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Byline: M. Omidian, M. Mapar Cutaneous leishmanasis (CL) may present with unusual clinical variants such as acute paronychial, annular, palmoplantar, zosteriform, erysipeloid, and sporotrichoid. The zosteriform variant has rarely been [...]

The prospect of being able to efficiently inject large plasmids in insulin-producing beta cells is very attractive for diabetes research. However, conventional transfection methods suffer from high cytotoxicity or low transfection efficiency, which negatively affect their outcome. In contrast, nanostraw electroporation is a gentle method that can provide a high transfection efficiency while maintaining high cell viability. While nanostraw electroporation has gone through some method optimization in the past, such as tuning the pulse frequency, amplitude, and duration, the effect of other parameters has not been thoroughly investigated. Here, we demonstrate efficient transfection of clonal beta cells and investigate the effect of voltage at a fixed inter-electrode distance, cell density, and cargo solution conductivity on transfection efficiency. We used GFP-encoding DNA plasmids stained with an intercalating dye to enable immediate analysis and assessment of the electrophoretic transport of cargo. Moreover, we ran simulations to assess how cargo buffer conductivity impacts the transfection efficiency by affecting the voltage drop on the nanostraws and cell membrane during electroporation. Both experiments and simulations show that MilliQ water as the cargo buffer yields the best transfection efficiency. We also show that the cell density should be adjusted to maximize the number of cells interfacing the nanostraws and avoid cell stacking. Finally, we compared the transfection efficiency when using nanostraws and nanopores. Whereas the amount of GFP plasmids injected using nanostraws is larger than for nanopores, the outcome in terms of GFP fluorescence 48 h after transfection was worse than for nanopores. Moreover, when using nanostraws, fewer cells were found on the substrate 48 h after transfection compared to when using nanopores. This suggests that injecting substantial amounts of plasmids in cells can affect their proliferation and/or viability, and that nanopore electroporation, as a simpler method, is an interesting alternative to nanostraws in achieving efficient and gentle clonal beta cell transfection., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Objectives: This study assessed the effects of two remineralizing agents namely MI Paste Plus containing casein phosphopeptide amorphous calcium phosphate fluoride (CPP-ACFP) and Remin Pro containing hydroxyapatite, fluoride and xylitol (HFX) with/without erbium-doped yttrium aluminium garnet (Er:YAG) and CO 2 laser irradiation on demineralized enamel microhardness. Materials and Methods: In this in vitro study, 70 sound human premolars were mesiodistally sectioned, demineralized at a pH of 4.6 for 8 hours, and randomly divided into 7 remineralization groups (n=10): of (I) MI Paste Plus (CPP-ACFP), (II) Remin Pro (HFX), (III) MI Paste Plus+CO 2 laser (0.7 W power, 50 Hz), (IV) Remin Pro+CO 2 laser, (V) MI Paste Plus+Er:YAG laser (1 W power, 10 Hz), (VI) Remin Pro+Er:YAG laser, and (VII) negative control. The Vickers hardness number of specimens was then measured. The groups were compared by one-way ANOVA and Tukey's test (α=0.05). Results: The mean microhardness was 319.8±49.9, 325.3±44.6, 359.4±35.7, 296.4±33.7, 319.9±58.1, 358.9±28.4, and 240.0±41.6 kg/mm2 in groups 1 to 7, respectively. The difference in microhardness was significant among the groups (P<0.0001). Pairwise comparisons revealed significant differences in microhardness between all groups (P≤0.03) except between groups 1 and 2, 1 and 5, 2 and 5, and 3 and 6 (P>0.05). Conclusion: Both Remin Pro (containing HFX) and MI Paste Plus (containing CPP-ACFP) can cause enamel remineralization. MI Paste Plus+CO 2 laser irradiation and Remin Pro+Er:YAG laser irradiation were significantly more effective than the application of each remineralizing agent alone ., Competing Interests: None declared., (Copyright © 2024 The Authors. Published by Tehran University of Medical Sciences.)

