Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Population Diversity at the Single-Cell Level.

Population-scale single-cell genomics is a transformative approach for unraveling the intricate links between genetic and cellular variation. This approach is facilitated by cutting-edge experimental methodologies, including the development of high-throughput single-cell multiomics and advances in multiplexed environmental and genetic perturbations. Examining the effects of natural or synthetic genetic variants across cellular contexts provides insights into the mutual influence of genetics and the environment in shaping cellular heterogeneity. The development of computational methodologies further enables detailed quantitative analysis of molecular variation, offering an opportunity to examine the respective roles of stochastic, intercellular, and interindividual variation. Future opportunities lie in leveraging long-read sequencing, refining disease-relevant cellular models, and embracing predictive and generative machine learning models. These advancements hold the potential for a deeper understanding of the genetic architecture of human molecular traits, which in turn has important implications for understanding the genetic causes of human disease. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 25 is August 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Therapeutic effect of intraperitoneal dexamethasone on noise-induced permanent threshold shift in mice model.

The purpose of the study was to which investigate whether dexamethasone, which has anti-inflammatory and immune response suppression roles, could treat noise-induced hearing loss caused by damage to hair cells in the cochlea. The experiment used 8-week-old CBA mice exposed to white noise at an intensity of 110 dB SPL for 2 h, with hearing loss confirmed by the auditory brainstem response test. Dexamethasone was administered by intraperitoneal injection for 5 days, and the therapeutic effect was investigated for 3 weeks. The experimental groups were 3 mg/kg of dexamethasone (3 mpk) and 10 mg/kg of dexamethasone (10 mpk), and the control group was a saline-administered group. The results showed that compared to the control group, the hearing threshold value was recovered by 10 dB SPL compared to the saline group from the 14th day in the 3 mpk group. In the 10 mpk group, thresholds were recovered from the 7th day compared to the saline group. This difference was similar at 4 kHz, and in the case of the 10 mpk group, the threshold was recovered by 20 dB SPL compared to the saline group. The study also confirmed the restoration of nerve cell activity and showed a recovery effect of about 20 µV in the amplitude value change in the 10 mpk group. In conclusion, the study suggests that dexamethasone has a therapeutic effect for noise-induced hearing loss by increasing the activity of nerve cells and showing a recovery effect from hair cells damaged by noise.

Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.

Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb mld biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.

Clinical Implications of Cardiac Symptoms and Electrocardiographic Abnormalities for Advanced Liver Fibrosis in Patients with Nonalcoholic Fatty Liver Disease.

Background and Objectives: Advanced liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD) can be a major predictor of cardiovascular disease (CVD) events and cardiac complications. However, the clinical significance of cardiac symptoms and abnormal electrocardiography (ECG) findings in patients with NAFLD associated with advanced liver fibrosis is unclear. Therefore, our study was aimed to evaluate the clinical implications based on the association between cardiac symptoms with ECG abnormalities for advanced liver fibrosis in patients with NAFLD. Materials and Methods: Of 31,795 participants who underwent health checkups, 6293 were diagnosed with NAFLD using ultrasound and inclusion criteria in a retrospective cross-sectional study. Advanced liver fibrosis was assessed based on a low NAFLD fibrosis score (NFS) and fibrosis-4 index (Fib-4) cut-off values (COVs). Cardiac data were assessed using a cardiac symptom questionnaire and 12-lead electrocardiography (ECG). Results: Among 6293 NAFLD patients with NAFLD, 304 (4.8%) experienced cardiac symptoms. NFS and Fib-4 indicated higher rates of advanced fibrosis in the cardiac-symptomatic group than in the non-symptomatic group (NFS: 7.3 vs. 4.1%; Fib-4: 7.8 vs. 3.7%; both p < 0.001). Cardiac symptoms were independently associated with advanced liver fibrosis using a step-wise-adjusted model and NFS and Fib-4 (final adjusted odds ratio (aOR), 1.40; 95% CI, 1.06-1.85; p = 0.018 for NFS; aOR, 1.67; 95%, 1.30-2.15; p < 0.001 for Fib-4). Cardiac symptoms with abnormal ECG findings independently predicted advanced liver fibrosis (aOR, 2.43; 95% CI, 1.72-3.39; p < 0.001 for NFS; aOR, 3.02; 95% CI, 2.19-4.15; p < 0.001 for Fib-4). Conclusions: Patients who have had cardiac symptoms and some ECG abnormalities may have a higher association with advanced liver fibrosis.

Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica.

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

Chlororesistoflavins A and B, Chlorinated Benzopyrene Antibiotics Produced by the Marine-Derived Actinomycete Streptomyces sp. Strain EG32.

As part of a collaborative biomedical investigation of actinomycete bacteria isolated from sediments collected along the northern coast of Egypt (Mediterranean Sea), we explored the antibacterial metabolites from a bacterium identified as a Streptomyces sp., strain EG32. HPLC analysis and antibacterial testing against methicillin-resistant Staphylococcus aureus (MRSA) resulted in the identification of six compounds related to the resistoflavin and resistomycin class. Two of these metabolites were the chlorine-containing analogues chlororesistoflavins A (1) and B (2). The absolute configurations of the lone stereogenic center (C-11b) in these metabolites were assigned by analysis of their ECD spectra. Interestingly, the ECD spectrum of chlororesistoflavin A (1) shows a Cotton effect of the n-π* transition antipodal to that of the parent natural product, a consequence of 1,3-allylic strain induced by the adjacent bulky chlorine atom that distorts the coplanarity of the carbonyl group with the π-system. The chiroptical analysis thus resolves the paradox and uniformly aligns the configuration of all analogues as identical to that reported for natural resistoflavin. Chlororesistoflavins A (1) and B (2) exhibited antibacterial activity against MRSA with a minimum inhibitory concentration of 0.25 and 2.0 µg/mL, respectively.

Discovery and Biosynthesis of Tetrachlorizine Reveals Enzymatic Benzylic Dehydrogenation via an ortho-Quinone Methide.

Ortho-quinone methides (o-QMs) are reactive intermediates in biosynthesis that give rise to a variety of intra- and intermolecular cyclization/addition products in bacteria, fungi, and plants. Herein, we report a new metabolic deviation of an o-QM intermediate in a benzylic dehydrogenation reaction that links the newly described marine bacterial natural products dihydrotetrachlorizine and tetrachlorizine. We discovered these novel dichloropyrrole-containing compounds from actinomycete strain AJS-327 that unexpectedly harbors in its genome a biosynthetic gene cluster (BGC) of striking similarity to that of chlorizidine, another marine alkaloid bearing a different carbon skeleton. Heterologous expression of the homologous flavin-dependent oxidoreductase enzymes Tcz9 and Clz9 revealed their native functions in tetrachlorizine and chlorizidine biosynthesis, respectively, supporting divergent oxidative dehydrogenation and pyrrolizine-forming reactions. Swapping these berberine bridge enzyme-like oxidoreductases, we produced cyclized and dehydrogenated analogs of tetrachlorizine and chlorizidine, including a dearomatized chlorizidine analog that stabilizes an o-QM via conjugation with a 3H-pyrrolizine ring.

Marinoterpins A-C: Rare Linear Merosesterterpenoids from Marine-Derived Actinomycete Bacteria of the Family Streptomycetaceae.

The chemical examination of two undescribed marine actinobacteria has yielded three rare merosesterterpenoids, marinoterpins A-C (1-3, respectively). These compounds were isolated from the culture broth extracts of two marine-derived actinomycetes associated with the family Streptomycetaceae, (our strains were CNQ-253 and AJS-327). The structures of the new compounds were determined by extensive interpretation of 1D and 2D NMR, MS, and combined spectroscopic data. These compounds represent new chemical motifs, combining quinoline-N-oxides with a linear sesterterpenoid side chain. Additionally, consistent in all three metabolites is the rare occurrence of two five-ring ethers, which were derived from an apparent cyclization of methyl group carbons to adjacent hydroxy-bearing methylene groups in the sesterterpenoid side chain. Genome scanning of AJS-327 allowed for the identification of the marinoterpin (mrt) biosynthetic cluster, which consists of 16 open-reading frames that code for a sesterterpene pyrophosphate synthase, prenyltransferase, type II polyketide synthase, anthranilate:CoA-ligase, and several tailoring enzymes apparently responsible for installing the N-oxide and bis-tetrahydrofuran ring motifs.

Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration.

Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.

Multi-cell ECM compaction is predictable via superposition of nonlinear cell dynamics linearized in augmented state space.

Cells interacting through an extracellular matrix (ECM) exhibit emergent behaviors resulting from collective intercellular interaction. In wound healing and tissue development, characteristic compaction of ECM gel is induced by multiple cells that generate tensions in the ECM fibers and coordinate their actions with other cells. Computational prediction of collective cell-ECM interaction based on first principles is highly complex especially as the number of cells increase. Here, we introduce a computationally-efficient method for predicting nonlinear behaviors of multiple cells interacting mechanically through a 3-D ECM fiber network. The key enabling technique is superposition of single cell computational models to predict multicellular behaviors. While cell-ECM interactions are highly nonlinear, they can be linearized accurately with a unique method, termed Dual-Faceted Linearization. This method recasts the original nonlinear dynamics in an augmented space where the system behaves more linearly. The independent state variables are augmented by combining auxiliary variables that inform nonlinear elements involved in the system. This computational method involves a) expressing the original nonlinear state equations with two sets of linear dynamic equations b) reducing the order of the augmented linear system via principal component analysis and c) superposing individual single cell-ECM dynamics to predict collective behaviors of multiple cells. The method is computationally efficient compared to original nonlinear dynamic simulation and accurate compared to traditional Taylor expansion linearization. Furthermore, we reproduce reported experimental results of multi-cell induced ECM compaction.

Sarcopenia Is a New Risk Factor of Nonalcoholic Fatty Liver Disease in Patients with Inflammatory Bowel Disease.

BACKGROUND AND AIM: Recently, sarcopenia has been proposed as an additional risk factor of nonalcoholic fatty liver disease (NAFLD), and there have been no studies in patients with inflammatory bowel disease (IBD). We aimed to analyze the clinical associations between sarcopenia and NAFLD in IBD patients. METHODS: From January 2004 to December 2017, a total of 488 IBD patients, with CT results, were classified according to the presence of NAFLD. Sarcopenia was assessed based on the muscle volume calculated by the total psoas muscle area in the third lumbar region divided by the square of the patient's height (m2). RESULTS: Among the 443 included patients, NAFLD was diagnosed in 49 patients (11.1%). Sarcopenia was noted in 34.9%; it was more common in the NAFLD group (51.0 vs. 33.0%; p = 0.019). In multivariate analysis, metabolic syndrome (odds ratio [OR], 8.63), hyperuricemia (OR, 4.66), small bowel resection (OR, 3.45), and sarcopenia (OR, 2.99) were significant risk factors of NAFLD in IBD patients. In addition, sarcopenia was an independent risk factor after adjustment for age, sex, and other metabolic factors (OR, 2.26). CONCLUSIONS: The prevalence of nonalcoholic fatty liver in IBD patients was 11.1%, and sarcopenia was an independent risk factor.

Verrucosamide, a Cytotoxic 1,4-Thiazepane-Containing Thiodepsipeptide from a Marine-Derived Actinomycete.

A new cytotoxic thiodepsipeptide, verrucosamide (1), was isolated along with the known, related cyclic peptide thiocoraline, from the extract of a marine-derived actinomycete, a Verrucosispora sp., our strain CNX-026. The new peptide, which is composed of two rare seven-membered 1,4-thiazepane rings, was elucidated by a combination of spectral methods and the absolute configuration was determined by a single X-ray diffraction study. Verrucosamide (1) showed moderate cytotoxicity and selectivity in the NCI 60 cell line bioassay. The most susceptible cell lines were MDA-MB-468 breast carcinoma with an LD50 of 1.26 µM, and COLO 205 colon adenocarcinoma with an LD50 of 1.4 µM. Also isolated along with verrucosamide were three small 3-hydroxy(alkoxy)-quinaldic acid derivatives that appear to be products of the same biosynthetic pathway.

Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality.

