Genomic context sensitizes regulatory elements to genetic disruption.

Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

Mouse genome rewriting and tailoring of three important disease loci.

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

Self-Supported WO3@RuO2 Nanowires for Electrocatalytic Acidic Water Oxidation.

Developing catalysts with high catalytic activity and stability in acidic media is crucial for advancing hydrogen production in proton exchange membrane water electrolyzers (PEMWEs). To this end, a self-supported WO3@RuO2 nanowire structure was grown in situ on a titanium mesh using hydrothermal and ion-exchange methods. Despite a Ru loading of only 0.098 wt %, it achieves an overpotential of 246 mV for the oxygen evolution reaction (OER) at a current density of 10 mA·cm-2 in acidic 0.5 M H2SO4 while maintaining excellent stability over 50 h, much better than that of the commercial RuO2. After the establishment of the WO3@RuO2 heterostructure, a reduced overpotential of the rate-determining step from M-O* to M-OOH* is confirmed by the DFT calculation. Meanwhile, its enhanced OER kinetics are also greatly improved by this self-supported system in the absence of the organic binder, leading to a reduced interface resistance between active sites and electrolytes. This work presents a promising approach to minimize the use of noble metals for large-scale PEMWE applications.

Suture passer combined with two-hole laparoscopic peritoneal dialysis catheterization in patients undergoing peritoneal dialysis.

BACKGROUND: Laparoscopic techniques are being widely applied for peritoneal dialysis (PD) catheter (PDC) placement. The suture passer is a novel fixation tool that aims to reduce catheter migration. We compared the clinical value of the suture passer combined with two-hole laparoscopic PDC placement to open surgical placement by evaluating preoperative and postoperative conditions, as well as the onset of complications in both groups. METHODS: A retrospective study was conducted including 169 patients who underwent PDC placement surgery from January 2021 to May 2023. Based on the method employed, patients were divided into two groups: the suture passer combined with a two-hole laparoscopy group (SLG) and the open surgical group (SG). Comprehensive patient information, including general data, preoperative and postoperative indicators, peritoneal function after surgery, and the incidence rate of complications, were collected and analyzed. RESULTS: The SLG showed a statistically significant decrease in operative time, intraoperative blood loss, and 6-month postoperative drift rate compared to the SG (p < 0.05). No statistically significant differences were observed between the two groups in terms of sex, age, primary disease, hospitalization time, hospitalization costs, preoperative and postoperative examination indicators, peritonitis, and omental wrapping. CONCLUSIONS: Suture passer combined with two-hole laparoscopic PDC placement, characterized by simplicity and facilitating secure catheter fixation, was deemed safe and effective for patients undergoing PD. It reduces the catheter migration rate and improved surgical comfort. Overall, this technique demonstrates favorable outcomes in clinical practice.

Interactions between MFAP5 + fibroblasts and tumor-infiltrating myeloid cells shape the malignant microenvironment of colorectal cancer.

BACKGROUND: The therapeutic targeting of the tumor microenvironment (TME) in colorectal cancer (CRC) has not yet been fully developed and utilized because of the complexity of the cell-cell interactions within the TME. The further exploration of these interactions among tumor-specific clusters would provide more detailed information about these communication networks with potential curative value. METHODS: Single-cell RNA sequencing, spatial transcriptomics, and bulk RNA sequencing datasets were integrated in this study to explore the biological properties of MFAP5 + fibroblasts and their interactions with tumor-infiltrating myeloid cells in colorectal cancer. Immunohistochemistry and multiplex immunohistochemistry were performed to confirm the results of these analyses. RESULTS: We profiled heterogeneous single-cell landscapes across 27,414 cells obtained from tumors and adjacent tissues. We mainly focused on the pro-tumorigenic functions of the identified MFAP5 + fibroblasts. We demonstrated that tumor-resident MFAP5 + fibroblasts and myeloid cells (particularly C1QC + macrophages) were positively correlated in both spatial transcriptomics and bulk RNA-seq public cohorts. These cells and their interactions might shape the malignant behavior of CRC. Intercellular interaction analysis suggested that MFAP5 + fibroblasts could reciprocally communicate with C1QC + macrophages and other myeloid cells to remodel unfavorable conditions via MIF/CD74, IL34/CSF1R, and other tumor-promoting signaling pathways. CONCLUSION: Our study has elucidated the underlying pro-tumor mechanisms of tumor-resident MFAP5 + fibroblasts and provided valuable targets for the disruption of their properties.

