Molecular Signatures of Senescence in Periodontitis: Clinical Insights.

Most of the elderly population is afflicted by periodontal diseases, creating a health burden worldwide. Cellular senescence is one of the hallmarks of aging and associated with several chronic comorbidities. Senescent cells produce a variety of deleterious secretions, collectively termed the senescence-associated secretory phenotype (SASP). This disrupts neighboring cells, leading to further senescence propagation and inciting chronic inflammation, known as "inflammaging." Detrimental repercussions within the tissue microenvironment can trigger senescence at a younger age, accelerate biological aging, and drive the initiation or progression of diseases. Here, we investigated the biological signatures of senescence in healthy and diseased gingival tissues by assessing the levels of key senescence markers (p16, lipofuscin, and ß-galactosidase) and inflammatory mediators (interleukin [IL]-1ß, IL-6, IL-8, matrix metalloproteinase [MMP]-1, MMP-3, and tumor necrosis factor-α). Our results showed significantly increased senescence features including p16, lipofuscin, and ß-galactosidase in both epithelial and connective tissues of periodontitis patients compared with healthy sites in all age groups, indicating that an inflammatory microenvironment can trigger senescence-like alterations in younger diseased gingival tissues as well. Subsequent analyses using double staining with specific cell markers noted the enrichment of ß-galactosidase in fibroblasts and macrophages. Concurrently, inflammatory mediators consistent with SASP were increased in the gingival biopsies obtained from periodontitis lesions. Together, our findings provide the first clinical report revealing susceptibility to elevated senescence and inflammatory milieu consistent with senescence secretome in gingival tissues, thus introducing senescence as one of the drivers of pathological events in the oral mucosa and a novel strategy for targeted interventions.

ß-Galactosidase-guided self-assembled 68Ga nanofibers probe for micro-PET tumor imaging.

ß-galactosidase (ß-gal) has high activity in various malignancies, which is suitable for targeted positron emission tomography (PET) imaging. Meanwhile, ß-gal can successfully guide the formation of nanofibers, which enhances the intensity of imaging and extends the imaging time. Herein, we designed a ß-galactosidase-guided self-assembled PET imaging probe [68Ga]Nap-NOTA-1Gal. We envisage that ß-gal could recognize and cleave the target site, bringing about self-assembling to form nanofibers, thereby enhancing the PET imaging effect. The targeting specificity of [68Ga]Nap-NOTA-1Gal for detecting ß-gal activity was examined using the control probe [68Ga]Nap-NOTA-1. Micro-PET imaging showed that tumor regions of [68Ga]Nap-NOTA-1Gal were visible after injection. And the tumor uptake of [68Ga]Nap-NOTA-1Gal was higher than [68Ga]Nap-NOTA-1 at all-time points. Our results demonstrated that the [68Ga]Nap-NOTA-1Gal can be used for the purpose of a new promising PET probe for helping diagnose cancer with high levels of ß-gal activity.

Visualize intracellular ß-galactosidase using an asymmetric near-infrared fluorescent probe with a large Stokes shift.

ß-galactosidase (ß-Gal) is an important biomarker of cell senescence and primary ovarian cancer. Therefore, it is of great significance to construct a near-infrared fluorescent probe with deep tissue penetration and a high signal-to-noise ratio for visualization of ß-galactosidase in biological systems. However, most near-infrared probes tend to have small Stokes shifts and low signal-to-noise ratios due to crosstalk between excitation and emission spectra. Using d-galactose residues as specific recognition units and near-infrared dye TJ730 as fluorophores, a near-infrared fluorescence probe SN-CR with asymmetric structure was developed for the detection of ß-Gal. The probe has a fast reaction equilibrium time (<12 min) with ß-Gal, excellent biocompatibility, near-infrared emission (738 nm), low detection limit (0.0029 U/mL), and no crosstalk between the excitation spectrum and emission spectrum (Stokes shifts 142 nm) of the probe. Cell imaging studies have shown that SN-CR can visually trace ß-Gal in different cells and distinguish ovarian cancer cells from other cells.

Methods to Study Myc-Regulated Cellular Senescence: An Update.

Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated ß-galactosidase activity (SA-ß-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.

A sensitive fluorescent probe for ß-galactosidase activity detection and application in ovarian tumor imaging.

