Search Results (106)

A restriction enzyme PsaI, an isoschizomer of the type II restriction endonuclease HindIII, has been purified to homogeneity from Gram-negative bacilli Pseudomonas anguilliseptica KM9 found in a wastewater treatment plant in Poland. Experimental data revealed that R.PsaI is highly active in the presence of Co²⁺, Mg²⁺, and Zn²⁺ and reached a maximal level of activity between 2.5 and 10 mM while its activity was significantly decreased in the presence of Ca²⁺, Fe²⁺, Mn²⁺, and Ni²⁺. Moreover, we found that the purified R.PsaI did not require NaCl for enzyme activity. Restriction cleavage analysis followed by sequencing confirmed 5′-AAGCTT-3′ as the recognition site. The genes for restriction–modification system PsaI were identified and characterized. Downstream of the psaIM gene, we noticed an ORF that shares extensive similarity with recombinase family protein specifically involved in genome rearrangements. Sequence analysis revealed that the PsaI R-M gene complex showed striking nucleotide sequence similarity (>98%) with the genes of the PanI R-M system from a P. anguilliseptica MatS1 strain identified in a soil sample from Sri Lanka. Full article

PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a’s collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10² copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management. Full article

Trichomonas vaginalis, the protozoan responsible for trichomoniasis, encounters fluctuating levels of metal cations in the male urogenital tract, notably zinc (Zn²⁺) and cadmium (Cd²⁺), which may induce genotoxic stress. While zinc is a key physiological component of the male reproductive tract, both Zn²⁺ and Cd²⁺ can become genotoxic at elevated concentrations. However, their effect on DNA repair mechanisms in T. vaginalis remains poorly understood. This study characterizes, for the first time, the expression and modulation of the recombinase TvRAD51, a homologous recombination (HR) key enzyme, in response to UV irradiation and sublethal concentrations of Zn²⁺ (1.6 mM) and Cd²⁺ (0.1 mM). In silico analyses confirmed the presence and conserved structure of the tvrad51 gene and its interaction with HR-related proteins, such as TvBLM and TvBRCA2. Quantitative RT-PCR, Western blot, and immunofluorescence assays revealed that TvRAD51 is upregulated at both transcript and protein levels following UV- and cation-induced DNA damage, with distinct temporal expression patterns for Zn²⁺ and Cd²⁺ exposure. Notably, TvRAD51 showed nuclear localization at early time points post-exposure, suggesting active participation in DNA repair processes. These findings demonstrate that TvRAD51 is a central component of the genotoxic stress response in T. vaginalis, potentially contributing to parasite survival and adaptation in hostile environments through homologous recombination repair pathways. Full article

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a severe neurodevelopmental disorder caused by a deficiency in the X-linked gene calcium/calmodulin-dependent serine protein kinase (CASK). A better understanding of the role of CASK in the pathophysiology of neurodevelopmental disorders may provide insights into novel therapeutic and diagnostic strategies for MICPCH syndrome and other neurodegenerative diseases. To investigate this, we generated CASK knockout (KO) cerebellar granule (CG) cell culture from CASK floxed (CASK^flox/flox) mice by infecting lentiviruses expressing codon-improved Cre recombinase (iCre). We performed RNA-sequencing (RNA-seq) on these cells and found that CASK-KO CG cells underwent apoptosis by activating intracellular Jun N-terminal kinase (JNK) signaling and upregulating reactive oxygen species (ROS)-related gene expression. We also performed mouse gait analysis and limb clasping behavior experiments on trans-heterozygous CASK-KO and Hprt-eGFP (CASK^+/- Hprt^eGFP/+) mice. The CASK^+/- Hprt^eGFP/+ mice exhibited cerebellar ataxic phenotypes as judged by the scores of these experiments compared to the CASK wild-type control (CASK^+/+ Hprt^eGFP/+) mice. Interestingly, the administration of the JNK inhibitor, JNK-IN-8, in CASK-KO CG cell cultures increased CG cell survival by reducing ROS generation. Moreover, injection of JNK-IN-8 into the cerebellum of CASK^+/- Hprt^eGFP/+ mice suppressed CG cell death and alleviated cerebellar ataxic phenotypes in vivo. In conclusion, JNK-IN-8 suppresses the cell death and activation of the ROS pathway in CASK-KO CG cells in both in vitro and in vivo models, suggesting its potential as a therapeutic strategy for cerebellar neurodegeneration in MICPCH syndrome. Full article

