Physiological State Of Cells Research Articles

Distribution of bacterial single cell parameters and their estimation from turbidity detection times

The stochastic growth of homogeneous bacterial populations in the wells of a microtiter plate was studied as a function of the random initial cell number and their random individual lag times. These significantly affected the population growth in the well, while the maximum specific growth rate of the population was constant (or its variance was negligible) for each well.We showed the advantages of the mathematical assumption that a transformation of the single cell lag time, called the single cell physiological state (or, more accurately, that of the sub-population generated by the single cell) follow the Beta distribution. Simulations demonstrated what patterns would such assumption generate for the distribution of the detection times observed in the wells. An estimation procedure was developed, based on the beta-assumption, that resulted in an explicit expression for the expected value of the single cell physiological state as a function of measured “time to detection” values using turbidity experiments. The method was illustrated using laboratory data with Escherichia coli, Salmonella enterica subsp. enterica strains. The results gave a basis to quantify the difference between the studied organisms in terms of their single-cell kinetics.

Morphological and physiological-biochemical variability of spore-forming bacteria isolated from the agrocoenosis of winter wheat

From an agrocoenosis of winter wheat (Triticum aestivum L.; phylloplane and rhizosphere of the root system; typical chernozem, soil column measuring up to 40 cm), using the classical microbiological methods, we had isolated soil bacteria and characterized them according to the morphological features as representatives of Gram-positive and spore-forming bacteria of Bacillus sp. genus. In the earing-swelling phase of grain, the screening studies found non-pigmented forms of colonies of bacterial isolates, 19 of which were classified to colonial-morphological diversity of R-type with the diameter of 7 to 13 mm. The analysis of physiological condition of cells of populations of soil isolates revealed technologic specificity according to parameters of spore formation in different conditions and incubation time (up to 48–72 h). We observed 90.0% of free spores in axenic cultures as early as after 72 h of cultivation and no more than 10.0% of prospores in the studied monoisolates with stable morphologic traits. Isolates Н10 and Н45 demonstrated the ability to grow in higher cultivation temperatures (+37…+40 °С). According to environmental рН, isolates were able to grow in рН ranging 4.5–8.0. Differential diagnostic testing revealed that as the source of carbon, with formation of acid, soil isolates used arabinose, xylose, mannitol, glucose, galactose, fructose, maltose, sorbitol, glycerin, dextrin, starch, rhamnose and dulcite (with development of alkaline). There was observed active use of mineral forms of nitrogen: ammonium salt and nitrates, aminoacids and proteins. The isolates hydrolyzed casein, gelatin, starch, and litmus was being reduced in the young during growth in milk with litmus. They also exerted catalase activity and were oxidase-positive. Biochemical testing using API test system determined that the studied isolated bacteria differed by a range of fermentation carbohydrates, reduction of nitrates. In the conditions of submerged fermentation, isolates Н38 and Н40 grew in heightened temperature ranges of cultivation (40 °С) for 48 h (according to fact of spore development). Therefore, according to the key morphologic and biochemical traits, strains Н3, Н10, Н13, Н36, Н38, Н40, Н43, Н45 were similar to such of reference strain B. subtilis 8A, and were identified to Bacillus sp., species B. subtilis.

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell–cell interactions for infection, immunology, and cancer research.

Adaptation of Pseudomonas helmanticensis to fat hydrolysates and SDS: fatty acid response and aggregate formation.

An essential part of designing any biotechnological process is examination of the physiological state of producer cells in different phases of cultivation. The main marker of a bacterial cell's state is its fatty acid (FA) profile, reflecting membrane lipid composition. Consideration of FA composition enables assessment of bacterial responses to cultivation conditions and helps biotechnologists understand the most significant factors impacting cellular metabolism. In this work, soil SDS-degrading Pseudomonas helmanticensis was studied at the fatty acid profile level, including analysis of rearrangement between planktonic and aggregated forms. The set of substrates included fat hydrolysates, SDS, and their mixtures with glucose. Such media are useful in bioplastic production since they can help incrementally lower overall costs. Conventional gas chromatography-mass spectrometry was used for FA analysis. Acridine orange-stained aggregates were observed by epifluorescence microscopy. The bacterium was shown to change fatty acid composition in the presence of hydrolyzed fats or SDS. These changes seem to be driven by the depletion of metabolizable substrates in the culture medium. Cell aggregation has also been found to be a defense strategy, particularly with anionic surfactant (SDS) exposure. It was shown that simple fluidity indices (such as saturated/unsaturated FA ratios) do not always sufficiently characterize a cell's physiological state, and morphological examination is essential in cases where complex carbon sources are used.