The development of non-invasive and sensitive optical probes for in vivo bioimaging of cancer-related enzymes is desirable for early diagnosis and effective cancer therapy. ß-galactosidase (ß-gal) is regarded as a key ovarian cancer biomarker, owing to its overexpression in primary ovarian cancer. Herein, we designed a sensitive near-infrared (NIR) probe (DCMCA-ßgal) for the detection and real-time imaging of ß-gal activity in ovarian tumors, thereby achieving the visualization of ovarian tumors by ß-gal activity detection. DCMCA-ß-gal could be triggered by ß-gal, resulting in the release of a NIR chromophore, DCM-NH2; the linear range of fluorescent response to ß-gal concentration was 0-1.2 U with a low detection limit of 1.26 × 10-3 U mL-1. We used DCMCA-ß-gal to detect and visualize ß-gal activity in SKOV3 human ovarian cancer cells, as well as for real-time imaging of ß-gal activity in ovarian cancer mouse models. DCMCA-ß-gal possessed high sensitivity, "turn-on" NIR emission, a large spectral shift, and high photostability in a dynamic living system and thus could serve as a highly sensitive sensor for real-time tracking of ß-gal activity in vivo and ovarian tumor imaging.

No effect of the endurance training status on senescence despite reduced inflammation in skeletal muscle of older individuals.

The aim of the present study was to determine if the training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle in older compared with younger subjects, with a specific focus on satellite cells. Analyses were conducted on skeletal muscle and cultured satellite cells from vastus lateralis biopsies (n = 34) of male volunteers divided into four groups: young sedentary (YS), young trained cyclists (YT), old sedentary (OS), and old trained cyclists (OT). The senescence state and inflammatory profile were evaluated by telomere dysfunction-induced foci (TIF) quantification, senescence-associated ß-galactosidase (SA-ß-Gal) staining, and quantitative (q)RT-PCR. Independently of the endurance training status, TIF levels (+35%, P < 0.001) and the percentage of SA-ß-Gal-positive cells (+30%, P < 0.05) were higher in cultured satellite cells of older compared with younger subjects. p16 (4- to 5-fold) and p21 (2-fold) mRNA levels in skeletal muscle were higher with age but unchanged by the training status. Aging induced higher CD68 mRNA levels in human skeletal muscle (+102%, P = 0.009). Independently of age, both trained groups had lower IL-8 mRNA levels (-70%, P = 0.011) and tended to have lower TNF-α mRNA levels (-40%, P = 0.10) compared with the sedentary subjects. All together, we found that the endurance training status did not slow down senescence in skeletal muscle and satellite cells in older compared with younger subjects despite reduced inflammation in skeletal muscle. These findings highlight that the link between senescence and inflammation can be disrupted in skeletal muscle.

Visualization of endogenous ß-galactosidase activity in living cells and zebrafish with a turn-on near-infrared fluorescent probe.

ß-Galactosidase (ß-gal) is an important biomarker for primary ovarian cancers. Developing noninvasive bioimaging probes for studying the activity of ß-gal is highly desirable for cancer diagnosis. Herein, a turn-on near-infrared (NIR) fluorescent probe， 2-((6-(((2S, 3R, 4S, 5R, 6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran -2-yl)oxy)-2,3-dihydro-1H-xanthen-4-yl)methylene)malononitrile named DXM-ßgal, was rationally designed based on enzymatic reaction for the detection of ß-gal activity both in vitro and in vivo. Upon incubating with ß-gal, DXM-ßgal displayed a significant fluorescence enhancement at 640 nm, accompanying by a color change of solution color from red to purple. DXM-ßgal exhibited high selectivity and sensitively to ß-gal with low limit of detection (2.92 × 10-4 U mL-1). Besides, based on its advantages of long-wavelength emission and excellent biocompatibility, DXM-ßgal was successfully applied to imaging ß-gal in living cells and zebrafish. Given these prominent properties, we believe that DXM-ßgal will be a potential tool for investigating ß-gal activity in biomedical research.

Contribution of senescence in human endometrial stromal cells during proliferative phase to embryo receptivity†.

