Cooperative assembly confers regulatory specificity and long-term genetic circuit stability.

A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits "wired" using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.

Modular design of synthetic receptors for programmed gene regulation in cell therapies.

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

Here to stay: Writing lasting epigenetic memories.

In this issue of Cell, Nuñez et al. develop CRISPRoff, a programmable epigenetic memory writer capable of establishing specific gene silencing programs that are stably maintained across cell division and differentiation. The singular dCas9 fusion offers a simple, reliable, and general tool for genome-wide screens, multiplexed editing, and potential therapeutics.

Engineering Epigenetic Regulation Using Synthetic Read-Write Modules.

Chemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating "read-write" architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.

A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation.

The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-up approach to investigating chromatin-mediated transcriptional regulation and introduces chromatin-based components and systems for synthetic biology and cellular engineering.

A synthetic biology framework for programming eukaryotic transcription functions.

Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.

Human herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: network analysis identifies interacting pathways.

Aggregation of TAR DNA-binding protein 43 kDa (TDP-43) is thought to drive the pathophysiology of amyotrophic lateral sclerosis and some frontotemporal dementias. TDP-43 is normally a nuclear protein that in neurons translocates to the cytoplasm and can form insoluble aggregates upon activation of the integrated stress response (ISR). Viruses evolved to control the ISR. In the case of Herpesvirus 8, the protein ORF57 acts to bind protein kinase R, inhibit phosphorylation of eIF2α and reduce activation of the ISR. We hypothesized that ORF57 might also possess the ability to inhibit aggregation of TDP-43. ORF57 was expressed in the neuronal SH-SY5Y line and its effects on TDP-43 aggregation characterized. We report that ORF57 inhibits TDP-43 aggregation by 55% and elicits a 2.45-fold increase in the rate of dispersion of existing TDP-43 granules. These changes were associated with a 50% decrease in cell death. Proteomic studies were carried out to identify the protein interaction network of ORF57. We observed that ORF57 directly binds to TDP-43 as well as interacts with many components of the ISR, including elements of the proteostasis machinery known to reduce TDP-43 aggregation. We propose that viral proteins designed to inhibit a chronic ISR can be engineered to remove aggregated proteins and dampen a chronic ISR.

A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response.

Recently, more than 1000 large intergenic noncoding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediates apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulate their localization to sets of previously active genes.

Outcomes of a novel algorithm for levator muscle plication surgery in congenital blepharoptosis.

AIM: to assess the outcomes of a novel algorithm for the calculation of the amount levator muscle plication in congenital blepharoptosis surgery. METHODS: this retrospective comparative study included 34 patients with congenital ptosis subjected to levator muscle plication surgery during the period from October 2021 to November 2022. They were divided into two groups. Group A: the amount of levator muscle plication was calculated by a traditional formula [(amount of ptosis x 3) + 9 mm in cases with good levator function or (amount of ptosis x 3) + 11 mm in cases with fair levator function]. Group B: the amount of levator muscle plication was calculated by a novel nomogram [the result of the traditional formula was modified by subtracting 4 mm if the calculated amount was ≥ 15 mm or subtracting 3 mm if the calculated amount was < 15 mm]. Demographic data, baseline ptosis characteristics and postoperative results at 1st week, 1st month, 3rd month and 6th month were compared between the groups. Primary outcome measure was postoperative Marginal Reflex Distance (MRD1). Secondary outcome measures were lid contour, lid crease and any reported complications. RESULTS: Group A included 20 eyes of 18 patients while Group B included 20 eyes of 16 patients. The mean amount of levator muscle plication was 16.98 ± 2.44 mm and 13.48 ± 2.42 mm in group A and group B respectively. The difference between the two groups was highly statistically significant (p < 0.001). Mean MRD1 at the 1st postoperative week was 4.95 ± 0.37 mm in group A and 4.08 ± 0.64 mm in group B. This difference was highly statistically significant (P < 0.001). Overcorrection was seen in 8 (40%) eyes in group A and 1 (5%) eye in group B. The difference was statistically significant between the two groups (p = 0.008). Undercorrection was seen in only 1 (5%) eye in group B. No other complications were reported. Surgical success was achieved in 12 (60%) eyes in group A versus 18 (90%) eyes in group B. The difference between the two groups was statistically significant (p = 0.03). CONCLUSION: our novel nomogram for the calculation of the amount levator muscle plication in congenital blepharoptosis surgery is effective in achieving a satisfactory postoperative MRD1.