Hydrogen regulates the M1/M2 polarization of alveolar macrophages in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a respiratory disease with high morbidity and mortality worldwide, so far there is no ideal treatment method. Previous studies have shown that hydrogen (H2) is involved in the treatment of COPD as an antioxidant. In this study, the effect of H2 on M1/M2 polarization of alveolar macrophages in COPD rats was observed, and its anti-inflammatory mechanism was further elucidated. Methods: Twenty-four Sprague-Dawley rats were randomly divided into three groups including the control, COPD and H2 group. A rat model of COPD was established by cigarette exposure combined with lipopolysaccharide (LPS) induction. H2 therapy was administered 2 hours per day for 14 days. Lung function and pathology were assessed. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and IL-10 in bronchoalveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA, protein expression and immunoreactivity of inducible nitric oxide synthase (iNOS) and arginase (Arg)-1 in lung were observed by quantitative real-time PCR, western blot and immunohistochemistry. Results: Compared with the control rats, there were a significant decline in lung function, a marked inflammatory infiltration and pulmonary parenchymal remodeling and the increases of IL-6, TNF-α and TGF-ß1 levels in BALF and lung tissue, but a lower expression of IL-10 in COPD rats. The iNOS mRNA and protein expression, as well as its optical density (OD), were increased significantly in lung tissue, while those of Arg-1 decreased significantly. H2 treatment improved the lung function and the parenchymal inflammation, reversed the increased levels of IL-6, TNF-α and TGF-ß1, and the lower IL-10. Meanwhile, H2 also down-regulated the expression of iNOS, but up-regulated expression of Arg-1 in lung tissue. Conclusion: H2 reduces inflammation in the lung of COPD, which may be related to its inhibition of M1 type polarization and activation of M2 type polarization of alveolar macrophage.

Liver Parenchyma Transection-First Approach for Laparoscopic Left Hemihepatectomy: A Propensity Score Matching Analysis.

BACKGROUND: Laparoscopic major liver resection, such as laparoscopic left hemihepatectomy (LLH), is still perceived as a complicated procedure due to technical difficulties and slow learning curve. The study introduced an optimized procedure using the liver parenchyma transection-first approach and investigated its advantages on surgical outcomes by comparison with the conventional hilar dissection approach for LLH. METHODS: Between January 2015 and May 2019, 96 patients who underwent laparoscopic left hemihepatectomy for hepatocellular carcinoma (HCC) were enrolled in the study. Among these, 41 patients underwent the liver parenchyma transection-first approach (LP-first group) and the other 55 underwent the conventional hilar dissection approach (conventional group). A 1:1 propensity score matching (PSM) was performed to compare the perioperative and long-term oncological outcomes of the two groups. RESULTS: After 1:1 PSM, 37 patients in each group were selected for further analysis. The LP-first group was associated with shorter median operative time (210 vs 235 min, P = 0.035) and less blood loss (200 vs 300 mL, P = 0.410). In addition, no statistical differences were found in overall complications between the two groups (8.1% vs 24.3%, P = 0.058). There were no significant differences between the two groups in terms of 1-year and 3-year disease-free survival (DFS, P = 0.608) and overall survival (OS, P = 0.414). CONCLUSION: The prior liver parenchyma approach for LLH is safe and reproducible in selected patients, which showed improved perioperative outcomes and comparable long-term oncological outcomes compared with the conventional approach.

Patient perspectives on surveillance after head and neck cancer treatment: A systematic review.