Successful assisted reproductive technology pregnancy depends on the viability of embryos and endometrial receptivity. However, the literature has neglected effects of the endometrial environment during the proliferative phase on implantation success or failure. Human endometrial stromal cells (hESCs) were isolated from endometrial tissues sampled at oocyte retrieval during the proliferative phase from women undergoing infertility treatment. Primary hESC cultures were used to investigate the relationship between stemness and senescence induction in this population and embryo receptivity. Patients were classified as receptive or non-receptive based on their pregnancy diagnosis after embryo transfer. Biomarkers of cellular senescence and somatic stem cells were compared between each sample. hESCs from non-receptive patients exhibited significantly higher (P < 0.01) proportions of senescent cells, mRNA expressions of CDKN2A and CDKN1A transcripts (P < 0.01), and expressions of genes encoding the senescence-associated secretory phenotype (P < 0.05). hESCs from receptive patients had significantly higher (P < 0.01) mRNA expressions of ABCG2 and ALDH1A1 transcripts. Our findings suggest that stemness is inversely associated with senescence induction in hESCs and, by extension, that implantation failure in infertility treatment may be attributable to a combination of senescence promotion and disruption of this maintenance function in this population during the proliferative phase of the menstrual cycle. This is a promising step towards potentially improving the embryo receptivity of endometrium. The specific mechanism by which implantation failure is prefigured by a loss of stemness among endometrial stem cells, and cellular senescence induction among hESCs, should be elucidated in detail in the future.

Activatable Formation of Emissive Excimers for Highly Selective Detection of ß-Galactosidase.

Small-molecular fluorescence sensors have become promising detection tools in many fields attributing to their high sensitivity, excellent temporal and spatial resolution, and low cytotoxicity. However, high concentration or aggregation-induced fluorescence quenching effect has usually hindered the development of traditional fluorescence dyes. Herein, a new fluorophore cyanovinylene dye BMZ with excimer emission property has been constructed. It shows an obvious enhanced and red-shift emission upon aggregation in aqueous solution, which overmatches the conventional pyrene with longer absorption and emission wavelengths. Using this unique optical property, a new fluorescence probe BMZ-Gal has been developed for trapping of ß-galactosidase (ß-Gal) activity with high selectivity, low limit of detection of 0.17 U, and rapid recognition, which is based on the ß-Gal-induced formation of red-shift emitting excimer. ß-Gal has a strong affinity for BMZ-Gal, which is verified through the Michaelis-Menten constants (Km, 1.87 µM) and the hydrolysis efficiencies (Kcat/Km, 1.78 × 103 M-1 s-1). Furthermore, the assay system has been successfully used for detecting endogenous ß-Gal in living ovarian cancer cells and can passively targeted to identify ß-Gal in organelle level and determine its subcellular location with satisfactory accuracy. We anticipate that the new fluorophore cyanovinylene dye herein may inaugurate new opportunities for the development of excimer emission sensors.

Specific Near-Infrared Probe for Ultrafast Imaging of Lysosomal ß-Galactosidase in Ovarian Cancer Cells.

Reactivity based fluorescent probes have been widely investigated as a powerful and noninvasive tool for disease diagnosis in recent years. ß-Galactosidase (ß-gal), one of the typical lysosomal glycosidases, is reported to be a vital biomarker overexpressed in primary ovarian cancer cells. Fluorescent probes with excellent performance for endogenous ß-gal detection offer a unique option for visualization and diagnosis of primary ovarian cancer cells. Herein, a near-infrared fluorescent probe Lyso-Gal with lysosome-targeting ability was developed for lysosomal ß-gal detection and imaging in ovarian cancer cells (SKOV-3 cells). Lyso-Gal exhibits weak fluorescence in aqueous solution but emits bright NIR fluorescence at 725 nm after incubation with ß-gal. Highly selective imaging of ovarian cancer cells has been achieved upon incubation with Lyso-Gal for only 1 min. The detection time is extremely short. In comparison with a similar hemicyanine probe, Hx-Gal, without lysosome-targeting ability, Lyso-Gal realizes endogenous ß-gal visualization in lysosomes and shows brighter fluorescence than Hx-Gal in SKOV-3 cells. This work demonstrates the potential of Lyso-Gal for detection of primary ovarian cancer cells by using ß-gal as the biomarker.

In vivo ratiometric tracking of endogenous ß-galactosidase activity using an activatable near-infrared fluorescent probe.

Herein, we developed a dual-channel and light-up near-infrared fluorescent probe for ratiometric sensing of ß-galactosidase (ß-gal) activity. The well-designed probe, which shows ratiometric optical response with a significant red-shift (from 575 nm to 730 nm), was successfully applied to detect endogenous ß-gal activity in SKOV-3 cells and tumor-bearing mice.

Age-associated liver alterations in wild populations of Austrolebias minuano, a short-lived Neotropical annual killifish.