Recent innovations in microscopy techniques are paving the way for label-free studies of single nanoscopic biological entities such as viruses, lipid-nanoparticle drug carriers, and even proteins. One such technique is waveguide evanescent-field microscopy, which offers a relatively simple, yet sensitive, way of achieving label-free light scattering-based imaging of nanoparticles on surfaces. Herein, we extend the application of this technique by incorporating microfluidic liquid control and adapting the design for use with inverted microscopes by fabricating a waveguide on a transparent substrate. We furthermore formulate analytical models describing scattering and fluorescence intensities from single spherical and shell-like objects interacting with evanescent fields. The models are then applied to analyze scattering and fluorescence intensities from adsorbed polystyrene beads and to temporally resolve cholera-toxin B (CTB) binding to individual surface-immobilized glycosphingolipid G M1 containing vesicles. We also propose a self-consistent means to quantify the thickness of the CTB layer, revealing that protein-binding to individual vesicles can be characterized with sub-nm precision in a time-resolved manner., Competing Interests: Fredrik Höök, Björn Agnarsson and Mattias Sjöberg are co-founders of the company Nanolyze AB, which commercializes waveguide-based microscopy systems. All other authors declare no competing interests., (Published by Optica Publishing Group under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.)

Objective: In recent years, dental composite resins such as tooth-colored restoration are frequently used to restore dental cavities, coronal fractures, and congenital defects. This study aimed to evaluate the microleakage of two methacrylate-based composites (GC Kalore and Luna SDI) in class II restorations., Materials and Methods: In this experimental study, a total of 18 intact human premolars previously extracted for periodontal and orthodontic reasons were randomly divided into two groups. Similar class II cavities (box only) were prepared on all teeth and restored with two different composites. In group 1, a bonding agent (Single Bond 2-SB2; 3M ESPE) and Luna SDI composite in mesial cavities and GC Kalore composite in distal cavities were used. In group 2, Single Bond 2 and GC Kalore composite in mesial cavities and Luna SDI composite in distal cavities were applied. They were then subjected to 2000 thermal cycles in a water bath between 5-55°C (dwell time: 30 seconds in every bath and transfer time: 10 seconds). Then, they were immersed in a 2% basic fuchsin dye solution for 24 hours. After rinsing with water, they were sectioned mesiodistally and evaluated for microleakage using a stereomicroscope., Results: Independent t -test (Mann-Whitney test) showed no statistically significant difference for microleakage in mesial and distal class II restorations between GC Kalore composite and Luna SDI composite ( p  = 1.000) ( p = 0.852). A total of 83.4% of the Luna SDI composite samples and 66.6% of the GC Kalore composite had a microleakage score of ≤3 in class II cavities., Conclusion: In the present study, marginal microleakage was found mainly at the gingival floor extending to 1/3 of the axial wall for the Luna SDI composite and GC Kalore composite. Furthermore, no statistically significant difference was found between the microleakage of the Class II cavities restored with Luna SDI composite and GC Kalore composite., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2022 Kooshan Moradi et al.)

Microbes have diverse metabolic capabilities and differences in these phenotypes are critical for differentiating strains, species, and broader taxa of microorganisms. Recent advances in liquid chromatography-mass spectrometry (LC-MS) allow researchers to track the complex combinations of molecules that are taken up by each cell type and to quantify the rates that individual metabolites enter or exit the cells. This metabolomics-based approach allows complex metabolic phenotypes to be captured in a single assay, enables computational models of microbial metabolism to be constructed, and can serve as a diagnostic approach for clinical microbiology. Unfortunately, metabolic phenotypes are directly affected by the molecular composition of the culture medium and many traditional media are subject to molecular-level heterogeneity. Herein, we show that commercially sourced Mueller Hinton (MH) medium, a Clinical and Laboratory Standards Institute (CLSI) approved medium for clinical microbiology, has significant lot-to-lot and supplier-to-supplier variability in the concentrations of individual nutrients. We show that this variability does not affect microbial growth rates but does affect the metabolic phenotypes observed in vitro -including metabolic phenotypes that distinguish six common pathogens. To address this, we used a combination of isotope-labeling, substrate exclusion, and nutritional supplementation experiments using Roswell Park Memorial Institute (RPMI) medium to identify the specific nutrients used by the microbes to produce diagnostic biomarkers, and to formulate a Biomarker Enrichment Medium (BEM) as an alternative to complex undefined media for metabolomics research, clinical diagnostics, antibiotic susceptibility testing, and other applications where the analysis of stable microbial metabolic phenotypes is important., (Copyright © 2022 Mapar, Rydzak, Groves and Lewis.)

