Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale.

Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.

Imprinted X chromosome inactivation at the gamete-to-embryo transition.

In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.

Global supply chains amplify economic costs of future extreme heat risk.

Evidence shows a continuing increase in the frequency and severity of global heatwaves1,2, raising concerns about the future impacts of climate change and the associated socioeconomic costs3,4. Here we develop a disaster footprint analytical framework by integrating climate, epidemiological and hybrid input-output and computable general equilibrium global trade models to estimate the midcentury socioeconomic impacts of heat stress. We consider health costs related to heat exposure, the value of heat-induced labour productivity loss and indirect losses due to economic disruptions cascading through supply chains. Here we show that the global annual incremental gross domestic product loss increases exponentially from 0.03 ± 0.01 (SSP 245)-0.05 ± 0.03 (SSP 585) percentage points during 2030-2040 to 0.05 ± 0.01-0.15 ± 0.04 percentage points during 2050-2060. By 2060, the expected global economic losses reach a total of 0.6-4.6% with losses attributed to health loss (37-45%), labour productivity loss (18-37%) and indirect loss (12-43%) under different shared socioeconomic pathways. Small- and medium-sized developing countries suffer disproportionately from higher health loss in South-Central Africa (2.1 to 4.0 times above global average) and labour productivity loss in West Africa and Southeast Asia (2.0-3.3 times above global average). The supply-chain disruption effects are much more widespread with strong hit to those manufacturing-heavy countries such as China and the USA, leading to soaring economic losses of 2.7 ± 0.7% and 1.8 ± 0.5%, respectively.

DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.

SynergyX: a multi-modality mutual attention network for interpretable drug synergy prediction.

Discovering effective anti-tumor drug combinations is crucial for advancing cancer therapy. Taking full account of intricate biological interactions is highly important in accurately predicting drug synergy. However, the extremely limited prior knowledge poses great challenges in developing current computational methods. To address this, we introduce SynergyX, a multi-modality mutual attention network to improve anti-tumor drug synergy prediction. It dynamically captures cross-modal interactions, allowing for the modeling of complex biological networks and drug interactions. A convolution-augmented attention structure is adopted to integrate multi-omic data in this framework effectively. Compared with other state-of-the-art models, SynergyX demonstrates superior predictive accuracy in both the General Test and Blind Test and cross-dataset validation. By exhaustively screening combinations of approved drugs, SynergyX reveals its ability to identify promising drug combination candidates for potential lung cancer treatment. Another notable advantage lies in its multidimensional interpretability. Taking Sorafenib and Vorinostat as an example, SynergyX serves as a powerful tool for uncovering drug-gene interactions and deciphering cell selectivity mechanisms. In summary, SynergyX provides an illuminating and interpretable framework, poised to catalyze the expedition of drug synergy discovery and deepen our comprehension of rational combination therapy.

Fast and sensitive CRISPR detection by minimized interference of target amplification.

Despite the great potential of CRISPR-based detection, it has not been competitive with other market diagnostics for on-site and in-home testing. Here we dissect the rate-limiting factors that undermine the performance of Cas12b- and Cas13a-mediated detection. In one-pot testing, Cas12b interferes with loop-mediated isothermal amplification by binding to and cleaving the amplicon, while Cas13a directly degrades the viral RNA, reducing its amplification. We found that the protospacer-adjacent motif-interacting domain engineered Cas12b accelerated one-pot testing with 10-10,000-fold improved sensitivity, and detected 85 out of 85 SARS-CoV-2 clinical samples with a sensitivity of 0.5 cp µl-1, making it superior to wild-type Cas12b. In parallel, by diminishing the interference of Cas13a with viral RNA, the optimized Cas13a-based assay detected 86 out of 87 SARS-CoV-2 clinical samples at room temperature in 30 min with a sensitivity of 0.5 cp µl-1. The relaxed reaction conditions and improved performance of CRISPR-based assays make them competitive for widespread use in pathogen detection.

Efficient targeted insertion of large DNA fragments without DNA donors.

Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.

Brain repair mechanisms after cell therapy for stroke.

