Non-nucleated Cells Research Articles

Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury.

Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies in vivo. Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate damaged volumes in vivo nor image non-nucleated cell types or compartments). Here, we develop modular fluorogenic probes that reveal the whole cytosolic volume of damaged cells, with near-zero background fluorescence so that no washing is needed. We identify a specific disulfonated fluorogenic probe type that only enters cells with damaged membranes, then is enzymatically activated and marks them. The esterase probe MDG1 is a reliable tool to reveal live cells that have been permeabilized by biological, biochemical, or physical membrane damage, and it can be used in multicolor microscopy. We confirm the modularity of this approach by also adapting it for improved hydrolytic stability, as the redox probe MDG2. We conclude by showing the unique performance of MDG probes in revealing axonal membrane damage (which DNA fluorogens cannot achieve) and in discriminating damage on a cell-by-cell basis in embryos in vivo. The MDG design thus provides powerful modular tools for wash-free in vivo imaging of membrane damage, and indicates how designs may be adapted for selective delivery of drug cargoes to these damaged cells: offering an outlook from selective diagnosis toward therapy of membrane-compromised cells in disease.

Platelet Indices: Is it a Reliable Biomarker in Viral Infections?

Platelets are non-nucleated blood cells that are essential for maintaining hemostasis. While platelet activation and increased platelet numbers (thrombocytosis) are associated with a higher risk of thrombotic events, low platelet counts (thrombocytopenia) and a variety of platelet function abnormalities are associated with a higher risk of bleeding. Platelets and their activation state have been proven in recent years to influence innate and adaptive immune responses, and low platelet counts have been identified as a surrogate indicator for poor prognosis in septic patients. Platelet activation frequently occurs in conjunction with viral infections. Viruses, on the other hand, can interact directly with platelets and megakaryocytes, modulating their function. All these factors contribute to platelet activation, which leads to increased platelet consumption and elimination, as well as thrombocytopenia, which is common during viral infection Platelets and their secreted products have been demonstrated to suppress infection and aid virus persistence in the presence of certain viruses, making platelets a double-edged sword during viral infections. In this review we have highlighted about what is currently known about platelet interactions with various types of viruses, virus effects on platelet activation, as well as platelet-mediated regulation of innate and adaptive immune responses.

Dysregulated platelet function in COVID-19 patients

More than two year-experience in monitoring patients with severe novel coronavirus disease revealed an increased risk of macroand microcirculatory thrombosis, clinically manifested by heart attack, stroke, thromboses of rare localizations (thrombosis of the hepatic veins, mesenteric veins, cerebral sinuses, portal vein), being the main cause of adverse outcomes. Thromboembolic complications, expressed as sepsis-induced coagulopathy, disseminated intravascular coagulation, venous and arterial thromboembolism, pulmonary embolism, microthrombosis, and thrombotic microangiopathy were noted to affect various organs such as the lungs, heart, kidneys, and brain. Earlier, we showed that hypercoagulability is closely related to inflammatory diseases and hemostasis dysregulation. Endothelial injury and dysfunction have been identified as critical pathways to thrombosis, and other mechanisms occurring in the microvasculature were described such as endothelial activation, cytokine storm, and formation of neutrophil extracellular traps (NETs). However, a role of platelets in severe COVID-19 has not been examined. Platelets are small non-nucleated cells most known as the central mediators of hemostasis. However, upon activation, platelets release a variety of immunomodulatory cytokines and chemokines involved in regulating immune response. Because platelets are involved in both immunity and coagulation, they play a central role in immunothrombosis, a physiological process wherein immune cells induce microthrombogenesis both to prevent spread of pathogens and facilitate their clearance. Moreover, platelets can directly interact with viral receptors including those related to SARS-CoV-2. Platelet dysfunction includes both thrombocytopenia and platelet hyperactivation. It should be noted that persistent thrombocytopenia is usually correlated with mortality being associated with thrombosis of the cerebral sinuses, splanchnic system, autoimmune reactions, as well as with administered heparin and vaccines. Therefore, here we review a role of platelets in the pathogenesis of COVID-19.

Overview of the Mechanisms of Oxidative Stress: Impact in Inflammation of the Airway Diseases.

