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Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.

In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa. Retinitis pigmentosa is mainly caused by mutations that initially affect survival of rod photoreceptors, leading to secondary loss of cones. Here the authors use gene editing to prevent rod degeneration, leading to survival of cones and improved vision in mice.

Salmonella is a foodborne pathogen that enters the host’s body through contaminated food and water, leading to gastroenteritis and systemic diseases. It is a significant veterinary and human pathogen capable of infecting both humans and animals, with substantial impacts on public health, human well-being, and the economic development of the livestock and poultry farming industry. Small non-coding RNAs (sRNAs), typically 50–500 nucleotides (nt) in length, have been identified in various bacteria, including Escherichia coli , Brucella , Pseudomonas aeruginosa , and Salmonella . These sRNAs play crucial roles in regulating diverse physiological processes within bacteria. This review emphasizes recent advances in understanding how sRNAs regulate the virulence of Salmonella spp, such as the discovery of novel sRNAs like SaaS and new regulatory mechanisms of known sRNAs like RyhB-1/RyhB-2 and SdsR/Spot 42. It also outlines critical future directions, including exploring the multifaceted functions of sRNAs in lifestyle or infection phase transitions, fully elucidating their roles in regulating the host immune response, studying the combined actions of multiple sRNAs on host pathogenesis and expanding research to more Salmonella serotypes and diverse animal models. Through these efforts, this review aims to enhance our understanding of Salmonella sRNAs and their infection mechanisms.

Salmonella enterica serovar Typhimurium ( S . Typhimurium), a prevalent zoonotic pathogenic bacterium, has its pathogenicity and intracellular parasitic mechanisms intricately linked to a range of virulence factors. S . Typhimurium infects the host mainly through the type III secretion system (T3SS), with SPI-1-encoded T3SS mediating bacterial internalization and host cytoskeletal remodeling, while SPI-2-encoded T3SS maintains the stability of bacterial-containing vesicles (SCVs) by inhibiting lysosomal fusion. Moreover, the type VI secretion system (T6SS) aids S . Typhimurium in competing with other microorganisms and facilitates its ability to traverse the mucus layer for host colonization, with flagella also contributing significantly to this process. Various effector proteins, secreted by S . Typhimurium, help preserve membrane stability for intracellular survival. To sustain intracellular viability, S . Typhimurium stabilizes its membranes by releasing multiple effector proteins. It sustains the homeostasis of its intracellular living environment by hijacking the host’s glycolytic metabolism and suppressing immune responses. At the same time, S . Typhimurium also uses the bidirectional regulation of the autophagy pathway and the co-regulation of apoptosis and pyroptosis programs to construct complex immune escape routes. Our primary objective is to integrate cutting-edge research on S . Typhimurium’s parasitism within host cells and the bidirectional mechanisms governing its coexistence with the host. Our focus lies on secretion systems, metabolic reprogramming, and immune evasion, which could provide new perspectives for developing approaches to combat and manage S . Typhimurium.

Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggested that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.

Background: Salmonella Typhimurium poses a substantial health risk to both humans and animals. This study evaluated the potential of using the Salmonella Typhimurium ΔsptP mutant as a live-attenuated vaccine candidate by constructing it through homologous recombination and assessing its key biological properties, including growth characteristics, immunogenicity, and protective efficacy. Methods: We generated the ΔsptP mutant through targeted gene deletion, ensuring the preservation of the bacterial strain’s growth and stability. In vitro and in vivo assays were performed to compare the invasive capabilities between the mutant and the wild-type strains. Specifically, we examined the invasion into RAW264.7 murine macrophages and mice. Furthermore, the virulence of the mutant was evaluated by determining the median lethal dose (LD50). To evaluate immunogenicity and protection, mice were immunized with 2 × 104 CFUs of the ΔsptP mutant, followed by a booster immunization, and then challenged with a virulent strain. Results: The ΔsptP mutant exhibited no significant changes in growth characteristics or genetic stability compared to the wild-type strain. However, it demonstrated a significantly diminished capacity for invasion in both murine macrophages and mice. The LD50 for the mutant was 39.92-fold higher than that of the wild-type, indicating a marked reduction in virulence. Mice immunized with the ΔsptP mutant and administered a booster immunization exhibited 87.5% protection against challenge with a virulent strain, as compared to the PBS control group. Moreover, the mutant induced IgG antibody levels comparable to those induced by the wild-type strain. Conclusions: The ΔsptP mutant of Salmonella Typhimurium exhibits markedly reduced virulence while retaining robust immunogenicity and protective efficacy. These findings suggest that the ΔsptP mutant is a promising candidate for a live-attenuated vaccine, potentially providing an effective strategy to prevent Salmonella Typhimurium infections.