Cell-based therapies hold great promise for brain repair after stroke. While accumulating evidence confirms the preclinical and clinical benefits of cell therapies, the underlying mechanisms by which they promote brain repair remain unclear. Here, we briefly review endogenous mechanisms of brain repair after ischemic stroke and then focus on how different stem and progenitor cell sources can promote brain repair. Specifically, we examine how transplanted cell grafts contribute to improved functional recovery either through direct cell replacement or by stimulating endogenous repair pathways. Additionally, we discuss recently implemented preclinical refinement methods, such as preconditioning, microcarriers, genetic safety switches, and universal (immune evasive) cell transplants, as well as the therapeutic potential of these pharmacologic and genetic manipulations to further enhance the efficacy and safety of cell therapies. By gaining a deeper understanding of post-ischemic repair mechanisms, prospective clinical trials may be further refined to advance post-stroke cell therapy to the clinic.

Evolutionary origin and gradual accumulation with plant evolution of the LACS family.

BACKGROUND: LACS (long-chain acyl-CoA synthetase) genes are widespread in organisms and have multiple functions in plants, especially in lipid metabolism. However, the origin and evolutionary dynamics of the LACS gene family remain largely unknown. RESULTS: Here, we identified 1785 LACS genes in the genomes of 166 diverse plant species and identified the clades (I, II, III, IV, V, VI) of six clades for the LACS gene family of green plants through phylogenetic analysis. Based on the evolutionary history of plant lineages, we found differences in the origins of different clades, with Clade IV originating from chlorophytes and representing the origin of LACS genes in green plants. The structural characteristics of different clades indicate that clade IV is relatively independent, while the relationships between clades (I, II, III) and clades (V, VI) are closer. Dispersed duplication (DSD) and transposed duplication (TRD) are the main forces driving the evolution of plant LACS genes. Network clustering analysis further grouped all LACS genes into six main clusters, with genes within each cluster showing significant co-linearity. Ka/Ks results suggest that LACS family genes underwent purifying selection during evolution. We analyzed the phylogenetic relationships and characteristics of six clades of the LACS gene family to explain the origin, evolutionary history, and phylogenetic relationships of different clades and proposed a hypothetical evolutionary model for the LACS family of genes in plants. CONCLUSIONS: Our research provides genome-wide insights into the evolutionary history of the LACS gene family in green plants. These insights lay an important foundation for comprehensive functional characterization in future research.

A Platinum-Aluminum Bimetallic Salen Complex for Pro-senescence Cancer Therapy.

Cell senescence is defined as irreversible cell cycle arrest, which can be triggered by telomere shortening or by various types of genotoxic stress. Induction of senescence is emerging as a new strategy for the treatment of cancer, especially when sequentially combined with a second senolytic drug capable of killing the resulting senescent cells, however severely suffering from the undesired off-target side effects from the senolytic drugs. Here, we prepare a bimetalic platinum-aluminum salen complex (Alumiplatin) for cancer therapy-a combination of pro-senesence chemotherapy with in situ senotherapy to avoid the side effects. The aluminum salen moiety, as a G-quadruplex stabilizer, enhances the salen's ability to induce cancer cell senescence and this phenotype is in turn sensitive to the cytotoxic activity of the monofunctional platinum moiety. It exhibits an excellent capability for inducing senescence, a potent cytotoxic activity against cancer cells both in vitro and in vivo, and an improved safety profile compared to cisplatin. Therefore, Alumiplatin may be a good candidate to be further developed into safe and effective anticancer agents. This novel combination of cell senescence inducers with genotoxic drugs revolutionizes the therapy options of designing multi-targeting anticancer agents to improve the efficacy of anticancer therapies.

Causal effects of gut microbiota on sepsis and sepsis-related death: insights from genome-wide Mendelian randomization, single-cell RNA, bulk RNA sequencing, and network pharmacology.

BACKGROUND: Gut microbiota alterations have been implicated in sepsis and related infectious diseases, but the causal relationship and underlying mechanisms remain unclear. METHODS: We evaluated the association between gut microbiota composition and sepsis using two-sample Mendelian randomization (MR) analysis based on published genome-wide association study (GWAS) summary statistics. Sensitivity analyses were conducted to validate the robustness of the results. Reverse MR analysis and integration of GWAS and expression quantitative trait loci (eQTL) data were performed to identify potential genes and therapeutic targets. RESULTS: Our analysis identified 11 causal bacterial taxa associated with sepsis, with increased abundance of six taxa showing positive causal relationships. Ten taxa had causal effects on the 28-day survival outcome of septic patients, with increased abundance of six taxa showing positive associations. Sensitivity analyses confirmed the robustness of these associations. Reverse MR analysis did not provide evidence of reverse causality. Integration of GWAS and eQTL data revealed 76 genes passing the summary data-based Mendelian randomization (SMR) test. Differential expression of these genes was observed between sepsis patients and healthy individuals. These genes represent potential therapeutic targets for sepsis. Molecular docking analysis predicted potential drug-target interactions, further supporting their therapeutic potential. CONCLUSION: Our study provides insights for the development of personalized treatment strategies for sepsis and offers preliminary candidate targets and drugs for future drug development.

