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Background Electrocautery is a common surgical technique and is often used during shoulder arthroplasty to elevate or transect the subscapularis tendon. The relative amount of tissue damage caused by cautery as opposed to sharp transection is not currently known. The purpose of this study was to examine local tissue damage resulting from electrocautery vs. sharp transection with a scalpel. We hypothesized that the electrosurgical unit would cause higher collateral tissue damage and cell death compared with sharp transection. Methods Twelve cadaveric ovine shoulders were randomized to either the electrosurgical or sharp transection group. The infraspinatus tendon was isolated, and a partial-thickness transection was made using either a monopolar electrosurgical device (Bovie) or No. 10 scalpel blade. Tendon explants were then visualized with confocal microscopy to evaluate tissue architecture. A live/dead assay was performed using microscopy imaging analysis software. Comparisons between Bovie and scalpel transection were made using the Mann-Whitney U test, and the cell death percentage at standardized distances from the transection site was compared between groups using a mixed-model analysis. Significance was defined at P Results The cellular and tendon fibril architecture was well maintained beyond the scalpel transection site, whereas Bovie transection disrupted the architecture beyond its transection path. The percentage of dead cells in the Bovie group (74.9% ± 31.2%) was significantly higher than that in the scalpel group (27.6% ± 29.9%, P = .0004). Compared with the transection site, the cell death percentage after Bovie transection significantly declined at 2.5 mm whereas that after scalpel transection significantly declined at 1 mm from the transection site. Conclusion There was a significantly higher dead cell percentage in the Bovie transection group, indicating extensive damage beyond the local incision site, compared with sharp transection. Electrosurgical transection of the ovine infraspinatus tendon ex vivo caused higher cell death and greater tissue architecture disruption compared with sharp scalpel transection.

The deficiency of dead cell clearance is a prominent pathogenic factor in systemic lupus erythematosus (SLE). In this study, the overexpression of miR-210-5p resulted in the accumulation of secondary necrotic cells (SNECs) in macrophages through the reduction of protein degradation. The upreguation of miR-210-5p inhibited NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and SNEC clearance. miR-210-5p overexpression suppressed Sp1 and HSCARG expression, and the knockdown of SP1 and HSCARG inhibited NOX expression and superoxide production in macrophages. Furthermore, patients with active SLE expressed a higher level of miR-210-5p and lower expression of SP1 and HSCARG in peripheral blood mononuclear cells. In summary, our findings indicate that the upregulation of miR-210-5p increases the accumulation of SNECs through a decrease in the Sp1-and HSCARG-mediated NOX activity and ROS generation in macrophages. Our results also suggest that targeting miR-210-5p may have therapeutic potential for SLE.

Single‐cell sorting of Escherichia coli was conducted to analyze the effect of osmotic stress–induced injury on the colony‐forming rate (CFR). Injury caused a time delay in the start of colony formation and a decrease in the final level of CFR. Surprisingly, dead cells also inhibited cell growth possibly due to the release of insoluble factors., Osmotic stress‐induced injured cells of Escherichia coli were prepared by sorting live cells onto tryptic soy agar (TSA) containing 10–50% sucrose. The time course of colony‐forming rate (CFR%) was analyzed. A time delay in colony formation indicated a sublethal effect. The final CFR level at 24 h indicated the relative number of culturable cells irrespective of injury. A value of (100‐CFR)% at 24 h indicated a lethal effect. When cells were grown on TSA containing 10% sucrose, the time delay was 4 h and the lethal effect was 4%. However, dead cells inhibited the growth of live cells. Physical contact with insoluble matter derived from dead cells or dead cells themselves might have caused growth inhibition. These findings highlight a novel perspective on colony count methods in practical situations, such as when sampling foods containing a high concentration of sucrose.

