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Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta ( Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated Hexb tdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS. Microglia have key roles in central nervous system (CNS) disease and homeostasis but their study can be challenging. Prinz and colleagues identify hexosaminidase subunit beta ( Hexb ) to be specifically expressed by microglia and stable even under inflammatory conditions.

Enteroviruses are positive-sense single-stranded RNA viruses and common pathogens that are responsible for diverse public health diseases. To facilitate the study of the virus biology and pathogenesis of enterovirus, we developed a rapid method for construction of the enteroviral cDNA clones including enterovirus A71 (EV-A71) and coxsackievirus B5 (CVB5). As described for EV-A71, the full-length cDNA of CVB5 was amplified by long-distance PCR and cloned into a T7 promoter-containing plasmid using directional seamless cloning technology. The virus was successfully rescued by single transfection into cells stably expressing T7 polymerase and exhibited characteristics similar to the parental virus. Next, through systematic construction and the optimization of the EV-A71 and CVB5 reporter viruses, we successfully generated two novel reporter virus panels with high virus titers, rapid replication, and relatively stable genetic inheritance across passages using the new fluorescence proteins mScarlet3-H and the smallest miRFP670nano3. Analysis of critical determinants for the reporter virus construction revealed that reporter gene sizes, genomic insertion sites, and the usage of protease recognition sites are crucial parameters. The EV-A71 and CVB5 reporter viruses enable antiviral drug evaluation, as demonstrated by our identification of gemcitabine as a broad-spectrum inhibitor of both viruses. These systems also facilitate the functional interrogation of host factors, exemplified by our discovery that METTL3 promotes EV-A71 and CVB5 replication. These reverse genetic tools, including infectious cDNA clones and reporter viruses, will advance basic enterovirus biology and accelerate antiviral drug discovery.

Until recently, complex multi-parameters were required for the isolation and identification of haematopoietic stem cells, complicating study of their biology in situ ; here the authors have found that expression of a single gene, Hoxb5 , defines haematopoietic stem cells with long-term reconstitution capacity, and that these cells are mainly found in direct contact with endothelial cells. Haematopoietic stem cell niche characterized Until recently, the isolation and recognition of haematopoietic stem cells (HSCs) has been a complex process involving the manipulation of multiple parameters, and this complicates the study of HSC biology in situ . In particular, it has been difficult to establish their relationship to the HSC niche, and how their self-renewal and differentiation properties are modulated by their environment. Here Irving Weissman and colleagues demonstrate that expression of a single gene, Hoxb5 , defines cells with long-term reconstitution capacity, and show that these cells are mainly found directly in contact with endothelial cells. Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation 1 , complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal 2 , 3 , 4 , differentiation, ageing, niche 5 , and diversity 6 , 7 , 8 . Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 ( Hoxb5 , also known as Hox-2.1 ), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5 + HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7–35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5 + ) are directly attached to VE-cadherin + cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments 1 , 2 . However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins—including gene expression, chromatin occupancy and three-dimensional conformation—we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait. Skate-specific changes in the epigenome and its three-dimensional organization contributed to the evolution of the batoid fin morphology.

To investigate the impact of different 5ʹ untranslated regions (UTRs) on mRNA vaccine translation efficiency, five dual-reporter gene expression plasmids with different 5ʹUTRs were constructed. The corresponding mRNA transcripts were transcribed and capped in vitro. By comparing the expression levels of reporter genes with different 5ʹUTRs, we identified the 5ʹUTR associated with the highest expression level. Subsequently, HIVgp145 mRNA vaccines containing various 5ʹUTRs were constructed and verified. The results demonstrated that mRNA 3 (β-globin 5ʹUTR) displayed the greatest number of green fluorescence-positive cells and the highest luciferase fluorescence intensity in the reporter gene expression system. Further, among the HIVgp145 mRNA vaccines with different 5ʹUTRs, mRNA 7 (β-globin 5ʹUTR) exhibited the highest level of expression. These findings indicate that it is feasible to use the 5ʹUTR of β-globin in an mRNA vaccine, laying the foundation for animal immunogenicity testing.