Bloodstream infections (BSIs) cause >500,000 infections and >80,000 deaths per year in North America. The length of time between the onset of symptoms and administration of appropriate antimicrobials is directly linked to mortality rates. It currently takes 2-5 days to identify BSI pathogens and measure their susceptibility to antimicrobials - a timeline that directly contributes to preventable deaths. To address this, we demonstrate a rapid metabolic preference assay (MPA) that uses the pattern of metabolic fluxes observed in ex-vivo microbial cultures to identify common pathogens and determine their antimicrobial susceptibility profiles. In a head-to-head race with a leading platform (VITEK 2, BioMérieux) used in diagnostic laboratories, MPA decreases testing timelines from 40 hours to under 20. If put into practice, this assay could reduce septic shock mortality and reduce the use of broad spectrum antibiotics., (© 2022. The Author(s).)

Background: Cancer diagnostic probe (CDP) had been developed to detect involved breast cavity side margins in real-time (Miripour et al. Bioeng Transl Med. e10236.). Here, we presented the results of the in vivo human model CDP studies on non-neoadjuvant cases., Methods: This study is a prospective, blind comparison to a gold standard, and the medical group recruited patients. CDP and frozen data were achieved before the permanent pathology experiment. The main outcome of the study is surgical margin status. From November 2018 to April 2020, 202 patients were registered, and 188 were assigned for the study. Breast-conserving surgery at any age or gender, re-surgery due to re-currency, or involved margins are acceptable. Patients must be non-neoadjuvant. The reliability of CDP scoring had been evaluated by the pathology of the scored IMs. Then, three models of the study were designed to compare CDP with the frozen sections. Receiver operating characteristic (ROC) curves and AUC were measured based on the permanent postoperative pathology gold standard., Results: A matched clinical diagnostic categorization between the pathological results of the tested IMs and response peaks of CDP on 113 cases, was reported (sensitivity = 97%, specificity = 89.3%, accuracy = 92%, positive predictive value (PPV) = 84.2%, and negative predictive value (NPV) = 98%). Study A showed the independent ability of CDP for IM scoring (sensitivity = 80%, specificity = 90%, accuracy = 90%, PPV = 22.2%, and NPV = 99.2%). Study B showed the complementary role of CDP to cover the missed lesions of frozen sections (sensitivity = 93.8%, specificity = 91%, accuracy = 91%, PPV = 55.6%, and NPV = 99.2%). Study C showed the ability of CDP in helping the pathologist to reduce his/her frozen miss judgment (specificity = 92%, accuracy = 93%, PPV = 42.1%, and NPV = 100%). Results were reported based on the post-surgical permanent pathology gold standard., Conclusion: CDP scoring ability in intra-operative margin detection was verified on non-neoadjuvant breast cancer patients. Non-invasive real-time diagnosis of IMs with pathological values may make CDP a distinct tool with handheld equipment to increase the prognosis of breast cancer patients., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Root hair formation in Arabidopsis thaliana is a well-established model system for epidermal patterning and morphogenesis in plants. Over the last decades, many underlying regulatory genes and well-established networks have been identified by thorough genetic and molecular analysis. In this study, we used a forward genetic approach to identify genes involved in root hair development in Arabis alpina , a related crucifer species that diverged from A. thaliana approximately 26-40 million years ago. We found all root hair mutant classes known in A. thaliana and identified orthologous regulatory genes by whole-genome or candidate gene sequencing. Our findings indicate that the gene-phenotype relationships regulating root hair development are largely conserved between A. thaliana and A. alpina . Concordantly, a detailed analysis of one mutant with multiple hairs originating from one cell suggested that a mutation in the SUPERCENTIPEDE1 ( SCN1 ) gene is causal for the phenotype and that AaSCN1 is fully functional in A. thaliana . Interestingly, we also found differences in the regulation of root hair differentiation and morphogenesis between the species, and a subset of root hair mutants could not be explained by mutations in orthologs of known genes from A. thaliana . This analysis provides insight into the conservation and divergence of root hair regulation in the Brassicaceae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mapar, Chopra, Stephan, Schrader, Sun, Schneeberger, Albani, Coupland and Hülskamp.)

