Exploring the Thermostability of CRISPR-Cas12b using Molecular Dynamics Simulations.

CRISPR (clustered regularly interspaced short palindromic repeat)-based diagnostics are at the forefront of rapid detection platforms of infectious diseases. The integration of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) with CRISPR-Cas protein systems has led to the creation of advanced one-pot assays. The sensitivity of these assays has been bolstered by the utilization of a thermophilic Cas12 protein, BrCas12b, and its engineered variant, which exhibits enhanced thermal stability and allows for broader operation temperatures of the assay. Here, we perform all-atom molecular dynamics (MD) simulations on wild-type and mutant BrCas12b to reveal the mechanism of stabilization conferred by the mutation. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings not only offer a comprehensive insight into the dynamic alterations induced by mutations but reveal important motions in BrCas12b, important for the rational design of diagnostic and therapeutic platforms of Cas12 proteins.

Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution.

Macrophage efferocytosis prevents apoptotic cell (AC) accumulation and triggers inflammation-resolution pathways. The mechanisms linking efferocytosis to resolution often involve changes in macrophage metabolism, but many gaps remain in our understanding of these processes. We now report that efferocytosis triggers an indoleamine 2,3-dioxygenase-1 (IDO1)-dependent tryptophan (Trp) metabolism pathway that promotes several key resolution processes, including the induction of pro-resolving proteins, such interleukin-10, and further enhancement of efferocytosis. The process begins with upregulation of Trp transport and metabolism, and it involves subsequent activation of the aryl hydrocarbon receptor (AhR) by the Trp metabolite kynurenine (Kyn). Through these mechanisms, macrophage IDO1 and AhR contribute to a proper resolution response in several different mouse models of efferocytosis-dependent tissue repair, notably during atherosclerosis regression induced by plasma low-density lipoprotein (LDL) lowering. These findings reveal an integrated metabolism programme in macrophages that links efferocytosis to resolution, with possible therapeutic implications for non-resolving chronic inflammatory diseases, notably atherosclerosis.

Design and synthesis of novel Thiazolo[5,4-b]pyridine derivatives as potent and selective EGFR-TK inhibitors targeting resistance Mutations in non-small cell lung cancer.

A novel series of substituted thiazolo[5,4-b]pyridine analogues were rationally designed and synthesized via a multi-step synthetic pathway, including Suzuki cross-coupling reaction. The anticancer activity of all forty-five synthesized derivatives was evaluated against HCC827, H1975, and A549 cancer cell lines utilizing the standard MTT assay. A significant number of the thiazolo[5,4-b]pyridine derivatives exhibited potent anticancer activity. Notably, compounds 10b, 10c, 10h, 10i, and 10k emerged as the most promising anticancer agents. The lead compound, N-(3-(6-(2-aminopyrimidin-5-yl)thiazolo[5,4-b]pyridin-2-yl)-2-methylphenyl)-2,5-difluorobenzenesulfonamide (10k), displayed remarkable potency with IC50 values of 0.010 µM, 0.08 µM, and 0.82 µM against the HCC827, NCI-H1975 and A-549 cancer cell lines, respectively, which were comparable to the clinically approved drug Osimertinib. Importantly, the potent derivatives 10b, 10c, 10h, 10i, and 10k exhibited selective cytotoxicity towards cancer cells and showing no toxicity against the normal BEAS-2B cell line at concentrations exceeding 35 µM. Mechanistic studies revealed that the active compound 10k acts as an EGFR-TK autophosphorylation inhibitor in HCC827 cells. Furthermore, apoptosis assays demonstrated that compound 10k induced substantial early apoptosis (31.9 %) and late apoptosis (8.8 %) in cancer cells, in contrast to the control condition exhibiting only 2.0 % early and 1.6 % late apoptosis. Molecular docking simulations of the synthesized compounds revealed that they formed essential hinge interactions and established hydrogen bonding with Cys797, indicating potential target engagement. These findings highlight the potential of the synthesized thiazolo [(Woodburn, 1999; Zigrossi et al., 2022) 5,45,4-b]pyridine derivatives as promising anticancer agents, warranting further investigation for the development of novel targeted therapies against non-small cell lung cancer.

Endoscopic excision of breast fibroadenoma through inframammary fold: Feasibility, safety and medium-term outcomes.