Cell Division Autoantigen 1 (CDA1) has been shown to play a role in enhancing transforming growth factor beta (TGFβ) signaling, leading to fibrosis in diabetic kidney disease (DKD) using mouse strains with global CDA1 gene deletion. In these models, diabetes has been induced, leading to DKD in the absence of CDA1. It is still unknown whether inhibition of CDA1 activity after onset of diabetes in the presence of CDA1 can attenuate renal fibrosis in vivo. Thus, we examined the effect of inducing genetic deletion of CDA1 in adulthood in mice using a tamoxifen-activated estrogen receptor fused cyclization recombinase (ERCre)-Locus of cross-over in P1 (LoxP) system. Male mice at 6–8 weeks of age were rendered diabetic with streptozotocin (STZ) or injected with buffer alone to serve as non-diabetic controls. Five weeks later, genetic deletion of CDA1 was induced by tamoxifen administration in CDA1Flox/ERCre mice, with mice injected with vehicle to serve as CDA1 wildtype controls. Kidney tissues were analyzed 5 weeks after deletion of CDA1. Tamoxifen administration reduced CDA1 gene expression by ~80% in CDA1Flox/ERCre mice. Renal levels of phosphorylated Smad3 and expression of profibrotic genes as well as accumulation of extracellular matrix proteins (ECMs) such as collagens III and IV were increased in diabetic mice, and induced deletion of CDA1 led to attenuation of these parameters. Therefore, targeting CDA1 after onset of diabetes in mice where CDA1 was initially expressed is able to attenuate diabetes-associated renal injury, providing the impetus to target this pathway in order to reduce diabetic kidney disease. Full article

Background: In eukaryotes with a double-stranded linear DNA genome, the loss of terminal DNA during replication is inevitable due to an end-replication problem; here, telomeres serve as a buffer against DNA loss. Thus, the activation of the telomere maintenance mechanism (TMM) is a prerequisite for malignant transformation. Methods: We compared neurofibroma (NF, benign) and malignant peripheral nerve sheath tumors (MPNSTs) occurring in the same patient with type 1 neurofibromatosis, where each NF–MPNST pair shared the same genetic background and differentiation lineage; this minimizes the genetic bias and contrasts only those changes that are related to malignant transformation. A total of 20 NF–MPNST pairs from 20 NF1 patients were analyzed. Whole-transcriptome sequencing (WTS) was conducted to profile the transcriptional relationship, and whole-genome sequencing (WGS) was performed to measure the telomere length. Results: We identified 22 differentially expressed genes (DEGs) during the malignant transformation of MPNSTs. Among them, NELL2 activated PAX7, which sequentially activated RAD52, the recombinase of RAD52-dependent alternative lengthening of telomeres (ALT). RAD52 elongated MPNSTs–telomeres (p = 0.017). Otherwise, neither NELL2 nor PAX7 affected telomere length (p = 0.647 and p = 0.354, respectively). RAD52 increased MPNSTs–telomeres length, independently of NELL2 and PAX7 in multiple analyses (p = 0.021). The group with increased telomere length during the malignant transformation showed inferior overall survival (OS) (HR = 3.809, p = 0.038) to the group without increased telomere length. Accordingly, the group with increased PAX7 showed inferior OS (HR = 4.896, p = 0.046) and metastasis-free survival (MFS) (HR = 9.129, p = 0.007) in comparison to the group without increased PAX7; the group with increased RAD52 showed inferior MFS (HR = 8.669, p = 0.011) in comparison to the group without increased RAD52. Conclusions: We suggest that the NELL2-PAX7 transcriptional cascade activates RAD52-dependent ALT to increase telomere length during the malignant transformation of MPNSTs, resulting in a poor prognosis. Full article

