Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution.

In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization over large volumes has been largely restricted to labeling of proteins. But volumetric nucleic acid labeling may represent a far greater scientific and clinical opportunity, enabling detection of not only diverse coding RNA variants but also non-coding RNAs. Moreover, scaling immunohistochemical detection to large tissue volumes has limitations due to high cost, limited renewability/availability, and restricted multiplexing capability of antibody labels. With the goal of versatile, high-content, and scalable molecular phenotyping of intact tissues, we developed a method using carbodiimide-based chemistry to stably retain RNAs in clarified tissue, coupled with amplification tools for multiplexed detection. The resulting technology enables robust measurement of activity-dependent transcriptional signatures, cell-identity markers, and diverse non-coding RNAs in rodent and human tissue volumes. The growing set of validated probes is deposited in an online resource for nucleating related developments from across the scientific community.

On-Demand Thio-Succinimide Hydrolysis for the Assembly of Stable Protein-Protein Conjugates.

Chemical post-translational protein-protein conjugation is an important technique with growing applications in biotechnology and pharmaceutical research. Maleimides represent one of the most widely employed bioconjugation reagents. However, challenges associated with the instability of first- and second-generation maleimide technologies are yet to be fully addressed. We report the development of a novel class of maleimide reagents that can undergo on-demand ring-opening hydrolysis of the resulting thio-succinimide. This strategy enables rapid post-translational assembly of protein-protein conjugates. Thio-succinimide hydrolysis, triggered upon application of chemical, photochemical, or enzymatic stimuli, allowed homobifunctional bis-maleimide reagents to be applied in the production of stable protein-protein conjugates, with complete temporal control. Bivalent and bispecific protein-protein dimers constructed from small binders targeting antigens of oncological importance, PD-L1 and HER2, were generated with high purity, stability, and improved functionality compared to monomeric building blocks. The modularity of the approach was demonstrated through elaboration of the linker moiety through a bioorthogonal propargyl handle to produce protein-protein-fluorophore conjugates. Furthermore, extending the functionality of the homobifunctional reagents by temporarily masking reactive thiols included in the linker allowed the assembly of higher order trimeric and tetrameric single-domain antibody conjugates. The potential for the approach to be extended to proteins of greater biochemical complexity was demonstrated in the production of immunoglobulin single-domain antibody conjugates. On-demand control of thio-succinimide hydrolysis combined with the facile assembly of chemically defined homo- and heterodimers constitutes an important expansion of the chemical methods available for generating stable protein-protein conjugates.

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

IDO-1 inhibition improves outcome after fluid percussion injury in adult male rats.

The enzyme indoleamine 2,3 dioxygenase 1 (IDO1) catalyzes the rate-limiting step in the kynurenine pathway (KP) which produces both neuroprotective and neurotoxic metabolites. Neuroinflammatory signals produced as a result of pathological conditions can increase production of IDO1 and boost its enzymatic capacity. IDO1 and the KP have been implicated in behavioral recovery after human traumatic brain injury (TBI), but their roles in experimental models of TBI are for the most part unknown. We hypothesized there is an increase in KP activity in the fluid percussion injury (FPI) model of TBI, and that administration of an IDO1 inhibitor will improve neurological recovery. In this study, adult male Sprague Dawley rats were subjected to FPI or sham injury and received twice-daily oral administration of the IDO1 inhibitor PF-06840003 (100 mg/kg) or vehicle control. FPI resulted in a significant increase in KP activity, as demonstrated by an increased ratio of kynurenine: tryptophan, in the perilesional neocortex and ipsilateral hippocampus 3 days postinjury (DPI), which normalized by 7 DPI. The increase in KP activity was prevented by PF-06840003. IDO1 inhibition also improved memory performance as assessed in the Barnes maze and anxiety behaviors as assessed in open field testing in the first 28 DPI. These results suggest increased KP activity after FPI may mediate neurological dysfunction, and IDO1 inhibition should be further investigated as a potential therapeutic target to improve recovery.

Effects of Repeated Treatment with the Monoacylglycerol Lipase Inhibitor MJN110 on Pain-Related Depression of Nesting and Cannabinoid 1 Receptor Function in Male and Female Mice.

