Bacterial filaments recover by successive and accelerated asymmetric divisions that allow rapid post-stress cell proliferation.

Filamentation is a reversible morphological change triggered in response to various stresses that bacteria might encounter in the environment, during host infection or antibiotic treatments. Here we re-visit the dynamics of filament formation and recovery using a consistent framework based on live-cells microscopy. We compare the fate of filamentous Escherichia coli induced by cephalexin that inhibits cell division or by UV-induced DNA-damage that additionally perturbs chromosome segregation. We show that both filament types recover by successive and accelerated rounds of divisions that preferentially occur at the filaments' tip, thus resulting in the rapid production of multiple daughter cells with tightly regulated size. The DNA content, viability and further division of the daughter cells essentially depends on the coordination between chromosome segregation and division within the mother filament. Septum positioning at the filaments' tip depends on the Min system, while the nucleoid occlusion protein SlmA regulates the timing of division to prevent septum closure on unsegregated chromosomes. Our results not only recapitulate earlier conclusions but provide a higher level of detail regarding filaments division and the fate of the daughter cells. Together with previous reports, this work uncovers how filamentation recovery allows for a rapid cell proliferation after stress treatment.

Simultaneous detection of SO2 and viscosity in drug-induced inflammation in live cells and zebrafish.

The viscosity within cells is a crucial microenvironmental factor, and sulfur dioxide (SO2 ) has essential functions in regulating cellular apoptosis and inflammation. Some evidence has been confirmed that changes in viscosity and overexposure of SO2 within the cell may cause detrimental effects including, but not limited to, respiratory and cardiovascular illnesses, inflammation, fatty liver, and various types of cancer. Therefore, precise monitoring of SO2 and viscosity in biological entities holds immense practical importance. Therefore, in this research, we developed a versatile fluorescent TCF-Cou that enables the dual detection of SO2 and viscosity in the living system. Probe TCF-Cou possessed a response to viscosity and SO2 through red and green emissions. The alteration of SO2 and viscosity levels in live cells and zebrafish were also monitored using probe TCF-Cou. We hope that this fluorescent probe could be a potential tool for revealing the related pathological and physiological processes through monitoring the changes in SO2 and viscosity.

Excited-State Dynamics in Borylated Arylisoquinoline Complexes in Solution and in cellulo.

Two four-coordinate organoboron N,C-chelate complexes with different functional terminals on the PEG chains are studied with respect to their photophysical properties within human MCF-7 cells. Their excited-state properties are characterized by time-resolved pump-probe spectroscopy and fluorescence lifetime microscopy. The excited-state relaxation dynamics of the two complexes are similar when studied in DMSO. Aggregation of the complexes with the carboxylate terminal group is observed in water. When studying the light-driven excited-state dynamics of both complexes in cellulo, i. e., after being taken up into human MCF-7 cells, both complexes show different features depending on the nature of the anchoring PEG chains. The lifetime of a characteristic intramolecular charge-transfer state is significantly shorter when studied in cellulo (360±170 ps) as compared to in DMSO (∼960 ps) at 600 nm for the complexes with an amino group. However, the kinetics of the complexes with the carboxylate group are in line with those recorded in DMSO. On the other hand, the lifetimes of the fluorescent state are almost identical for both complexes in cellulo. These findings underline the importance to evaluate the excited-state properties of fluorophores in a complex biological environment in order to fully account for intra- and intermolecular effects governing the light-induced processes in functional dyes.

Live Cells versus Fixated Cells: Kinetic Measurements of Biomolecular Interactions with the LigandTracer Method and Surface Plasmon Resonance Microscopy.

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.

Effects of Saccharomyces cerevisiae live cells and culture on growth and productive performance in lactating Nili-Ravi buffaloes.