High-power and narrow-linewidth nanosecond pulsed intracavity crystalline Raman laser operating at 1.7 µm.

We report on a high-power and narrow-linewidth nanosecond pulsed intracavity crystalline Raman laser at 1.7 µm. Driven by an acousto-optically Q-switched 1314 nm two-crystal Nd:YLF laser, the highly efficient cascaded YVO4 Raman laser at 1715nm was obtained within the well-designed L-shaped resonator. Thanks to the absence of spatial hole burning in the stimulated Raman scattering process, significant spectral purification of second-Stokes radiation was observed by incorporating a fused silica etalon in the high-Q fundamental cavity. Under the repetition rate of 4 kHz, the highest average output power for single longitudinal mode operation was up to 2.2 W with the aid of precision vibration isolation and precision temperature controlling, corresponding to the pulse duration of ∼2.8 ns and the spectral linewidth of ∼330 MHz. Further increasing the launched pump power, the second-Stokes laser tended toward be always multimode, and the maximum average output power amounted to 4.8 W with the peak power of ∼0.8 MW and the spectral linewidth of ∼0.08 nm. The second-Stokes emission was near diffraction limited with M2 < 1.4 across the whole pump power range.

Ten-watt-level all-solid-state eye-safe intracavity Raman laser.

We demonstrate the first ten-watt-level eye-safe intracavity crystalline Raman laser, to the best of our knowledge. The efficient high-power eye-safe Raman laser is intracavity-pumped by an acousto-optically Q-switched 1314 nm two-crystal Nd:YLF laser. Benefiting from the unique bi-axial properties of KGW crystal, two sets of eye-safe dual-wavelength Raman lasers operating at 1461, 1645 nm and 1490, 1721nm are achieved by rotating the Raman crystal. Under the launched pump power of 84.9 W and the repetition rate of 4 kHz, the maximum first-Stokes output powers of 7.9 W at 1461 nm and 8.2 W at 1490 nm are acquired with the second-Stokes output powers of 1.4 W at 1645 nm and 1.5 W at 1721nm, respectively, leading to the eye-safe dual-wavelength Raman output powers of up to 9.3 and 9.7 W. Meanwhile, the pulse durations at the wavelengths of 1461, 1490, 1645, 1721nm are determined to be 4.8, 5.5, 4.3, and 3.6 ns, respectively, which give rise to the peak powers approaching about 410, 370, 80, 100 kW. These Stokes emissions are found to be near diffraction limited with M2 < 1.6 across the entire output power range.

Improved nested U-structure for accurate nailfold capillary segmentation.

Changes in the structure and function of nailfold capillaries may be indicators of numerous diseases. Noninvasive diagnostic tools are commonly used for the extraction of morphological information from segmented nailfold capillaries to study physiological and pathological changes therein. However, current segmentation methods for nailfold capillaries cannot accurately separate capillaries from the background, resulting in issues such as unclear segmentation boundaries. Therefore, improving the accuracy of nailfold capillary segmentation is necessary to facilitate more efficient clinical diagnosis and research. Herein, we propose a nailfold capillary image segmentation method based on a U2-Net backbone network combined with a Transformer structure. This method integrates the U2-Net and Transformer networks to establish a decoder-encoder network, which inserts Transformer layers into the nested two-layer U-shaped architecture of the U2-Net. This structure effectively extracts multiscale features within stages and aggregates multilevel features across stages to generate high-resolution feature maps. The experimental results demonstrate an overall accuracy of 98.23 %, a Dice coefficient of 88.56 %, and an IoU of 80.41 % compared to the ground truth. Furthermore, our proposed method improves the overall accuracy by approximately 2 %, 3 %, and 5 % compared to the original U2-Net, Res-Unet, and U-Net, respectively. These results indicate that the Transformer-U2Net network performs well in nailfold capillary image segmentation and provides more detailed and accurate information on the segmented nailfold capillary structure, which may aid clinicians in the more precise diagnosis and treatment of nailfold capillary-related diseases.

