A comprehensive survey of dimensionality reduction and clustering methods for single-cell and spatial transcriptomics data.

In recent years, the application of single-cell transcriptomics and spatial transcriptomics analysis techniques has become increasingly widespread. Whether dealing with single-cell transcriptomic or spatial transcriptomic data, dimensionality reduction and clustering are indispensable. Both single-cell and spatial transcriptomic data are often high-dimensional, making the analysis and visualization of such data challenging. Through dimensionality reduction, it becomes possible to visualize the data in a lower-dimensional space, allowing for the observation of relationships and differences between cell subpopulations. Clustering enables the grouping of similar cells into the same cluster, aiding in the identification of distinct cell subpopulations and revealing cellular diversity, providing guidance for downstream analyses. In this review, we systematically summarized the most widely recognized algorithms employed for the dimensionality reduction and clustering analysis of single-cell transcriptomic and spatial transcriptomic data. This endeavor provides valuable insights and ideas that can contribute to the development of novel tools in this rapidly evolving field.

Synthesis of struvite-enriched slow-release fertilizer using magnesium-modified biochar: Desorption and leaching mechanisms.

To improve the retention and slow-release abilities of nitrogen (N) and phosphorus (P), an 82 %-purity struvite fertilizer (MAP-BC) was synthesized using magnesium-modified biochar and a solution with a 2:1 concentration ratio of NH4+ to PO43- at a pH of 8. Batch microscopic characterizations and soil column leaching experiments were conducted to study the retention and slow-release mechanisms and desorption kinetics of MAP-BC. The slow-release mechanism revealed that the dissolution rate of high-purity struvite was the dominant factor of NP slow release. The re-adsorption of NH4+ and PO43- by biochar and unconsumed MgO prolonged slow release. Mg2+ ionized by MgO could react with PO43- released from struvite to form Mg3(PO4)2. The internal biochar exhibited electrostatic attraction and pore restriction towards NH4+, while magnesium modification and nutrient loading formed a physical antioxidant barrier that ensured long-term release. The water diffusion experiment showed a higher cumulative release rate for PO43- compared to NH4+, whereas in soil column leaching, the trend was reversed, suggesting that soil's competitive adsorption facilitated the desorption of NH4+ from MAP-BC. During soil leaching, cumulative release rates of NH4+ and PO43- from chemical fertilizers were 3.55-3.62 times faster than those from MAP-BC. The dynamic test data for NH4+ and PO43- in MAP-BC fitted the Ritger-Peppas model best, predicting release periods of 163 days and 166 days, respectively. The leaching performances showed that MAP-BC reduced leaching solution volume by 5.58 % and significantly increased soil large aggregates content larger than 0.25 mm by 24.25 %. The soil nutrients retention and pH regulation by MAP-BC reduced leaching concentrations of NP. Furthermore, MAP-BC significantly enhanced plant growth, and it is more suitable as a NP source for long-term crops. Therefore, MAP-BC is expected to function as a long-term and slow-release fertilizer with the potential to minimize NP nutrient loss and replace part of quick-acting fertilizer.

Deep learning models incorporating endogenous factors beyond DNA sequences improve the prediction accuracy of base editing outcomes.

Adenine base editors (ABEs) and cytosine base editors (CBEs) enable the single nucleotide editing of targeted DNA sites avoiding generation of double strand breaks, however, the genomic features that influence the outcomes of base editing in vivo still remain to be characterized. High-throughput datasets from lentiviral integrated libraries were used to investigate the sequence features affecting base editing outcomes, but the effects of endogenous factors beyond the DNA sequences are still largely unknown. Here the base editing outcomes of ABE and CBE were evaluated in mammalian cells for 5012 endogenous genomic sites and 11,868 genome-integrated target sequences, with 4654 genomic sites sharing the same target sequences. The comparative analyses revealed that the editing outcomes of ABE and CBE at endogenous sites were substantially different from those obtained using genome-integrated sequences. We found that the base editing efficiency at endogenous target sites of both ABE and CBE was influenced by endogenous factors, including epigenetic modifications and transcriptional activity. A deep-learning algorithm referred as BE_Endo, was developed based on the endogenous factors and sequence information from our genomic datasets, and it yielded unprecedented accuracy in predicting the base editing outcomes. These findings along with the developed computational algorithms may facilitate future application of BEs for scientific research and clinical gene therapy.