OBJECTIVES: Current guidelines advise post-treatment surveillance of head and neck cancer (HNC) patients should involve scheduled appointments with a variety of practitioners. Increasing numbers of HNC survivors raise the burden to provide efficient and effective care. With resource limitation, there is growing importance to identify how surveillance can be justified and optimised for survivors. This systematic review presents current evidence on patient perspectives of post-treatment HNC surveillance, aiming to inform future work putting patient priorities at the forefront of surveillance planning. DESIGN: MEDLINE, Embase, the Cochrane Library, NIHR Dissemination Centre, The Kings Fund Library, Clinical Evidence, NHS Evidence and NICE Clinical Evidence were searched to identify publications regarding patient perspectives of HNC post-treatment surveillance. Studies not reporting on both surveillance and patient perspectives were excluded. RESULTS: Three thousand five hundred fifty-eight citations were screened and 49 full-text articles reviewed. Sixteen studies were included in the final review. Three authors reviewed all articles prior to final analysis to ensure all met inclusion criteria. Most evidence was low quality. Study models returned included cross-sectional surveys, structured interviews and one systematic review. Overall, positive perceptions of HNC surveillance were mostly related to increased reassurance. Negative perceptions predominantly focused on anxiety and fear of recurrence, but a lack of psychological support and inadequate access to certain aspects of care were also reported. CONCLUSIONS: This systematic review demonstrates that patients' perceptions of surveillance after HNC are mostly positive, feeling it provides reassurance. However, several studies report unmet needs, particularly regarding managing anxiety.

A study on the treatment of brain tumors with BNCT using several moderators with different thicknesses.

Boron neutron capture therapy (BNCT) is an effective binary radiation therapy that depends on nuclear capture reactions. In recent years, BNCT can be performed without a reactor owing to the development of accelerator-based neutron sources. A new BNCT irradiation facility is proposed, which is based on a 15 mA 2.5 MeV proton accelerator with a 100 µm thickness natural lithium target as a neutron converter. A great quantity of studies has shown that neutron beams with different spectra have unique therapeutic effects on tumors. An appropriate neutron beam for BNCT is obtained by Beam Shaping Assembly (BSA) and the moderator plays a main role in determining the BSA outlet beam spectrum. To figure out the dose distribution in phantom with various kinds of neutron spectrum modes during BNCT, a series of cases are calculated by MCNPX code. The results give a database for treatment of brain tumors with BNCT by using different moderators.

Overexpression of programmed cell death ligand 1 reduces LPS-induced inflammatory cytokine upregulation and enhances osteo/odontogenic-differentiation of human dental pulp stem cells via upregulation of CCCTC-binding factor.

OBJECTIVE: The aim of this study was to explore the effect and mechanism of programmed cell death ligand 1 (PD-L1) in promoting the proliferation and osteo/odontogenic-differentiation of human dental pulp stem cells (hDPSCs) by mediating CCCTC-binding factor (CTCF) expression. DESIGN: The interaction between PD-L1 and CTCF was verified through co-immunoprecipitation. hDPSCs transfected with PD-L1 overexpression and CTCF knockdown vectors were treated with lipopolysaccharide or an osteogenic-inducing medium. Inflammatory cytokines and osteo/odontogenic-differentiation related genes were measured. Osteo/odontogenic-differentiation of hDPSCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining. RESULTS: Overexpression of PD-L1 inhibited LPS-induced pro-inflammatory cytokine upregulation, cell proliferation, ALP activity, and calcium deposition in hDPSCs and elevated the expression of osteo/odontogenic-differentiation related genes; however, such expression patterns could be reversed by CTCF knockdown. Co-immunoprecipitation results confirmed the binding of PD-L1 to CTCF, indicating that PD-L1 overexpression in hDPSCs increases CTCF expression, thus inhibiting the inflammatory response and increasing osteo/odontogenic-differentiation of hDPSCs. CONCLUSION: PD-L1 overexpression in hDPSCs enhances the proliferation and osteo/odontogenic-differentiation of hDPSCs and inhibit the inflammatory response by upregulating CTCF expression.

Genomic context sensitizes regulatory elements to genetic disruption.

Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis. Thus it remains unclear to what extent enhancer function at native loci relies on surrounding genomic context. Using the Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the murine Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting activity of payloads delivered at the endogenous Igf2/H19 locus or ectopically at Hprt revealed that the Igf2/H19 locus includes additional, previously unknown long-range regulatory elements. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate the relationship between locus architecture and function.

Cellular dynamics in pig-to-human kidney xenotransplantation.