Artificial intelligence evaluation of coronary computed tomography angiography for coronary stenosis classification and diagnosis.

BACKGROUND: Ruling out obstructive coronary artery disease (CAD) using coronary computed tomography angiography (CCTA) is time-consuming and challenging. This study developed a deep learning (DL) model to assist in detecting obstructive CAD on CCTA to streamline workflows. METHODS: In total, 2929 DICOM files and 7945 labels were extracted from curved planar reformatted CCTA images. A modified Inception V3 model was adopted. To validate the artificial intelligence (AI) model, two cardiologists labelled and adjudicated the classification of coronary stenosis on CCTA. The model was trained to differentiate the coronary artery into binary stenosis classifications <50% and ≥50% stenosis. Using the quantitative coronary angiography (QCA) consensus results as a reference standard, the performance of the AI model and CCTA radiology readers was compared by calculating Cohen's kappa coefficients at patient and vessel levels. The net reclassification index was used to evaluate the net benefit of the DL model. RESULTS: The diagnostic accuracy of the AI model was 92.3% and 88.4% at the patient and vessel levels, respectively. Compared with CCTA radiology readers, the AI model had a better agreement for binary stenosis classification at both patient and vessel levels (Cohen kappa coefficient: .79 vs. .39 and .77 vs. .40, p < .0001). The AI model also exhibited significantly improved model discrimination and reclassification (Net reclassification index = .350; Z = 4.194; p < .001). CONCLUSIONS: The developed AI model identified obstructive CAD, and the model results correlated well with QCA results. Incorporating the model into the reporting system of CCTA may improve workflows.

Controllable orbital-angular-momentum Hall effect by engineering intrinsic orbit-orbit interaction.

We report both theoretically and experimentally a process of optical intrinsic orbit-orbit interaction with a vortex-antivortex structure nested in a freely propagating light field. The orbit-orbit interaction is originating from the coupling between different vortices and antivortices. Based on this process, we reveal the resultant controllable orbital-angular-momentum Hall effect by considering a typical structure, which comprises a vortex-antivortex pair and another vortex (or antivortex) as a controllable knob. The intrinsic Hall effect can be spatially manipulated by appropriately engineering the orbit-orbit interaction, namely arranging the initial distribution of these elements. This work can find interesting potential applications. For example, it provides an effective technique for controllable paired photon generation.

Nanosecond pulsed deep-red Raman laser based on the Nd:YLF dual-crystal configuration.

A highly powerful nanosecond pulsed deep-red laser was demonstrated by intracavity second-harmonic generation of an actively Q-switched Nd:YLF dual-crystal-based KGW Raman laser in a critically phase-matched lithium triborate (LBO) crystal. The first-Stokes fields at 1461 and 1490 nm driven by the 1314 nm fundamental laser were firstly produced by accessing the Raman shifts of 768 and 901 cm-1 in the KGW crystal, respectively, and thereafter converted to the deep-red emission lines at 731 and 745 nm by finely tuning the phase-matching angle of the LBO crystal and carefully realigning the resonator. Integrating the benefits of the Nd:YLF dual-crystal configuration and the meticulously designed L-shaped resonator, this deep-red laser system delivered the maximum average output powers of 5.2 and 7.6 W with the optical power conversion efficiencies approaching 6.3% and 9.2% under the optimal pulse repetition frequency of 4 kHz, respectively. The pulse durations of 6.7 and 5.5 ns were acquired with the peak powers up to approximately 190 and 350 kW, respectively, and the resultant beam qualities were determined to be near-diffraction-limited with M2 ≈ 1.5.

Multitask deep learning for prediction of microvascular invasion and recurrence-free survival in hepatocellular carcinoma based on MRI images.