INTRODUCTION: Endoscopic-assisted surgery for breast tumors has the advantage of inconspicuous scars, less breast volume loss, and nipple areolar distortion. A novel endoscopic-assisted technique through inframammary fold for excision of fibroadenomas is presented. MATERIALS AND SURGICAL TECHNIQUE: Endoscopic-assisted excision of fibroadenoma(s) through inframammary fold was performed in four patients after informed written consent via three ports (12, 5, and 5 mm). Breast Cancer Treatment Outcome Score-12 (BCTOS-12) was used to evaluate patient satisfaction after surgery. DISCUSSION: No intraoperative and wound complication was noted. On median follow-up of 26.5 months, patients reported satisfactory responses to aesthetic and functional outcomes. No scar related complications were noted. Endoscopic-assisted excision of fibroadenoma through inframammary fold can be a safe and feasible option with good aesthetic outcomes.

Black seed assisted synthesis, characterization, free radical scavenging, antimicrobial and anti-inflammatory activity of iron oxide nanoparticles.

Iron nanoparticles comprise a significant class of inorganic nanoparticles, which discover applications in various zones by prudence of their few exciting properties. This study achieved the green synthesis of iron oxide nanoparticles (IONPs) by black cumin seed (Nigella sativa) extract, which acts as a reducing and capping agent. The iron nanoparticles and black cumin extract were synthesized in three different concentrations: (01:01, 02:04,01:04). UV-visible spectroscopy, XRD, FTIR, and AFM characterized the synthesized iron oxide nanoparticles. UV-visible spectra show the maximum absorbance peak of 01:01 concentration at 380 nm. The other concentrations, such as 02:04, peaked at 400 nm and 01:04 at 680 nm, confirming the formation of iron oxide nanoparticles. AFM analysis reveals the spherical shape of iron oxide nanoparticles. The XRD spectra reveal the (fcc) cubic crystal structure of the iron oxide nanoparticles. The FTIR analysis's peaks at 457.13, 455.20, and 457.13 cm-1 depict the characteristic iron nanoparticle synthesis. The black cumin extract-mediated iron oxide nanoparticles show substantial antibacterial, antifungal, antioxidant and anti-inflammatory activity in a dose-dependent manner.

Crosstalk between efferocytic myeloid cells and T-cells and its relevance to atherosclerosis.

The interplay between myeloid cells and T-lymphocytes is critical to the regulation of host defense and inflammation resolution. Dysregulation of this interaction can contribute to the development of chronic inflammatory diseases. Important among these diseases is atherosclerosis, which refers to focal lesions in the arterial intima driven by elevated apolipoprotein B-containing lipoproteins, notably low-density lipoprotein (LDL), and characterized by the formation of a plaque composed of inflammatory immune cells, a collection of dead cells and lipids called the necrotic core, and a fibrous cap. As the disease progresses, the necrotic core expands, and the fibrous cap becomes thin, which increases the risk of plaque rupture or erosion. Plaque rupture leads to a rapid thrombotic response that can give rise to heart attack, stroke, or sudden death. With marked lowering of circulating LDL, however, plaques become more stable and cardiac risk is lowered-a process known as atherosclerosis regression. A critical aspect of both atherosclerosis progression and regression is the crosstalk between innate (myeloid cells) and adaptive (T-lymphocytes) immune cells. Myeloid cells are specialized at clearing apoptotic cells by a process called efferocytosis, which is necessary for inflammation resolution. In advanced disease, efferocytosis is impaired, leading to secondary necrosis of apoptotic cells, inflammation, and, most importantly, defective tissue resolution. In regression, efferocytosis is reawakened aiding in inflammation resolution and plaque stabilization. Here, we will explore how efferocytosing myeloid cells could affect T-cell function and vice versa through antigen presentation, secreted factors, and cell-cell contacts and how this cellular crosstalk may contribute to the progression or regression of atherosclerosis.

Orthodontic treatment in adults: Challenges, outcomes, and factors affecting compliance and satisfaction.