MJN110 inhibits the enzyme monoacylglycerol lipase (MAGL) to increase levels of the endocannabinoid 2-arachidonoylglycerol , an endogenous high-efficacy agonist of cannabinoid 1 and 2 receptors (CB1/2R). MAGL inhibitors are under consideration as candidate analgesics, and we reported previously that acute MJN110 produced partial antinociception in an assay of pain-related behavioral depression in mice. Given the need for repeated analgesic administration in many pain patients and the potential for analgesic tolerance during repeated treatment, this study examined antinociceptive effects of repeated MJN110 on pain-related behavioral depression and CB1R-mediated G-protein function. Male and female ICR mice were treated daily for 7 days in a 2 × 2 design with (a) 1.0 mg/kg/d MJN110 or its vehicle followed by (b) intraperitoneal injection of dilute lactic acid (IP acid) or its vehicle as a visceral noxious stimulus to depress nesting behavior. After behavioral testing, G-protein activity was assessed in lumbar spinal cord (LSC) and five brain regions using an assay of CP55,940-stimulated [35S]GTPÉ£S activation. As reported previously, acute MJN110 produced partial but significant relief of IP acid-induced nesting depression on day 1. After 7 days, MJN110 continued to produce significant but partial antinociception in males, while antinociceptive tolerance developed in females. Repeated MJN110 also produced modest decreases in maximum levels of CP55,940-induced [35S]GTPÉ£S binding in spinal cord and most brain regions. These results indicate that repeated treatment with a relatively low antinociceptive MJN110 dose produces only partial and sex-dependent transient antinociception associated with the emergence of CB1R desensitization in this model of IP acid-induced nesting depression. SIGNIFICANCE STATEMENT: The drug MJN110 inhibits monoacylglycerol lipase (MAGL) to increase levels of the endogenous cannabinoid 2-arachidonoylglycerol and produce potentially useful therapeutic effects including analgesia. This study used an assay of pain-related behavioral depression in mice to show that repeated MJN110 treatment produced (1) weak but sustained antinociception in male mice, (2) antinociceptive tolerance in females, and (3) modest cannabinoid-receptor desensitization that varied by region and sex. Antinociceptive tolerance may limit the utility of MJN110 for treatment of pain.

Crossing the Solubility Rubicon: 15-Crown-5 Facilitates the Preparation of Water-Soluble Sulfo-NHS Esters in Organic Solvents.

The Sulfo-NHS ester is a mainstay reagent for facilitating amide bond formation between carboxylic acids and amine functionalities in water. However, the preparation of Sulfo-NHS esters currently requires hydrophobic carboxylic acids, which are poorly water-soluble, to first be reacted with the N-hydroxysulfosuccinimide sodium salt, which is insoluble in organic solvents. The mutually incompatible solvation requirements thus complicate the synthesis of Sulfo-NHS esters. As a simple, rapid, and cost-effective solution to this problem, we report that the use of 15-crown-5 to complex the sodium cation of N-hydroxysulfosuccinimide sodium salt circumnavigates these solvation incompatibility issues by rendering the N-hydroxysulfosuccinimide salt soluble in organic solvents, resulting in a cleaner esterification reaction and thus improved yields of activated ester product. We also demonstrate that the resultant "crowned" Sulfo-NHS-ester remains water-soluble and is no less reactive than its classic "uncrowned" Sulfo-NHS counterpart when used in bioconjugation reactions between protein amine-functionalities and hydrophobic carboxylic acids.

Low Molecular Weight Shuttle Molecules Enhance Polychloramide Antimicrobial Activity.