Photodynamic theranostics integrating synchronous photodynamic therapy and real-time monitoring of therapeutic efficacy has attracted great interest in clinical cancer management. Recently, a series of cationic molecules have been reported as self-reporting photosensitizers for unimolecular photodynamic theranostics. However, the design rationality and mechanisms for such multifunctional photosensitizers are not well understood and need further exploration. In this work, we have designed and synthesized three multicationic AIEgen photosensitizers with similar core structures, but which are end-capped with pyridinium groups (BPCI and TPCI) or quaternary ammonium groups (TPCB), respectively. The tetra-pyridinium-tethered TPCI exhibits superior efficiency in reactive oxygen species generation and dead cell identification compared to both bis-pyridinium-tethered BPCI and tetra-ammonium-tethered TPCB. The structure–activity relationship of all three AIEgens has been studied in detail, which provides great insights into designing the new generation of photosensitizers for photodynamic theranostics. Strikingly, all the multicationic AIEgens have large two-photon absorption cross-sections, and TPCI can effectively kill Caenorhabditis elegans upon white light irradiation and differentiate the dead nematodes from the living ones through the redistribution of its two-photon fluorescence, demonstrating great potential in two-photon fluorescence imaging-guided photodynamic theranostics.

The clearance of apoptotic cells by professional and non-professional phagocytes — a process termed ‘efferocytosis’ — is essential for the maintenance of tissue homeostasis. Accordingly, defective efferocytosis underlies a growing list of chronic inflammatory diseases. Although much has been learnt about the mechanisms of apoptotic cell recognition and uptake, several key areas remain incompletely understood. This Review focuses on new discoveries related to how phagocytes process the metabolic cargo they receive during apoptotic cell uptake; the links between efferocytosis and the resolution of inflammation in health and disease; and the roles of efferocytosis in host defence. Understanding these aspects of efferocytosis sheds light on key physiological and pathophysiological processes and suggests novel therapeutic strategies for diseases driven by defective efferocytosis and impaired inflammation resolution. Clearing away dead cells — a process known as efferocytosis — is crucial for normal tissue homeostasis and is impaired in several pathological processes. This Review describes new insights into how efferocytes deal with the engulfed dead cell cargo, how efferocytosis supports the resolution of inflammation and how this understanding is informing new therapeutic strategies.

Background To evaluate the accuracy and safety of micro radial and arcuate keratotomy incisions constructed by a femtosecond laser system with a curved contact patient interface in porcine eyes. Methods Partial thickness micro radial and arcuate keratotomy incisions were constructed in porcine eyes with a femtosecond laser system and evaluated for precision of depth, quality, and consistency. Optical coherence tomography was used to determine the accuracy and precision of incision depth. Corneal endothelial safety was assessed by a fluorescent live/dead cell viability assay to demonstrate laser-induced endothelial cell loss. Quality was evaluated by ease of opening and examination of interfaces. Results In two micro radial incision groups, intended incision depths of 50% and 80% resulted in mean achieved depths of 50.01% and 77.69%, respectively. In three arcuate incision groups, intended incision depths of 80%, 600 μm or 100 μm residual uncut bed thickness resulted in mean achieved depths of 80.16%, 603.03 μm and residual bed of 115 μm, respectively. No loss of endothelial cell density occurred when the residual corneal bed was maintained at a minimum of 85–116 µm. The incisions were easy to open, and interfaces were smooth. Conclusions A femtosecond laser system with curved contact interface created precise and reproducible micro radial and arcuate keratotomy incisions. Accuracy and precision of the incision depth and preservation of endothelial cell density demonstrated the effectiveness and safety of the system.

In vitro cell culture relies on that the cultured cells thrive and behave in a physiologically relevant way. A standard method to evaluate their behavior is to perform chemical staining in which fluorescent probes are added to the cell culture for further imaging and analysis. However, such technique is invasive and sometimes even toxic to cells, hence, alternative methods are requested. Here, we describe an analysis method for detecting and discriminating live, dead, and apoptotic cells using deep learning. Such an approach will be less labor-intensive than traditional chemical staining procedures and will enable cell imaging with minimal impact.