Aging processes have become an attractive field for researchers and annual fish have been used as biological models. However, the study on the changes in age-associated markers during the normal aging in wild populations of annual fish remains open. Austrolebias is a genus of Neotropical annual killifishes, distributed mainly in ephemeral pools across grassland floodplains of temperate South America and represent an emerging biological model for aging research, but studies investigating rapid aging and senescence in this genus of annual fish are almost non-existent. This study was undertaken to examine the changes in age-associated liver markers at the different developmental stages in wild populations of Austrolebias minuano. We demonstrate that A. minuano has a number of liver alterations of different severities throughout the life cycle, suggesting that these changes tend to increase with age. Our results revealed that > 70% of the analyzed livers presented alterations. Thus, our study should instigate new approaches on aging using Neotropical annual fish, and could be useful to improve the knowledge already provided by consecrated biological aging models as e.g. Nothobranchius killifishes.

Recent Advances of Molecular Optical Probes in Imaging of ß-Galactosidase.

ß-Galactosidase (ß-Gal), as a lysosomal hydrolytic enzyme, plays an important physiological role in catalyzing the hydrolysis of glycosidic bonds which convert lactose into galactose. Moreover, upregulation of ß-Gal is often correlated with the occurrence of primary ovarian cancers and cell senescence. Thereby, detection of ß-Gal activity is relevant to cancer diagnosis. Optical imaging possesses high spatial and temporal resolution, high sensitivity, and real-time imaging capability. These properties are beneficial for the detection of ß-Gal in living systems. This Review summarizes the recent progress in development of molecular optical probes for near-infrared fluorescence (NIRF), bioluminescence (BL), chemiluminescence (CL), or photoacoustic (PA) imaging of ß-Gal in biological systems. The challenges and opportunities in the probe design for detection of ß-Gal are also discussed.

Ratiometric fluorescent probes with a self-immolative spacer for real-time detection of ß-galactosidase and imaging in living cells.

Ratiometric fluorescent probes with a self-immolative spacer for ß-galactosidase (ß-gal) were developed. They function by ß-gal-cleaving the ß-galactoside bond of fluorescent substrates, followed by self-immolation to liberate the amino group of fluorophore. Thus, a remarkable variation in the photophysical properties was observed and the corresponding ratiometric detection of ß-gal was realized. Our studies demonstrated that the GNPN exhibited high sensitivity for recognition of ß-gal, with a detection limit as low as 0.17 U L-1. GNPN can rapidly quantify ß-gal enzyme activity; the emission ratio F545/F475 for the GNPN reached maxima after approximately 4 min, which was one of the shortest response time ever reported. Furthermore, we demonstrated that these probes possess excellent biocompatibility and can be used to visualize the endogenous ß-gal in ovarian cancer cells.

Screening Method for Identifying Toxicants Capable of Inducing Astrocyte Senescence.

Cellular senescence is a tumor-suppressive mechanism which leads to near irreversible proliferative arrest. However, senescent cells can cause tissue dysfunction, in large part because they express a senescence-associated secretory phenotype (SASP) involving secretion of, amongst other factors, proinflammatory cytokines known to compromise neuronal health. Therefore, established neurotoxicants may cause neurotoxicity in vivo, in part by triggering mitotic cells in the brain to undergo senescence and adopt an inflammatory SASP which in turn could cause deleterious effects to surrounding neurons. To begin to address this hypothesis, we examined whether we could screen known neurotoxicants for their ability to cause astrocytes (a mitotic cell type especially important for maintaining neuronal health) to undergo senescence. For this purpose, we utilized inducible pluripotent stem cell-derived human astrocytes and screened an 80 compound neurotoxicant library provided by the Biomolecular Screening Branch of the NIEHS National Toxicology Program. Here we present a screening method based on induction of the senescent marker, senescent-associated beta-galactosidase (SA-ß-gal). We describe in detail an automated method for the unbiased quantitation of percentage of SA-ß-gal + astrocytes. Although our results suggest that conducting an SA-ß-gal senescence screen using human inducible pluripotent stem cell-derived astrocytes may be feasible, they also highlight challenges that likely preclude its adaptation to high-throughput. We also explore the possibility of using primary mouse astrocytes for this purpose and explain why this platform is problematic and very unlikely to yield meaningful results, even in small screens with compound replicates.

Annexin A1 peptide is able to induce an anti-parasitic effect in human placental explants infected by Toxoplasma gondii.