Investigated were the influences of succinimide (SI), 5,5-dimethylhydantoin (DMH), and 3-(hydroxymethyl)-5,5-dimethylhydantoin (HDMH) on the biocidal activity of chlorinated, water-soluble polyamide prepared by the reaction of isopropylamine with poly(styrene-alt-maleic anhydride). The resulting polymer was a negatively charged, water-soluble polymer bearing a carboxylic acid and an isopropylamide moiety on nearly every repeat unit. Subsequent treatment with NaOCl chlorinated the polymers to up to 4.4% Cl while inflicting some polymer chain scission. SI, DMH, or HDMH increased the biocidal activity of polychloramides toward planktonic Escherichia coli and Staphylococcus aureus. Independent solution studies confirmed that oxidative chlorine spontaneously transferred from aqueous polychloramides to small molecules. We concluded that SI, DMH, and HDMH acted as shuttles that extracted oxidative Cl from the polymer chloramides and transported oxidative Cl more efficiently to microbial surfaces. Succinimide was the most effective shuttle. These results warn that some low molecular weight soluble molecules in antimicrobial testing solutions may exaggerate the effectiveness of the polymer or immobilized antimicrobial agents.

Reference-free thio-succinimide isomerization characterization by electron-activated dissociation.

RATIONALE: Isomerism can be an important aspect in pharmaceutical drug development. Identification of isomers can provide insights into drug pharmacology and contribute to better design of drug molecules. The general approaches to differentiate isomers include Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and circular dichroism. Additionally, a commonly used method to differentiate isomers is liquid chromatography coupled with mass spectrometry (LC-MS). Notably, LC-MS is routinely applied to leucine and isoleucine differentiation to facilitate protein sequencing. This work focuses on isomer differentiation of widely employed thio-succinimide structure bridging the antibody backbone and linker-payload of antibody-drug conjugates (ADCs). Thio-succinimide hydrolysis stabilizes the payload-protein structure while generating a pair of constitutional isomers: thio-aspartyl and thio-isoaspartyl. METHODS: This paper introduces a hybrid method using ligand binding assay (LBA) and liquid chromatography coupled with tandem MS (LC-MS/MS) to reveal isomerization details of thio-succinimide hydrolysis over time in plasma samples incubated with ADC. Application of two orthogonal dissociation methods, collision-induced dissociation (CID) and electron-activated dissociation (EAD) revealed different MS/MS spectra for this pair of isomers. This observation enables a unique approach in distinguishing thio-succinimide hydrolysis isomers. RESULTS: We observed signature [R1 + Thio + 57 + H]+, [R2 + Succ + H2O - 57 + H]+, and [R2 + Succ + H2O - 44 + 2H]2+ product ions (Succ = succinimide) that differentiated thio-aspartyl and thio-isoaspartyl isomers using EAD. A newly discovered [R2 + ThioSucc + H2O - 44 + 2H]2+ ion also served as additional evidence that further supported our findings. CONCLUSIONS: This study is a first-to-date identification of thio-succinimide hydrolysis isomers without using synthesized reference materials. This approach should be applicable to all thio-succinimide-linked molecules. Correct identification of thio-succinimide hydrolysis isomers may eventually benefit the development of ADCs in the future.

A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

RATIONALE: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. METHODS: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. RESULTS: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. CONCLUSIONS: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

AIMS: Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). METHODS: Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. RESULTS: In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased, and bladder MAGL was decreased after MAGL inhibitor treatment. CONCLUSIONS: MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

Deep eutectic solvent extraction combined with magnetic bead ligand fishing for identification of α-glucosidase inhibitors from Pueraria lobata.

In this study, a deep eutectic solvent (DES) extraction combined with a magnetic bead ligand affinity analytical method was developed and used for α-glucosidase inhibitor identification from Pueraria lobata. Several critical parameters affecting the analysis performance, including the type of DES, molar ratio, water amount, pH, salt concentration, and volume of DES, were investigated. The selected analytical sample preparation conditions were as follows. The composition of DES is choline chloride-1,4-butanediol (1:3), the water content is 40%, pH is 7.0 and the volume of extraction solution is 2 mL. The obtained sample extraction solution was analyzed directly using α-glucosidase immobilized magnetic beads (GMBs). Three α-glucosidase inhibitors in Pueraria lobata, including puerarin, daidzin, and daidzein, were identified. Luteolin was used as a positive control to evaluate the method's selectivity. Results showed it could selectively bond to the GMBs in the DES. As the affinity analysis was performed directly in a DES, the solution-removing process could be avoided. The intra-day and inter-day precisions of the method are 5.21% and 6.38%, respectively. The solvent amount was 1/50-1/2000 of that used in traditional methods.