BACKGROUND: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. METHODS: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. FINDINGS: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. CONCLUSIONS: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. FUNDING: This work was supported by NIH RM1HG009491 and DP5OD033430.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

In-situ constructing self-supported NiO/RuO2 heterostructure for reinforced alkaline hydrogen evolution reaction.

Rationally designing a strongly coupled heterostructure with rich functional sites and high catalytic stability is essential for efficient energy conversion. This work synthesizes a self-supported NiO/RuO2 heterostructure for hydrogen production via facile dealloying following an in-situ electrochemical oxidation method. It only requires 88 ± 1 mV to drive a current density of -100 mA/cm2 in the alkaline electrolyte during hydrogen evolution reaction (HER), outperforming NiO, RuO2, and Pt foil. The higher anodic potential applied to the dealloyed ribbons results in lower overpotentials and faster reaction kinetics. Meanwhile, the catalytic activity and stability of the individual NiO can be significantly improved once coupled with a small amount of heterogeneous RuO2. The strong synergistic effect between NiO and RuO2 contributes to exposing abundant active sites, optimizing electronic structure, facilitating charge transfer at the interface, and most importantly, maintaining structural stability. These advantages make the self-supported NiO/RuO2 heterostructure a promising candidate for replacing the Pt-based catalysts.

Element immiscibility assisted Ru@Ni3B as an efficient electrocatalyst toward alkaline and acidic hydrogen evolution reaction.

Based on the element immiscibility of Ni-Ru, xRu@Ni3B (x = 0, 0.2, 0.5, 1.0) were facilely synthesized through a one-step dealloying method. Of them, 1.0Ru@Ni3B requires overpotentials of 40 ± 0.2 and 72 ± 0.3 mV to reach a current density of -20 mA cm-2 for acidic and alkaline hydrogen evolution reaction, respectively, which are close to or even better than those of metallic Pt foil. In addition, it could maintain superior catalytic and chemical stability after 24 hours of testing. This work provides a promising strategy for improving the atomic utilization efficiency of highly active noble metals toward the hydrogen evolution reaction (HER).

Tumour Seeding Following Core Biopsy of a Mucoepidermoid Carcinoma of the Parotid: Radiological Evidence and a Surgical Approach to En-Bloc Dissection of the Mass and Tract.

Tumour seeding along the needle tract following core needle biopsy of the parotid is a recognised complication. We present a unique case of mucoepidermoid carcinoma of the parotid in an 18-year-old patient with associated tumour seeding within the core needle biopsy tract. Tumour seeding was confirmed both histologically and radiologically on magnetic resonance imaging as early as 35 days post-biopsy. The patient was treated successfully with a combination of surgery and adjuvant proton beam therapy. This case also visually demonstrates a surgical approach to en-block excision of the mass and tract.

Electrochemical incorporation of heteroatom into surface reconstruction induced Ni vacancy of NixO nanosheet for enhanced water oxidation.

Surface reconstruction of non-oxide oxygen evolution reaction (OER) electrocatalysts has been intensively studied to improve their catalytic performances. However, further modification of the reconstructed active surfaces for better catalytic performances has not been reported. In this work, NiSe nanorods are prepared on nickel foam (NiSe@NF) as the pre-catalyst for electrochemical OER. It is revealed that non-stoichiometric NiO nanosheets with abundant Ni vacancies (NixO) are formed on the surfaces of NiSe nanorods (NixO/NiSe@NF) via in-situ electrochemical oxidation. Furthermore, the OER performances are obviously improved after heteroatom Fe is incorporated electrochemically into NixO nanosheets ((FeNi)O/NiSe@NF). For OER to have a current density of 20 mA cm-2 in 1 M KOH solution, the as-prepared (FeNi)O/NiSe@NF electrode only needs an overpotential of 268 mV. Density functional theory (DFT) calculations reveal that the formation of Ni vacancy can increase the free energy of *OH. More importantly, the incorporation of heteroatom Fe into Ni vacancy can significantly decrease the free energy of *O, which enables Fe-NiO to have the lowest theoretical overpotential for OER in this work. The present work provides a facile and universal strategy to modify the reconstructed active oxides' surfaces for higher electrocatalytic performances.

A conditional counterselectable Piga knockout in mouse embryonic stem cells for advanced genome writing applications.

Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.