An experimental work was conducted to evaluate the effects of Saccharomyces cerevisiae live cells and its culture on dry matter intake (DMI), milk yield, milk composition, body condition score, selected blood metabolites, feed conversion efficiency (FCE), nutrient digestibility, body weight gain, and economics of milk production in lactating multiparous Nili-Ravi buffaloes. In total, 20 buffaloes of age 5 years ± 6 months and weighing 550 ± 20 kg were selected and assigned to four dietary treatments (n=5 buffalo/treatment) under completely randomized design. The dietary treatments include treatment 1 (T1) control, treatment 2 (T2) 5g/head live yeast, treatment 3 (T3) 5g/head yeast culture, and treatment 4 (T4) 10 g/head yeast culture per day for 60 days excluding 14 days as an adjustment period. The results indicated that T4 showed significant (p<0.05) improvement in DMI, milk yield and components, blood glucose level, digestibility of nutrients, and body weight gain while significant decrease in blood urea nitrogen as compared to other treatment groups. Body condition score was not affected among treatments. In conclusion, yeast culture supplementation significantly improved (p <0.05) milk yield, milk composition, DMI, body weight gain, blood glucose level, and digestibility while significantly decreased blood urea level as compare to control. Economic return was also improved. BCS was not improved. Comparatively, yeast culture showed significant improvement in growth and productive performance as compare to live yeast. Meanwhile, 10-g yeast culture showed better results as compare to 5-g yeast culture.

Using Biological Photophysics to Map the Excited-State Topology of Molecular Photosensitizers for Photodynamic Therapy.

This study employs TLD1433, a RuII -based photodynamic therapy (PDT) agent in human clinical trials, as a benchmark to establish protocols for studying the excited-state dynamics of photosensitizers (PSs) in cellulo, in the local environment provided by human cancer cells. Very little is known about the excited-state properties of any PS in live cells, and for TLD1433, it is terra incognita. This contribution targets a general problem in phototherapy, which is how to interrogate the light-triggered, function-determining processes of the PSs in the relevant biological environment, and establishes methodological advances to study the ultrafast photoinduced processes for TLD1433 when taken up by MCF7 cells. We generalize the methodological developments and results in terms of molecular physics by applying them to TLD1433's analogue TLD1633, making this study a benchmark to investigate the excited-state dynamics of phototoxic compounds in the complex biological environment.

DNA Nanotechnology-Empowered Live Cell Measurements.

The systematic analysis and precise manipulation of a variety of biomolecules should lead to unprecedented findings in fundamental biology. However, conventional technology cannot meet the current requirements. Despite this, there has been progress as DNA nanotechnology has evolved to generate DNA nanostructures and circuits over the past four decades. Many potential applications of DNA nanotechnology for live cell measurements have begun to emerge owing to the biocompatibility, nanometer addressability, and stimulus responsiveness of DNA. In this review, the DNA nanotechnology-empowered live cell measurements which are currently available are summarized. The stability of the DNA nanostructures, in a cellular microenvironment, which is crucial for accomplishing precise live cell measurements, is first summarized. Thereafter, measurements in the extracellular and intracellular microenvironment, in live cells, are introduced. Finally, the challenges that are innate to, and the further developments that are possible in this nascent field are discussed.

Endogenous Proteins Modulation in Live Cells with Small Molecules and Light.

Protein modulation by light illumination enables the investigation of biological function in high spatiotemporal precision. Compared to genetic methods, the small molecule approach is uniquely suited for modulating endogenous proteins. Endogenous protein modulation in live cells with small molecules and light has recently advanced on three distinctive frontiers: i) the infrared-light-induced or localized decaging of small molecules by photolysis, ii) the visible-light-induced photocatalytic releasing of small molecules, and iii) the small-molecule-ligand-directed caging for photomodulation of proteins. Together, these methods provide powerful chemical biology tool kits for spatiotemporal modulation of endogenous proteins with potential therapeutic applications. This Concept article aims to inspire organic chemists and chemical biologists to delve into this burgeoning endogenous protein modulation field for new biological discoveries.

