with the original Tracking-seq. Tracking-seq2 exhibits enhanced sensitivity in profiling off-target sites of diverse genome editors-including Cas9, Cas12a, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Critically, Tracking-seq2 is directly applicable to clinically relevant primary human cell types, such as T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Furthermore, our findings reveal that genomic variations drive distinct off-target heterogeneity across different individuals, highlighting the necessity for personalized safety assessment in clinical genome editing applications. Tracking-seq2 provides a robust platform for sensitive off-target detection in primary cells, with sensitivity comparable to or exceeding current state-of-the-art methods.

Humans↗