The challenges and prospects of Escherichia coli as an organic acid production host under acid stress.

Organic acids have a wide range of applications and have attracted the attention of many industries, and their large-scale applications have led fermentation production to low-cost development. Among them, the microbial fermentation method, especially using Escherichia coli as the production host, has the advantages of fast growth and low energy consumption, and has gradually shown better advantages and prospects in organic acid fermentation production. However, when the opportunity comes, the acidified environment caused by the acid products accumulated during the fermentation process also challenges E. coli. The acid sensitivity of E. coli is a core problem that needs to be solved urgently. The addition of neutralizers in traditional operations led to the emergence of osmotic stress inadvertently, the addition of strong acid substances to recover products in the salt state not only increases production costs, but the discharged sewage is also harmful to the environment. This article summarizes the current status of the application of E. coli in the production of organic acids, and based on the impact of acid stress on the physiological state of cells and the impact of industrial production profits, put forward some new conjectures that can make up for the deficiencies in existing research and application. At this point, the diversified transformation of E. coli has become a chassis microbe that is more suitable for industrial fermentation, enhancing industrial application value. • E. coli is a potential host for high value-added organic acids production. • Classify the damage mechanism and coping strategies of E. coli when stimulated by acid molecules. • Multi-dimensional expansion tools are needed to create acid-resistant E. coli chassis.

Characterization of cell cycle and apoptosis in Chinese hamster ovary cell culture using flow cytometry for bioprocess monitoring

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.

Subtropical Freshwater Cyanobacterial Blooms as Hydrogen Peroxide Hot Spots

In aquatic environments, chemical and biological processes represent major sources of reactive oxygen species (ROS) such as hydrogen peroxide. Diurnal patterns of hydrogen peroxide in surface water may suggest photochemical processes affect ROS dynamics. We hypothesized that thin surface hydrogen peroxide maxima could be detected through millimeter-scale depth profiling. These maxima were previously overlooked because they cannot be detected using standard surface water sampling techniques. Combined field and laboratory experiments demonstrated that cyanobacterial blooms could create extremely high concentrations of hydrogen peroxide in a thin layer of surface water (5–60 mm). Hydrogen peroxide concentrations were higher within surface scums of Microcystis aeruginosa than in areas without scum. The cyanobacterial scum increased the temperature of the ambient water, and hydrogen peroxide concentrations were higher at locations exposed to sunlight that at locations in the shade. We found that scum samples directly collected from the field could release hydrogen peroxide to the surrounding water when the biomass was exposed by the light, while 20 laboratory cultures tested did not. The production of hydrogen peroxide from cyanobacteria may depend on the physiological condition of cells. Overall, we identified thin surface water layers during subtropical freshwater cyanobacterial blooms as hydrogen peroxide hot spots.

Abnormal function of telomere protein TRF2 induces cell mutation and the effects of environmental tumor‑promoting factors (Review).