Inflammation of the human lung is mediated in response to different stimuli (e.g., physical, radioactive, infective, pro-allergenic or toxic) such as cigarette smoke and environmental pollutants. They often promote an increase in inflammatory activities in the airways that manifest themselves as chronic diseases (e.g., allergic airway diseases, asthma, chronic bronchitis/chronic obstructive pulmonary disease (COPD) or even lung cancer). Increased levels of oxidative stress (OS) reduce the antioxidant defenses, affect the autophagy/mitophagy processes, and the regulatory mechanisms of cell survival, promoting inflammation in the lung. In fact, OS potentiate the inflammatory activities in the lung, favoring the progression of chronic airway diseases. OS increases the production of reactive oxygen species (ROS), including superoxide anions (O2-), hydroxyl radicals (OH) and hydrogen peroxide (H2O2), by the transformation of oxygen through enzymatic and non-enzymatic reactions. In this manner, OS reduces endogenous antioxidant defenses in both nucleated and non-nucleated cells. The production of ROS in the lung can derive from both exogenous insults (cigarette smoke or environmental pollution) and endogenous sources such as cell injury and/or activated inflammatory and structural cells. In this review, we describe the most relevant knowledge concerning the functional interrelation between the mechanisms of OS and inflammation in airway diseases.

Role of platelet in Parkinson's disease: Insights into pathophysiology & theranostic solutions.

Parkinson's disease (PD) is the second-most-common neurodegenerative disease characterized by motor and non-motor dysfunctions, which currently affects about 10 million people worldwide. Gradual death and progressive loss of dopaminergic neurons in the pars compacta region of substantia nigra result in striatal dopamine deficiency in PD. Specific mutation with further aggregation of α-synuclein in the intraneuronal inclusion bodies is considered the neuropathological hallmark of this disease. PD is often associated with various organelle dysfunctions inside a dopaminergic neuron, including mitochondrial damage, proteasomal impairment, and production of reactive oxygen species, thus causing subsequent neuronal death. Apart from several genetic and non-genetic risk factors, emerging research establishes an association between cardiovascular diseases, including coronary heart disease, myocardial infarction, congestive heart failure, and ischemic stroke with PD. The majority of these cardiovascular diseases have an origin from atherosclerosis, where endothelial dysfunction following thrombus formation is significantly regulated by blood platelet. This non-nucleated cell fragment expresses not only neuron-specific molecules and receptors but also several PD-specific biomarkers such as α-synuclein, parkin, PTEN-induced kinase-1, tyrosine hydroxylase, dopamine transporter, thus making platelet a suitable peripheral model for PD. Besides its similarity with a dopaminergic neuron, platelet structural alterations, as well as functional abnormalities, are also evident in PD. However, the molecular mechanism behind platelet dysfunction is still elusive and quite controversial. This state-of-the-art review describes the detailed mechanism of platelet impairment in PD, addressing the novel platelet-associated therapeutic drug candidates for plausible PD management.

Methodological considerations for circulating long noncoding RNA quantification.

In the past decade, significant resources have been invested in long noncoding RNA (lncRNA) research. Despite the knowledge available, we are far from incorporation of lncRNA into clinical practice. Here, we emphasize the technical challenges in the field, hoping to provoke a response leading to new consensus and guidelines.

Large-Scale Profiling on lncRNAs in Human Platelets: Correlation with Platelet Reactivity.

Recently, long noncoding RNAs (lncRNAs) have been key regulators for both mRNAs and proteins in nucleated cells. However, the expression profiles of lncRNAs in non-nucleated cells such as platelets are currently unclear. In this study, we determined the expression profiles of lncRNAs in human platelets. We found that 6109 lncRNAs were expressed in human platelets. Interestingly, 338 lncRNAs were differentially expressed in hyperreactive and hyporeactive platelets. Bioinformatics’ analysis revealed that these aberrantly expressed lncRNAs might be related to platelet activity and other platelet functions. To provide a proof of concept, we measured the expression levels of PARLncRNA-1, a down-regulated lncRNA of hyperreactive platelets, in platelets from 12 patients with acute myocardial infarction and their controls. We found that the lncRNA was also significantly down-regulated in platelets from patients, which was partially reversed by treatment with aspirin a known antiplatelet drug. LncRNAs may represent a novel class of modulators for platelet functions.

Detecting Circadian Rhythms in Human Red Blood Cells by Dielectrophoresis.