Piglet diarrhea poses significant economic losses to the pig industry, posing a worldwide challenge that urgently needs to be addressed in pig breeding practices. Porcine rotavirus (PoRV) is an important viral diarrhea pathogen in piglets, with a high incidence rate and a tendency to cause growth retardation. To enhance the sensitivity and specificity of PoRV detection, we sequenced the NSP3 gene of G5 and G9 genotypes of rotavirus A (RVA), enabling simultaneous detection of the two serotypes. Subsequently, we developed a rapid PoRV detection method using a combination of recombinase-aided amplification (RAA) and CRISPR/Cas12a. In this method, Cas12a binds to RAA amplification products, guided by CRISPR-derived RNA (crRNA), which activates its cleavage activity and releases fluorescence by cutting FAM-BHQ-labeled single-stranded DNA (ssDNA). In the optimized reaction system, the recombinant plasmid PoRV can achieve a highly sensitive reaction within 30 min at 37 °C, with a detection limit as low as 2.43 copies/μL, which is ten times higher in sensitivity compared to the qPCR method. Results from specificity testing indicate that no cross-reactivity was observed between the RAA-CRISPR/Cas12a analysis of PoRV and other viral pathogens, including PoRV G3, PoRV G4, porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV). In the clinical sample detection using the RAA-CRISPR/Cas12a method and qPCR, Cohen’s Kappa value reached as high as 0.952. Furthermore, this approach eliminates the need for large-scale instrumentation, offering a visual result under an ultraviolet lamp through fluorescence signal output. Full article

A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA. Full article

Progression of metabolic dysfunction-associated steatites liver disease (MASLD) to steatohepatitis (MASH) is driven by stress-inducing lipids that promote liver inflammation and fibrosis, and MASH can lead to cirrhosis and hepatocellular carcinoma. Previously, we showed coordinated defenses regulated by transcription factors, nuclear factor erythroid 2-related factor-1 (Nrf1) and -2 (Nrf2), protect against hepatic lipid stress. Here, we investigated protective effects of hepatocyte Nrf1 and Nrf2 against MASH-linked liver fibrosis and tumorigenesis. Male and female mice with flox alleles for genes encoding Nrf1 (Nfe2l1), Nrf2 (Nfe2l2), or both were fed a MASH-inducing diet enriched with high fat, fructose, and cholesterol (HFFC) or a control diet for 24–52 weeks. During this period, hepatocyte Nrf1, Nrf2, or combined deficiency for ~7 days, ~7 weeks, and ~35 weeks was induced by administering mice hepatocyte-targeting adeno-associated virus (AAV) expressing Cre recombinase. The effects on MASH, markers of liver fibrosis and proliferation, and liver tumorigenesis were compared to control mice receiving AAV-expressing green fluorescent protein. Also, to assess the impact of Nrf1 and Nrf2 induction on liver fibrosis, HFFC diet-fed C57bl/6J mice received weekly injections of carbon tetrachloride, and from week 16 to 24, mice were treated with the Nrf2-activating drug bardoxolone, hepatocyte overexpression of human NRF1 (hNRF1), or both, and these groups were compared to control. Compared to the control diet, 24-week feeding with the HFFC diet increased bodyweight as well as liver weight, steatosis, and inflammation. It also increased hepatocyte proliferation and a marker of liver damage, p62. Hepatocyte Nrf1 and combined deficiency increased liver steatosis in control diet-fed but not HFFC diet-fed mice, and increased liver inflammation under both diet conditions. Hepatocyte Nrf1 deficiency also increased hepatocyte proliferation, whereas combined deficiency did not, and this also occurred for p62 level in control diet-fed conditions. In 52-week HFFC diet-fed mice, 35 weeks of hepatocyte Nrf1 deficiency, but not combined deficiency, resulted in more liver tumors in male mice, but not in female mice. In contrast, hepatocyte Nrf2 deficiency had no effect on any of these parameters. However, in the 15-week CCL4-exposed and 24-week HFFC diet-fed mice, Nrf2 induction with bardoxolone reduced liver steatosis, inflammation, fibrosis, and proliferation. Induction of hepatic Nrf1 activity with hNRF1 enhanced the effect of bardoxolone on steatosis and may have stimulated liver progenitor cells. Physiologic Nrf1 delays MASLD progression, Nrf2 induction alleviates MASH, and combined enhancement synergistically protects against steatosis and may facilitate liver repair. Full article

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs’ eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs. Full article

