Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus.

Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay's performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.

Seasonal timing of fluorescence and photosynthetic yields at needle and canopy scales in evergreen needleleaf forests.

The seasonal timing and magnitude of photosynthesis in evergreen needleleaf forests (ENFs) has major implications for the carbon cycle and is increasingly sensitive to changing climate. Earlier spring photosynthesis can increase carbon uptake over the growing season or cause early water reserve depletion that leads to premature cessation and increased carbon loss. Determining the start and the end of the growing season in ENFs is challenging due to a lack of field measurements and difficulty in interpreting satellite data, which are impacted by snow and cloud cover, and the pervasive "greenness" of these systems. We combine continuous needle-scale chlorophyll fluorescence measurements with tower-based remote sensing and gross primary productivity (GPP) estimates at three ENF sites across a latitudinal gradient (Colorado, Saskatchewan, Alaska) to link physiological changes with remote sensing signals during transition seasons. We derive a theoretical framework for observations of solar-induced chlorophyll fluorescence (SIF) and solar intensity-normalized SIF (SIFrelative) under snow-covered conditions, and show decreased sensitivity compared with reflectance data (~20% reduction in measured SIF vs. ~60% reduction in near-infrared vegetation index [NIRv] under 50% snow cover). Needle-scale fluorescence and photochemistry strongly correlated (r2 = 0.74 in Colorado, 0.70 in Alaska) and showed good agreement on the timing and magnitude of seasonal transitions. We demonstrate that this can be scaled to the site level with tower-based estimates of LUEP and SIFrelative which were well correlated across all sites (r2 = 0.70 in Colorado, 0.53 in Saskatchewan, 0.49 in Alaska). These independent, temporally continuous datasets confirm an increase in physiological activity prior to snowmelt across all three evergreen forests. This suggests that data-driven and process-based carbon cycle models which assume negligible physiological activity prior to snowmelt are inherently flawed, and underscores the utility of SIF data for tracking phenological events. Our research probes the spectral biology of evergreen forests and highlights spectral methods that can be applied in other ecosystems.

Gestodene, a novel positive allosteric modulator of PAR1, enhances PAR1-mediated human platelet aggregation.

Background: Protease-activated receptor 1 (PAR1) is expressed in human platelets and can be activated by low concentrations of thrombin. Vorapaxar, a selective antagonist of PAR1, inhibits thrombin-induced calcium mobilization in human platelet, which is associated with an increased risk of bleeding. Conversely, the administration of a positive allosteric modulator (PAM) of PAR1 may pose a substantial risk of thrombosis due to inducing excessive platelet activation. In this study, we discovered a novel PAM of PAR1 and investigated the effect of enhanced PAR1 activation by PAM of PAR1 on platelet activation. Methods: To find PAMs of PAR1, a cell-based screen was performed in HT29 cells, and finally, gestodene, an oral contraceptive drug (OC), was identified as a novel PAM of PAR1. The mechanism of action of gestodene and its effects on platelet activation were investigated in human megakaryocytic leukemia cell line MEG-01 cells and human platelet. Results: Gestodene enhanced both thrombin- and PAR1-activating peptide (AP)-induced intracellular calcium levels in a dose-dependent manner without altering PAR2 and PAR4 activity. Gestodene significantly increased PAR1-AP-induced internalization of PAR1 and phosphorylation of ERK1/2, and the enhancing effects were significantly blocked by vorapaxar. Furthermore, gestodene potently increased PAR1-AP induced morphological changes in MEG-01 cells. Remarkably, in human blood, gestodene exerted a robust augmentation of PAR1-AP-induced platelet aggregation, and vorapaxar effectively attenuated the gestodene-induced enhancement of platelet aggregation mediated by PAR1. Conclusion: Gestodene is a selective PAM of PAR1 and suggest one possible mechanism for the increased risk of venous thromboembolism associated with OCs containing gestodene.

Fluorescence and electron transfer of Limnospira indica functionalized biophotoelectrodes.