For most people, the first step in treatment is to take out the tumor (surgery), so precise and fast diagnosis of any sign of high-risk and neoplastic cells, especially in surgical cavity margins, is significant. The frozen pathology method is the conventional standard of intraoperative diagnosis, but the low number of slides prepared from non-fixed tissues prevents us from achieving a perfect diagnosis. Although many improvements in intraoperative margin detection were achieved, still real-time detection of neoplastic lesions is crucial to improving diagnostic quality. Functionalized carbon nanotubes grown on the electrode needles lively and selectively determine the H 2 O 2 released from cancer/atypical cells through reverse Warburg effect and hypoxia assisted glycolysis pathways in a quantitative electrochemical manner. The study was carried out on cell lines, 57 in vivo mice models with breast cancer, and 258 fresh in vitro samples of breast cancer tumors. A real-time electrotechnical system, named cancer diagnostic probe (CDP) (US Patent Pub. No.: US 2018/02991 A1, US 2021/0007638 A1, and US 2021/0022650 A1 [publications], and US 10,786,188 B1 [granted]), has been developed to find pre-neoplastic/neoplastic cells in vivo in a quantitative electrochemical manner by tracing hypoxia glycolysis byproducts. Matched pathological evaluations with response peaks of CDP were found based on the presence of neoplasia (from atypia to invasive carcinoma) in live breast tissues. The ability of CDP to find neoplastic lesions in mice models in vivo and fresh breast tumors in vitro was verified with sensitivity and specificity of 95% and 97%, respectively. The system may help a surgeon assistant system for usage in the operating room after passing many trials and standard examinations in the future., Competing Interests: Four USA patents (one granted; US Patent US10,786,188 B1, and three publications; US Patent App. US2018/0299401 A1, US2021/0007638 A1, and US2021/0022650 A1) have been published based on this work. M.A. is a member of the scientific advisory board of Arya Nano biosensor Manufacturer Co., a company that is commercializing CDP technology. The remaining authors declare that they have no competing interests., (© 2021 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

The genetic and molecular analysis of trichome development in Arabidopsis thaliana has generated a detailed knowledge about the underlying regulatory genes and networks. However, how rapidly these mechanisms diverge during evolution is unknown. To address this problem, we used an unbiased forward genetic approach to identify most genes involved in trichome development in the related crucifer species Arabis alpina In general, we found most trichome mutant classes known in A. thaliana We identified orthologous genes of the relevant A. thaliana genes by sequence similarity and synteny and sequenced candidate genes in the A. alpina mutants. While in most cases we found a highly similar gene-phenotype relationship as known from Arabidopsis , there were also striking differences in the regulation of trichome patterning, differentiation, and morphogenesis. Our analysis of trichome patterning suggests that the formation of two classes of trichomes is regulated differentially by the homeodomain transcription factor AaGL2 Moreover, we show that overexpression of the GL3 basic helix-loop-helix transcription factor in A. alpina leads to the opposite phenotype as described in A. thaliana Mathematical modeling helps to explain how this nonintuitive behavior can be explained by different ratios of GL3 and GL1 in the two species., Competing Interests: The authors declare no conflict of interest.

Infection with the hepatitis C virus is a major public health concern which can lead to carcinoma and liver failure. It has been shown that single nucleotide polymorphisms can affect the level of gene activity of tumor necrosis factor (TNF) which has an important role, especially in viral infections which can lead to apaptosis of infected hepatocellular cells. We investigated the impact of three possible genotypes for rs1800629 or A/G single nucleotide polymorphism located downstream of TNFα gene promoter in groups of control (n=76) and chronic hepatitis C patients (n=89) focusing on the response to treatment among sensitive and resistant groups. Genomic DNA was extracted from 500 μl prepheral whole blood and PCR and RFLP were used to amplify the region of interest and genotyping. With statistical analyzes a p-value <0.05 was considered meaningful. There was no significant difference in distribution of the possible three genotypes among healthy individuals and patients (P=0.906, OR=1.194, CI=0.063-22.790). However, the frequency of the G allele was higher in patients whereas A allele was more common among healthy individuals (p<0.0001). Further studies with more samples appears to be necessary.