Simultaneous multiplex genome loci editing of Halomonas bluephagenesis using an engineered CRISPR-guided base editor.

Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various products. However, the simultaneous editing of multiple loci in H. bluephagenesis TD remains a significant challenge. Herein, we report the development of a multiple loci genome editing system, named CRISPR-deaminase-assisted base editor (CRISPR-BE) in H. bluephagenesis TD. This system comprises two components: a cytidine (CRISPR-cBE) and an adenosine (CRISPR-aBE) deaminase-based base editor. CRISPR-cBE can introduce a cytidine to thymidine mutation with an efficiency of up to 100 % within a 7-nt editing window in H. bluephagenesis TD. Similarly, CRISPR-aBE demonstrates an efficiency of up to 100 % in converting adenosine to guanosine mutation within a 7-nt editing window. CRISPR-cBE has been further validated and successfully employed for simultaneous multiplexed editing in H. bluephagenesis TD. Our findings reveal that CRISPR-cBE efficiently inactivated all six copies of the IS1086 gene simultaneously by introducing stop codon. This system achieved an editing efficiency of 100 % and 41.67 % in inactivating two genes and three genes, respectively. By substituting the Pcas promoter with the inducible promoter PMmp1, we optimized CRISPR-cBE system and ultimately achieved 100 % editing efficiency in inactivating three genes. In conclusion, our research offers a robust and efficient method for concurrently modifying multiple loci in H. bluephagenesis TD, opening up vast possibilities for industrial applications in the future.

LysSYL: a broad-spectrum phage endolysin targeting Staphylococcus species and eradicating S. aureus biofilms.

BACKGROUND: Staphylococcus aureus and its single or mixed biofilm infections seriously threaten global public health. Phage therapy, which uses active phage particles or phage-derived endolysins, has emerged as a promising alternative strategy to antibiotic treatment. However, high-efficient phage therapeutic regimens have yet to be established. RESULTS: In this study, we used an enrichment procedure to isolate phages against methicillin-resistant S. aureus (MRSA) XN108. We characterized phage SYL, a new member of the Kayvirus genus, Herelleviridae family. The phage endolysin LysSYL was expressed. LysSYL demonstrated stability under various conditions and exhibited a broader range of efficacy against staphylococcal strains than its parent phage (100% vs. 41.7%). Moreover, dynamic live/dead bacterial observation demonstrated that LysSYL could completely lyse MRSA USA300 within 10 min. Scan and transmission electron microscopy revealed evident bacterial cell perforation and deformation. In addition, LysSYL displayed strong eradication activity against single- and mixed-species biofilms associated with S. aureus. It also had the ability to kill bacterial persisters, and proved highly effective in eliminating persistent S. aureus when combined with vancomycin. Furthermore, LysSYL protected BALB/c mice from lethal S. aureus infections. A single-dose treatment with 50 mg/kg of LysSYL resulted in a dramatic reduction in bacterial loads in the blood, liver, spleen, lungs, and kidneys of a peritonitis mouse model, which resulted in rescuing 100% of mice challenged with 108 colony forming units of S. aureus USA300. CONCLUSIONS: Overall, the data provided in this study highlight the strong therapeutic potential of endolysin LysSYL in combating staphylococcal infections, including mono- and mixed-species biofilms related to S. aureus.

Iron overload increases the sensitivity of endometriosis stromal cells to ferroptosis via a PRC2-independent function of EZH2.

Given the high concentration of iron in the micro-environment of ovarian endometriosis, it is plausible to hypothesize that ectopic endometrial cells may be more susceptible to undergoing ferroptosis. Manipulation of ferroptosis has been explored as a potential therapeutic strategy to treat related diseases. In this study, we examined the impact on ectopic endometrial stromal cells (EESCs) of iron overload and an inducer of ferroptosis. We found that the iron concentration in the ovarian endometriosis was much higher than control samples. Treatment of cultured EESCs with ferric ammonium citrate (FAC) increase the sensitivity to undergo ferroptosis. By analyzing the RNA-seq results, it was discovered that zeste 2 polycomb repressive complex 2 subunit (EZH2) was significantly downregulated in ferroptosis induced EESCs. Moreover, overexpression of EZH2 effectively prevented the induction of ferroptosis. In addition, the activity or expression of EZH2 is directly and specifically inhibited by the methyltransferase inhibitor GSK343, which raises the sensitivity of stromal cells to ferroptosis. Taken together, our findings revealed that EZH2 act as a suppressor in the induced cell ferroptosis through a PRC2-independent methyltransferase mechanism. Therefore, blocking EZH2 expression and inducing ferroptosis may be effective treatment approaches for ovarian endometriosis.