Recent studies have found that somatic gene mutations and environmental tumor-promoting factors are both indispensable for tumor formation. Telomeric repeat-binding factor (TRF)2 is the core component of the telomere shelterin complex, which plays an important role in chromosome stability and the maintenance of normal cell physiological states. In recent years, TRF2 and its role in tumor formation have gradually become a research hot topic, which has promoted in-depth discussions into tumorigenesis and treatment strategies, and has achieved promising results. Some cells bypass elimination, due to either aging, apoptosis via mutations or abnormal prolongation of the mitotic cycle, and enter the telomere crisis period, where large-scale DNA reorganization occurs repeatedly, which manifests as the precancerous cell cycle. Finally, at the end of the crisis cycle, the mutation activates either the expression level of telomerase or activates the alternative lengthening of telomere mechanism to extend the local telomeres. Under the protection of TRF2, chromosomes are gradually stabilized, immortal cells are formed and the stagewise mutation-driven transformation of normal cells to cancer cells is completed. In addition, TRF2 also shares the characteristics of environmental tumor-promoting factors. It acts on multiple signal transduction pathway-related proteins associated with cell proliferation, and affects peripheral angiogenesis, inhibits the immune recognition and killing ability of the microenvironment, and maintains the stemness characteristics of tumor cells. TRF2 levels are abnormally elevated by a variety of tumor control proteins, which are more conducive to the protection of telomeres and the survival of tumor cells. In brief, the various regulatory mechanisms which tumor cells rely on to survive are organically integrated around TRF2, forming a regulatory network, which is conducive to the optimization of the survival direction of heterogeneous tumor cells, and promotes their survival and adaptability. In terms of clinical application, TRF2 is expected to become a new type of cancer prognostic marker and a new tumor treatment target. Inhibition of TRF2 overexpression could effectively cut off the core network regulating tumor cell survival, reduce drug resistance, or bypass the mutation under the pressure of tumor treatment selection, which may represent a promising therapeutic strategy for the complete eradication of tumors in the clinical setting. Based on recent research, the aim of the present review was to systematically elaborate on the basic structure and functional characteristics of TRF2 and its role in tumor formation, and to analyze the findings indicating that TRF2 deficiency or overexpression could cause severe damage to telomere function and telomere shortening, and induce DNA damage response and chromosomal instability.

The Discovery, Development and Demonstration of Three Caged Compounds.

An overview of the history, mechanistic aspects and applications is provided for p-hydroxyphenacyl (pHP) and benzoin photoremovable protecting groups, which release biologically important leaving groups upon photolysis with UV light. Also discussed is (7-diethylaminocoumarin-4-yl)methyl (DEACM), a photoremovable protecting group that absorbs visible light. These are followed by the α-keto amides and naphtho- and benzothiophene-2-carboxanilides as caging groups, which eliminate leaving groups via photochemically produced zwitterionic intermediates. Also covered are amino-1,4-benzoquinones, which upon exposure to green and red wavelengths of light photorearrange to an unstable photoproduct that subsequently eliminates leaving groups in aqueous media. Selected examples are given that use these photoremovable protecting (caging) groups for the light-activated release of biologically important substrates under physiological conditions in cells and tissue as practical applications in biology, biochemistry and physiology. These caging groups have found significant applications because their photochemistry is efficient and a single coproduct is formed in addition to the photoreleased substrate.

Lipid droplets in the nervous system.

Lipid droplets are dynamic intracellular lipid storage organelles that respond to the physiological state of cells. In addition to controlling cell metabolism, they play a protective role for many cellular stressors, including oxidative stress. Despite prior descriptions of lipid droplets appearing in the brain as early as a century ago, only recently has the role of lipid droplets in cells found in the brain begun to be understood. Lipid droplet functions have now been described for cells of the nervous system in the context of development, aging, and an increasing number of neuropathologies. Here, we review the basic mechanisms of lipid droplet formation, turnover, and function and discuss how these mechanisms enable lipid droplets to function in different cell types of the nervous system under healthy and pathological conditions.

Nanofluidics for Simultaneous Size and Charge Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived membrane structures that circulate in body fluids and show considerable potential for noninvasive diagnosis. EVs possess surface chemistries and encapsulated molecular cargo that reflect the physiological state of cells from which they originate, including the presence of disease. In order to fully harness the diagnostic potential of EVs, there is a critical need for technologies that can profile large EV populations without sacrificing single EV level detail by averaging over multiple EVs. Here we use a nanofluidic device with tunable confinement to trap EVs in a free-energy landscape that modulates vesicle dynamics in a manner dependent on EV size and charge. As proof-of-principle, we perform size and charge profiling of a population of EVs extracted from human glioblastoma astrocytoma (U373) and normal human astrocytoma (NHA) cell lines.