Cyanobacteria play a crucial role in global carbon and nitrogen cycles through photosynthesis, making them valuable subjects for understanding the factors influencing their light utilization efficiency. Photosynthetic microorganisms offer a promising avenue for sustainable energy conversion in the field of photovoltaics. It was demonstrated before that application of an external electric field to the microbial biofilm or cell improves electron transfer kinetics and, consequently, efficiency of power generation. We have integrated live cyanobacterial cultures into photovoltaic devices by embedding Limnospira indica PCC 8005 cyanobacteria in agar and PEDOT:PSS matrices on the surface of boron-doped diamond electrodes. We have subjected them to varying external polarizations while simultaneously measuring current response and photosynthetic performance. For the latter, we employed Pulse-Amplitude-Modulation (PAM) fluorometry as a non-invasive and real-time monitoring tool. Our study demonstrates an improved light utilization efficiency for L. indica PCC 8005 when immobilized in a conductive matrix, particularly so for low-intensity light. Simultaneously, the impact of electrical polarization as an environmental factor influencing the photosynthetic apparatus diminishes as matrix conductivity increases. This results in only a slight decrease in light utilization efficiency for the illuminated sample compared to the dark-adapted state.

Mechanistic Insight Into the Conformational Changes of Cas8 Upon Binding to Different PAM Sequences in the Transposon-Encoded Type I-F CRISPR-Cas System.

The INTEGRATE system is a gene-editing approach that offers advantages over the widely used CRISPR-Cas9 system. It does not introduce double strand breaks in the target DNA but rather integrates the desired DNA sequence directly into it. The first step in the integration process is PAM recognition, which is critical to understanding and optimizing the system. Experimental testing revealed varying integration efficiencies of different PAM mutants, and computational simulations were carried out to gain mechanistic insight into the conformational changes of Cas8 during PAM recognition. Our results showed that the interaction between Arg246 and guanine at position (-1) of the target strand is critical for PAM recognition. We found that unfavorable interactions in the 5'-AC-3' PAM mutant disrupted this interaction and may be responsible for its 0% integration efficiency. Additionally, we discovered that PAM sequences not only initiate the integration process but also regulate it through an allosteric mechanism that connects the N-terminal domain and the helical bundle of Cas8. This allosteric regulation was present in all PAMs tested, even those with lower integration efficiencies, such as 5'-TC-3' and 5'-AC-3'. We identified the Cas8 residues that are involved in this regulation. Our findings provide valuable insights into PAM recognition mechanisms in the INTEGRATE system and can help improve the gene-editing technology.

Characterizing offshore polar ocean soundscapes using ecoacoustic intensity and diversity metrics.

Polar offshore environments are considered the last pristine soundscapes, but accelerating climate change and increasing human activity threaten their integrity. In order to assess the acoustic state of polar oceans, there is the need to investigate their soundscape characteristics more holistically. We apply a set of 14 ecoacoustic metrics (EAMs) to identify which metrics are best suited to reflect the characteristics of disturbed and naturally intact polar offshore soundscapes. We used two soundscape datasets: (i) the Arctic eastern Fram Strait (FS), which is already impacted by anthropogenic noise, and (ii) the quasi-pristine Antarctic Weddell Sea (WS). Our results show that EAMs when applied in concert can be used to quantitatively assess soundscape variability, enabling the appraisal of marine soundscapes over broad spatiotemporal scales. The tested set of EAMs was able to show that the eastern FS, which is virtually free from sea ice, lacks seasonal soundscape dynamics and exhibits low acoustic complexity owing to year-round wind-mediated sounds and anthropogenic noise. By contrast, the WS exhibits pronounced seasonal soundscape dynamics with greater soundscape heterogeneity driven in large part by the vocal activity of marine mammal communities, whose composition in turn varies with the prevailing seasonal sea ice conditions.

Deficiency of Peptidylglycine-alpha-amidating Monooxygenase, a Cause of Sarcopenic Diabetes Mellitus.