Dielectrophoresis (DEP) enables the measurement of population-level electrophysiology in many cell types by examining their interaction with an externally applied electric field. Here we describe the application of DEP to the measurement of circadian rhythms in a non-nucleated cell type, the human red blood cell. Using DEP, population-level electrophysiology of ~20,000 red blood cells can be measured from start to finish in less than 3min, and can berepeated over several days to reveal cell-autonomous daily regulation of membrane electrophysiology. This method is amenable to the characterization of circadian rhythms by altering entrainment and free-run conditions or through pharmacological perturbation.

A rapid spectrophotometric method to identify inhibitors of human erythropoiesis

Erythropoiesis is a complex physiological process by which erythroid progenitors proliferate and differentiate into nonnucleated red blood cells. Several methods can be used to monitor in vitro the differentiation of erythroid precursors, and hence the toxic effects of drugs, chemicals, or pollutants. One of the most commonly available assay of erythropoiesis is the microscopic observation of differentiated cells after benzidine staining, which forms a blue complex with hemoglobin. However, this method is laborious and does not provide accurate results since it heavily relies on the reader's interpretation. Moreover, benzidine is a carcinogen and a highly reactive molecule which forces the reader to microscopically count differentiated and non-differentiated cells within a short time frame (5 min).Here we have developed a simple, inexpensive, in-vitro spectrophotometric assay to measure erythroid differentiation using K562 cell line as a model. Materials needed included 96-well round-bottomed microplates and a microplate reader. Remarkably, carcinogenic benzidine was replaced by its isomeric tetramethyl derivative, the 3,3′, 5,5′- tetramethylbenzidine (TMB), which presents several advantages: it is cheap, not mutagenic and a ready-to-use chromogenic substrate. A small volume (50 μl) of TMB added to the samples forms a blue complex in 15 min, and the reaction can be easily stopped and stabilized by the addition of H2SO4. The yellow precipitate is then solubilized, and the absorbance is measured at 450 nm. In addition, the suitability of the assay to determine the effects of compounds on erythroid differentiation was further tested with known inhibitors (artemisinin derivatives) of K562 differentiation.Overall, the reported methodology permits to measure in an accurate and reproducible manner the K562 differentiation and can be used for medium throughput screenings (MTS) of compounds or environmental toxics with potential erythro-toxicity and ability to inhibit erythroid differentiation.

Characterizing Naked Nuclei Frequency and Movement in Primary AML Cell Culture Using an ECM-Based Model

INTRODUCTIONNaked nuclei (NN) are observed upon examining bone marrow aspirate slides from healthy individuals and from those with hematologic malignancies, but are not well understood. Without characteristic findings like cytoplasm or plasma membrane, NN are considered remnants of slide preparation or are discounted as cells of undetermined significance. NN have been associated with poor prognosis in several solid cancers. Understanding the significance of NN in hematologic malignancies such as acute myelogenous leukemia (AML) may elucidate valuable diagnostic and prognostic knowledge. Decellularized Wharton's jelly matrix (DWJM) is an extra cellular matrix (ECM)-based in vitro model that shares similar elements with the bone marrow ECM and can be used as scaffolding to culture leukemia cells. We hypothesize that NN exist in AML and interact with other cells, suggesting potential biological relevance within the bone marrow microenvironment.METHODSPrimary AML samples obtained by leukapheresis were cultured in suspension with growth media or in the presence of DWJM submerged in growth media. In samples grown with DWJM, cells that were non-adherent to the matrix were collected first and then adherent cells were isolated by treating DWJM with collagenase. Live cells were stained with CellVue Maroon (CVM) for membrane, CellTracker Green (CTG) for cytoplasm, and Hoechst 33342 for nucleus, followed by analysis with Amnis/Luminex ImageStream-X imaging flow cytometer (Figure 1A). NN were defined as events positive for nuclear stain and negative for cytoplasmic and membrane stains (Figure 1B). 3-D movement of adherent AML cells in DWJM was captured in real time using confocal microscopy. Fixed cells from leukemia cell line K562 served as a control for movement. NN (Hoechst positive only), non-nucleated (Hoechst negative/CTG positive), and nucleated cells (Hoechst and CTG positive) were identified by fluorescent labeling. NN were also observed after isolation by cell sorting. Cell speed, cell displacement from origin, and change in distance to closest neighboring cell over time were measured. Additionally, flow sorted NN were examined by immunohistochemistry (IHC).RESULTSAdherent populations contained significantly more NN than non-adherent and suspension populations. The frequency of NN in matrix adherent cells ranged from 0.4-2.4% at day 3 and 0.5-5.4% at day 7 of culture (Figure 1C). Through confocal microscopic analysis, we observed NN, nucleated, and non-nucleated cells moving at speeds ranging from 0.002-0.08 µm/sec. Fixed cells showed no discernible movement in DWJM. The average speed of NN [0.019 mm/s, SD 0.011] significantly differed from the average speed of nucleated and non-nucleated cells [0.027 mm/s, SD 0.016] (p=0.004) (Figure 1E). To demonstrate directional movement, we measured change in distance between NN and closest neighboring nucleated or non-nucleated cells over time. Cells (nucleated and non-nucleated) and NN moved closer to each other over time suggesting directional movement (p=0.001) (Figure 1D). NN also showed movement in DWJM after isolation by cell sorting. IHC analysis showed sorted NN stained positive for nuclear lamin A/C, which are considered markers of nuclear membrane (Figure 1F).CONCLUSIONSOur findings confirm that NN are present in primary AML cells cultured in vitro using ourECM-based model and that they can be isolated using flow cytometry. Additionally, NN display directional movement in DWJM suggesting that they interact with other cells and may be biologically relevant structures in the bone marrow microenvironment. [Display omitted] DisclosuresBaran: AstraZeneca/Acerta: Research Funding. Chu: Pfizer: Current equity holder in publicly-traded company; Acerta/AstraZeneca: Research Funding; TG Therapeutics: Research Funding.