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. The construction of SVA virus carrying foreign reporter gene provides a powerful tool in virus research. However, it is often fraught with rescuing a recombinant picornavirus harboring a foreign gene or maintaining the stability of foreign gene in the virus genome. Here, we successfully generated recombinant SVA GD05/2017 viruses (V-GD05-clone) expressing the green fluorescent protein (iLOV), red fluorescent protein (RFP), or NanoLuc luciferase (Nluc). These recombinant viruses have comparable growth kinetics to the parental virus. Genetic stability analysis indicated that V-GD05-iLOV was highly stable in retaining iLOV gene for more than 10 passages, while V-GD05-RFP and V-GD05-Nluc lost the foreign genes in five passages. In addition, high-intensity fluorescent signals were found in the V-GD05-RFP- and V-GD05-iLOV-infected cells by fluorescence observation and flow cytometry analysis, and the luciferase activity assay could quantitatively monitor the replication of V-GD05-Nluc. In order to identify the porcine cell receptor for SVA, anthrax toxin receptor 1 (ANTXR1) was knocked out or overexpressed in the ST-R cells. The ANTXR1 knock-out cells lost the ability for SVA infection, while overexpression of ANTXR1 significantly increased the cell permissivity. These results confirmed that ANTXR1 was the receptor for SVA to invade porcine cells as reported in the human cells. Overall, this study suggests that these SVA reporter viruses will be useful tools in elucidating virus pathogenesis and developing control measures.Key points• We successfully generated SVA viruses expressing the iLOV, RFP, or Nluc.• The iLOV was genetically stable in the V-GD05-iLOV genome over ten passages.• ANTXR1 was the receptor for SVA to invade porcine cells.

Various whole cell-based biosensors have been reported in the literature for the last 20 years and these reports have shown great potential for their use in the areas of pollution detection in environmental and in biomedical diagnostics. Unlike other reviews of this growing field, this mini-review argues that: (1) the selection of reporter genes and their regulatory proteins are directly linked to the performance of celllular biosensors; (2) broad enhancements in microelectronics and information technologies have also led to improvements in the performance of these sensors; (3) their future potential is most apparent in their use in the areas of medical diagnostics and in environmental monitoring; and (4) currently the most promising work is focused on the better integration of cellular sensors with nano and micro scaled integrated chips. With better integration it may become practical to see these cells used as (5) real-time portable devices for diagnostics at the bedside and for remote environmental toxin detection and this in situ application will make the technology commonplace and thus as unremarkable as other ubiquitous technologies.

Summary A critical step in generating gene‐edited plants is the removal of CRISPR‐related transgenes from T0 plants and their progenies, a process that is generally time‐consuming and labour‐intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self‐elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T1 plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY‐CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T0 generation and its progenies. We then combined the RUBY‐CRISPR unit with several TKC variants to develop four RUBY‐TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non‐TKC, TKC alone and RUBY‐CRISPR, our TKC2s were much more efficient in the generation of transgene‐free edited progenies by up to 100% in the T0 generation. TKC2s not only omit the need for screening of the plants with transgenes in the T0 generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene‐free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.

The current diagnosis of acute kidney injury involves the measurement of renal biomarkers, such as serum creatinine, which provide a crude means of detecting cellular stress and injury. To determine whether Ngal expression provides an alternate renal biomarker capable of detecting the initial phases of renal injury, Paragas et al . have developed an Ngal reporter mouse that offers a noninvasive and real-time method for the continuous and quantitative reporting of cell stress and injury at the injury site. Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal ( Lcn2 ) locus. The Ngal -Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal -Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal -Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal -Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.

This study compares media coverage of the Palestinian-Israeli conflict between Al Jazeera and Western media outlets such as the BBC, CNN, and The New York Times. The study aims to discover viewpoint gaps between Palestinian and Western news coverage, assess the role of Aljazeera local reporters in promoting world awareness, and investigate biases in media stories from both sides. A qualitative content analysis examined 14 news articles—seven from Al Jazeera and seven from Western sources—that covered significant events during the 2023-2024 Gaza conflict. The study examined variations in how conflict-related events are portrayed, language usage, and narrative framing. The findings show essential differences in viewpoint between Al Jazeera and Western media. Al Jazeera's reporting is typically more humane and passionate, highlighting Palestinian suffering and placing blame for violence on Israeli troops; in contrast, news from Western media tends to be largely ignorant of the sufferings of the innocent Palestinians who continue to become victims of endless indiscriminate bombings. Moreover, Aljazeera local reporters are essential in influencing attitudes worldwide because they provide personal stories of the war in contrast to Western media's more detached, objective reporting style. These results emphasize the significance of comprehending media bias and viewpoint gaps in conflict reporting and draw attention to the particular contribution that local reporters make in offering a more thorough analysis of the Palestinian-Israeli conflict.