Colorimetric sensing assay based on aptamer-gold nanoparticles for rapid detection of salivary melatonin to monitor circadian rhythm sleep disorders.

Salivary melatonin is a clinically used biomarker for diagnosing circadian rhythm sleep disorders. Current melatonin detection assays are complex, expensive, and in many cases do not adequately measure low levels of salivary melatonin. Precisely measuring melatonin levels at multiple time points is crucial for determining dim light melatonin onset to evaluate its circadian fluctuation as well as the extent of circadian disruption and consequently adapt treatment regimens. Moreover, melatonin low levels in saliva challenges the reliability of routine clinical testing. This paper presents the development of a novel, highly sensitive, yet cost-effective, colorimetric assay for the rapid detection of salivary melatonin utilizing aptamer-AuNPs. Among several types of the aptamer tested, the 36-mer MLT-A-2 aptamer-AuNP probe showed the highest sensitivity with a melatonin limit of detection of 0.0011 nM along with a limit of quantification of 0.0021 nM in saliva. Moreover, our assay showed preferential interaction with melatonin when tested in presence of other structurally similar counter-targets. Taken together, this study provides new parameters for a melatonin assay that meets adequate levels of sensitivity and selectivity. The developed colorimetric assay could be adapted in a point-of-care system for profiling salivary melatonin levels at multiple time points during 24 h, crucial for accurately diagnosing and monitoring circadian rhythm sleep disorders and beyond.

Structural properties and binding mechanism of DNA aptamers sensing saliva melatonin for diagnosis and monitoring of circadian clock and sleep disorders.

Circadian desynchrony with the external light-dark cycle influences the rhythmic secretion of melatonin which is among the first signs of circadian rhythm sleep disorders. An accurate dim light melatonin onset (established indicator of circadian rhythm sleep disorders) measurement requires lengthy assays, and antibody affinities alterations, especially in patients with circadian rhythm disorders whose melatonin salivary levels vary significantly, making antibodies detection mostly inadequate. In contrast, aptamers with their numerous advantages (e.g., target selectivity, structural flexibility in tuning binding affinities, small size, etc.) can become preferable biorecognition molecules for salivary melatonin detection with high sensitivity and specificity. This study thoroughly characterizes the structural property and binding mechanism of a single-stranded DNA aptamer full sequence (MLT-C-1) and its truncated versions (MLT-A-2, MLT-A-4) to decipher its optimal characteristics for saliva melatonin detection. We use circular dichroism spectroscopy to determine aptamers' conformational changes under different ionic strengths and showed that aptamers display a hairpin loop structure where few base pairs in the stem play a significant role in melatonin binding and formation of aptamer stabilized structure. Through microscale thermophoresis, aptamers demonstrated a high binding affinity in saliva samples (MLT-C-1F Kd = 12.5 ± 1.7 nM; MLT-A-4F Kd = 11.2 ± 1.6 nM; MLT-A-2F Kd = 2.4 ± 2.8 nM; limit-of-detection achieved in pM, highest sensitivity attained for MLT-A-2F aptamer with the lowest detection limit of 1.35 pM). Our data suggest that aptamers are promising as biorecognition molecules and provide the baseline parameters for the development of an aptamer-based point-of-care diagnostic system for melatonin detection and accurate profiling of its fluctuations in saliva.

Bioprinting of alginate-carboxymethyl chitosan scaffolds for enamel tissue engineeringin vitro.