CONTEXT: Peptidylglycine-α-amidating monooxygenase (PAM) is a critical enzyme in the endocrine system responsible for activation, by amidation, of bioactive peptides. OBJECTIVE: To define the clinical phenotype of carriers of genetic mutations associated with impaired PAM-amidating activity (PAM-AMA). DESIGN: We used genetic and phenotypic data from cohort studies: the Malmö Diet and Cancer (MDC; 1991-1996; reexamination in 2002-2012), the Malmö Preventive Project (MPP; 2002-2006), and the UK Biobank (UKB; 2012). SETTING: Exome-wide association analysis was used to identify loss-of-function (LoF) variants associated with reduced PAM-AMA and subsequently used for association with the outcomes. PATIENTS OR OTHER PARTICIPANTS: This study included n∼4500 participants from a subcohort of the MDC (MDC-Cardiovascular cohort), n∼4500 from MPP, and n∼300,000 from UKB. MAIN OUTCOME MEASURES: Endocrine-metabolic traits suggested by prior literature, muscle mass, muscle function, and sarcopenia. RESULTS: Two LoF variants in the PAM gene, Ser539Trp (minor allele frequency: 0.7%) and Asp563Gly (5%), independently contributed to a decrease of 2.33 [95% confidence interval (CI): 2.52/2.15; P = 2.5E-140] and 0.98 (1.04/0.92; P = 1.12E-225) SD units of PAM-AMA, respectively. The cumulative effect of the LoF was associated with diabetes, reduced insulin secretion, and higher levels of GH and IGF-1. Moreover, carriers had reduced muscle mass and function, followed by a higher risk of sarcopenia. Indeed, the Ser539Trp mutation increased the risk of sarcopenia by 30% (odds ratio 1.31; 95% CI: 1.16/1.47; P = 9.8E-06), independently of age and diabetes. CONCLUSION: PAM-AMA genetic deficiency results in a prediabetic sarcopenic phenotype. Early identification of PAM LoF carriers would allow targeted exercise interventions and calls for novel therapies that restore enzymatic activity.

Sustainable soil rehabilitation with multiple network structures of layered double hydroxide beads: Immobilization of heavy metals, fertilizer release, and water retention.

The remediation of heavy metal-contaminated soils necessitated a holistic approach that encompassed water and fertilizer conservation alongside soil property restoration. This study introduced the synthesis of (poly)acrylamide-layered double hydroxide gel spheres (PAM-LDH beads), which were designed to simultaneously immobilize heavy metals, control the release of fertilizers, and enhance soil water retention. Laboratory soil experiments under diverse conditions highlighted the superior performance of PAM-LDH beads in the immobilization of hexavalent chromium (Cr(VI)). The layered double hydroxide (LDH) component was identified as the key player in Cr(VI) immobilization, with anion exchange being the predominant mechanism. Notably, the encapsulated urea within the beads was released independently of environmental influences, governed by a concentration gradient across the beads surface. This release process was characterized by an initial phase of absorptive swelling followed by a diffusive phase. The impact on plant growth was assessed, revealing that PAM-LDH beads significantly curtailed Cr(VI) accumulation and alleviated its phytotoxic effects. Changes in the carbon (C) and nitrogen (N) content of the plants suggested that the urea encapsulated within the beads served as a nutrient source, contributing to soil fertility. Moreover, the water-holding capacity and soil-water characteristic curves of PAM-LDH beads suggested that these superabsorbent beads could delay soil water evaporation. The observed shifts in microbial community structure provided evidence for the enhancement of soil carbon and nitrogen cycles, indicative of improved soil properties.

Endometabolic profiling of pigmented glacier ice algae: the impact of sample processing.