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

ß-arrestins (ßarr1 and ßarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that ß-arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into ßarr1 and ßarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on arrestins, whereas the two ß-arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

A novel phenanthridine and terpyridine based D-π-A fluorescent probe for the ratiometric detection of Cd2+ in environmental water samples and living cells.

A "turn-on" Donor-π-Acceptor (D-π-A) containing phenanthridine-functionalized extended π-conjugate terpyridine, 5-(4'-([2,2':6',2''-terpyridin]-4'-yl)-[1,1'-biphenyl]4-yl)7,8,13,14-tetrahydrodibenzo [a, i] phenanthridine (TBTP) was synthesised. It shows strong selectivity for the detection of toxic Cd2+ without interference from other metal ions. In the presence of Cd2+, the absorption of the TBTP changes dramatically along with the fluorescent emission with the large Stokes shift of 6300 cm-1. When the compound TBTP is exposed to UV light, its colour changes from blue to orange over the addition of Cd2+. Adding other transition metal ions has no effect. This is based on the mechanism of intramolecular charge transfer. The detection limit for Cd2+ was found to be around 1.181 × 10-8 M. An investigation of the sensing mechanism includes job plot, NMR titration, DFT calculation, and HRMS analyses. Excitingly, the recognition of Cd2+ in CH3CN: H2O (8:2, v/v) medium is quantitative without interference from Zn2+, which is a common interferent for Cd2+. Furthermore, the probe was used for detecting Cd2+ in real water samples and cell imaging in living cells was also performed.

Screening of dopamine in living cells and animal model via graphene quantum dots anchored 3D macroporous nonenzymatic sensor.

A series of three-dimensional copper oxide (CuO) inverse opals anchored with carboxylated graphene quantum dots (CuO/cGQDs) have been fabricated for non-enzymatic tracking of dopamine (DA). Heterostructures composed of various building blocks are promising to construct versatile biosensing platforms. The optimal CuO/cGQDs modified electrode demonstrates sensitivities of 243.45 µA mM-1 cm-2 (50 nM-1888.5 µM) with the practical detection limit as low as 0.5 nM in mimic physiological environment (at + 0.45 V vs. Ag/AgCl). The extraordinary tolerance to various interferents enables the practical detection of intracellular DA amount in human neural cells. On this basis, the proposed biosensor attains precise evaluation of antipsychotic drug effects on stimulated DA release. Particularly, it successfully spots fluctuation of DA in plasma and cerebrospinal fluid in murine model of Parkinson's disease, which serves as a crucial tool to understand neuropathology and symptomatology of DA-related diseases. This study developed a reliable sensing platform and is expected to be applied to physiological and pathological studies.

AIE+ESIPT Active Hydroxybenzothiazole for Intracellular Detection of Cu2+: Anticancer and Anticounterfeiting Applications.

Here, in the present work, a new hydroxybenzothiazole derivative (HBT 2) with AIE+ESIPT features was synthesized by Suzuki-Miyora coupling of HBT 1 with 4-formylphenylboronic acid. The AIE and ESIPT features were confirmed by optical, microscopic (AFM) and dynamic light scattering (DLS) techniques. The yellow fluorescent aggregates of HBT 2 can specifically detect Cu2+/Cu+ ions with limits of detection as low as 250 nM and 69 nM. The Job's plot revealed the formation of a 1:1 complex. The Cu2+ complexation was further confirmed by optical, NMR, AFM and DLS techniques. HBT 2 was also used for the detection of Cu2+ ions in real water samples collected from different regions of Punjab. HBT 2 was successfully used for the bio-imaging of Cu2+ ions in live A549 and its anticancer activity was checked on different cancer cell lines, such as MG63, and HeLa, and normal cell lines such as L929. We successfully utilized HBT 2 to develop security labels for anticounterfeiting applications.

Label-free classification of dead and live colonic adenocarcinoma cells based on 2D light scattering and deep learning analysis.