MR imaging findings of stage I intravenous leiomyomatosis: a retrospective single-center study in 19 cases.

OBJECTIVES: The aim was to explore the magnetic resonance imaging (MRI) features of stage-I intravenous leiomyomatosis (IVL). MATERIALS AND METHODS: From January 2019 to January 2023, clinical, pathological, and MRI data were collected from 19 cases confirmed by surgical pathology. Two radiologists retrospectively measured the tumor sizes, T1WIs, T2WIs, and ADC values and evaluated contrast-enhanced T1WIs, DWIs, complications and parauterine infiltrations. The number of tumor cells and the total nuclear area were measured. The percentage of tumor cell area out of the total area was used as the tumor cell density. RESULTS: Nineteen patients with stage-I IVL aged 33 to 66 years (mean age: 46 ± 7.6 years) were included in this study. All 19 cases were located in the myometrium or parametrium, with a mean diameter of 11.2 ± 4.8 cm. Among these cases, 14 (73.6%) were associated with leiomyoma, and six (31.6%) involved the broad ligament. Isointensity was observed in the T1WIs of 12 cases (63.2%), while slight hypointensity was seen in five patients (26.3%). Isointensity was observed in the on T2WIs of four cases (21.1%), and iso- or slight hyperintensity was observed in 15 cases (78.9%). A significant difference was detected between the normalized T2WIs of IVL and myometrium (p < 0.001). A Pearson correlation test showed demonstrated a negative correlation between the ADC and tumor cell density values (r = - 0.946, p < 0.001). Tortuous vessels were present in 17 cases (89.5%) within or next to the lesions, and multiple winding cord-like filling defects were seen in 11 cases (57.9%) within the tortuous vessels on the T2WIs. CONCLUSION: Identifying the characteristic MRI features of stage-I IVL helped improve the diagnostic accuracy achieves for this rare tumor. Stage-I IVL often presents as a large mass accompanied by leiomyoma, and it easily invades the broad ligament. TIWI signals exhibited isointensity, and T2WI signals contained iso- or slight hyperintensity. Tortuous vessels were present within or next to the lesions, and multiple winding cord-like filling defects were observed within the tortuous vessels on the T2WIs.

Polyphyllin I inhibits endometriosis in vitro by inducing apoptosis and autophagy via the inactivation of AKT/mTOR signalling pathway.

Previous research has indicated that Polyphyllin I (PPI) possesses potent anticancer properties. However, its impact on endometriosis remains unexplored. This study aims to investigate the inhibitory effects of PPI on ectopic endometrial stromal cells (EESCs). The CCK-8 and flow cytometry results respectively showed that the cell viability of EESCs decreased and the number of apoptotic cells increased in a dosage dependent of PPI. Wound healing and transwell assays demonstrated a notable reduction in cell motility and migration ability in the PPI group. Moreover, the Western blot analysis revealed a decrease in p62 levels and an increase in LC3-II expression following PPI administration. Additionally, the protein levels of p-Akt and p-mTOR were observed to decrease with increasing concentrations of PPI, indicating the potential of PPI to induce autophagy in EESCs through modulation of the Akt/mTOR signalling pathway. Consequently, PPI holds promise as a targeted therapeutic agent for the management of endometriosis.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Genomic Epidemiology and Phenotypic Characterization of Staphylococcus aureus from a Tertiary Hospital in Tianjin Municipality, Northern China.