Whole-brain spatial organization of hippocampal single-neuron projectomes.

Mapping single-neuron projections is essential for understanding brain-wide connectivity and diverse functions of the hippocampus (HIP). Here, we reconstructed 10,100 single-neuron projectomes of mouse HIP and classified 43 projectome subtypes with distinct projection patterns. The number of projection targets and axon-tip distribution depended on the soma location along HIP longitudinal and transverse axes. Many projectome subtypes were enriched in specific HIP subdomains defined by spatial transcriptomic profiles. Furthermore, we delineated comprehensive wiring diagrams for HIP neurons projecting exclusively within the HIP formation (HPF) and for those projecting to both intra- and extra-HPF targets. Bihemispheric projecting neurons generally projected to one pair of homologous targets with ipsilateral preference. These organization principles of single-neuron projectomes provide a structural basis for understanding the function of HIP neurons.

Single-cell profiling of the microenvironment in human bone metastatic renal cell carcinoma.

Bone metastasis is of common occurrence in renal cell carcinoma with poor prognosis, but no optimal treatment approach has been established for bone metastatic renal cell carcinoma. To explore the potential therapeutic targets for bone metastatic renal cell carcinoma, we profile single cell transcriptomes of 6 primary renal cell carcinoma and 9 bone metastatic renal cell carcinoma. We also include scRNA-seq data of early-stage renal cell carcinoma, late-stage renal cell carcinoma, normal kidneys and healthy bone marrow samples in the study to better understand the bone metastasis niche. The molecular properties and dynamic changes of major cell lineages in bone metastatic environment of renal cell carcinoma are characterized. Bone metastatic renal cell carcinoma is associated with multifaceted immune deficiency together with cancer-associated fibroblasts, specifically appearance of macrophages exhibiting malignant and pro-angiogenic features. We also reveal the dominance of immune inhibitory T cells in the bone metastatic renal cell carcinoma which can be partially restored by the treatment. Trajectory analysis showes that myeloid-derived suppressor cells are progenitors of macrophages in the bone metastatic renal cell carcinoma while monocytes are their progenitors in primary tumors and healthy bone marrows. Additionally, the infiltration of immune inhibitory CD47+ T cells is observed in bone metastatic tumors, which may be a result of reduced phagocytosis by SIRPA-expressing macrophages in the bone microenvironment. Together, our results provide a systematic view of various cell types in bone metastatic renal cell carcinoma and suggest avenues for therapeutic solutions.

Macro CD5L+ deteriorates CD8+T cells exhaustion and impairs combination of Gemcitabine-Oxaliplatin-Lenvatinib-anti-PD1 therapy in intrahepatic cholangiocarcinoma.

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+-CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

Impact of blood-brain barrier disruption on clinical features and treatment response in patients with newly diagnosed autoimmune encephalitis.

AIMS: Blood-brain barrier (BBB) breakdown is an essential mechanism in inflammatory diseases of the central nervous system. However, the association between BBB integrity and autoimmune encephalitis (AE) has not been investigated. Our study aimed to analyze this relationship in patients with AE between BBB integrity with clinical manifestations and therapeutic responses. METHODS: Our study enrolled 147 patients with AE who were newly diagnosed at the First Affiliated Hospital of Zhengzhou University between August 2015 and December 2021. Patients were classified into normal or damaged BBB groups based on cerebrospinal fluid (CSF) albumin/serum albumin (QAlb). To evaluate the severity of the illness, we used the modified Rankin Scale (mRS) and the Clinical Assessment Scale for Autoimmune Encephalitis (CASE). RESULTS: We found a higher proportion of males, higher CSF protein, immunoglobulin IgG, and 24-h intrathecal IgG synthesis rate in the damaged BBB group. The improvement rate was lower in the damaged BBB group, but we found that double- or triple-combination immunotherapy had better clinical outcomes than single immunotherapy. In addition, there was a positive correlation between the CASE score and mRS score, and a positive correlation between the CASE score or mRS score and QAlb on admission. CONCLUSIONS: BBB integrity is closely related to the clinical features and treatment responses of newly diagnosed AE. Patients with AE and a damaged BBB may benefit from combination immunotherapy.

Integrated proteomic and phosphoproteomic data-independent acquisition data evaluate the personalized drug responses of primary and metastatic tumors in colorectal cancer.