This study was conducted to investigate annexin A1 (ANXA1) functions in human placental explants infected with Toxoplasma gondii (T. gondii). We examined the first and third trimester placental explants infected with T. gondii (n = 7 placentas/group) to identify the number and location of parasites, ANXA1 protein, potential involvement of formyl peptide receptors (FPR1 and FPR2), and COX-2 expressions by immunohistochemistry. Treatments with Ac2-26 mimetic peptide of ANXA1 were performed to verify the parasitism rate (ß-galactosidase assay), prostaglandin E2 levels (ELISA assay), and ANXA1, FPR1 and COX-2 expression in third trimester placentas. Placental explants of third trimester expressed less ANXA1 and were more permissive to T. gondii infection than first trimester placentas that expressed more ANXA1. Ac2-26 treatment increases endogenous ANXA1 and decreases parasitism rate, COX-2, and prostaglandin E2 levels. Altogether, these data provide further insight into the anti-parasitic and anti-inflammatory effects of ANXA1 in placentas infected with T. gondii.

Manipulating Femtoliter to Picoliter Droplets by Pins for Single Cell Analysis and Quantitative Biological Assay.

Herein, we developed an automated and flexible system for performing miniaturized liquid-liquid reactions and assays in the femtoliter to picoliter range, by combining the contact printing and the droplet-based microfluidics techniques. The system mainly consisted of solid pins and an oil-covered hydrophilic micropillar array chip fixed on an automated x- y- z translation stage. A novel droplet manipulation mode called "dipping-depositing-moving" (DDM) was proposed, which was based on the programmable combination of three basic operations, dipping liquids and depositing liquids with the solid pins and moving the two-dimensional oil-covered hydrophilic pillar microchip. With the DDM mode, flexible generation and manipulation of small droplets with volumes down to 179 fL could be achieved. For overcoming the scale phenomenon specially appeared in picoliter-scale droplets, we used a design of water moat to protect the femtoliter to picoliter droplets from volume loss through the cover oil during the droplet generation, manipulation, reaction and assay processes. Moreover, we also developed a precise quantitative method, quantitative droplet dilution method, to accurately measure the volumes of femtoliter to picoliter droplets. To demonstrate its feasibility and adaptability, we applied the present system in the determination of kinetics parameter for matrix metalloproteinases (MMP-9) in 1.81 pL reactors and the measurement the activity of ß-galactosidase in single cells (HepG2 cells) in picoliter droplet array. The ultrasmall volumes of the droplet reactors avoided the excessive dilution to the reaction solutions and enabled the highly sensitive measurement of enzyme activity in the single cell level.

Antibacterial mechanism of artemisinin / beta-cyclodextrins against methicillin-resistant Staphylococcus aureus (MRSA).

A new inhibitor to overcome the multi-drug resistance of MRSA was developed in this study. Artemisinin (ART) was encapsulated in beta-cyclodextrin (ß-CD) in order to enhance the solubility of ART. The molecular encapsulation of ART was confirmed by using Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC), X-Ray Diffraction (XRD), and nuclear magnetic resonance (1H NMR) methods. The phase solubility study showed the relationship between ART solubility and ß-CD concentration. The antibacterial activity of ART/ß-CDs inclusion complexes (ART/ß-CDs-IC) (20 mg/mL) against MRSA was distinguished, with the inhibition rate of 99.94% after 4 days. The antibacterial mechanism research indicated the membrane permeability of MRSA can be increased by the complexes. Besides, the respiratory metabolism of MRSA was inhibited via Embden-Meyerhof-Parnas (EMP) pathway. The conclusion was further confirmed by measuring the contents of three enzymes in the irreversible reaction in EMP pathway. The obtained results enable the potential use of ART/ß-CDs-IC in the field of antimicrobial.

ortho-Chlorination of phenoxy 1,2-dioxetane yields superior chemiluminescent probes for in vitro and in vivo imaging.

A recent methodology, developed by our group, has enabled a dramatic improvement in the emissive nature of the excited species, formed during the chemiexcitation of dioxetanes under physiological conditions. This approach has resulted in the discovery of distinct phenoxy-dioxetane luminophores that produce a chemiluminescence signal via a direct-mode of emission. Here, we show a significant pKa effect of our new phenoxy-dioxetanes on their chemiexcitation and on their ability to serve as chemiluminescent turn-ON probes for biological applications. Using an appropriate phenoxy-dioxetane probe with a direct-mode of emission, we were able to image ß-galactosidase activity, in cancer cells and in tumor-bearing mice. To the best of our knowledge, this is the first example to demonstrate in vitro and in vivo endogenous enzymatic chemiluminescence images obtained by a single-component phenoxy-dioxetane probe. We anticipate that our strategy, for the design and synthesis of such distinct luminophores, will assist in providing new effective turn-ON probes for non-invasive intravital chemiluminescence imaging techniques.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated ß-galactosidase (SA-ß-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal ß-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry (IHC). The major limitation of the SA-ß-Gal assay is the requirement of fresh or frozen samples. Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-ß-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.