Study on the intervention of platelet biological activity by ultrasound

Platelets are non-nucleated blood effector cells, which plays an important role in coagulation, hemostasis, and thrombosis. However, platelets are extremely susceptible to activation by external stimuli, which in turn damages the platelet's natural biological activity and affects its biological function. Platelet biological activity has become a hotspot in the field of vascular diseases. In this study, ultrasound parameters (ultrasound intensity and duration time) were used to intervene in the biological activity of platelets. The response of platelets to ultrasound energy was explored from the aspects of platelet morphology, aggregation ability and particle release (the expression of P-selectin and the number of particles). The results showed that the ultrasound intensity of 0.25 W/cm 2 (1 MHz, 60 s) had no effect on the morphology, aggregation ability and particle release of platelets. When the ultrasonic intensity was increased to greater than 0.25 W/cm 2, the generation of platelet pseudopods, morphological changes, increase of particle release, as well as effect on aggregation were observed. When the ultrasound duration time was 60 s (1 MHz, 0.25 W/cm 2), it had no effect on the biological activity of platelets. However, when the ultrasound time was greater than 60 s, the morphology, aggregation ability and microparticles release would been induced with no effect on the secretion of CD62P and total protein components. Therefore, when the ultrasound parameters were 1 MHz and 0.25 W/cm 2 with 60 s duration time, the ultrasound energy had no effect on the biological activity of platelets. The results in this study are of great significant for ultrasound energy intervention for the treatment of platelet-related diseases.

A data-driven approach to modeling cancer cell mechanics during microcirculatory transport

In order to understand the effect of cellular level features on the transport of circulating cancer cells in the microcirculation, there has been an increasing reliance on high-resolution in silico models. Accurate simulation of cancer cells flowing with blood cells requires resolving cellular-scale interactions in 3D, which is a significant computational undertaking warranting a cancer cell model that is both computationally efficient yet sufficiently complex to capture relevant behavior. Given that the characteristics of metastatic spread are known to depend on cancer type, it is crucial to account for mechanistic behavior representative of a specific cancer’s cells. To address this gap, in the present work we develop and validate a means by which an efficient and popular membrane model-based approach can be used to simulate deformable cancer cells and reproduce experimental data from specific cell lines. Here, cells are modeled using the immersed boundary method (IBM) within a lattice Boltzmann method (LBM) fluid solver, and the finite element method (FEM) is used to model cell membrane resistance to deformation. Through detailed comparisons with experiments, we (i) validate this model to represent cancer cells undergoing large deformation, (ii) outline a systematic approach to parameterize different cell lines to optimally fit experimental data over a range of deformations, and (iii) provide new insight into nucleated vs. non-nucleated cell models and their ability to match experiments. While many works have used the membrane-model based method employed here to model generic cancer cells, no quantitative comparisons with experiments exist in the literature for specific cell lines undergoing large deformation. Here, we describe a phenomenological, data-driven approach that can not only yield good agreement for large deformations, but explicitly detail how it can be used to represent different cancer cell lines. This model is readily incorporated into cell-resolved hemodynamic transport simulations, and thus offers significant potential to complement experiments towards providing new insights into various aspects of cancer progression.