Mass spectrometry (MS)-based proteomics and phosphoproteomics are powerful methods to study the biological mechanisms, diagnostic biomarkers, prognostic analysis, and drug therapy of tumors. Data-independent acquisition (DIA) mode is considered to perform better than data-dependent acquisition (DDA) mode in terms of quantitative reproducibility, specificity, accuracy, and identification of low-abundance proteins. Mini patient derived xenograft (MiniPDX) model is an effective model to assess the response to antineoplastic drugs in vivo and is helpful for the precise treatment of cancer patients. Kinases are favorable spots for tumor-targeted drugs, and their functional completion relies on signaling pathways through phosphorylating downstream substrates. Kinase-phosphorylation networks or edge interactions are considered more credible and permanent for characterizing complex diseases. Here, we provide a workflow for personalized drug response assessment in primary and metastatic colorectal cancer (CRC) tumors using DIA proteomic data, DIA phosphoproteomic data, and MiniPDX models. Three kinase inhibitors, afatinib, gefitinib, and regorafenib, are tested pharmacologically. The process mainly includes the following steps: clinical tissue collection, sample preparation, hybrid spectral libraries establishment, MS data acquisition, kinase-substrate network construction, in vivo drug test, and elastic regression modeling. Our protocol gives a more direct data basis for individual drug responses, and will improve the selection of treatment strategies for patients without the druggable mutation.

Effects of five-year field aged zeolite on grain yield and reactive gaseous N losses in alternate wetting and drying paddy system.

Clinoptilolite zeolite has been widely used in agricultural production systems for enhancing water and fertilizer savings, mitigating greenhouse gas emissions, and increasing yield. However, there is little information on field-aged effects of zeolite on reactive gaseous N losses under alternate wetting and drying irrigation (AWD). We conducted a five-year field experiment to investigate field-aged effect of natural zeolite addition at 0 (Z0), 5 (Z5), and 10 (Z10) t ha-1 on reactive gaseous N losses (NH3, N2O), N-related global warming potential (GWPN), soil properties and grain yield under two irrigation regimes (CF: continuous flooding irrigation; AWD) in the 4th (2020) and 5th (2021) years since its initial application in 2017. As compared with CF, AWD did not significantly affect grain yield and NH3 volatilization but increased seasonal N2O emissions by 46 %-71 % over two years. Zeolite increased rice yield for five consecutive years. Z10 reduced averaged cumulative NH3 volatilization and GWPN by 23 % and 26 %, compared to zeolite-free treatment, respectively, in the 4th and 5th years. Soil NH4+-N was increased with the increased rate of Z application under both CF and AWD. Z10 increased soil NH4+-N by 27 %-38 % and NO3--N by 14 %-22 % in five years, compared to Z0, respectively. Compared to AWD without zeolite, the addition of 10 t ha-1 zeolite under AWD lowered NH3 volatilization, cumulative N2O emissions, and GWPN by an average of 28 %, 29 %, and 30 % in two years, respectively. IAWDZ10 did not differ from ICFZ0 on reactive gaseous N losses but significantly lowered reactive gaseous losses relative to IAWDZ0. Therefore, zeolite addition could mitigate the reactive gaseous N losses and GWPN for at least five years after initial application, which is beneficial to promoting zeolite application and ensuring sustainable agriculture.

Effects of acid modified biochar on potassium uptake, leaching and balance in an alternate wetting and drying paddy ecosystem.