Bicuspid Aortic Valve Function and Aortopathy on Presentation and Progression in Children: Does Sex Difference Have Any Implications?

Bicuspid aortic valve (BAV) is the most common congenital heart defect. It can be accompanied by aortic regurgitation or stenosis with aortopathies. Studies in adults showed a sex difference, but there are limited number of reports in the pediatric population. To evaluate the difference in bicuspid aortic valve morphology and functionality between sexes, and the presence and progression of aortopathies, a retrospective chart review study was performed at a tertiary referral care center in the Midwest. In our study, we analyzed a cohort of 476 pediatric patients diagnosed with BAV who presented between January 2007 and February 2018. During the follow-up period spanning 2 to 10 years, male patients (n = 314, 66%) had larger aortic valve annulus (AVA) and sinus of Valsalva (SOV) at the time of initial presentation with more likelihood for progression. In the subgroup analysis, the larger SOV in males was observed in isolated BAV patients without genetic syndromes or cardiac malformations, and there were no significant differences between both sexes in the ascending aorta dimension, valve functionality, valve morphology, and the need for intervention in any of the studied groups. As such, these findings may alter the follow-up focus and frequency for patients with BAV, particularly before adulthood, and warrant further studies.

Structural Mapping of Protein Aggregates in Live Cells Modeling Huntington's Disease.

While protein aggregation is a hallmark of many neurodegenerative diseases, acquiring structural information on protein aggregates inside live cells remains challenging. Traditional microscopy does not provide structural information on protein systems. Routinely used fluorescent protein tags, such as Green Fluorescent Protein (GFP), might perturb native structures. Here, we report a counter-propagating mid-infrared photothermal imaging approach enabling mapping of secondary structure of protein aggregates in live cells modeling Huntington's disease. By comparing mid-infrared photothermal spectra of label-free and GFP-tagged huntingtin inclusions, we demonstrate that GFP fusions indeed perturb the secondary structure of aggregates. By implementing spectra with small spatial step for dissecting spectral features within sub-micrometer distances, we reveal that huntingtin inclusions partition into a ß-sheet-rich core and a ɑ-helix-rich shell. We further demonstrate that this structural partition exists only in cells with the [RNQ+] prion state, while [rnq-] cells only carry smaller ß-rich non-toxic aggregates. Collectively, our methodology has the potential to unveil detailed structural information on protein assemblies in live cells, enabling high-throughput structural screenings of macromolecular assemblies.

Enhancing nutritional niche and host defenses by modifying the gut microbiome.

The gut microbiome is essential for processing complex food compounds and synthesizing nutrients that the host cannot digest or produce, respectively. New model systems are needed to study how the metabolic capacity provided by the gut microbiome impacts the nutritional status of the host, and to explore possibilities for altering host metabolic capacity via the microbiome. Here, we colonized the nematode Caenorhabditis elegans gut with cellulolytic bacteria that enabled C. elegans to utilize cellulose, an otherwise indigestible substrate, as a carbon source. Cellulolytic bacteria as a community component in the worm gut can also support additional bacterial species with specialized roles, which we demonstrate by using Lactobacillus plantarum to protect C. elegans against Salmonella enterica infection. This work shows that engineered microbiome communities can be used to endow host organisms with novel functions, such as the ability to utilize alternate nutrient sources or to better fight pathogenic bacteria.

QTc prolongation during levofloxacin and triazole combination chemoprophylaxis: Prevalence and predisposing risk factors in a cohort of hematopoietic cell transplantation recipients.