[EN] Accurate detection of H. pylori in different environmental and clinical samples is essential for public health studies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete exclusion of dead cells amplification signals. The use of DNA intercalating dyes has been proposed. However, its efficacy is still not well determined. In this study, we aimed to test the suitability of PMA and PEMAXTM dyes used prior to qPCR for only detecting viable cells of H. pylori. Their efficiency was evaluated with cells submitted to different disinfection treatments and confirmed by the absence of growth on culture media and by LIVE/DEAD counts. Our results indicated that an incubation period of 5 min for both, PMA and PEMAXTM, did not affect viable cells. Our study also demonstrated that results obtained by using intercalating dyes may vary depending on the cell stress conditions. In all dead cell¿s samples, both PMA and PEMAXTM pre-qPCR treatments decreased the amplification signal (>103 Genomic Units (GU)), although none of them allowed for its disappearance confirming that intercalating dyes, although useful for screening purposes, cannot be considered as universal viability markers. To investigate the applicability of the method specifically to detect H. pylori cells in environmental samples, PMA-qPCR was performed on samples containing the different morphological and viability states that H. pylori can acquire in environment. The optimized PMA-qPCR methodology showed to be useful to detect mostly (but not only) viable forms, regardless the morphological state of the cell., This work was supported by the Conselleria de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, under project AICO/2018/273, and by the Spanish Ministerio de Ciencia e Innovacion PID2019-105691RB-I00 Grant and by the Spanish Ministry of Economy and Competitiveness AGL2014/53875-R Grant.

Recently, cell sheet engineering has emerged as one of the most accentuated approaches of tissue engineering and cardiac tissue is the pioneering application area of cell sheets with clinical use. In this study, we cultured rat cardiomyoblasts (H9C2 cell line) to obtain cell sheets by using three different approaches; using (1) thermo-responsive tissue culture plates, (2) high cell seeding density/high serum content and (3) ascorbic acid treatment. To compare the outcomes of three methods, morphologic examination, immunofluorescent stainings and live/dead cell assay were performed and the effects of serum concentration and ascorbic acid treatment on cardiac gene expressions were examined. The results showed that cardiomyoblast sheets were successfully obtained in all approaches without losing their integrity and viability. Also, the results of RT-PCR analysis showed that the types of tissue culture surface, cell seeding density, serum concentration and ascorbic acid treatment affect cardiac gene expressions of cells in cell sheets. Although three methods were succeeded, ascorbic acid treatment was found as the most rapid and effective method to obtain cell sheets with cardiac characteristics. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-019-00325-2) contains supplementary material, which is available to authorized users.

A mathematical model, validated on experimental data aiming at describing and predicting soil bacteria growth on an essential limited substrate in batch pure cultures is proposed as an extension of the Monod’s one in revisiting the way where the optical density is modelled. This model takes into account viable cell growth, substrate consumption, cell mortality, non-viable cell accumulation in the culture medium and partial dead cell recycling into substrate. The least squares method is used to identify model parameters. The model is extended and validated for mixed cultures proving, for artificially reconstituted soil ecosystems, that there is only competition for the substrate. Mathematics Subject Classification: 34D23, 35N25, 37B25, 49K40, 00A71.

Ti–xZr (x = 5, 15, 25, 35, 45% wt%) alloys with low elastic modulus and high mechanical strength were fabricated as a novel implant material. The biocompatibility of the Ti–xZr alloys was evaluated by osteoblast-like cell line (MG63) in terms of cytotoxicity, proliferation, adhesion, and osteogenic induction using CCK-8 and live/dead cell assays, electron microscopy, and real-time PCR. The Ti–xZr alloys were non-toxic and showed superior biomechanics compared to commercially pure titanium (cpTi). Ti–45Zr had the optimum strength/elastic modulus ratio and osteogenic activity, thus is a promising to used as dental implants.