Straw biochar amended soils reduce fertilizer losses and alleviate soil K-exhaustion, while decrease grain yield due to its high pH. H2SO4-modified biochar has been studied as a means to enhance the advantages of biochar and address yield decrease. However, little information is available on its effects on aboveground K uptake, soil K fixation, K leaching, and utilization in paddy rice systems, especially under water stress. A 3-year field experiment was conducted with two irrigation regimes (continuously flooded irrigation, ICF and alternate wetting and drying irrigation, IAWD) as main plots and 0 (control), 20 t ha-1 biochar (B20), and 20 t ha-1 acid-modified biochar (B20A-M) as subplots. The results showed that IAWD significantly decreased water percolation by 9.26 %-14.74 % but increased K leaching by 10.84 %-15.66 %. Compared to B0, B20 and B20A-M significantly increased K leaching by 32.40 % and 30.42 % in 2019, while decreased it by 11.60 %-14.01 % in 2020 and 2021. Both B20 and B20A-M significantly improved aboveground K uptake by 3.45 %-6.71 % throughout the three years. B20 reduced grain yield in 2019 and increased it in 2020 and 2021, while B20A-M increased grain yield throughout the three years. Apparent K balance (AKB) from pre-transplanting to post-harvest over the three years suggested that IAWD significantly increased the risk of soil K depletion but B20 and B20A-M significantly increased AKB, thereby addressing the depletion of it. IAWDB20A-M have a comparable AKB with ICFB20A-M, but had up to 18.3 % and 21.61 % higher AKB than IAWDB20 and ICFB20. Therefore, the use of H2SO4 modified biochar could produce higher grain yield with lower K leaching for addition in IAWD paddy systems, which is beneficial to mitigate soil K depletion and ensure a sustainable agricultural production.

Single-cell spatial transcriptome reveals cell-type organization in the macaque cortex.

Elucidating the cellular organization of the cerebral cortex is critical for understanding brain structure and function. Using large-scale single-nucleus RNA sequencing and spatial transcriptomic analysis of 143 macaque cortical regions, we obtained a comprehensive atlas of 264 transcriptome-defined cortical cell types and mapped their spatial distribution across the entire cortex. We characterized the cortical layer and region preferences of glutamatergic, GABAergic, and non-neuronal cell types, as well as regional differences in cell-type composition and neighborhood complexity. Notably, we discovered a relationship between the regional distribution of various cell types and the region's hierarchical level in the visual and somatosensory systems. Cross-species comparison of transcriptomic data from human, macaque, and mouse cortices further revealed primate-specific cell types that are enriched in layer 4, with their marker genes expressed in a region-dependent manner. Our data provide a cellular and molecular basis for understanding the evolution, development, aging, and pathogenesis of the primate brain.

Pharmaco-proteogenomic characterization of liver cancer organoids for precision oncology.

Organoid models have the potential to recapitulate the biological and pharmacotypic features of parental tumors. Nevertheless, integrative pharmaco-proteogenomics analysis for drug response features and biomarker investigation for precision therapy of patients with liver cancer are still lacking. We established a patient-derived liver cancer organoid biobank (LICOB) that comprehensively represents the histological and molecular characteristics of various liver cancer types as determined by multiomics profiling, including genomic, epigenomic, transcriptomic, and proteomic analysis. Proteogenomic profiling of LICOB identified proliferative and metabolic organoid subtypes linked to patient prognosis. High-throughput drug screening revealed distinct response patterns of each subtype that were associated with specific multiomics signatures. Through integrative analyses of LICOB pharmaco-proteogenomics data, we identified the molecular features associated with drug responses and predicted potential drug combinations for personalized patient treatment. The synergistic inhibition effect of mTOR inhibitor temsirolimus and the multitargeted tyrosine kinase inhibitor lenvatinib was validated in organoids and patient-derived xenografts models. We also provide a user-friendly web portal to help serve the biomedical research community. Our study is a rich resource for investigation of liver cancer biology and pharmacological dependencies and may help enable functional precision medicine.

CATI: an efficient gene integration method for rodent and primate embryos by MMEJ suppression.

The efficiency of homology-directed repair (HDR) plays a crucial role in the development of animal models and gene therapy. We demonstrate that microhomology-mediated end-joining (MMEJ) constitutes a substantial proportion of DNA repair during CRISPR-mediated gene editing. Using CasRx to downregulate a key MMEJ factor, Polymerase Q (Polq), we improve the targeted integration efficiency of linearized DNA fragments and single-strand oligonucleotides (ssODN) in mouse embryos and offspring. CasRX-assisted targeted integration (CATI) also leads to substantial improvements in HDR efficiency during the CRISPR/Cas9 editing of monkey embryos. We present a promising tool for generating monkey models and developing gene therapies for clinical trials.

A systematic pan-cancer analysis reveals the clinical prognosis and immunotherapy value of C-X3-C motif ligand 1 (CX3CL1).