BACKGROUND: QTc interval prolongation has been reported when combining fluoroquinolones and triazoles for chemoprophylaxis in cancer patients. Herein, we aimed to identify the prevalence and contributing factors to QTc prolongation in hematopoietic cell transplantation (HCT) recipients who received these agents during the neutropenic phase. METHODS: This is a retrospective medical chart review conducted at a university hospital in Lebanon from 2017 to 2020. It included all adult HCT inpatients on antimicrobial prophylaxis with fluoroquinolones and triazoles and whose baseline ECG monitoring done prior to chemoprophylaxis administration, then on day-3 and day-6 of therapy, were available. RESULTS: Overall, 68 HCT recipients met our inclusion criteria, of which 22% developed QTc prolongation. Based on bivariate analysis, female gender contributed to QTc prolongation (P = 0.001). There was a trend to QTc prolongation in patients with predisposing thyroid disease (P = 0.12), grade 2 vomiting and diarrhea (P = 0.16, P = 0.46, respectively), baseline hypokalemia (P = 0.18) and hypocalcemia (P = 0.3), hypomagnesemia on day-3 (P = 0.21) and day-6 hyponatremia (P = 0.36). Patients receiving two or more drugs with a known or probable risk of QTc prolongation (other than the fluoroquinolone/ triazole combination) were more prone to experience a prolonged QTc interval (P = 0.09). None of the patients that had QTc prolongation died or developed serious arrhythmias. CONCLUSION: The prevalence of QTc prolongation was 22% among HCT recipients on fluoroquinolone and triazole prophylaxis, yet we did not identify any independent risk factors for this issue. None of the patients that had QTc interval prolongation died or developed serious arrhythmias.

The Changing Face of the Cornea in a Case of Juvenile Glaucoma and Subclinical Keratoconus.

We report on a 23-year-old man who presented with bilateral subclinical keratoconus and juvenile glaucoma (JG). With intraocular pressures (IOPs) of 30 and 28 mmHg, both eyes were consecutively operated by adjusted trabeculotomy, leading to a remarkable decrease in IOP to well below the mean for this surgery in JG. Meanwhile, most keratoconus indices clearly progressed in the first 5 months postoperatively, with increases in corneal hysteresis, associated with a remarkable drop in the corneal resistance factor. During the following years, IOP remained low, and all changes (except the increase in corneal hysteresis) reverted to near preoperative levels through the follow-up course of 5 years. This report complements a few existing reports that show the coincidence of keratoconus and JG, and, more importantly, documents a novel pattern of remarkable and prolonged corneal changes following surgical lowering of IOP in eyes with these changes. Postoperative biomechanical disturbances in the cornea and possibly limbus are proposed in cases of JG and subclinical keratoconus.

Chromatin regulation at the frontier of synthetic biology.

As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.

Outcomes of adjusted trabeculotomy in cases with juvenile glaucoma.

PURPOSE: To evaluate surgical and visual outcomes of modified adjusted trabeculotomy in juvenile glaucoma (JG) cases. METHODS: A retrospective case series; medical records of 43 patients (69 eyes) JG cases operated by adjusted trabeculotomy between 2011 and 2018. Those who completed a minimum of 1 year of regular follow-up, and up to 5 years were included in the study. Intraocular pressure (IOP), number of medications, cup/disc ratio (CDR), and visual acuity (VA) at baseline, postoperative 1, 3, 5 years were evaluated. Success required IOP ≤ 18 mmHg and a minimum of 20% reduction, without medications (full), or with medications (qualified). RESULTS: Numbers of patients (eyes) who completed 1, 3, and 5 follow-up years were as follows: 26 (43), 15 (27), and 11 (19). Median (range) age at surgery was 21.5 (8-43) years. Mean (range) IOP was significantly (P < .001) reduced from 23.4 ± 8.8 (11.0-46.0) mmHg to 11.5 ± 3.5 (7.0-28.0), 10.9 ± 3.6 (6.0-24.0), and 11.4 ± 3.0 (7.0-17.0) mmHg at 1, 3, and 5 years, with reduction of medication scores. At years 1, 3, and 5, complete success rates were 89.5%, 86.8%, and 89.5%, and qualified success rates were 97.4%, 94.7%, and 97.4%. Median (range) LogMAR VA significantly (P < .001) improved from 0.3 (0-2.8) to 0.17 (0-2.8) and 0.17 (0-2.8) at 1 and 5 years. Median (range) CDR was significantly (P < .001) reduced from 0.85 (0.3-1.0) to 0.85 (0.1-1.0), 0.7 (0.05-1.0), and 0.7 (0.05-0.9) at 1, 3, and 5 years. CONCLUSIONS: Adjusted trabeculotomy could effect remarkable IOP lowering for up to 5 years postoperatively in JG eyes, and seems to be an effective, low-risk surgical modality for treating such eyes. It can be associated with cupping reversibility, and visual improvement on the long-term. Good surgical technique and postoperative care are imperative to achieve a successful outcome.