Tissue engineering offers a great potential in regenerative dentistry and to this end, three dimensional (3D) bioprinting has been emerging nowadays to enable the incorporation of living cells into the biomaterials (such a mixture is referred as a bioink in the literature) to create scaffolds. However, the bioinks available for scaffold bioprinting are limited, particularly for dental tissue engineering, due to the complicated, yet compromised, printability, mechanical and biological properties simultaneously imposed on the bioinks. This paper presents our study on the development of a novel bioink from carboxymethyl chitosan (CMC) and alginate (Alg) for bioprinting scaffolds for enamel tissue regeneration. CMC was used due to its antibacterial ability and superior cell interaction properties, while Alg was added to enhance the printability and mechanical properties as well as to regulate the degradation rate. The bioinks with three mixture ratios of Alg and CMC (2-4, 3-3 and 4-2) were prepared, and then printed into the calcium chloride crosslinker solution (100 mM) to form a 3D structure of scaffolds. The printed scaffolds were characterized in terms of structural, swelling, degradation, and mechanical properties, followed by theirin vitrocharacterization for enamel tissue regeneration. The results showed that the bioinks with higher concentrations of Alg were more viscous and needed higher pressure for printing; while the printed scaffolds were highly porous and showed a high degree of printability and structural integrity. The hydrogels with higher CMC ratios had higher swelling ratios, faster degradation rates, and lower compressive modulus. Dental epithelial cell line, HAT-7, could maintain high viability in the printed constructs after 1, 7 and 14 d of culture. HAT-7 cells were also able to maintain their morphology and secrete alkaline phosphatase after 14 d of culture in the 3D printed scaffolds, suggesting the capacity of these cells for mineral deposition and enamel-like tissue formation. Among all combinations Alg4%-CMC2% and in a less degree 2%Alg-4%CMC showed the higher potential to promote ameloblast differentiation, Ca and P deposition and matrix mineralizationin vitro. Taken together, Alg-CMC has been illustrated to be suitable to print scaffolds with dental epithelial cells for enamel tissue regeneration.

When the clock ticks wrong with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the coronavirus family that causes the novel coronavirus disease first diagnosed in 2019 (COVID-19). Although many studies have been carried out in recent months to determine why the disease clinical presentations and outcomes can vary significantly from asymptomatic to severe or lethal, the underlying mechanisms are not fully understood. It is likely that unique individual characteristics can strongly influence the broad disease variability; thus, tailored diagnostic and therapeutic approaches are needed to improve clinical outcomes. The circadian clock is a critical regulatory mechanism orchestrating major physiological and pathological processes. It is generally accepted that more than half of the cell-specific genes in any given organ are under circadian control. Although it is known that a specific role of the circadian clock is to coordinate the immune system's steady-state function and response to infectious threats, the links between the circadian clock and SARS-CoV-2 infection are only now emerging. How inter-individual variability of the circadian profile and its dysregulation may play a role in the differences noted in the COVID-19-related disease presentations, and outcome remains largely underinvestigated. This review summarizes the current evidence on the potential links between circadian clock dysregulation and SARS-CoV-2 infection susceptibility, disease presentation and progression, and clinical outcomes. Further research in this area may contribute towards novel circadian-centred prognostic, diagnostic and therapeutic approaches for COVID-19 in the era of precision health.

Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma.

Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

Gemini surfactant-based nanoparticles T-box1 gene delivery as a novel approach to promote epithelial stem cells differentiation and dental enamel formation.

Enamel is the highest mineralized tissue in the body protecting teeth from external stimuli, infections, and injuries. Enamel lacks the ability to self-repair due to the absence of enamel-producing cells in the erupted teeth. Here, we reported a novel approach to promote enamel-like tissue formation via the delivery of a key ameloblast inducer, T-box1 gene, into a rat dental epithelial stem cell line, HAT-7, using non-viral gene delivery systems based on cationic lipids. We comparatively assessed the lipoplexes prepared from glycyl-lysine-modified gemini surfactants and commercially available 1,2-dioleoyl-3-trimethylammonium-propane lipids at three nitrogen-to phosphate (N/P) ratios of 2.5, 5 and 10. Our findings revealed that physico-chemical characteristics and biological activities of the gemini surfactant-based lipoplexes with a N/P ratio of 5 provide the most optimal outcomes among those examined. HAT-7 cells were transfected with T-box1 gene using the optimal formulation then cultured in conventional 2D cell culture systems. Ameloblast differentiation, mineralization, bio-enamel interface and structure were assessed at different time points over 28 days. Our results showed that our gemini transfection system provides superior gene expression compared to the benchmark agent, while keeping low cytotoxicity levels. T-box1-transfected HAT-7 cells strongly expressed markers of secretory and maturation stages of the ameloblasts, deposited minerals, and produced enamel-like crystals when compared to control cells. Taken together, our gemini surfactant-based T-box1 gene delivery system is effective to accelerate and guide ameloblastic differentiation of dental epithelial stem cells and promote enamel-like tissue formation. This study would represent a significant advance towards the tissue engineering and regeneration of dental enamel.