Aim: The umbilical cord (UC) is a rich source of mesenchymal stem cell (MSC) isolation. Since the MSCs isolated from here have high self-renewal capacity and differentiation potential, production through biofabrication is essential for clinical treatments. For the cells to be stored for a long time and presented ready for use, encapsulation is required. In this study, UC-MSC cells were encapsulated with alginate using three different methods: alginate drop, alginate coating, and alginate sphere. Methods: The cell viability, live/dead cell ratio, and colony formation capacities of the encapsulated cells were examined for 14 days. Results: In the study, it was found that the most effective method was the alginate sphere form and that the structure of the cells should be preserved by injecting them into biomaterials in encapsulation. Colony formation potential was found to be high in biomaterials with alginate spheres. Conclusion: As a result, the preservation of UC-MSC cells with alginate sphere encapsulation via biofabrication and their clinical use availability may be beneficial for treating of many diseases.

Phagocytic clearance of dead cells and debris is critical for inflammation resolution and maintenance of tissue homeostasis. Consequently, defective clearance of dead cells and debris is associated with initiation and exacerbation of several autoimmune disorders and chronic inflammatory diseases such as atherosclerosis. The progressive loss of dead cell clearance capacity within the atherosclerotic plaque leads to accumulation of necrotic cells, chronic non-resolving inflammation, and expansion of the necrotic core, which triggers atherosclerotic plaque rupture and clinical manifestation of acute thrombotic cardiovascular adverse events. In this review, we describe the fundamental molecular and cellular mechanisms of dead cell clearance and how it goes awry in atherosclerosis. Finally, we highlight novel therapeutic strategies that enhance dead cell and debris clearance within the atherosclerotic plaque to promote inflammation resolution and atherosclerotic plaque stabilization.

Hoijman et al. show that in early vertebrate development, engulfment of apoptotic cells is mediated by epithelial cells covering the embryo, which use specialized protrusions to distribute apoptotic bodies and support their efficient clearance.

A mannose-modified perylene monoimide derivative PMI-Man was developed, which shows highly selective binding to double-stranded DNA molecules, potent live/dead cell imaging, and histological imaging via both confocal and light microscopies. This approach can be used to develop a universal colorful staining method for human tissues for both confocal and light microscopies.

The use of Beneficial Microorganisms for Corals (BMCs) to increase the resistance of corals to environmental stress has proven to be effective in laboratory trials. Because direct inoculation of BMCs in larger tanks or in the field can be challenging, a delivery mechanism is needed for efficient transmission of the BMC consortium. Packaged delivery mechanisms have been successfully used to transmit probiotics to other organisms, including humans, lobsters, and fish. Here, we tested a method for utilizing rotifers of the species Brachionus plicatilis for delivery of BMCs to corals of the species Pocillopora damicornis. Epifluorescence microscopy combined with a live/dead cell staining assay was used to evaluate the viability of the BMCs and monitor their in vivo uptake by the rotifers. The rotifers efficiently ingested BMCs, which accumulated in the digestive system and on the body surface after 10 min of interaction. Scanning electron microscopy confirmed the adherence of BMCs to the rotifer surfaces. BMC-enriched rotifers were actively ingested by P. damicornis corals, indicating that this is a promising technique for administering coral probiotics in situ. Studies to track the delivery of probiotics through carriers such as B. plicatilis, and the provision or establishment of beneficial traits in corals are the next proof-of-concept research priorities.

Here, a method for label-free, real-time interrogation, monitoring, detection and sorting of biological rare cells in magnetically-suspended heterogeneous samples is developed. To achieve this, heterogeneous populations of cells were levitated and confined in a microcapillary channel. This strategy enables spatiotemporal differential magnetic levitation of rare fragile dead cells equilibrating at different heights based on the balance between magnetic and corrected gravitational forces. In addition, sorting of fragile rare dead cell populations is monitored in real-time. This technique provides a broadly applicable label-free tool for high resolution, real-time research, as well as forensic evidence processing of rape kits. This method is validated with forensic mock samples dating back to 2003, isolating sperm from epithelial fraction with >90% efficiency and >97% purity. Overall, this method reduces the processing time by over 20-fold down to 20 minutes, eliminating centrifugation and labels, and providing an inexpensive and a high-yield alternative to the current centrifuge-based differential extraction techniques. It can potentially facilitate the forensic downstream genomic analyses, accelerating the identification of suspects, and advancing public safety.