INTRODUCTION: Glacier ice algae, mainly Ancylonema alaskanum and Ancylonema nordenskiöldi, bloom on Greenland Ice Sheet bare ice surfaces. They significantly decrease surface albedo due to their purple-brown pigmentation, thus increasing melt. Little is known about their metabolic adaptation and factors controlling algal growth dynamics and pigment formation. A challenge in obtaining such data is the necessity of melting samples, which delays preservation and introduces bias to metabolomic analysis. There is a need to evaluate the physiological response of algae to melting and establish consistent sample processing strategies for metabolomics of ice microbial communities. OBJECTIVES: To address the impact of sample melting procedure on metabolic characterization and establish a processing and analytical workflow for endometabolic profiling of glacier ice algae. METHODS: We employed untargeted, high-resolution mass spectrometry and tested the effect of sample melt temperature (10, 15, 20 °C) and processing delay (up to 49 h) on the metabolome and lipidome, and complemented this approach with cell counts (FlowCam), photophysiological analysis (PAM) and diversity characterization. RESULTS AND CONCLUSION: We putatively identified 804 metabolites, with glycerolipids, glycerophospholipids and fatty acyls being the most prominent superclasses (> 50% of identified metabolites). Among the polar metabolome, carbohydrates and amino acid-derivatives were the most abundant. We show that 8% of the metabolome is affected by melt duration, with a pronounced decrease in betaine membrane lipids and pigment precursors, and an increase in phospholipids. Controlled fast melting at 10 °C resulted in the highest consistency, and is our recommendation for future supraglacial metabolomics studies.

Enhancing Microplastic Removal from Natural Water through Coagulant Aids.

Microplastic (MP) pollution poses a significant environmental challenge, underscoring the need for improved water treatment methods. This study investigates the effectiveness of coagulation, flocculation, and sedimentation processes for removing microbeads, focusing on key factors that influence removal efficiency. Among the coagulants tested, polyaluminium chloride (PAC) demonstrated superior performance by enhancing the aggregation of microplastics with flocs. Optimal treatment conditions were determined to be 0.4 mmol/L PAC and 3 mg/L polyacrylamide (PAM) at pH 8 (before adding PAC), with rapid stirring at 240 rpm for 1 min, followed by slow stirring at 35 rpm for 13 min, and a sedimentation period of 25 min. Under these conditions, removal efficiencies exceeded 95 % for a range of microbeads (10-1000 µm: Polystyrene (PS), Polypropylene (PP), Polyvinyl chloride (PVC), Polyamide (PA), Polyethylene (PE), and Polyurethane (PU)) from natural water samples. Without PAM, PAC alone achieved a 97 % removal rate for PS microbeads. The addition of PAM maintained high removal efficiency, while aluminium sulphate and ferric chloride were less effective, with removal rates of 67% and 48% for PS microbeads, respectively. PAM enhanced MP removal across various coagulants and microbead types, with maximum efficiency observed at PAM concentrations of ≥3 mg/L. The treatment also demonstrated that organic matter in Regent's Park pond water could further improve MP removal. Size significantly impacts removal efficiency: larger microbeads (1 mm to >250 µm) were removed more effectively (95 %) compared to smaller ones (10 to <250 µm), which had a lower removal rate of 49 %. Denser microbeads like PVC (density 1.38 g/cm³) settled more efficiently than lighter microbeads such as PE (density 0.97 g/cm³). These findings suggest a need for advanced technologies to better remove lighter, smaller MPs from water.

Extrusion-based 3D printing for development of complex capsular systems for advanced drug delivery.

This review explores the feasibility of extrusion-based 3D printing techniques for producing complex dosage forms (such as capsular shells/devices) that provide controlled drug release and targeted delivery. The current discussion explores how extrusion-based 3D printing techniques, particularly Fused Deposition Modelling (FDM) and Pressure-Assisted Modelling (PAM), offer significant advantages in fabricating such complex dosage forms. This technology enables the fabrication of single-, dual-, or multi-compartment capsular systems with customized designs/geometry of the capsular shell to achieve delayed, sustained, or pulsatile drug release. The impact of customized design/geometry on the biopharmaceutical performances of loaded therapeutics is comprehensively discussed. The potential of 3D printing techniques for different specialized drug delivery purposes like gastric floating, implants, suppositories, and printfills are also addressed. This technique has the potential to significantly improve the therapeutic outcomes, and patient adherence to medication regimens, and pave the way for personalized medicine.

Predicting invasive disease-free survival in ER-positive, HER2-negative early breast cancer using the PAM50 risk-of-recurrence score: a retrospective analysis using single-center long-term follow-up data of postmenopausal Japanese patients.