BACKGROUND: The demand for orthodontic treatment among adults has witnessed a substantial rise in recent years. This study aims to explore the complexities of adult orthodontics, focusing on challenges faced, treatment outcomes, and the influence of factors such as age, gender, and education on patient compliance and satisfaction. METHODS: A multi-dimensional approach was employed, combining a review of clinical records with structured patient surveys. Descriptive statistics summarized demographic characteristics, treatment duration, and orthodontic problems addressed. Inferential statistics included Pearson correlation, Chi-squared tests, and analysis of variance to examine age compliance, gendersatisfaction, and education-orthodontic problem relationships. Qualitative analysis enriched findings, and statistical software facilitated data processing. RESULTS: The analysis revealed a statistically significant negative correlation between age and compliance (r = -0.28, P < 0.05), indicating that younger participants demonstrated higher compliance rates. Gender emerged as a significant factor influencing patient satisfaction (P = 0.024), with females reporting notably higher levels of satisfaction than males. Furthermore, participants with advanced education levels (Master's/Ph.D.) were significantly more likely to have orthodontic issues related to malocclusion (P = 0.041). CONCLUSION: The study provides an insight into the multi-dimensional aspects of adult orthodontics, recognizing the challenges, compliance, and satisfaction levels. Tailored approaches considering age, gender, and education are essential. This research contributes to a deeper understanding of orthodontic treatment in adults and its potential implications for enhanced patient care.

PAM-free diagnostics with diverse type V CRISPR-Cas systems.

Type V CRISPR-Cas effectors have revolutionized molecular diagnostics by facilitating the detection of nucleic acid biomarkers. However, their dependence on the presence of protospacer adjacent motif (PAM) sites on the target double-stranded DNA (dsDNA) greatly limits their flexibility as diagnostic tools. Here we present a novel method named PICNIC that solves the PAM problem for CRISPR-based diagnostics with just a simple ∼10-min modification to contemporary CRISPR-detection protocols. Our method involves the separation of dsDNA into individual single-stranded DNA (ssDNA) strands through a high- temperature and high-pH treatment. We then detect the released ssDNA strands with diverse Cas12 enzymes in a PAM-free manner. We show the utility of PICNIC by successfully applying it for PAM-free detection with three different subtypes of the Cas12 family- Cas12a, Cas12b, and Cas12i. Notably, by combining PICNIC with a truncated 15-nucleotide spacer containing crRNA, we demonstrate PAM-independent detection of clinically important single- nucleotide polymorphisms with CRISPR. We apply this approach to detect the presence of a drug-resistant variant of HIV-1, specifically the K103N mutant, that lacks a PAM site in the vicinity of the mutation. Additionally, we successfully translate our approach to clinical samples by detecting and genotyping HCV-1a and HCV-1b variants with 100% specificity at a PAM-less site within the HCV genome. In summary, PICNIC is a simple yet groundbreaking method that enhances the flexibility and precision of CRISPR-Cas12-based diagnostics by eliminating the restriction of the PAM sequence.

Design, synthesis, and biological evaluation of novel 2,3-Di-O-Aryl/Alkyl sulfonate derivatives of l-ascorbic acid: Efficient access to novel anticancer agents via in vitro screening, tubulin polymerization inhibition, molecular docking study and ADME predictions.

A series of novel l-ascorbic acid derivatives bearing aryl and alkyl sulfonate substituents were synthesized and characterized. In vitro anticancer evaluation against MCF-7 (breast) and A-549 (lung) cancer cell lines revealed potent activity for most of the compounds, with 2b being equipotent to the standard drug colchicine against MCF-7 (IC50 = 0.04 µM). Notably, compound 2b displayed 89-fold selectivity for MCF-7 breast cancer over MCF-10A normal breast cells. Derivatives with two sulfonate groups (2a-g, 3a-g) exhibited superior potency over those with one sulfonate (4a-c,5g, 6b). Compounds 2b and 2c potently inhibited tubulin polymerization in A-549 cancer cells (73.12 % and 62.09 % inhibition, respectively), substantiating their anticancer potential through microtubule disruption. Molecular docking studies showed higher binding scores and affinities for these compounds at the colchicine-binding site of α, ß-tubulin compared to colchicine itself. In-silico ADMET predictions indicated favourable drug-like properties, with 2b exhibiting the highest binding affinity. These sulfonate derivatives of l-ascorbic acid represents promising lead scaffolds for anticancer drug development.

Unlocking the Potential: Investigating Dental Practitioners' Willingness to Embrace Artificial Intelligence in Dental Practice.