Bacterial natural products remain an important source of new medicines. DNA sequencing has revealed that a majority of natural product biosynthetic gene clusters (BGCs) maintained in bacterial genomes have yet to be linked to the small molecules whose biosynthesis they encode. Efforts to discover the products of these orphan BGCs are driving the development of genome mining techniques based on the premise that many are transcriptionally silent during normal laboratory cultivation. Here, we employ comparative transcriptomics to assess BGC expression among four closely related strains of marine bacteria belonging to the genus Salinispora The results reveal that slightly more than half of the BGCs are expressed at levels that should facilitate product detection. By comparing the expression profiles of similar gene clusters in different strains, we identified regulatory genes whose inactivation appears linked to cluster silencing. The significance of these subtle differences between expressed and silent BGCs could not have been predicted a priori and was only revealed by comparative transcriptomics. Evidence for the conservation of silent clusters among a larger number of strains for which genome sequences are available suggests they may be under different regulatory control from the expressed forms or that silencing may represent an underappreciated mechanism of gene cluster evolution. Coupling gene expression and metabolomics data established a bioinformatic link between the salinipostins and their associated BGC, while genetic manipulation established the genetic basis for this series of compounds, which were previously unknown from Salinispora pacifica.

Robotic Localization Based on Planar Cable Robot and Hall Sensor Array Applied to Magnetic Capsule Endoscope.

Recently an active locomotive capsule endoscope (CE) for diagnosis and treatment in the digestive system has been widely studied. However, real-time localization to achieve precise feedback control and record suspicious positioning in the intestine is still challenging owing to the limitation of capsule size, relatively large diagnostic volume, and compatibility of other devices in clinical site. To address this issue, we present a novel robotic localization sensing methodology based on the kinematics of a planar cable driven parallel robot (CDPR) and measurements of the quasistatic magnetic field of a Hall effect sensor (HES) array. The arrangement of HES and the Levenberg-Marquardt (LM) algorithm are applied to estimate the position of the permanent magnet (PM) in the CE, and the planar CDPR is incorporated to follow the PM in the CE. By tracking control of the planar CDPR, the position of PM in any arbitrary position can be obtained through robot forward kinematics with respect to the global coordinates at the bedside. The experimental results show that the root mean square error (RMSE) for the estimated position value of PM was less than 1.13 mm in the X, Y, and Z directions and less than 1.14° in the θ and φ orientation, where the sensing space could be extended to ±70 mm for the given 34 × 34 mm2 HES array and the average moving distance in the Z-direction is 40 ± 2.42 mm. The proposed method of the robotic sensing with HES and CDPR may advance the sensing space expansion technology by utilizing the provided single sensor module of limited sensible volume.

Highly Reproducible Large-Area Perovskite Solar Cell Fabrication via Continuous Megasonic Spray Coating of CH3 NH3 PbI3.

A simple, low-cost, large area, and continuous scalable coating method is proposed for the fabrication of hybrid organic-inorganic perovskite solar cells. A megasonic spray-coating method utilizing a 1.7 MHz megasonic nebulizer that could fabricate reproducible large-area planar efficient perovskite films is developed. The coating method fabricates uniform large-area perovskite film with large-sized grain since smaller and narrower sized mist droplets than those generated by existing ultrasonic spray methods could be generated by megasonic spraying. The volume flow rate of the CH3 NH3 PbI3 precursor solution and the reaction temperature are controlled, to obtain a high quality perovskite active layer. The devices reach a maximum efficiency of 16.9%, with an average efficiency of 16.4% from 21 samples. The applicability of megasonic spray coating to the fabrication of large-area solar cells (1 cm2 ), with a power conversion efficiency of 14.2%, is also demonstrated. This is a record high efficiency for large-area perovskite solar cells fabricated by continuous spray coating.