Protein assembly systems in natural and synthetic biology.

The traditional view of protein aggregation as being strictly disease-related has been challenged by many examples of cellular aggregates that regulate beneficial biological functions. When coupled with the emerging view that many regulatory proteins undergo phase separation to form dynamic cellular compartments, it has become clear that supramolecular assembly plays wide-ranging and critical roles in cellular regulation. This presents opportunities to develop new tools to probe and illuminate this biology, and to harness the unique properties of these self-assembling systems for synthetic biology for the purposeful manipulation of biological function.

Sphingomonas solaris sp. nov., isolated from a solar panel in Boston, Massachusetts.

Solar panel surfaces, although subjected to a range of extreme environmental conditions, are inhabited by a diverse microbial community adapted to solar radiation, desiccation and temperature fluctuations. This is the first time a new bacterial species has been isolated from this environment. Strain R4DWNT belongs to the genus Sphingomonas and was isolated from a solar panel surface in Boston, MA, USA. Strain R4DWNT is a Gram-negative, non-motile and rod-shaped bacteria that tested positive for oxidase and catalase and forms round-shaped, shiny and orange-coloured colonies. It is mesophilic, neutrophilic and non-halophilic, and presents a more stenotrophic metabolism than its closest neighbours. The major fatty acids in this strain are C18:1ω7c/C18:1ω6c, C16:1ω7c/C16:1ω6c, C14:0 2OH and C16:0. Comparison of 16S rRNA gene sequences revealed that the closest type strains to R4DWNT are Sphingomonas fennica, Sphingomonas formosensis, Sphingomonas prati, Sphingomonas montana and Sphingomonas oleivorans with 96.3, 96.1, 96.0, 95.9 and 95.7 % pairwise similarity, respectively. The genomic G+C content of R4DWNT is 67.9 mol%. Based on these characteristics, strain R4DWNT represents a novel species of the genus Sphingomonas for which the name Sphingomonas solaris sp. nov. is proposed with the type strain R4DWNT (=CECT 9811T=LMG 31344T).

LncRNAs in molluscan and mammalian stages of parasitic schistosomes are developmentally-regulated and coordinately expressed with protein-coding genes.

Despite the low level expression of some long noncoding RNAs (lncRNAs), the differential expression of specific lncRNAs plays important roles during the development of many organisms. Schistosomes, parasitic flatworms that are responsible for schistosomiasis, infects over 200 million people resulting in chronic disease and hundreds of thousands of deaths. Schistosomes have a complex life cycle that transitions between molluscan and mammalian hosts. In a molluscan snail host, the sporocyst stage develops over 5 weeks undergoing asexual reproduction to give rise to free-swimming and infectious cercariae that penetrate human skin and eventually mature into egg producing worms in mammals. The tight integration of the sporocyst to the snail host hepatopancreas hinders the -omics study in the molluscan stage, so the sporocyst transcriptome has only been examined for lncRNAs in immature in vitro samples. Here we analyzed the in vivo mature sporocyst transcriptome to identify 4,930 total lncRNAs between the molluscan and mammalian stages of the parasite. We further demonstrate that the lncRNAs are differentially expressed in a development-dependent manner. In addition, we constructed a co-expression correlation network between lncRNAs and protein-coding (PC) genes that was used to identify clusters of lncRNA transcripts with potential functional relevance. We also describe lncRNA-lncRNA and lncRNA-kinome correlations that identify lncRNAs with prospective roles in gene regulation. Finally, our results show clear differential expression patterns of lncRNAs in host-dependent development stages of S. mansoni and ascribe potential functional roles in development based on predicted intracellular interaction.