BACKGROUND AND AIMS: Accurate preoperative prediction of microvascular invasion (MVI) and recurrence-free survival (RFS) is vital for personalised hepatocellular carcinoma (HCC) management. We developed a multitask deep learning model to predict MVI and RFS using preoperative MRI scans. METHODS: Utilising a retrospective dataset of 725 HCC patients from seven institutions, we developed and validated a multitask deep learning model focused on predicting MVI and RFS. The model employs a transformer architecture to extract critical features from preoperative MRI scans. It was trained on a set of 234 patients and internally validated on a set of 58 patients. External validation was performed using three independent sets (n = 212, 111, 110). RESULTS: The multitask deep learning model yielded high MVI prediction accuracy, with AUC values of 0.918 for the training set and 0.800 for the internal test set. In external test sets, AUC values were 0.837, 0.815 and 0.800. Radiologists' sensitivity and inter-rater agreement for MVI prediction improved significantly when integrated with the model. For RFS, the model achieved C-index values of 0.763 in the training set and ranged between 0.628 and 0.728 in external test sets. Notably, PA-TACE improved RFS only in patients predicted to have high MVI risk and low survival scores (p < .001). CONCLUSIONS: Our deep learning model allows accurate MVI and survival prediction in HCC patients. Prospective studies are warranted to assess the clinical utility of this model in guiding personalised treatment in conjunction with clinical criteria.

Repositioning baloxavir marboxil as VISTA agonist that ameliorates experimental asthma.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), a novel negative checkpoint regulator, plays an essential role in allergic pulmonary inflammation in mice. Treatment with a VISTA agonistic antibody could significantly improve asthma symptoms. Thus, for allergic asthma treatment, VISTA targeting may be a compelling approach. In this study, we examined the functional mechanism of VISTA in allergic pulmonary inflammation and screened the FDA-approved drugs for VISTA agonists. By using mass cytometry (CyTOF), we found that VISTA deficiency primarily increased lung macrophage infiltration in the OVA-induced asthma model, accompanied by an increased proportion of M1 macrophages (CD11b+F4/80+CD86+) and a decreased proportion of M2 macrophages (CD11b+F4/80+CD206+). Further in vitro studies showed that VISTA deficiency promoted M1 polarization and inhibited M2 polarization of bone marrow-derived macrophages (BMDMs). Importantly, we discovered baloxavir marboxil (BXM) as a VISTA agonist by virtual screening of FDA-approved drugs. The surface plasmon resonance (SPR) assays revealed that BXM (KD = 1.07 µM) as well as its active form, baloxavir acid (BXA) (KD = 0.21 µM), could directly bind to VISTA with high affinity. Notably, treatment with BXM significantly ameliorated asthma symptoms, including less lung inflammation, mucus secretion, and the generation of Th2 cytokines (IL-5, IL-13, and IL-4), which were dramatically attenuated by anti-VISTA monoclonal antibody treatment. BXM administration also reduced the pulmonary infiltration of M1 macrophages and raised M2 macrophages. Collectively, our study indicates that VISTA regulates pulmonary inflammation in allergic asthma by regulating macrophage polarization and baloxavir marboxil, and an old drug might be a new treatment for allergic asthma through targeting VISTA.

Plaque features of the middle cerebral artery are associated with periprocedural complications of intracranial angioplasty and stenting.

PURPOSE: The identification of plaque features in the middle cerebral artery (MCA) may help minimize periprocedural complications and select patients suitable for percutaneous transluminal angioplasty and stenting (PTAS). However, relevant research is lacking. METHODS: We retrospectively included patients with symptomatic MCA stenosis who received PTAS. All patients underwent intracranial vessel wall MRI (VWMRI) before surgery. Periprocedural complications (PC) included ischemic and hemorrhagic stroke within 30 days. Stenosis location, MCA shape, plaque eccentricity and distribution, plaque thickness and length, and enhancement ratio were compared between patients with and without PC. RESULTS: Sixty-six patients were included in the study, of which 12.1% (8/66) had PC. Of the eight patients with PC, seven (87.5%) had superior wall plaques. In the non-PC group (n = 58), nine (17%) patients had superior wall plaques. Compared with patients without PC, those with PC had more frequent superior wall plaques (17% vs 87.5%, p < 0.001) and s-shaped MCAs (19% vs 50%, p = 0.071), different stenosis locations (p = 0.012), thicker plaques (1.58 [1.35, 2.00] vs 1.98 [1.73, 2.43], p = 0.038), and less frequent inferior wall plaques (79.2% vs 12.5%, p < 0.001). Multivariate analysis showed that only the presence of superior wall plaques (OR = 41.54 [2.31, 747.54]) was independently associated with PC. CONCLUSION: MCA plaque features were highly correlated with PC in patients with symptomatic MCA stenosis who underwent PTAS.