Monitoring of the Electrototation of Cells Stimulated By the Ionophore

Monitoring of the Electrototation of Cells Stimulated By the Ionophore

Toward an Understanding of Extracellular tRNA Biology.

Extracellular RNAs (exRNAs) including abundant full length tRNAs and tRNA fragments (tRFs) have recently garnered attention as a promising source of biomarkers and a novel mediator in cell-to-cell communication in eukaryotes. Depending on the physiological state of cells, tRNAs/tRFs are released to the extracellular space either contained in extracellular vesicles (EVs) or free, through a mechanism that is largely unknown. In this perspective article, we propose that extracellular tRNAs (ex-tRNAs) and/or extracellular tRFs (ex-tRFs) are relevant paracrine signaling molecules whose activity depends on the mechanisms of release by source cells and capture by recipient cells. We speculate on how ex-tRNA/ex-tRFs orchestrate the effects in target cells, depending on the type of sequence and the mechanisms of uptake. We further propose that tRNA modifications may be playing important roles in ex-tRNA biology.

A Hybrid Continuum-Particle Approach for Fluid-Structure Interaction Simulation of Red Blood Cells in Fluid Flows

Transport of cells in fluid flow plays a critical role in many physiological processes of the human body. Recent developments of in vitro techniques have enabled the understanding of cellular dynamics in laboratory conditions. However, it is challenging to obtain precise characteristics of cellular dynamics using experimental method alone, especially under in vivo conditions. This challenge motivates new developments of computational methods to provide complementary data that experimental techniques are not able to provide. Since there exists a large disparity in spatial and temporal scales in this problem, which requires a large number of cells to be simulated, it is highly desirable to develop an efficient numerical method for the interaction of cells and fluid flows. In this work, a new Fluid-Structure Interaction formulation is proposed based on the use of hybrid continuum-particle approach, which can resolve local dynamics of cells while providing large-scale flow patterns in the vascular vessel. Here, the Dissipative Particle Dynamics (DPD) model for the cellular membrane is used in conjunction with the Immersed Boundary Method (IBM) for the fluid plasma. Our results show that the new formulation is highly efficient in computing the deformation of cells within fluid flow while satisfying the incompressibility constraints of the fluid. We demonstrate that it is possible to couple the DPD with the IBM to simulate the complex dynamics of Red Blood Cells (RBC) such as parachuting. Our key observation is that the proposed coupling enables the simulation of RBC dynamics in realistic arterioles while ensuring the incompressibility constraint for fluid plasma. Therefore, the proposed method allows an accurate estimation of fluid shear stresses on the surface of simulated RBC. Our results suggest that this hybrid methodology can be extended for a variety of cells in physiological conditions.

Integrin-α9β1 as a Novel Therapeutic Target for Refractory Diseases: Recent Progress and Insights

Integrins refer to heterodimers consisting of subunits α and β. They serve as receptors on cell membranes and interact with extracellular ligands to mediate intracellular molecular signals. One of the least-studied members of the integrin family is integrin-α9β1, which is widely distributed in various human tissues and organs. Integrin-α9β1 regulates the physiological state of cells through a variety of complex signaling pathways to participate in the specific pathological processes of some intractable diseases. In recent years, an increasing amount of research has focused on the role of α9β1 in the molecular mechanisms of different refractory diseases and its promising potential as a therapeutic target. Accordingly, this review introduces and summarizes recent research related to integrin-α9β1, describes the synergistic functions of α9β1 and its corresponding ligands in cancer, autoimmune diseases, nerve injury and thrombosis and, more importantly, highlights the potential of α9β1 as a distinctive target for the treatment of these intractable diseases.