Changes in Human Erythrocyte Exposed to Organophosphate Flame Retardants: Tris(2-chloroethyl) Phosphate and Tris(1-chloro-2-propyl) Phosphate.

Tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) are the main representatives of organophosphate flame retardants (OPFRs). The exposure of humans to OPFRs present in air, water, and food leads to their occurrence in the circulation. Thus far, no report has been published about the influence of these retardants on non-nucleated cells like mature erythrocytes. Therefore, the impact of TCEP and TCPP (in concentrations determined in human blood as well as potentially present in the human body after intoxication) on human erythrocytes was evaluated. In this study, the effect of TCEP and TCPP on the levels of methemoglobin, reduced glutathione (GHS), and reactive oxygen species (ROS), as well as the activity of antioxidative enzymes, was assessed. Moreover, morphological, hemolytic, and apoptotic alterations in red blood cells were examined. Erythrocytes were incubated for 24 h with retardants in concentrations ranging from 0.001 to 1000 μg/mL. This study has revealed that the tested flame retardants only in very high concentrations disturbed redox balance; increased ROS and methemoglobin levels; and induced morphological changes, hemolysis, and eryptosis in the studied cells. The tested compounds have not changed the activity of the antioxidative system in erythrocytes. TCPP exhibited a stronger oxidative, eryptotic, and hemolytic potential than TCEP in human red blood cells. Comparison of these findings with hitherto published data confirms a much lower toxicity of OPFRs in comparison with brominated flame retardants.

Activation Mediated By Peroxisome Proliferator-Activated Receptor (PPAR) Ligands and Brown Algae-Based Compounds: Different Mechanisms between Megakaryoblasts and Mature Platelets

Background: Nuclear receptors aretranscription factors that are activated by ligands and subsequently bind to regulatory regions in target genes. Recently ligand-activated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to promote the formation of platelets from megakaryocytes and accelerate platelet recovery after radiation-induced bone marrow injuries. The endogenous prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) are ligands of PPARγ. Accumulating evidences also suggest that PPAR activation is involved in inflammation responses and atherosclerosis. In the present study, we analyzed how PPAR ligands and some brown algae-based compounds affect platelet differentiation and functions in vitro.Methods: Platelets were obtained from plateletpheresis products by centrifugation. DAMI and Meg-1 cells, both human megakaryocytic cell lines, were also obtained. Western blotting for PPARγ, PPARα and COX-2 was performed, and bands were visualized by chemiluminescence. The effects of 15d-PGJ2, GW9662, Fucoidan, Seanol, GW6471 and Celecoxib were evaluated. Morphology analysis, platelet flow cytometry, total thrombus formation analysis system (T-TAS) and microarray expression analysis were also used in order to analyze the effects of the treatment on platelets.Results: Megakaryoblasts and mature platelets showed different results in the expression of PPARα, COX-2, glycoproteins and platelet activation markers. PGJ2 induced morphological differentiation evidences in DAMI and Meg-1 cells. PGJ2 increased the levels of CD41, CD42b, CD62p and PAC-1 both in DAMI and Meg-1 cells. In mature platelets, Fucoidan and Seanol decreased the expression of PPARα and increased COX-2 expression. Fucoidan elevated the expression of CD62p and CD63 in mature platelets, suggesting that Fucoidan causes platelet activation. In microarray analysis, fucoidan showed early growth response (EGR)-1 gene upregulation. Unfortunately thrombogenesity by T-TAS was not evident from the study.Conclusions: The mechanism of platelet production and activation is not fully understood. Understanding the mechanisms underlying megakaryocyte maturation and platelet production will facilitate the development of methods to increase the production of platelets from precursor cells. Although platelets are non-nucleated cells, they contain transcription factors, especially PPAR and NF-κB. From the present study, our findings support the hypothesis that PPAR, both PPARγ and PPARα play a role in platelet differentiation and activation, and exact mechanisms need to be defined related to prostaglandin pathways. Fucoidan may activate platelets via different pathways than PGJ2. DisclosuresNo relevant conflicts of interest to declare.