Combined WNT-activated deep-penetrating/plexiform melanocytoma: insights into clinicopathological and molecular characterization.

BACKGROUND: A combined deep-penetrating tumour redefined as WNT-activated deep-penetrating/plexiform melanocytoma (DPM), may pose challenging clinical and histological diagnoses. OBJECTIVES: To review the clinicopathological characteristics of combined DPMs and characterize the molecular profile of atypical and malignant forms. METHODS: The study included 51 patients with combined DPMs diagnosed at the Hospital Clinic of Barcelona and the University of Florence between 2012 and 2020. Clinical data, dermoscopy images (when available) and histological characteristics were reviewed. Immunohistochemistry for ß-catenin, LEF1, HMB45, Ki67, p16 and PRAME (preferentially expressed antigen in melanoma) was performed. Atypical forms underwent next-generation sequencing (NGS) panel analysis, including driver genes implicated in DPMs, TERT-promoter (p) mutations and the investigation of the 9p21 locus via fluorescence in situ hybridization. RESULTS: Among the 51 patients (32 females and 19 males, age range 4-74 years), 68% with available clinical data (15/22) were initially suspected of having melanoma. Except for one patient, complete excision resulted in no recurrences or metastases. One patient who had an incompletely excised combined DPM developed a lymph node melanoma metastasis 10 years later. In the 51 patients, 10 samples (20%) showed atypical histological features; 7 (14%) exhibited a significant loss of p16 expression; and 2 (4%) showed a high-proliferative index (Ki67 over 5%). NGS analysis in 11 patients revealed a double mutation BRAFV600E and exon 3 CTNNB1; no TERTp mutations were detected. CONCLUSIONS: Clinical suspicion of melanoma is common in combined DPMs, but malignant progression is infrequent in tumours lacking high-grade atypia or proliferation. These findings are congruent with the consideration of these lesions as intermediate-grade tumours or melanocytomas.

Identification and characterization of dystrophin-locus-derived testis-specific protein: A testis-specific gene within the intronic region of the rat dystrophin gene.

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

DPM2 serve as novel oncogene and prognostic marker transactivated by ESR1 in breast cancer.

Breast cancer (BRCA) is the most common malignancies worldwide with increasing rate. Dolichol phosphate mannose synthase (DPMS) is a critical mannosyltransferase involved in the posttranslational modification of proteins. At present, there is limited knowledge regarding the function of DPMS in breast cancer. In this study, silica analysis in multiple datasets found that dolichyl-phosphate mannosyltransferase subunit 2 (DPM2) is an unfavorable prognostic marker, suggesting its oncogenic role. Cell counting kit-8 and apoptosis assays show that DPM2-silenced cancer cells exhibit decreased growth potential and enhanced cell death rate. Further, transwell and wound healing assays show reduced invasion and migration capabilities in DPM2 knockdown groups, xenograft nude mice model demonstrated smaller tumor volume in DPM2 silenced BC cells. Then, the underlying downstream mechanism of DPM2 in BC was predicted and analyzed, highlighting classical tumorigenic pathways like JAK/STAT signaling pathway and oxidative phosphorylation activated in the cancer group. Finally, ChIP-seq analysis, expression correlation analysis, inhibitor treatment, and dual luciferase assays show that DPM2 is transcriptionally activated by estrogen receptor1 (ESR1). The results show that high expression of DPM2 mRNA is significantly correlated with shorter overall survival (OS) and disease-free survival (DFS) in breast cancer patients, and in vitro knockdown of DPM2 can significantly inhibit the malignant phenotypes of cells, including proliferation, invasion, migration, and apoptosis. These results suggest that DPM2 may play an important role in breast cancer. Altogether, we first uncovered the tumorigenic and prognostic role of DPM2 in breast cancer, cellular assays, and bioinformatics analysis highlighted DPM2 as oncogene via inhibited cancer-related signaling pathways in breast cancer. Besides, DPM2 is transcriptionally activated by ESR1, the signaling axis of ESR1/DPM2 provides a new strategy for BC-targeted therapy.

