Engineering of Injectable Antibiotic-laden Fibrous Microparticles Gelatin Methacryloyl Hydrogel for Endodontic Infection Ablation

This study aimed at engineering cytocompatible and injectable antibiotic-laden fibrous microparticles gelatin methacryloyl (GelMA) hydrogels for endodontic infection ablation. Clindamycin (CLIN) or metronidazole (MET) was added to a polymer solution and electrospun into fibrous mats, which were processed via cryomilling to obtain CLIN- or MET-laden fibrous microparticles. Then, GelMA was modified with CLIN- or MET-laden microparticles or by using equal amounts of each set of fibrous microparticles. Morphological characterization of electrospun fibers and cryomilled particles was performed via scanning electron microscopy (SEM). The experimental hydrogels were further examined for swelling, degradation, and toxicity to dental stem cells, as well as antimicrobial action against endodontic pathogens (agar diffusion) and biofilm inhibition, evaluated both quantitatively (CFU/mL) and qualitatively via confocal laser scanning microscopy (CLSM) and SEM. Data were analyzed using ANOVA and Tukey’s test (α = 0.05). The modification of GelMA with antibiotic-laden fibrous microparticles increased the hydrogel swelling ratio and degradation rate. Cell viability was slightly reduced, although without any significant toxicity (cell viability > 50%). All hydrogels containing antibiotic-laden fibrous microparticles displayed antibiofilm effects, with the dentin substrate showing nearly complete elimination of viable bacteria. Altogether, our findings suggest that the engineered injectable antibiotic-laden fibrous microparticles hydrogels hold clinical prospects for endodontic infection ablation.

An In Vitro Evaluation of Denture Cleansing Regimens against a Polymicrobial Denture Biofilm Model

Denture stomatitis (DS) is an inflammatory disease resulting from a polymicrobial biofilm perturbation at the denture surface–palatal mucosa interface. Recommendations made by dental health care professionals often lack clarity for appropriate denture cleaning. This study investigated the efficacy of brushing with off-the-shelf denture cleanser (DC) tablets (Poligrip®) vs. two toothpastes (Colgate® and Crest®) in alleviating the viable microorganisms (bacteria and fungi) in an in vitro denture biofilm model. Biofilms were grown on poly(methyl)methacrylate (PMMA) discs, then treated daily for 7 days with mechanical disruption (brushing), plus Poligrip® DC, Colgate® or Crest® toothpastes. Weekly treatment with Poligrip® DC on day 7 only was compared to daily modalities. All treatment parameters were processed to determine viable colony forming units for bacteria and fungi using the Miles and Misra technique, and imaged by confocal laser scanning microscopy (CLSM). Brushing with daily DC therapy was the most effective treatment in reducing the viable biofilm over 7 days of treatment. Brushing only was ineffective in controlling the viable bioburden, which was confirmed by CLSM imaging. This data indicates that regular cleansing of PMMA with DC was best for polymicrobial biofilms.

SLDP and LIPA mediate lipid droplet-plasma membrane tethering in Arabidopsis thaliana

Membrane contact sites (MCS) are inter-organellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here we identified and characterised three proteins that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localised SEED LD PROTEIN (SLDP) 1 and 2 and PM-localised LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and 2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA on the other hand is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterised by dynamically moving LDs in the cytosolic streaming. Further, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis thaliana.

Age-associated features of the expression level of apoptosis markers in cardiomyocytes of patients with dilated cardiomyopathy