BACKGROUND: Artificial intelligence (AI) holds significant promise for transforming healthcare delivery, including dentistry. However, the successful integration of AI into dental practice necessitates an understanding of dental professionals' perspectives, attitudes, and readiness to adopt AI technology. This study aimed to explore dental professionals' perceptions, attitudes, and practices regarding AI adoption in dentistry. METHODS: This cross-sectional study was conducted among 256 dental professionals using an online questionnaire. Participants were assessed for familiarity with AI technology, perceived barriers to adoption, attitudes towards AI, current usage patterns, and factors influencing adoption decisions. Data are analysed using descriptive statistics, including frequencies, percentages, means, and standard deviations. Inferential statistics, such as chi-square tests and regression analysis, were employed to examine associations between variables and identify predictors of AI adoption in dentistry. RESULTS: The study surveyed 256 dental professionals from various regions across India, primarily aged 30 to 50 years (mean age: 42.6), with a nearly equal gender split (male: 48.4%, female: 51.6%) and high educational attainment (67.8% with master's or doctoral degrees). Private practices were predominant (56.3%). The diagnostic algorithms and treatment planning software were well known (77.3% and 70.3% familiarity, respectively). Technical concerns (average score: 3.82 ± 0.68) were the main barriers to AI adoption, followed by financial considerations (average score: 3.45 ± 0.72), ethical and legal issues (average score: 3.21 ± 0.65), and organizational factors (average score: 3.67 ± 0.71). Despite these concerns, most participants had positive attitudes towards AI (70.3% agreed). Current usage varied, with diagnostic support and administrative tasks being the most common (44.5% and 82.8% usage, respectively). Perceived utility (average score: 4.12 ± 0.75) and ease of use (average score: 3.98 ± 0.69) significantly influenced adoption, as identified by regression analysis (perceived utility: ß = 0.342, p < 0.001; ease of use: ß = 0.267, p = 0.005). CONCLUSION: This study provides valuable insights into AI adoption in dentistry, highlighting the multifaceted nature of barriers and facilitators that influence dental professionals' adoption decisions. Strategies to promote AI adoption should address practical considerations, ethical concerns, and educational needs to facilitate the integration of AI technology into dental practices.

The structural, stability, electronic, optical and thermodynamic properties of MoX2 (X= S, Se, and Te) under hydrostatic pressures: a plasmon approach and first-principle study.

CONTEXT: The new equations have been developed for the structural and electronic properties using the plasmon calculations for the first time for 2-D MoX2 structures. Literature shows still an extensive study is required on the stability and optical properties of MoX2 under different hydrostatic pressures and thermal properties under different temperatures using the first principles, for electronic industrial applications. The stability is analyzed using binding energy and phonon calculations. The phase transition of metallization of MoX2 is discussed using band structure calculations under different hydrostatic pressures. The calculated work function shows the photoemission starts from the threshold frequency of 4.189×104 cm-1, 3.184×104 cm-1, and 3.651×104 cm-1, respectively, for MoS2, MoSe2, and MoTe2 materials. The optical properties such as refractive index n(0), and static dielectric permittivity ε(0) for three successive materials are calculated under different hydrostatic pressures, applicable for optoelectronic applications. The calculated theoretical and computational values agree well with each other and also agree with reported and experimental values. Some of the values are calculated for the first time. METHODS: The theoretical equations are derived using the molecular weight, effective valence electrons, and density of molecule of MoX2 structures. The simulation work is performed using GGA-PBE approximation in the CASTEP simulation package with DFT+D semi-empirical dispersion correction. An ultra-soft pseudopotential representation calculates the electronic and optical properties with a finite basis set kinetic energy cut-off of 381.0 eV. Each geometry has been optimized using Broyden, Fletcher, Goldfarb, and Shanno's (BFGS) algorithm for 100 iterations with a fixed basis quality variable cell method and finite electronic minimization parameters. The phonon calculations were performed using TDFT with a kinetic energy cut of 460 eV in a norm-conserving linear response method. The interpolation with a finite dispersion quality and q-vector grid spacing is performed.

Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing.

Micro computed tomography (Micro-CT) characterization of root and root canal morphology of mandibular first premolars: a systematic review and meta-analysis.