Staphylococcus aureus remains a dangerous pathogen and poses a great threat to public health worldwide. The prevalence of the S. aureus clonotype is temporally and geographically variable. The genomic and phenotypic characteristics of S. aureus isolates in Tianjin, which is among the four big municipalities in China, are unclear. In the present study, 201 nonduplicate S. aureus isolates, including 70 methicillin-resistant S. aureus (MRSA) and 131 methicillin-susceptible S. aureus (MSSA), were collected from 2015 to 2021 in a tertiary hospital in Tianjin. Whole-genome sequencing of S. aureus isolates was carried out to investigate bacterial molecular characteristics, genomic phylogeny, antimicrobial resistance (AMR) gene carriage, and virulence factor gene distribution. The antibiotic resistance profiles, hemolytic activities, and biofilm formation abilities of the S. aureus isolates were also determined. In total, 31 distinct sequence types (STs) and 68 spa types were identified. ST59 (15.9%, 32/201) was the predominant clonotype, followed by ST398 (14.9%, 30/201) and several other major STs (ST1, ST5, ST6, ST22, ST25, ST188, and the newly emerging ST5527). ST59 and ST5527 mainly included MRSA isolates, while ST398 and the other major STs mainly included MSSA isolates. The unique characteristics of the S. aureus isolates belonging to the major STs were determined. ST59 isolates exhibited strong hemolytic activity, and ST398 strains had high biofilm formation capacity, while ST5527 isolates presented the greatest AMR. The genomic epidemiology and phenotypic characteristics of S. aureus isolates determined in this study will help in disease control in nosocomial environments. IMPORTANCE Staphylococcus aureus is an important bacterium pathogen in tertiary hospitals, which provide rich medical resources. Tianjin is one of the four municipalities in China with a population of more than 13 million. However, the epidemiology and molecular characteristics of S. aureus isolates in Tianjin are unknown. In this study, the genomic and phenotypic analyses were performed to investigate 201 S. aureus isolates collected from a tertiary hospital in Tianjin over a time span of 6 years. The refined analysis of predominant clones ST59, ST398, the newly emerging clone ST5527, as well as other major clones, will undoubtedly aid in the control and prevention of infections caused by S. aureus in tertiary hospitals.

Upper cortical layer-driven network impairment in schizophrenia.

Schizophrenia is one of the most widespread and complex mental disorders. To characterize the impact of schizophrenia, we performed single-nucleus RNA sequencing (snRNA-seq) of >220,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls. In addition, >115,000 neurons were analyzed topographically by immunohistochemistry. Compositional analysis of snRNA-seq data revealed a reduction in abundance of GABAergic neurons and a concomitant increase in principal neurons, most pronounced for upper cortical layer subtypes, which was substantiated by histological analysis. Many neuronal subtypes showed extensive transcriptomic changes, the most marked in upper-layer GABAergic neurons, including down-regulation in energy metabolism and up-regulation in neurotransmission. Transcription factor network analysis demonstrated a developmental origin of transcriptomic changes. Last, Visium spatial transcriptomics further corroborated upper-layer neuron vulnerability in schizophrenia. Overall, our results point toward general network impairment within upper cortical layers as a core substrate associated with schizophrenia symptomatology.

Accessory Gene Regulator (agr) Allelic Variants in Cognate Staphylococcus aureus Strain Display Similar Phenotypes.

The accessory gene regulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.

Non-puerperal Uterine Inversion with endometrial polyps in an 11-year-old girl: A Case Report.

BACKGROUND: Non-puerperal uterine inversion is a rare condition with diagnostic and surgical challenges. Clinically, the inverted uterus appears as a mass protruding from the vagina and is often misdiagnosed as a malignant tumor and surgically removed. CASE: An 11-year-old girl was admitted to the emergency room due to spontaneous vaginal mass protrusion. The pudendum examination showed an irregular and dark red neoplasm protruding from the vagina. The final diagnosis was non-puerperal uterine inversion with an endometrial polyp. SUMMARY AND CONCLUSION: MRI is the key to the diagnosis of uterine inversion. Our review confirmed that the 11-year-old girl was the youngest in the world to suffer from non-puerperal uterine inversion.

Intercomparison of global terrestrial carbon fluxes estimated by MODIS and Earth system models.

Earth system models (ESMs) have been widely used to simulate global terrestrial carbon fluxes, including gross primary production (GPP) and net primary production (NPP). Assessment of such GPP and NPP products can be valuable for understanding the efficacy of certain ESMs in simulating the global carbon cycle and future climate impacts. In this work, we studied the model performance of 22 ESMs participating in the fifth and sixth phases of the Coupled Model Intercomparison Project (CMIP5 and CMIP6) by comparing historical GPP and NPP simulations with satellite data from MODIS and further evaluating potential model improvement from CMIP5 to CMIP6. In CMIP6, the average global total GPP and NPP estimated by the 22 ESMs are 16% and 13% higher than MODIS data, respectively. The multi-model ensembles (MME) of the 22 ESMs can fairly reproduce the spatial distribution, zonal distribution and seasonal variations of both GPP and NPP from MODIS. They perform much better in simulating GPP and NPP for grasslands, wetlands, croplands and other biomes than forests. However, there are noticeable differences among individual ESM simulations in terms of overall fluxes, temporal and spatial flux distributions, and fluxes by biome and region. The MME consistently outperforms all individual models in nearly every respect. Even though several ESMs have been improved in CMIP6 relative to CMIP5, there is still much work to be done to improve individual ESM and overall CMIP performance. Future work needs to focus on more comprehensive model mechanisms and parametrizations, higher resolution and more reasonable coupling of land surface schemes and atmospheric/oceanic schemes.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

[Data analysis and prospects of the national college students' life science competition].