BACKGROUND: The prognostic value of the risk-of-recurrence (ROR) score calculated using PAM50 has been validated using clinical trials and patient cohorts. This study aimed to investigate the prognostic value of the PAM50 ROR score in Japanese patients with early breast cancer using long-term follow-up data. METHODS: We enrolled postmenopausal patients with ER-positive, HER2-negative, stage I-II breast cancer who had undergone surgery at the Kyoto University Hospital between 2008 and 2014. The intrinsic subtype and ROR score were calculated using PAM50. The primary endpoint was invasive disease-free survival (IDFS). RESULTS: We enrolled 146 patients, of whom 47 (32%) patients had node-positive disease, and 36 (25%) had received neoadjuvant or adjuvant chemotherapy. The proportions of intrinsic subtypes for luminal A, luminal B, HER2-enriched, and basal-like subtypes were 67%, 27%, 3%, and 2%, respectively. The median follow-up duration was 8.4 (range 6.3-10.0) years, and 21 IDFS events were observed. Based on the ROR score, 37%, 33%, and 30% of the patients were classified as low, intermediate, and high risks, respectively. Patients in the high-risk group had a significantly worse 8-year IDFS rate than those in the low-to-intermediate-risk groups (75.1% vs. 91.6%, p = 0.04). The same trend was observed in patients with and without neoadjuvant or adjuvant chemotherapy. CONCLUSIONS: Using long-term follow-up data, this study showed that the ROR score can predict the prognosis of ER-positive, HER2-negative early breast cancer in Japanese postmenopausal patients. Further investigations are required to confirm the prognostic value of the ROR score in Asian populations.

Characterization of NiCas12b for In Vivo Genome Editing.

The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system represents the third family of CRISPR-Cas systems that are harnessed for genome editing. However, only a few nucleases have demonstrated activity in human cells, and their in vivo therapeutic potential remains uncertain. In this study, a green fluorescent protein (GFP)-activation assay is conducted to screen a panel of 15 Cas12b orthologs, and four of them exhibited editing activity in mammalian cells. Particularly noteworthy is the NiCas12b derived from Nitrospira sp., which recognizes a "TTN" protospacer adjacent motif (PAM) and facilitates efficient genome editing in various cell lines. Importantly, NiCas12b also exhibits a high degree of specificity, rendering it suitable for therapeutic applications. As proof of concept, the adeno-associated virus (AAV) is employed to introduce NiCas12b to target the cholesterol regulatory gene proprotein convertase subtilisin/ kexin type 9 (Pcsk9) in the mouse liver. After 4 weeks of injections, an impressive is observed over 16.0% insertion/deletion (indel) efficiency, resulting in a significant reduction in serum cholesterol levels. NiCas12b provides a novel option for both basic research and clinical applications.

Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica.

CRISPR/Cas9, presently the most widely used genome editing technology, has provided great potential for functional studies and plant breeding. However, the strict requirement for a protospacer adjacent motif (PAM) has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is limited. Recently, the engineered variants Cas9-NG, SpG, and SpRY, which recognize non-canonical PAMs, have been successfully tested in plants (mainly in rice, a monocot). In this study, we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important Brassica vegetables, Chinese cabbage (Brassica rapa spp. pekinensis) and cabbage (Brassica oleracea var. capitata). Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs, while SpG outperformed Cas9-NG at NGC and NGT PAMs. SpRY achieved efficient editing at almost all PAMs (NRN > NYN), albeit with some self-targeting activity at transfer (T)-DNA sequences. And SpRY-induced mutants were detected in cabbage plants in a PAM-less fashion. Moreover, an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical PAMs. Together, the toolboxes developed here induced successful genome editing in Chinese cabbage and cabbage. Our work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in Brassica. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00155-7.

Effect of medication adherence on quality of life, activation measures, and health imagine in the elderly people: a cross-sectional study.