Association of low skeletal muscle mass with advanced liver fibrosis in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Although low skeletal muscle mass (LSMM) is known to increase the risk of non-alcoholic fatty liver disease (NAFLD), limited reports have described the relationship between LSMM and advanced fibrosis. Here, we investigated the association between LSMM and advanced liver fibrosis in NAFLD patients. METHODS: Fatty liver was diagnosed using ultrasound, and appendicular skeletal muscle mass (ASM) was measured using bioelectrical impedance analysis. LSMM was defined in two ways: ASM/body weight percentage (LSMM-BW) and ASM/body mass index. Liver fibrosis stage was assessed by two models, the NAFLD fibrosis score and the Fibrosis-4 index, which determined low and high cutoff values (COVs). RESULTS: Of 10 711 NAFLD patients, 615 were diagnosed with LSMM-BW. LSMM patients were older (47.6 vs 52.5 years, P = 0.001) and had higher body mass index values (23.6 vs 29.1 kg/m2 , P < 0.001) and waist circumferences (80.1 vs 93.3 cm, P < 0.001) than non-LSMM patients. LSMM was an independent risk factor for advanced fibrosis assessed by a low COV for the Fibrosis-4 index regardless of its classification (adjusted for metabolic and lipid profiles and sex, odds ratio [OR], 1.27-2.01; all P < 0.05). LSMM was an independent risk factor for advanced fibrosis assessed by both COVs of NAFLD fibrosis score (adjusted for obesity, hypertension, lipid profile, and sex; OR, 1.64-2.01, P < 0.01 in the low COV group; OR, 2.68-3.12, P = 0.002 in the high COV group). CONCLUSIONS: Low skeletal muscle mass is associated with advanced fibrosis in NAFLD patients independent of metabolic risk factors.

Photopiperazines A-D, Photosensitive Interconverting Diketopiperazines with Significant and Selective Activity against U87 Glioblastoma Cells, from a Rare, Marine-Derived Actinomycete of the Family Streptomycetaceae.

Photopiperazines A-D (1-4), unsaturated diketopiperazine derivatives, were isolated from the culture broth of a rare, marine-derived actinomycete bacterium, strain AJS-327. This strain shows very poor 16S rRNA sequence similarity to other members of the actinomycete family Streptomycetaceae, indicating it is likely a new lineage within this group. The structures of the photopiperazines were defined by analysis of HR-ESI-TOF-MS spectra in conjunction with the interpretation of 1D and 2D NMR data. The photopiperazines are sensitive to light, causing interconversion among the four olefin geometrical isomers, which made purification of each isomer challenging. The photopiperazines are highly cytotoxic metabolites that show selective toxicity toward U87 glioblastoma and SKOV3 ovarian cancer cell lines.

Integration of Genomic Data with NMR Analysis Enables Assignment of the Full Stereostructure of Neaumycin B, a Potent Inhibitor of Glioblastoma from a Marine-Derived Micromonospora.

The microbial metabolites known as the macrolides are some of the most successful natural products used to treat infectious and immune diseases. Describing the structures of these complex metabolites, however, is often extremely difficult due to the presence of multiple stereogenic centers inherent in this class of polyketide-derived metabolites. With the availability of genome sequence data and a better understanding of the molecular genetics of natural product biosynthesis, it is now possible to use bioinformatic approaches in tandem with spectroscopic tools to assign the full stereostructures of these complex metabolites. In our quest to discover and develop new agents for the treatment of cancer, we observed the production of a highly cytotoxic macrolide, neaumycin B, by a marine-derived actinomycete bacterium of the genus Micromonospora. Neaumycin B is a complex polycyclic macrolide possessing 19 asymmetric centers, usually requiring selective degradation, crystallization, derivatization, X-ray diffraction analysis, synthesis, or other time-consuming approaches to assign the complete stereostructure. As an alternative approach, we sequenced the genome of the producing strain and identified the neaumycin gene cluster ( neu). By integrating the known stereospecificities of biosynthetic enzymes with comprehensive NMR analysis, the full stereostructure of neaumycin B was confidently assigned. This approach exemplifies how mining gene cluster information while integrating NMR-based structure data can achieve rapid, efficient, and accurate stereostructural assignments for complex macrolides.

The Importance of Being Inconsistent.

We review the role of self-consistency in density functional theory (DFT). We apply a recent analysis to both Kohn-Sham and orbital-free DFT, as well as to partition DFT, which generalizes all aspects of standard DFT. In each case, the analysis distinguishes between errors in approximate functionals versus errors in the self-consistent density. This yields insights into the origins of many errors in DFT calculations, especially those often attributed to self-interaction or delocalization error. In many classes of problems, errors can be substantially reduced by using better densities. We review the history of these approaches, discuss many of their applications, and give simple pedagogical examples.