Programmed Cell Death and Functional Activity of Platelets in Case of Oncohematologic Diseases.

Programmed cell death of non-nucleated blood cells - platelets - could be associated with pathophysiology of oncologic and oncohematologic diseases. It contributes to both bleedings (caused by the thrombocytopenia, which is induced by elimination of the platelets) and thrombosis (caused by the processes of blood coagulation on the surface of phosphatidylserine exposing platelets). Here we characterized functional responses of platelets from the patients with various oncological disorders undergoing chemotherapy and compared them to the platelets from the healthy donors and platelets pre-incubated with apoptosis inducer ABT-737. Some patients exhibited diminished capability of platelets to aggregate. Immunophenotyping of these platelets revealed their pre-activation in comparison to the platelets from the healthy donors. Calcium signaling analysis revealed that in the patient-derived platelets, as well as in the apoptotic platelets, intracellular calcium levels were increased in resting cells. However, moderate level of this increase together with weak expression of phosphatidylserine allows us to assume that apoptotic processes in the circulating platelets from the patients are limited.

The Scaffold Proteins Paxillin B and α-Actinin Regulate Septation in Aspergillus nidulans via Control of Actin Ring Contraction.

Cytokinesis, as the final step of cell division, plays an important role in fungal growth and proliferation. In the filamentous fungus Aspergillus nidulans, defective cytokinesis is able to induce abnormal multinuclear or nonnucleated cells and then result in reduced hyphal growth and abolished sporulation. Previous studies have reported that a conserved contractile actin ring (CAR) protein complex and the septation initiation network (SIN) signaling kinase cascade are required for cytokinesis and septation; however, little is known about the role(s) of scaffold proteins involved in these two important cellular processes. In this study, we show that a septum-localized scaffold protein paxillin B (PaxB) is essential for cytokinesis/septation in A. nidulans. The septation defects observed in a paxB deletion strain resemble those caused by the absence of another identified scaffold protein, α-actinin (AcnA). Deletion of α-actinin (AcnA) leads to undetectable PaxB at the septation site, whereas deletion of paxB does not affect the localization of α-actinin at septa. However, deletion of either α-actinin (acnA) or paxB causes the actin ring to disappear at septation sites during cytokinesis. Notably, overexpression of α-actinin acnA partially rescues the septum defects of the paxB mutant but not vice versa, suggesting AcnA may play a dominant role over that of PaxB for cytokinesis and septation. In addition, PaxB and α-actinin affect the septal dynamic localization of MobA, a conserved component of the SIN pathway, suggesting they may affect the SIN protein complex function at septa. Protein pull-down assays combined with liquid chromatography–mass spectrometry identification indicate that α-actinin AcnA and PaxB likely do not directly interact, but presumably belong to an actin cytoskeleton protein network that is required for the assembly and contraction of the CAR. Taken together, findings in this study provide novel insights into the roles of conserved scaffold proteins during fungal septation in A. nidulans.

Sex differences in circadian endocrine rhythms: Clinical implications.

Organisms have developed a highly conserved and tightly regulated circadian system, to adjust their daily activities to day/night cycles. This system consists of a central clock, which is located in the hypothalamic suprachiasmatic nucleus, and the peripheral clocks that are ubiquitously expressed in all tissues. Both the central and peripheral clocks communicate with each other and achieve circadian oscillations of gene expression through transcriptional/translational loops mediated by clock transcription factors. It is worth mentioning that circadian non-transcriptional/non-translational rhythms also occur in non-nucleated cells. Interestingly, sex has been identified as an important factor influencing the activity of the circadian system. Indeed, several sex differences have been documented in the anatomy, physiology and pathophysiology that pertain to circadian rhythms. In this review, we present the historical milestones of understanding circadian rhythms, describe the central and peripheral components of the circadian clock system, discuss representative examples of sexual dimorphism of circadian rhythms, and present the most relevant clinical implications.

Variations in Nuclear Number and Size in Vegetative Hyphae of the Edible Mushroom Lentinula edodes.