The Chinese national college students' life science competition has been held for three times, with good organization, large scale and high participation degree. The competition plays an important role in promoting life science education and research. This paper reports the form and status of the competition, statistically analyses the registration data and competition results by region and year, based on the previous three competitions. By combining new changes and understanding in the field of life science, we also indicate prospects on how to better promote the competition.

Exploration of the Evaluation and Optimization of Community Epidemic Prevention in Wuhan Based on a DEA Model.

Background-Communities played a key role in preventing the spread of coronavirus, not only during the threshold period of the epidemic but also in the normal stage of prevention. Scientifically evaluating the community's work is necessary for prevention in the normal period of the epidemic and can provide a reference for the management of different countries. Methods-Based on data envelopment analysis (DEA), this article used community worker data to evaluate the matching of service supply and demand during the epidemic period and used co-word analysis to analyze the content and the residents' demands for community service from the threshold period to the normal period of the epidemic. Results-According to the results of the DEA model, early in the epidemic, 13 of the 15 districts' DEA values were invalid, indicating that there was a shortage in community workers in Wuhan. The results of public opinion analysis showed that from the threshold to the normal period of the epidemic, the emphasis on community service gradually transformed from epidemic prevention to an integrated service, which effectively met the composite service needs of community residents for both prevention and life. Conclusions-In the face of public health emergencies, the government should ensure an adequate number of service personnel, mobilize the service resources, refine the service content, and adjust the incentive policy, which can help to improve the quality of residents' lives and the coordination degree of the prevention and control as part of the epidemic control in the emergency period and the social and economic recovery after the epidemic.

A Revised Surgical Strategy for the Distal Tibiofibular Interosseous Osteochondroma.

Osteochondroma is one of the most common benign bone tumor; however, the surgical treatment still remains a challenge for those that occur at the distal tibiofibular interosseous location. Previously, the transfibular approach has been successfully described, but the potential damage of the syndesmosis would give rise to the instability of the ankle joint and thus may result in the unfavorable long-term outcome. Here, a revised strategy which can protect the syndesmotic complex is introduced. From 2010 to 2017, eleven patients with the distal tibiofibular interosseous osteochondroma who underwent the revised surgery were collected. The distal fibular osteotomy and posterior tibial osteotomy were performed to keep the inferior syndesmosis intact for better stability of the ankle joint. Both the anterior tibiofibular ligaments (AITFL) and posterior tibiofibular ligaments (PITFL) have been preserved successfully, and thus, the stability of the ankle joint has been maintained due to our strategy. The VAS and AOFAS scores were utilized to assess the clinical outcome and function. Postoperatively, all the patients were pain-free and were able to wear the appropriate shoes at the last follow-up. Preoperative and postoperative AOFAS scores were 93.63 ± 6.91 and 47.27 ± 5.27 (P < 0.05), respectively. Moreover, the average VAS score was 1.73 ± 0.27 (compared with preoperative as 7.45 ± 2.15, P < 0.05), demonstrating obvious improvement after the operation. To our best knowledge, this is the first time to perform the resection of the distal tibial interosseous osteochondroma involving the fibula without interrupting the inferior syndesmotic complex especially the AITFL and PITFL. We believe that this strategy may pave a new way for optimized clinical outcome for these patients with distal tibiofibular interosseous osteochondroma. This clinical trial study is registered with number ChiCTR1900024690.

Specific histone modifications associate with alternative exon selection during mammalian development.

Alternative splicing (AS) is frequent during early mouse embryonic development. Specific histone post-translational modifications (hPTMs) have been shown to regulate exon splicing by either directly recruiting splice machinery or indirectly modulating transcriptional elongation. In this study, we hypothesized that hPTMs regulate expression of alternatively spliced genes for specific processes during differentiation. To address this notion, we applied an innovative machine learning approach to relate global hPTM enrichment to AS regulation during mammalian tissue development. We found that specific hPTMs, H3K36me3 and H3K4me1, play a role in skipped exon selection among all the tissues and developmental time points examined. In addition, we used iterative random forest model and found that interactions of multiple hPTMs most strongly predicted splicing when they included H3K36me3 and H3K4me1. Collectively, our data demonstrated a link between hPTMs and alternative splicing which will drive further experimental studies on the functional relevance of these modifications to alternative splicing.