INTRODUCTION: Mandibular first premolars are familiar with their varied root canal morphology, causing difficulties and challenges for successful endodontic procedures. This systematic review and meta-analysis aim to study the characterization of root and canal morphology of the first mandibular premolar using micro-computed tomography. METHODOLOGY: The literature search was conducted using electronic web databases like PubMed, Scopus, ScienceDirect and Cochrane with the chosen MeSH key words and data was retrieved until May 2023. Further to perform the statistical analysis, R v 4.3.1 software with "meta", 'metafor" "metaviz" " ggplot2" package was used, and results were represented by odds ratios (OR) and the percentage of forest plots along a 95 per cent confidence interval (CI). RESULTS: The total number of studies meeting the inclusion criteria was 13; these studies were conducted on mandibular first premolar using Micro-CT; the total sample size was 1817. To scan the sample, an X-ray micro-focus CT system (Siemens Inveon CT, Erlangen, Germany) was used in four studies and seven different machines were used in the respective studies. Mimics 10.01 software (Materialize, Leuven, Belgium) and NRecon v.1.6.9 software (Bruker, Kontich, Belgium) were commonly operated. The minimum and maximum voxel size ranges between 11.94 and 50 µm. Vertucci's classification was frequently used (9), while one study applied Ahmed et al. and Vertucci's classification. CONCLUSION: This systematic review provides essential information about the root and canal configurations, radicular grooves, accessory canals, and apical foramina through Micro-CT, aiming to improve the accuracy of endodontic treatment and help practitioners.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

Rett and Rett-related disorders: Common mechanisms for shared symptoms?

Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in the methyl-CpG binding protein-2 (MeCP2) gene that is characterized by epilepsy, intellectual disability, autistic features, speech deficits, and sleep and breathing abnormalities. Neurologically, patients with all three disorders display microcephaly, aberrant dendritic morphology, reduced spine density, and an imbalance of excitatory/inhibitory signaling. Loss-of-function mutations in the cyclin-dependent kinase-like 5 (CDKL5) and FOXG1 genes also cause similar behavioral and neurobiological defects and were referred to as congenital or variant Rett syndrome. The relatively recent realization that CDKL5 deficiency disorder (CDD), FOXG1 syndrome, and Rett syndrome are distinct neurodevelopmental disorders with some distinctive features have resulted in separate focus being placed on each disorder with the assumption that distinct molecular mechanisms underlie their pathogenesis. However, given that many of the core symptoms and neurological features are shared, it is likely that the disorders share some critical molecular underpinnings. This review discusses the possibility that deregulation of common molecules in neurons and astrocytes plays a central role in key behavioral and neurological abnormalities in all three disorders. These include KCC2, a chloride transporter, vGlut1, a vesicular glutamate transporter, GluD1, an orphan-glutamate receptor subunit, and PSD-95, a postsynaptic scaffolding protein. We propose that reduced expression or activity of KCC2, vGlut1, PSD-95, and AKT, along with increased expression of GluD1, is involved in the excitatory/inhibitory that represents a key aspect in all three disorders. In addition, astrocyte-derived brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), and inflammatory cytokines likely affect the expression and functioning of these molecules resulting in disease-associated abnormalities.

Programmable RNA detection with CRISPR-Cas12a.

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Systematic Photophysical Interaction Studies Between Newly Synthesised Oxazole Derivatives and Silver Nanoparticles: Experimental and DFT Approach.

In this study, the photophysical properties of oxazole derivatives such as 5-(furan-2-yl) -4-tosyloxazole (OX-1) and 5-(2-bromothiazol-4-yl)-4-tosyloxazoles (OX-2) were investigated using theoretical and experimental techniques. The ground and excited state dipole moments were empirically obtained utilising the solvatochromic shift technique and several solvatochromic correlations such as Lippert's, Bakhshiev's, KawskiChamma- Viallet's, and solvent polarity equations. The ground state dipole moments, HOMO-LUMO and molecule electrostatic potential map were also computed using ab initio calculations and evaluated using Gaussian 09 W software. Furthermore, spectroscopic interactions between newly synthesised dyes (OX-1 and OX-2) and freshly synthesised silver nanoparticles (size 40 nm) were studied. Increased absorbance and widening of absorption spectra for both dyes in the presence of varied quantities of silver nanoparticles show the potential of dye-nanoparticle interactions. Fluorescence quenching has been detected for both dyes in the presence of colloidal silver nanoparticles, indicating dynamic quenching, and a significant overlap between the absorption and emission spectra of the silver nanoparticle reveals that fluorescence quenching is also due to energy transfer.

Correction: Understanding liquid-liquid equilibria in binary mixtures of hydrocarbons with a thermally robust perarylphosphonium-based ionic liquid.

[This corrects the article DOI: 10.1039/D1RA06268A.].