Exploring the problems and coping strategies of pharmacy internship in large general hospitals in China: from the perspective of preceptors.

OBJECTIVE: The role of the Hospital Pharmacy Preceptor (HPP) is pivotal in upholding the excellence of experiential training and fostering the professional growth of pharmacy interns. However, there is a lack of studies that provide an overview of pharmacy internships from the perspective of HPP. This study explores the experience and expectations of HPPs regarding existing problems and possible coping strategies in intern teaching. METHODS: This is a qualitative study that was conducted through individual interviews and focus group discussions. HPPs were invited as participants from large-scale tertiary hospitals in representative provinces of mainland China. Interview and focus group discussion data were analyzed using thematic analysis to see emerging themes from the data. Nvivo 12 was utilized for data management and processing. RESULTS: Eight individual interviews and two focus group discussions were conducted, involving 14 HPPs as participants. Upon the examination of the interviews and focus group data, four themes were summarized regarding HPPs' perceptions: 1) current presenting problems; 2) possible coping strategies; 3) something HPPs should do; 4) something interns should do. CONCLUSION: This study found that from the HPPs' perspective, the hospital-based pharmacy internship still has some problems from policy to practice, which need to be addressed by the joint efforts of the state, schools, internship bases, pharmacy preceptors, and students.

Efficacy of an automatic electric toothbrush with nylon bristles in dental plaque removal: a cross-over randomized controlled trial.

OBJECTIVES: The objective of this single-use, five-treatment, five-period, cross-over randomized controlled trial (RCT) was to compare the efficacy in dental plaque removal of a new Y-shaped automatic electric toothbrush (Y-brush) compared to a U-shaped automatic electric toothbrush (U-brush), a manual toothbrushing procedure (for 45 and 120 s), and no brushing (negative control). MATERIALS AND METHODS: Eligible participants were volunteer students randomized to the treatments in the five periods of the study. The primary outcome measure was the reduction in full-mouth plaque score (FMPS) after brushing while the secondary outcome variable was a visual analogic scale (VAS) on subjective clean mouth sensation. Mixed models were performed for difference in FMPS and VAS. RESULTS: After brushing procedures, manual toothbrushing (120 s) showed a statistically significant reduction in FMPS than Y-brush (difference 36.9; 95%CI 29.6 to 44.1, p < 0.0001), U-brush (difference 42.3; 95%CI 35.1 to 49.6, p < 0.0001), manual brushing (45 s) (difference 13.8; 95%CI 6.5 to 21.1, p < 0.0001), and No brushing (difference 46.6; 95%CI 39.3 to 53.9, p < 0.0001). Y-brush was significantly more effective than No brushing (difference 9.8; 95%CI 2.5 to 17.0, p = 0.0030), while there was no significant difference compared to U- brush. Similar results were obtained for the differences in the Clean Mouth VAS. CONCLUSIONS: Y-brush was significantly more effective than no brushing (negative control) in removing dental plaque. When compared to manual toothbrushing for both 45 and 120 s, however, Y-brush was less effective in dental plaque removal. CLINICAL RELEVANCE: Modified design of automatic toothbrushing devices could improve plaque reduction, especially in patients with intellectual disabilities or motor difficulties.

The impact of armed conflicts on the nutritional status of children: Evidence from Northern Nigeria.

Armed conflicts often significantly exacerbate the magnitude and severity of malnutrition by increasing food insecurity. Evidence shows that malnutrition is among the leading causes of morbidity and mortality among children during conflicts. This study examines the impact of the armed conflicts in Northern Nigeria on nutritional status of children under the age of five. Three waves (2008, 2013, and 2018) of individual-level birth records data from the Nigeria Demographic and Health Survey (NDHS) dataset are spatially merged with information on conflict events drawn from the Armed Conflict Location and Events Dataset. All fatal incidents in the study region during the 5-year intervals 2004-2008, 2009-2013 and 2014-2018 are aggregated and mapped to the 2008, 2013 and 2018 NDHS clusters, respectively. A cluster is classified to be exposed to conflict if located within 5-10 km radius of an incident with at least 1 fatality. We use matching analysis in a difference-in-differences approach to estimate the effects of the conflicts on stunting, wasting, and underweight. We find that the impact of conflict exposure differs by the dimension of child nutritional status. While it significantly lowers the risk of stunting, it has no discernible significant effect on the likelihood of wasting or being underweight among under-fives. Though nutritional support/interventions in the conflict-affected areas are crucial and must be prioritised, an all-inclusive strategy for a long-term resolution of the conflict is needed to engender development, improve food security, reduce vulnerability to malnutrition, and improve the health and wellbeing of the residents of the region.