Emerging biotechnologies for evaluating disruption of stress, sleep, and circadian rhythm mechanism using aptamer-based detection of salivary biomarkers.

The internally driven 24-h cycle in humans, called circadian rhythm, controls physiological, metabolic, and hormonal processes, and is tied to the circadian clocks ticking in most of the cells and tissues. The central clock, located in suprachiasmatic nuclei of the hypothalamus, is directly influenced by external cues, particularly light, and entrains the peripheral clocks through neural and hormonal pathways to the external light-dark cycle. However, peripheral clocks also have self-sustained circadian rhythmicity and feeding is the potent synchronizer. The internal clock system regulates the sleep-wake cycle and maintains stress responses through the hypothalamus-pituitary-adrenal axis and autonomic pathways. Any misalignment in this complex network could lead to circadian clock disruption and endocrine and metabolic dysfunction that may induce inflammatory responses. The detrimental consequences of such dysfunction are broad and can lead to serious health problems; however, the extent of the circadian disruption is difficult to assess. New promising techniques based on biosensors and point-of-care devices using aptamers - single-stranded DNA or RNA biorecognition molecules that can measure biomarkers of stress, sleep, and circadian rhythms in bodily fluids such as saliva with high sensitivity and specificity - can provide timely and accurate diagnosis and allow for effective implementation of behavioral and therapeutic interventions. This review provides detailed insight into the complex crosstalk between stress, sleep, and circadian rhythm, their relationship with the body's homeostasis, and the consequences of circadian dysregulation. The review also summarizes the mechanisms of aptamer-based biosensors and/or point-of-care devices developed to date for the detection of salivary biomarkers linked to stress, sleep, and circadian rhythm. Lastly, the review outlines the knowledge gaps in the literature related to the detection of lower concentrations of biomarkers in saliva and discusses the prospects of aptamer-based detection of salivary biomarkers from a high-precision perspective that is crucial for clinical diagnosis, at a time when circadian disruption is evident in unprecedented proportions across the globe.

Calcium Sets the Clock in Ameloblasts.

BACKGROUND: Stromal interaction molecule 1 (STIM1) is one of the main components of the store operated Ca2+ entry (SOCE) signaling pathway. Individuals with mutated STIM1 present severely hypomineralized enamel characterized as amelogenesis imperfecta (AI) but the downstream molecular mechanisms involved remain unclear. Circadian clock signaling plays a key role in regulating the enamel thickness and mineralization, but the effects of STIM1-mediated AI on circadian clock are unknown. OBJECTIVES: The aim of this study is to examine the potential links between SOCE and the circadian clock during amelogenesis. METHODS: We have generated mice with ameloblast-specific deletion of Stim1 (Stim1 fl/fl/Amelx-iCre+/+, Stim1 cKO) and analyzed circadian gene expression profile in Stim1 cKO compared to control (Stim1 fl/fl/Amelx-iCre-/-) using ameloblast micro-dissection and RNA micro-array of 84 circadian genes. Expression level changes were validated by qRT-PCR and immunohistochemistry. RESULTS: Stim1 deletion has resulted in significant upregulation of the core circadian activator gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocation 1 (Bmal1) and downregulation of the circadian inhibitor Period 2 (Per2). Our analyses also revealed that SOCE disruption results in dysregulation of two additional circadian regulators; p38α mitogen-activated protein kinase (MAPK14) and transforming growth factor-beta1 (TGF-ß1). Both MAPK14 and TGF-ß1 pathways are known to play major roles in enamel secretion and their dysregulation has been previously implicated in the development of AI phenotype. CONCLUSION: These data indicate that disruption of SOCE significantly affects the ameloblasts molecular circadian clock, suggesting that alteration of the circadian clock may be partly involved in the development of STIM1-mediated AI.

Controlled Drug Delivery Systems for Oral Cancer Treatment-Current Status and Future Perspectives.