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder. While metformin has demonstrated the ability to inhibit cyst growth in animal models of ADPKD via activation of adenosine monophosphate-activated protein kinase (AMPK), its effectiveness in humans is limited due to its low potency. This study explored the impact of HL156A, a new and more potent AMPK activator, in a mouse model of ADPKD. Methods: To investigate whether HL156A inhibits the proliferation of renal cyst cells in ADPKD in vitro, exogenous human telomerase reverse transcriptase (hTERT)-immortalized renal cyst cells from ADPKD patients were treated with HL156A, and an MTT (dimethylthiazol-diphenyltetrazolium bromide) assay was performed. To assess the cyst-inhibitory effect of HL156A in vivo, we generated Pkd1 conditional knockout (KO) mice with aquaporin 2 (AQP2)-Cre, which selectively expresses Cre recombinase in the collecting duct. The effectiveness of HL156A in inhibiting cyst growth and improving renal function was confirmed by measuring the number of cysts and blood urea nitrogen (BUN) levels in the collecting duct-specific Pkd1 KO mice. Results: When cyst cells were treated with up to 20 µM of metformin or HL156A, HL156A reduced cell viability by 25% starting at a concentration of 5 µM, whereas metformin showed no effect. When AQP2-Cre male mice were crossed with Pkd1^flox/flox female mice, and when AQP2-Cre female mice were crossed with Pkd1^flox/flox male mice, the number of litters produced by both groups was comparable. In collecting duct-specific Pkd1 KO mice, HL156A was found to inhibit cyst growth, reducing both the number and size of cysts. Furthermore, it was confirmed that kidney function improved as HL156A treatment led to a reduction in elevated BUN levels. Lastly, it was observed that the increase in AMPK phosphorylation induced by HL156A decreased ERK phosphorylation and α-SMA expression. Conclusion: HL156A has potential as a drug that can restore kidney function in ADPKD patients by inhibiting cyst growth. Full article

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34⁺ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID. Full article

Mating in female Drosophila melanogaster causes midgut hypertrophy and reduced lifespan, and these effects are blocked by the drug mifepristone. Eip75B is a transcription factor previously reported to have pleiotropic effects on Drosophila lifespan. Because Eip75B null mutations are lethal, conditional systems and/or partial knock-down are needed to study Eip75B effects in adults. Previous studies showed that Eip75B is required for adult midgut cell proliferation in response to mating. To test the possible role of Eip75B in mediating the lifespan effects of mating and mifepristone, a tripartite FLP-recombinase-based conditional system was employed that provides controls for genetic background. Expression of a Hsp70-FLP transgene was induced in third instar larvae by a brief heat pulse. The FLP recombinase catalyzed the recombination and activation of an Actin5C-GAL4 transgene. The GAL4 transcription factor in turn activated expression of a UAS-Eip75B-RNAi transgene. Inhibition of Eip75B activity was confirmed by loss of midgut hypertrophy upon mating, and the lifespan effects of both mating and mifepristone were eliminated. In addition, the negative effects of mifepristone on egg production were eliminated. The data indicate that Eip75B mediates the effects of mating and mifepristone on female midgut hypertrophy, egg production, and lifespan. Full article

Presynaptic Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca²⁺ influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca²⁺ indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca²⁺ transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca²⁺ influx but increased Ca²⁺ influx via N-type VGCCs. Moreover, we tested whether the decrease in Ca²⁺ influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for β-neurexins, we found that the decrease in presynaptic Ca²⁺ transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca²⁺ influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs. Full article

Recently, we and others have shown that manipulating the activity of cholinergic interneurons (CIN) present in the NAc can modulate binge alcohol consumption. The present study is designed to examine the relationship between binge alcohol consumption and the activity of the CIN in real time by using an in vivo microendoscopic technique. We hypothesized that mice exposed to Drinking in the Dark (DID)—a recognized mouse model for binge drinking—would exhibit increased activity in the accumbal shell region (NAcSh). To test this hypothesis, male mice expressing Cre-recombinase in the cholinergic neurons were exposed to binge alcohol consumption (alcohol group), employing the DID method, and utilized in vivo calcium imaging to observe CIN activity in real time during alcohol consumption. The control (sucrose) group was exposed to 10% (w/v) sucrose. As compared to sucrose, mice in the alcohol group displayed a significant increase in the frequency and amplitude of discharge activity, which was measured using calcium transients in the CIN present in the NAcSh. In summary, our findings suggest that the activity of CIN in the NAcSh plays a crucial role in alcohol self-administration. These results emphasize the potential significance of targeting CIN activity as a therapeutic approach for addressing AUD. Full article