In basidiomycete fungi, the number of nuclei and their ploidy level per nucleus can vary tremendously among species; however, within species, nuclear number and ploidy levels are traditionally considered fixed in their vegetative hyphae. In the edible mushroom Lentinula edodes, the hyphae are classified as either monokaryotic or dikaryotic, with each monokaryotic hyphal cell containing one haploid nucleus, and each dikaryotic hyphal cell containing two haploid nuclei. The dikaryotic hyphae are the results of mating between two genetically distinct monokaryons with different mating types. In this study, we examined the nuclear number and size (a potential correlate to ploidy) of L. edodes mycelia throughout its vegetative growth. We found that the number of nuclei within individual hyphal cells varied widely from non-nucleated to uninucleated, dinucleated, and multinucleated. Additionally, different nuclei within the same cell appeared very different in size, with a maximum nucleus cross-sectional area of 4.94 μm2 and the minimum nucleus cross-sectional area at only 0.37 μm2. Moreover, as culture time increased, more cells appeared to be devoid of any nuclei, with transmission electron microscopy and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays of late-stage cultures showing autophagosomes fusing and dissolving the nuclei and resulting in a large number of TUNEL-positive DNA fragments in non-nucleated cells. These results indicated that non-nucleated cells were likely caused by autophagy and apoptosis-like activities within aging L. edodes hyphae.

Role of bacterial minicells in cancer therapy

Cancer is one of the most important diseases threatening human health. Frequently-used traditional cancer treatment methods, like radiotherapy, chemotherapy and surgery, have serious toxic side effects and limitations. The widely-used drug delivery carriers (liposomes, nanoparticles, etc.) have also possessed many issues such as drug leakage and incomplete loading in the late clinical stage. Currently, using tumor-targeting vectors to deliver anti-tumor drugs or small molecules is one of the promising strategies for mediating safe and effective tumor therapy. In recent years, bacterial-derived non-replicating minicells, which are nanoscale non-nucleated cells produced during abnormal bacterial division, have got more and more attention. With a diameter of 200-400 nm, minicells have a large drug loading capacity. Meanwhile, the surface of minicells are able to be modified to load the assembly of antibodies/ligands that bind to tumor cell surface specific antigens or receptors, which can significantly improve tumor targeting of minicells. This tumor-targeting nanomaterials of minicells not only are used to deliver anti-tumor chemotherapeutic drugs, functional nucleic acids or plasmids encoding functional small molecules to mammalian cells, but also greatly increase drug loading and reduce drug penetration. Thus, the use of minicells combining with chemical therapy could help reduce the toxicity and maximize the effectiveness of the drug in the body. This paper summarizes the research and development of production purification, drug loading, tumor cells targeting, and internalization process of minicells, as well as its use in the delivery of anti-tumor drugs, to provide some information for the development and utilization of minicell carriers.

Partial Characterization of the Inactivation Process of the Human Erythrocyte Mechano-Activated K+ Channel a (HEMKCA): Effect of Membrane Potential, Ca2+ and Rb+

With a lifespan of 120 days, Human Erythrocytes are non-nucleated cells, so these cells cannot undergo a process of classic apoptosis. In our laboratory, we have proposed a hypothesis to explain how the process of aging in these cells occurs. This hypothesis involves two novel mechanisms: a K+/Ca2+ exchanger, which is activated in response to changes in membrane potential; and a mechano-activated K+ channel (HEMKCA), which presents a sigmoid dependence of Po with the pressure applied to the membrane, an average unitary conductance of approximately 20pS and is modulated by the internal concentration of Ca2+. Here we present a partial characterization of the process of inactivation of HEMKCA. This channel presents an exponential drop of the open probability in time with a t=2.4 min at −60 mV. We tested the effect of various parameters on this process in order to characterize this inactivation. The development of the inactivation presented a U-shape complex dependence with the membrane potential at T=15 min, thus the Po decreased by 85 to 95% at Vm=+/−60mV, but no statistically significant difference was found at Vm=+/−80mV with respect to T=1min; this result is discussed in terms of the voltage dependence of the inactivation recovery. On the other hand, Rb+ delays the inactivation by a factor of 3.3 with respect to K+, from a tK=2.4min to a tRb=7.99 min, but it has no effect on the level of inactivation reached at the end of the record. Changes in cytoplasmic Ca2+ concentration did not affect the kinetics parameters of the inactivation. We conclude that these results are consistent with a C-type inactivation.