The measurement of cell viability plays an essential role in the area of cell biology. At present, the common methods for cell viability assay mainly on the responses of cells to different dyes. However, the additional steps of cell staining will consequently cause time-consuming and laborious efforts. Furthermore, the process of cell staining is invasive and may cause internal structure damage of cells, restricting their reuse in subsequent experiments. In this work, we proposed a label-free method to classify live and dead colonic adenocarcinoma cells by 2D light scattering combined with the deep learning algorithm. The deep convolutional network of YOLO-v3 was used to identify and classify light scattering images of live and dead HT29 cells. This method achieved an excellent sensitivity (93.6%), specificity (94.4%), and accuracy (94%). The results showed that the combination of 2D light scattering images and deep neural network may provide a new label-free method for cellular analysis.

KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements.

Design of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe.

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.

Saccharomyces cerevisiae as a probiotic feed additive to non and pseudo-ruminant feeding: a review.

The production of livestock and poultry faces major challenges to meet the global demand for meat and dairy products and eggs due to a steady increase in the world's population and the ban of antibiotics in animal production. This ban has forced animal nutritionists to seek for natural alternatives to antibiotics. In this context, the yeast Saccharomyces cerevisiae has received considerable attention in the last decade. It has been reported that feed supplementation with live yeast cells improve feed efficiency, enhance feed digestibility, increase animal performance, reduce the number of pathogenic bacteria, improve animal health and reduce the negative environmental impacts of livestock production. The current review sheds light on the effects of the use of live S. cerevisiae cells in the diets of nonruminant and pseudo-ruminant's animals and the mechanisms by which they exert its effects. This review work revealed that the addition of S. cerevisiae in poultry feed causes a phenomenon called competitive exclusion of pathogenic bacteria capable of causing disease adhere to the yeast surface, and so removing a large amount of harmful micro-organisms and allowing the Animal defend more effectively, the production of antimicrobial agents, the balancing the gut microbiota and stimulation of host adaptive immune system and improving gut morphological structure, thus these benefits are reflected on the overall poultry health. In addition, in the presence of live S. cerevisiae cells, the immunity of rabbits was improved due to the high number of white blood cell. In addition, apparent digestibility of acid and neutral detergent fibre was improved in horses and rabbits. Saccharomyces cerevisiae in pig diets augment mucosal immunity by increasing IgM and IgA activity against pathogens, enhance intestinal development and function, adsorb mycotoxins, modulate gut microbiota and reduce postweaning diarrhoea.

Non-Cytotoxic Dibenzyl and Difluoroborate Curcuminoid Fluorophores Allow Visualization of Nucleus or Cytoplasm in Bioimaging.

Curcumin, the most important secondary metabolite isolated from Curcuma longa, is known for its numerous purported therapeutic properties and as a natural dye. Herein, based on curcumin's intrinsic fluorescence, a search for improved curcumin-based fluorophores was conducted. Within the set of semi-synthetic curcumin derivatives i.e. mono (1), di (2), tri (3), tetra (4) benzylated and dibenzyl-fluoroborate (5), the fluorescence properties of 2 and 5 in solution outstood with a two-fold quantum yield compared to curcumin. Furthermore, all benzylated derivatives showed a favorable minimal cytotoxic activity upon screening at 25 µM against human cancer and non-tumoral COS-7 cell lines, with a reduction of its cytotoxic effect related to the degree of substitution. Fluorophores 2 and 5 are versatile bioimaging tools, as revealed by Confocal Fluorescence Microscopy (CFM), and showed permeation of living cell membranes of astrocytes and astrocytomas. When 2 is excited with a 405- (blue) or 543-nm (green) laser, it is possible to exclusively and intensively visualize the nucleus. However, the fluorescence emission fades as the laser wavelength moves towards the red region. In comparison, 5 allows selective visualization of cytoplasm when a 560-nm laser is used, showing emission in the NIR region, while it is possible to exclusively observe the nucleus at the blue region with a 405-nm laser.

A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells.