Oral squamous cell carcinoma (OSCC), which encompasses the oral cavity-derived malignancies, is a devastating disease causing substantial morbidity and mortality in both men and women. It is the most common subtype of the head and neck squamous cell carcinoma (HNSCC), which is ranked the sixth most common malignancy worldwide. Despite promising advancements in the conventional therapeutic approaches currently available for patients with oral cancer, many drawbacks are still to be addressed; surgical resection leads to permanent disfigurement, altered sense of self and debilitating physiological consequences, while chemo- and radio-therapies result in significant toxicities, all affecting patient wellbeing and quality of life. Thus, the development of novel therapeutic approaches or modifications of current strategies is paramount to improve individual health outcomes and survival, while early tumour detection remains a priority and significant challenge. In recent years, drug delivery systems and chronotherapy have been developed as alternative methods aiming to enhance the benefits of the current anticancer therapies, while minimizing their undesirable toxic effects on the healthy non-cancerous cells. Targeted drug delivery systems have the potential to increase drug bioavailability and bio-distribution at the site of the primary tumour. This review confers current knowledge on the diverse drug delivery methods, potential carriers (e.g., polymeric, inorganic, and combinational nanoparticles; nanolipids; hydrogels; exosomes) and anticancer targeted approaches for oral squamous cell carcinoma treatment, with an emphasis on their clinical relevance in the era of precision medicine, circadian chronobiology and patient-centred health care.

Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer.

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin's potential clinical use against MDR cancers.

The ubiquitin-conjugating enzyme, Ubc1, indirectly regulates SNF1 kinase activity via Forkhead-dependent transcription.

The SNF1 kinase in Saccharomyces cerevisiae is an excellent model to study the regulation and function of the AMP-dependent protein kinase (AMPK) family of serine-threonine protein kinases. Yeast discoveries regarding the regulation of this non-hormonal sensor of metabolic/environmental stress are conserved in higher eukaryotes, including poly-ubiquitination of the α-subunit of yeast (Snf1) and human (AMPKα) that ultimately effects subunit stability and enzyme activity. The ubiquitin-cascade enzymes responsible for targeting Snf1 remain unknown, leading us to screen for those that impact SNF1 kinase function. We identified the E2, Ubc1, as a regulator of SNF1 kinase function. The decreased Snf1 abundance found upon deletion of Ubc1 is not due to increased degradation, but instead is partly due to impaired SNF1 gene expression, arising from diminished abundance of the Forkhead 1/2 proteins, previously shown to contribute to SNF1 transcription. Ultimately, we report that the Fkh1/2 cognate transcription factor, Hcm1, fails to enter the nucleus in the absence of Ubc1. This implies that Ubc1 acts indirectly through transcriptional effects to modulate SNF1 kinase activity.

Secreted hCLCA1 is a signaling molecule that activates airway macrophages.

The CLCA gene family produces both secreted and membrane-associated proteins that modulate ion-channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The primary up-regulated human CLCA ortholog in airway inflammation is hCLCA1. Here we show that this protein can activate airway macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In a U-937 airway macrophage-monocyte cell line, conditioned media from HEK 293 cells heterologously expressing hCLCA1 (with or without fetal bovine serum) increased the levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-8). This effect was independent of the metalloprotease domain of hCLCA1. Primary porcine alveolar macrophages were similarly activated, demonstrating the effect was not cell line dependent. Similarly, immuno-purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was able to activate macrophages and induce pro-inflammatory response. This cytokine response increased with higher concentration of immuno-purified hCLCA1. These findings demonstrate the ability of hCLCA1 to function as a signaling molecule and activate macrophages, central regulators of airway inflammation.

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.

Mucosal adenovirus-vectored vaccine for measles.

Several problems associated with the available anti-measles vaccine emphasize the need for a single shot anti-measles vaccine which is efficacious by mucosal route of administration and functional in the presence of anti-measles neutralizing antibodies. To achieve these goals, we constructed two recombinant human adenoviruses (collectively designated Ad-F/H) carrying genes for measles virus (MV) fusion (F) and haemagglutinin (H) proteins. Single intranasal or intramuscular vaccination of mice and cotton rats with Ad-F/H elicited high MV-specific serum neutralizing-antibody titers. Furthermore, bronchoalveolar lavage samples from mice vaccinated intranasally with Ad-F/H showed a 100-fold increase in MV-specific IgA titers compared with intramuscularly vaccinated mice. Moreover, Ad-F/H vaccine administered intranasally, but not intramuscularly, completely protected challenged cotton rats from MV replication in the lungs.