It is now widely known that C-X3-C motif ligand 1 (CX3CL1) plays an essential part in the process of regulating pro-inflammatory cells migration across a wide range of inflammatory disorders, including a number of malignancies. However, there has been no comprehensive study on the correlation between CX3CL1 and cancers on the basis of clinical features. In order to investigate the potential function of CX3CL1 in the clinical prognosis and immunotherapy, I evaluated the expression of CX3CL1 in numerous cancer types, methylation levels and genetic alterations. I found CX3CL1 was differentially expressed in numerous cancer types, which indicated CX3CL1 may plays a potential role in tumor progression. Furthermore, CX3CL1 was variably expressed in methylation levels and gene alterations in most cancers according to The Cancer Genome Atlas (TCGA). CX3CL1 was robustly associated with clinical characteristics and pathological stages, suggesting that it was related to the degree of tumor malignancy and the physical function of patients. As determined by the Kaplan-Meier method of estimating survival, high CX3CL1 expression was associated with either favorable or unfavorable outcomes depending on the different types of cancer. It suggests the correlation between CX3CL1 and tumor prognosis. Significant positive correlations of CX3CL1 expression with CD4+ T cells, M1 macrophage cells and activated mast cells have been established in the majority of TCGA malignancies. Which indicates CX3CL1 plays an important role in tumor immune microenvironment. Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that the chemokine signaling pathway may shed light on the pathway for CX3CL1 to exert function. In a conclusion, our study comprehensively summarizes the potential role of CX3CL1 in clinical prognosis and immunotherapy, suggesting that CX3CL1 may represent a promising pharmacological treatment target of tumors.

Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation.

During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt-null embryos display gastrulation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG-DMR partially restores primitive streak elongation in Dnmt-null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.

Reading and writing of mRNA m6A modification orchestrate maternal-to-zygotic transition in mice.

N6-methyladenosine (m6A) modification has been shown to regulate RNA metabolism. Here, we investigate m6A dynamics during maternal-to-zygotic transition (MZT) in mice through multi-omic analysis. Our results show that m6A can be maternally inherited or de novo gained after fertilization. Interestingly, m6A modification on maternal mRNAs not only correlates with mRNA degradation, but also maintains the stability of a small group of mRNAs thereby promoting their translation after fertilization. We identify Ythdc1 and Ythdf2 as key m6A readers for mouse preimplantation development. Our study reveals a key role of m6A mediated RNA metabolism during MZT in mammals.

Cynomolgus monkey embryo model captures gastrulation and early pregnancy.

Human stem cell-derived blastoids display similar morphology and cell lineages to normal blastocysts. However, the ability to investigate their developmental potential is limited. Here, we construct cynomolgus monkey blastoids resembling blastocysts in morphology and transcriptomics using naive ESCs. These blastoids develop to embryonic disk with the structures of yolk sac, chorionic cavity, amnion cavity, primitive streak, and connecting stalk along the rostral-caudal axis through prolonged in vitro culture (IVC). Primordial germ cells, gastrulating cells, visceral endoderm/yolk sac endoderm, three germ layers, and hemato-endothelial progenitors in IVC cynomolgus monkey blastoids were observed by single-cell transcriptomics or immunostaining. Moreover, transferring cynomolgus monkey blastoids to surrogates achieves pregnancies, as indicated by progesterone levels and presence of early gestation sacs. Our results reveal the capacity of in vitro gastrulation and in vivo early pregnancy of cynomolgus monkey blastoids, providing a useful system to dissect primate embryonic development without the same ethical concerns and access challenges in human embryo study.

Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice.

Base editors have been reported to induce off-target mutations in cultured cells, mouse embryos and rice, but their long-term effects in vivo remain unknown. Here, we develop a Systematic evaluation Approach For gene Editing tools by Transgenic mIce (SAFETI), and evaluate the off-target effects of BE3, high fidelity version of CBE (YE1-BE3-FNLS) and ABE (ABE7.10F148A) in ~400 transgenic mice over 15 months. Whole-genome sequence analysis reveals BE3 expression generated de novo mutations in the offspring of transgenic mice. RNA-seq analysis reveals both BE3 and YE1-BE3-FNLS induce transcriptome-wide SNVs, and the numbers of RNA SNVs are positively correlated with CBE expression levels across various tissues. By contrast, ABE7.10F148A shows no detectable off-target DNA or RNA SNVs. Notably, we observe abnormal phenotypes including obesity and developmental delay in mice with permanent genomic BE3 overexpression during long-time monitoring, elucidating a potentially overlooked aspect of side effects of BE3 in vivo.