This study evaluated the antibacterial effects of short-time exposure of surface pre-reacted glass-ionomer (S-PRG) eluate on oral microcosm biofilm. Biofilms were treated with an S-PRG eluate at different concentrations (25%, 50%, and 100%), distilled water (DW), and 0.1% chlorhexidine (CHX) twice a day for 5 min repeatedly. After 7 days, the total and aciduric bacterial counts and biofilm dry weights were measured. An image analysis program calculated the red/green (R/G) ratios in the biofilm autofluorescence images. Microscopic analyses quantified the biofilm thickness and live/dead cell ratio and determined morphological changes in the biofilm. Bacterial counts and dry weights were not significantly different in the DW group for all S-PRG eluate concentrations. An increasing trend in the R/G ratio for 7 days biofilm treatment was observed for the S-PRG eluate and the DW groups. Furthermore, the live/dead cell ratios in the biofilm and the biofilm thickness of the S-PRG eluate groups were similar to those of the DW group. The bacteria morphology inside the biofilm changed only in the CHX group. Short-time S-PRG eluate treatment showed no significant antibacterial and antibiofilm effects. These results indicated that limited biofilm formation inhibition can be obtained by using only the S-PRG eluate.

High-resolution cell patterning technology is a versatile tool for studying tissue engineering and in vitro preclinical research, but simple methods that can be implemented easily are lacking. Here, we demonstrate a simple electrochemical approach for arranging cells into various patterns, enabling rapid and accurate localization for cell array formation. A voltage pulse is applied directly to the pre-patterned conducting substrate, causing cellular damage and rapidly forming live/dead cell arrays. The array platform, which focuses on assay miniaturization, is applicable to cell-based drug screening as well as high-content cell culture experiments.

Methods of stool assessment are mostly focused on next-generation sequencing (NGS) or classical culturing, but only rarely both. We conducted a series of experiments using a multi-method approach to trace the stability of gut microbiota in various donors over time, to find the best method for the proper selection of fecal donors and to find &ldquo, super-donor&rdquo, indicators. Ten consecutive stools donated by each of three donors were used for the experiments (30 stools in total). The experiments assessed bacterial viability measured by flow cytometry, stool culturing on different media and in various conditions, and NGS (90 samples in total). There were no statistically significant differences between live and dead cell numbers, however, we found a group of cells classified as not-dead-not-alive, which may be possibly important in selection of &ldquo, good&rdquo, donors. Donor C, being a regular stool donor, was characterized by the largest number of cultivable species (64). Cultivable core microbiota (shared by all donors) was composed of only 16 species. ANCOM analysis of NGS data highlighted particular genera to be more abundant in one donor vs. the others. There was a correlation between the not-dead-not-alive group found in flow cytometry and Anaeroplasma found by NGS, and we could distinguish a regular stool donor from the others. In this work, we showed that combining various methods of microbiota assessment gives more information than each method separately.

The response of microbial communities to releases of hydraulic fracturing flowback and produced water (PW) may influence ecosystem functions. However, knowledge of the effects of PW spills on freshwater microbiota is limited. Here, we conducted two separate experiments: 16S rRNA gene sequencing combined with random forests modelling was used to assess freshwater community changes in simulated PW spills by volume from 0.05% to 50%. In a separate experiment, live/dead cell viability in a freshwater community was tested during exposure to 10% PW by volume. Three distinct patterns of microbial community shifts were identified: (i) indigenous freshwater genera remained dominant in 5% PW. Microbial taxa including less abundant genera such as Cellvibrio were potential bioindicators for the degree of contamination with PW. Additionally, live cells were quickly damaged by adding 10% PW, but cell counts recovered in the following days. Our study shows that the responses of freshwater microbiota vary by spill size, and these responses show promise as effective fingerprints for PW spills in aquatic environments.