, the primary pathogen responsible for avian pullorum disease, has imposed substantial economic losses on the poultry industry. sRNAs, a class of small non-coding RNAs, have been identified in numerous bacterial species and serve as pivotal regulatory factors in bacteria. A bacterial infection assay was conducted to detect the differential transcription levels of sRNA12 in the macrophage cell HD11. Environmental stress tests, intracellular survival assays, target gene transcription analyses and chick virulence tests were conducted to compare the wild-type strain and the ΔsRNA12 deletion strain. A significant 7.5-fold increase in the transcription level of sRNA12 was observed during the invasion of host cells by bacteria. Under hyperosmotic conditions, the survival ability of the deletion strain was markedly reduced, while in a highly oxidative environment, it was significantly enhanced. Compared with the wild-type strain, the colonization ability of the ΔsRNA12 deletion strain in HD11 cells was enhanced by 3.5-fold. The transcription levels of most target genes of sRNA12, such as , , and , were significantly upregulated. The LD of the deletion strain in chicks was approximately three times lower than that of the wild-type strain. Moreover, the colonization abilities of the deletion strain in the liver, spleen, and cecum of chicks were significantly enhanced and it induced more severe organ lesions. The findings suggest that the deletion of sRNA12 enhances the virulence of . This research provides novel insights into elucidating the pathogenic mechanism of and the associated regulatory signaling pathways.

The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a −/− mouse that recapitulates features of Meckel syndrome including embryonic lethality and multiorgan defects. Cilia are absent in Cc2d2a −/− embryonic node and other somatic tissues; disruption of cilia-dependent Shh signalling appears to underlie exencephaly in mutant embryos. The Cc2d2a −/− mouse embryonic fibroblasts (MEFs) lack cilia, although mother centrioles and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a −/− MEFs, subdistal appendages are lacking or abnormal by transmission electron microscopy. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild-type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis. Mutations in the centrosome-cilia gene, Cc2d2a , result in Meckel and Joubert syndromes in humans. By creating Cc2d2a -mutant mice, Veleri et al. show that this gene encodes a component of subdistal appendages; ciliary structures thought to be required to anchor cilia to the microtubule network.

Brucellosis is a highly contagious zoonosis caused by Brucella. Brucella can invade and persist inside host cells, which results in chronic infection. We constructed AIR interference and overexpression lentiviruses to acquire AIR interference, overexpression, and rescue stable expression cell lines. We also established a Brucella melitensis 16M-infected macrophage model, which was treated with either the vehicle control or NAC (ROS scavenger N-acetylcysteine (NAC) for 0, 3, 6, 12, and 24 h. Confocal laser microscopy, transmission electron microscopy, fluorescence quantitative PCR, flow cytometry, ELISA, and Western blot were used to detect inflammation, cell autophagy and apoptosis-related protein expression levels, ROS levels, and the distribution of mitochondria. It was found that after interference and overexpression of AIR, ROS release was significantly changed, and mitochondria became abnormally aggregated. B. melitensis 16M activated the NLRP3/AIM2 inflammatory complex, and induced RAW264.7 cells to secrete IL-1β and IL-18 through the ROS pathway. B. melitensis 16M also altered autophagy-related gene expression, increased autophagy activity, and induced cell apoptosis through the ROS pathway. The results showed that after B. melitensis 16M infection, ROS induced apoptosis, inflammation, and autophagy while AIR inhibited autophagosome maturation and autophagy initiation. Autophagy negatively regulated the activation of inflammasomes and prevented inflammation from occurring. In addition, mitophagy could promote cell apoptosis.