Для понимания патогенеза дилатационной кардиомиопатии (ДКМП) необходимо установить молекулярно-клеточные механизмы старения миокарда, в том числе связанные с программируемой клеточной гибелью, молекулярные механизмы которого практически не изучены. Цель работы - изучение маркеров апоптоза в кардиомиоцитах у пациентов с ДКМП in vitro. В работе использовали метод первичных диссоциированных клеточных культур и метод иммунофлюоресцентной конфокальной лазерной микроскопии. Для моделирования клеточного старения использовали клетки 3-го и 14-го пассажей, соответствующие «молодым» и «старым» культурам. На молекулярном уровне старение клеток кардиомиоцитов сопровождалось повышением экспрессии р16 в 2 раза по сравнению с «молодыми культурами» как в контрольной, так и в группе с ДКМП. Также установлено, что экспрессия р16 в культурах, взятых от пациентов с патологией, была в 2 раза выше, чем в аналогичных культурах от здоровых пациентов. Экспрессия р21 была повышена в группе с ДКМП по сравнению с контрольной группой, однако при старении культуры экспрессия p21 не изменялась, оставаясь на высоком уровне. Наиболее значимые различия были получены при сравнении экспрессии Bax в культуре клеток кардиомиоцитов из группы с ДКМП в «молодой» культуре с нормой - в 3,2 раза. Старение клеток миокарда на молекулярном уровне проявлялось в повышении экспрессии белка Baх, именно он является запускающим механизмом митохондриального пути апоптоза. Возможно, этот путь клеточной гибели является превалирующем при ДКМП. To understand the pathogenesis of dilated cardiomyopathy (DCMP), it is necessary to establish the molecular-cellular mechanisms of myocardial aging, including those associated with programmed cell death, the molecular mechanisms of which have not been practically studied. The aim of this work is to study markers of apoptosis in cardiomyocytes of patients with DCMP in vitro. We used the method of primary dissociated cell cultures and the method of immunofluorescence confocal laser microscopy. Cells of the 3 and 14 passages, corresponding to «young» and «old» cultures, were used to simulate cellular senescence. Results. At the molecular level, aging of cardiomyocyte cells was accompanied by a twofold increase in the expression of p16 compared to «young cultures» both in the control group and in the group with DCMP. It was also found that the expression of p16 in cultures taken from patients with pathology was 2 times higher than in similar cultures from healthy patients. The expression of p21 was increased in the group with DCMP compared to the control; however, with aging of the culture, the expression of p21 did not change, remaining at a significant level. The most significant differences were obtained when comparing the expression of Bax in the cell culture of cardiomyocytes from the group with DCMP in a «young» culture compared with the norm, 3,2 times. Aging of myocardial cells at the molecular level was manifested in an increase in the expression of the Bax protein, which is the triggering mechanism of the mitochondrial apoptosis pathway. It is possible that this pathway of cell death is prevalent in DCMP.

Sugammadex affects GH/GHR’s signaling transduction on muscle cells by regulating the membrane-localized GHR level

Objectives Sugammadex (also known as bridion) is a modified γ-cyclodextrin, which is a reversal agent for the neuromuscular block. Growth hormone (GH) has an important biological effect on muscle, regulating muscle growth and development. In the current work, we explored the effect of Sugammadex on GH’s bioactivities. Methods Confocal laser scanning microscope (CLSM), flow cytometry, indirect immunofluorescence, Western-blot, and IP-WB were used to explore the effect of Sugammadex on GH’s bioactivities. Results We found that Sugammadex reduced the activity of GH on muscle cells, which down-regulated GH/GHR-mediated intracellular signaling pathway, such as Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5). We further study the potential biological mechanism by which Sugammadex down-regulated GH/GHR-mediated signaling pathway, a series of related experiments were conducted, and found that Sugammadex may inhibit the proliferation of C2C12 cell via regulating the membrane-localized GHR, which may be the underlying mechanism by which Sugammadex suppressed GHR-induced signaling transduction. This work has laid the theoretical and experimental basis for further exploring the relationship between Sugammadex and GH’s activity. Conclusions In conclusion, this study laid a foundation for further study on the relationship between Sugammadex and GH’s activity.

Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidence

The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

Phytohormone Priming of Tomato Plants Evoke Differential Behavior in Rhizoctonia solani During Infection, With Salicylate Priming Imparting Greater Tolerance Than Jasmonate

In the field of phytohormone defense, the general perception is that salicylate (SA)-mediated defense is induced against biotrophic pathogens while jasmonate (JA)-mediated defense functions against necrotrophic pathogens. Our goals were to observe the behavior of the necrotrophic pathogen Rhizoctonia solani in the vicinity, on the surface, and within the host tissue after priming the host with SA or JA, and to see if priming with these phytohormones would affect the host defense differently upon infection. It was observed for the first time, that R. solani could not only distinguish between JA versus SA-primed tomato plants from a distance, but surprisingly avoided SA-primed plants more than JA-primed plants. To corroborate these findings, early infection events were monitored and compared through microscopy, Scanning Electron Microscopy, and Confocal Laser Scanning Microscopy using transformed R. solani expressing green fluorescence protein gene (gfp). Different histochemical and physiological parameters were compared between the unprimed control, JA-primed, and SA-primed plants after infection. The expression of a total of fifteen genes, including the appressoria-related gene of the pathogen and twelve marker genes functioning in the SA and JA signaling pathways, were monitored over a time course during early infection stages. R. solani being traditionally designated as a necrotroph, the major unexpected observations were that Salicylate priming offered better tolerance than Jasmonate priming and that it was mediated through the activation of SA-mediated defense during the initial phase of infection, followed by JA-mediated defense in the later phase. Hence, the present scenario of biphasic SA-JA defense cascades during R. solani infection, with SA priming imparting maximum tolerance, indicate a possible hemibiotrophic pathosystem that needs to be investigated further.

Quantifying Phase Behaviour in Model Food Composites Using 3D Confocal Laser Scanning Microscopy

There is an increasing demand for the design of complex bio-composites with customized structural characteristics for use in processed food products. Phase behaviour of these mixtures determines textural properties, encouraging the pursue of a rapid technique that can accurately quantify it. The present work tests the efficacy of confocal laser scanning microscopy (CLSM) coupled with image analysis software (Imaris), for the quantification of phase behaviour in complex tertiary systems. In doing so, it develops phase separated gels of agarose and gelatin supporting inclusions of canola oil. The polysaccharide was replaced with whey protein isolate (WPI) and the topology of the tertiary dispersion with gelatin and canola oil was also examined. Reproducible phase volume estimates were obtained, including those of the lipid phase, which were a close match to the actual concentrations added to the hydrocolloid gel. The approach could offer an alternative to the rheological estimation, via theoretical blending law analysis, of phase volumes in bio-composites. Graphical Abstract

Triacrylamide-Based Adhesives Stabilize Bonds in Physiologic Conditions

In this study, an acrylamide-based adhesive was combined with a thiourethane-based composite to improve bond stability and reduce polymerization stress, respectively, of simulated composite restorations. The stability testing was conducted under physiologic conditions, combining mechanical and bacterial challenges. Urethane dimethacrylate was combined with a newly synthesized triacrylamide (TMAAEA) or HEMA (2-hydroxyethyl-methacrylate; control) to produce a 2-step total-etch adhesive system. Methacrylate-based composites (70 wt% silanized filler) were formulated, containing thiourethane oligomers at 0 (control) or 20 wt%. Standardized preparations in human third molars were restored; then, epoxy replicas were obtained from the occlusal surfaces before and after 7-d storage in water or with Streptococcus mutans biofilm, which was tested after storage in an incubator (static) or the bioreactor (mechanical challenge). Images were obtained from the replicas (scanning electron microscopy) and cross sections of the samples (confocal laser scanning microscopy) and then analyzed to obtain measurements of gap, bacterial infiltration, and demineralization. Microtensile bond strength of specimens stored in water or biofilm was assessed in 1-mm2 stick specimens. Data were analyzed with analysis of variance and Tukey’s test (α = 0.05). HEMA-based materials had greater initial gap measurements, indicating more efficient bonding for the acrylamide materials. When tested in water, the triacrylamide-based adhesive had smaller gaps in the incubator or bioreactor. In the presence of biofilm, there was less difference among materials, but the acrylamide/thiourethane combination led to statistically lower gap formation in the bioreactor. HEMA and TMAAEA-based adhesives produced statistically similar microtensile bond strengths after being stored in water for 7 d, but after the same period with biofilm-challenged specimens, the TMAAEA-based adhesives were the only ones to retain the initial bond strength values. The use of a stable multiacrylamide-based adhesive led to the preservation of the resin-dentin bonded interface after a physiologically relevant challenge. Future studies will include a multispecies biofilm model.