BACKGROUND: GPCRs are considered essential for various physiological processes and have been the most productive drug targets. Therefore, development of the methods of GPCR ligands screening is a high priority for pharmaceutical industries and research institutions. METHODS: We developed a potential method (piggyBac-TANGO) based on the TANGO and PRESTO-TANGO assays. The system was optimized with a piggyBac transposon as a transgene vehicle, and eGFP was used as a reporter instead of luciferase. The assay was validated in the HEK 293T and U87-MG cell lines and antagonist activities of the compounds were assessed. The transgene copy number and long-term stability were evaluated by qPCR. Then, we performed a DRD2-targeted screening for natural products using the piggyBac-TANGO assay. RESULTS: The validation assay showed that using the piggyBac transposon as a transgene vehicle produced high signal-to-background ratio and stable readout confirmed by investigation of the transgene copy number and long-term stability. Use of eGFP instead of luciferase as a reporter enabled to create a high throughput system suitable for live cells. Moreover, the piggyBac-TANGO assay permitted versatile detection of antagonist activity of compounds and was not limited to a particular cell type. With the use of the piggyBac-TANGO assay, we have successfully identified a novel agonist of DRD2. CONCLUSION: Thus, the results indicate that the piggyBac-TANGO method is a user-friendly, robust and imaging-based assay that provides a novel approach to high throughput GPCR-targeted ligand screening and drug development.

Single Virion Tracking Microscopy for the Study of Virus Entry Processes in Live Cells and Biomimetic Platforms.

The most widely-used assays for studying viral entry, including infectivity, cofloatation, and cell-cell fusion assays, yield functional information but provide low resolution of individual entry steps. Structural characterization provides high-resolution conformational information, but on its own is unable to address the functional significance of these conformations. Single virion tracking microscopy techniques provide more detail on the intermediate entry steps than infection assays and more functional information than structural methods, bridging the gap between these methods. In addition, single virion approaches also provide dynamic information about the kinetics of entry processes. This chapter reviews single virion tracking techniques and describes how they can be applied to study specific virus entry steps. These techniques provide information complementary to traditional ensemble approaches. Single virion techniques may either probe virion behavior in live cells or in biomimetic platforms. Synthesizing information from ensemble, structural, and single virion techniques ultimately yields a more complete understanding of the viral entry process than can be achieved by any single method alone.

Superlattice stacking by hybridizing layered double hydroxide nanosheets with layers of reduced graphene oxide for electrochemical simultaneous determination of dopamine, uric acid and ascorbic acid.

A self-assembled periodic superlattice material was obtained by integrating positively charged semiconductive sheets of a Zn-NiAl layered double hydroxide (LDH) and negatively charged layers of reduced graphene oxide (rGO). The material was used to modify a glassy carbon electrode which then is shown to be a viable sensor for the diagnostic parameters dopamine (DA), uric acid (UA) and ascorbic acid (AA). The modified GCE displays excellent electrocatalytic activity towards these biomolecules. This is assumed to be due to the synergistic effects of (a) excellent interfacial electrical conductivity that is imparted by direct neighboring of conductive rGO to semiconductive channels of LDHs, (b) the superb intercalation feature of LDHs, and (c) the enlarged surface with an enormous number of active sites. The biosensor revealed outstanding electrochemical performances in terms of selectivity, sensitivity, and wide linear ranges. Typically operated at working potentials of -0.10, +0.13 and + 0.27 V vs. saturated calomel electrode, the lower detection limits for AA, DA and UA are 13.5 nM, 0.1 nM, and 0.9 nM, respectively, at a signal-to-noise ratio of 3. The sensor was applied to real-time tracking of dopamine efflux from live human nerve cells. Graphical abstract Schematic of the preparation of a superlattice self-assembled material by integrating positively charged semiconductive sheets of Zn-NiAl layered double hydroxide (LDH) with negatively charged reduced graphene oxide (rGO) layers. It was applied to simultaneous electrochemical detection of dopamine (DA), uric acid and ascorbic acid.