Problem-oriented coping and resilience among Fibromyalgia patients who live under security threats and have undergone a Fibrotherapy intervention program.

Our study examined the association between problem-focused coping and resilience among fibromyalgia (FM) patients who live under constant security threats. Resilience is a coping resource and detrimentally affects FM female patients (FMPs) to get up and cope with life. A cohort of 96 FMPs ages 19-75 was subjected to a Fibrotherapy intervention program in the Rehabilitation Help Center in Sderot (Ezra Le'Marpeh), Israel. We examined levels of problem-oriented coping and levels of resilience among the sample. In addition, we assessed whether there is a correlation between their resilience level and their medical metrics. The research included medical metrics and physical metrics. A cohort of 16 FMPs who participated in the quantitative phase composed the qualitative sample. Data from the t-test showed improved mental resilience among all the sample, with a significantly higher level among problem-oriented FMPs. We conclude that resilience is acquired through problem-oriented coping strategies. Furthermore, the association between resilience and problem-oriented coping helped to improve health indicators since coping with the disease included entering a regime of physicals activity and maintaining a healthy lifestyle.

Synthesis of 3,4-Disubstituted Maleimide Derivatives via Phosphine-Catalyzed Isomerization of α-Succinimide-Substituted Allenoates Cascade γ'-Addition with Aryl Imines.

3,4-disubstituted maleimides find wide applications in various pharmacologically active compounds. This study presents a highly effective approach for synthesizing derivatives of 3,4-disubstituted maleimides through the direct isomerization of α-succinimide-substituted allenoates, followed by a cascade γ'-addition and aryl imines using PR3 as a catalyst. The resulting series of 3,4-disubstituted maleimides exhibited excellent stereoselectivities, achieving yields of up to 86%. To our knowledge, the phosphine-mediated γ'-addition reaction of allenoates is seldom reported.

Function of Presynaptic Inhibitory Cannabinoid CB1 Receptors in Spontaneously Hypertensive Rats and Its Modification by Enhanced Endocannabinoid Tone.

We studied whether the function of presynaptic inhibitory cannabinoid CB1 receptors on the sympathetic nerve fibres innervating resistance vessels is increased in spontaneously hypertensive rats (SHR) like in deoxycorticosterone (DOCA)-salt hypertension. An increase in diastolic blood pressure (DBP) was induced by electrical stimulation of the preganglionic sympathetic neurons or by phenylephrine injection in pithed SHR and normotensive Wistar-Kyoto rats (WKY). The electrically (but not the phenylephrine) induced increase in DBP was inhibited by the cannabinoid receptor agonist CP55940, similarly in both groups, and by the endocannabinoid reuptake inhibitor AM404 in SHR only. The effect of CP55940 was abolished/reduced by the CB1 receptor antagonist AM251 (in both groups) and in WKY by endocannabinoid degradation blockade, i.e., the monoacylglycerol lipase (MAGL) inhibitor MJN110 and the dual fatty acid amide hydrolase (FAAH)/MAGL inhibitor JZL195 but not the FAAH inhibitor URB597. MJN110 and JZL195 tended to enhance the effect of CP55940 in SHR. In conclusion, the function of presynaptic inhibitory CB1 receptors depends on the hypertension model. Although no differences occurred between SHR and WKY under basal experimental conditions, the CB1 receptor function was better preserved in SHR when the endocannabinoid tone was increased by the inhibition of MAGL or the endocannabinoid transporter.