BACKGROUND: Usually, old age brings a poor quality of life due to illness and frailty. To prolong their lives and ensure their survival, all elderly patients with chronic diseases must adhere to their medications. In our study, we investigate medication adherence for elderly patients and its impact on the general health of the patient. METHODS: We implemented a cross-sectional survey-based study with four sections in April 2022 in Saudi Arabia. Data about the participants' demographic characteristics, the Morisky Medication Adherence Scale, Patient Activation Measure (PAM) 13, and EQ-5D-5 L. RESULTS: A total of 421 patients participated in this study, their mean age was 60.4 years, and most of them were males. Most of our population is living independently 87.9%. The vast majority of people have a low adherence record in the Morisky Medication Adherence Scale (8-MMAS) classes (score = < 6). Moreover, the average PAM13 score is 51.93 (Level2) indicating a low level of confidence and sufficient knowledge to take action. Our analysis showed a significant correlation between socioeconomic status and medication adherence. Also, there was an association between housing status and medication adherence. On the other hand, we found no correlation between medication adherence and quality of life (QOL) by EQ-5D-5 L. CONCLUSION: Medication adherence is directly affected by living arrangements, as patients who live with a caretaker who can remind them to take their medications at the appropriate times have better medication adherence than those who live alone. Medication adherence was also significantly influenced by socioeconomic status, perhaps as a result of psychological effects and the belief of the lower-salaried population that they would be unable to afford the additional money required to cure any comorbidities that arose as a result of the disease. On the other hand, we did not find any correlation between medication adherence and quality of life. Finally, awareness of the necessity of adherence to medication for the elderly is essential.

The Role of the PI3K/Akt/mTOR Axis in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies globally, representing a significant public health problem with a poor prognosis. The development of efficient therapeutic strategies for HNSCC prevention and treatment is urgently needed. The PI3K/AKT/mTOR (PAM) signaling pathway is a highly conserved transduction network in eukaryotic cells that promotes cell survival, growth, and cycle progression. Dysfunction in components of this pathway, such as hyperactivity of PI3K, loss of PTEN function, and gain-of-function mutations in AKT, are well-known drivers of treatment resistance and disease progression in cancer. In this review, we discuss the major mutations and dysregulations in the PAM signaling pathway in HNSCC. We highlight the results of clinical trials involving inhibitors targeting the PAM signaling pathway as a strategy for treating HNSCC. Additionally, we examine the primary mechanisms of resistance to drugs targeting the PAM pathway and potential therapeutic strategies.

Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

The Takeda G protein-coupled receptor 5 (TGR5) is activated endogenously by primary and secondary bile acids. This receptor is considered a candidate target for addressing inflammatory and metabolic disorders. We have targeted TGR5 with structure-based methods for ligand finding using the recently solved experimental structures, as well as structures obtained from molecular dynamics simulations. Through addressing the orthosteric as well as a putative allosteric site, we identified agonists and positive allosteric modulators. While the predicted binding locations were not in line with their efficacy, our work contributes activating small-molecule ligands that we have thoroughly characterized in vitro.

Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

PLGA nanoparticle-delivered Leishmania antigen and TLR agonists as a therapeutic vaccine against cutaneous leishmaniasis in BALB/c mice.

Leishmaniasis, caused by Leishmania (L.) species, remains a neglected infection. Therapeutic vaccination presents a promising strategy for its treatment. In this study, we aimed to develop a therapeutic vaccine candidate using Leishmaniaantigens (SLA) and Toll-like receptor (TLR) 7/8 agonist (R848) encapsulated into the poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Moreover, TLR1/2 agonist (Pam3CSK4) was loaded onto the NPs. The therapeutic effects of these NPs were evaluated in L. major-infected BALB/c mice. Footpad swelling, parasite load, cellular and humoral immune responses, and nitric oxide (NO) production were analyzed. The results demonstrated that the PLGA NPs (SLA-R848-Pam3CSK4) therapeutic vaccine effectively stimulated Th1 cell responses, induced humoral responses, promoted NO production, and restricted parasite burden and lesion size.Our findings suggest that vaccination with SLA combined with TLR1/2 and TLR7/8 agonists in PLGA NPs as a therapeutic vaccine confers strong protection againstL. majorinfection. These results represent a novel particulate therapeutic vaccine against Old World cutaneous leishmaniasis.

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.