Woody breast myopathy broiler show age-dependent adaptive differential gene expression in Pectoralis major and altered in-vivo triglyceride kinetics in adipogenic tissues

A study was conducted to understand the differentially expressed genes in Pectoralis (P) major under woody breast (WB) myopathy condition in a high yielding broiler strain using RNA-sequencing at the growing (d 21) and finishing (d 42 and d 56) grow-out ages. Follow-up study was conducted to understand the in vivo triglyceride (TG) synthesis (d 49) occurring in adipogenic tissues using deuterium oxide (2H2O) as a metabolic tracer. Results indicated the top physiological systems affected in myopathy broiler were related to the musculo-skeletal system (d 21, 42, and 56) and cardiovascular system (d 42 and 56). Ubiquitin-specific proteases are expressed higher in myopathy broiler at d 21 (OTUD1) and d 42 (SACS) that potentially indicated higher degradation of muscle protein occurring at those ages. While genes related to transcription factors and muscle cell differentiation (ZNF234, BTG2) and muscle growth (IGF1) were upregulated with myopathy broiler suggesting concurrent muscle fiber regeneration. The downregulation of PYGB and MGAM genes related to carbohydrate transport and metabolism at d 42 potentially indicated nutrient-deficient state of myopathy affected fibers; whereas the nutrient-deficient physiological state of cells seemed to be counteracted by up-regulation of genes related to carbohydrate (ALDOB, GPD1L2) at d 56. There was a reduced (P < 0.05) in vivo TG synthesis in liver of the myopathy broiler (0.123 %/hr) compared to non-myopathy broiler (0.197 %/hr). The majority of TG synthesized in liver with myopathy broiler could conceivably be delivered to P. major (rather than to abdominal fat pad storage) to fulfil the increased energy need of muscle cells (via TG lipolysis and fatty acid [FA] oxidation). The increased utilization of FAs in the WB affected muscle could result in reduced secretion of FAs into blood circulation leading to sub-optimal availability of FAs for re-esterification for TG synthesis in liver. Results indicated that myopathy broiler at later age (d 56) of grow-out period were synchronously going through adaptive physiological processes of feedback responses to adverse cellular states.

Deep-Learning Based Label-Free Classification of Activated and Inactivated Neutrophils for Rapid Immune State Monitoring.

The differential count of white blood cells (WBCs) is one widely used approach to assess the status of a patient’s immune system. Currently, the main methods of differential WBC counting are manual counting and automatic instrument analysis with labeling preprocessing. But these two methods are complicated to operate and may interfere with the physiological states of cells. Therefore, we propose a deep learning-based method to perform label-free classification of three types of WBCs based on their morphologies to judge the activated or inactivated neutrophils. Over 90% accuracy was finally achieved by a pre-trained fine-tuning Resnet-50 network. This deep learning-based method for label-free WBC classification can tackle the problem of complex instrumental operation and interference of fluorescent labeling to the physiological states of the cells, which is promising for future point-of-care applications.

Adenosine triphosphate (ATP) as a metric of microbial biomass in aquatic systems: new simplified protocols, laboratory validation, and a reflection on data from the literature

AbstractThe use of adenosine triphosphate (ATP) as a universal biomass indicator is built on the premise that ATP concentration tracks biomass rather than the physiological condition of cells. However, reportedly high variability in ATP in response to environmental conditions is the main reason the method has not found widespread application. To test possible sources of this variability, we used the diatom Thalassiosira weissflogii as a model and manipulated its growth rate through nutrient limitation and through exposure to three different temperatures (15°C, 20°C, and 25°C). We simplified the ATP protocol with hot‐water or chemical extraction methods, modified a commercially available luciferin‐luciferase assay, and employed single‐photon counting in a scintillation counter, all of which increased sensitivity and throughput. Per‐cell ATP levels remained relatively constant despite changes in growth rates by approximately 10‐fold in the batch culture (i.e., nutrient limitation) experiments, and approximately 2‐fold in response to temperature. The re‐examination of related literature values revealed that average cellular ATP levels differed little among taxonomic groups of aquatic microbes, even at the domain level, and correlated well with bulk properties such as elemental carbon or nitrogen. Fulfilling multiple cellular functions in addition to being the universal energy currency requires ATP to be maintained in a millimolar concentration range. Consequently, ATP relates directly to live cytoplasm volume, while elemental carbon and nitrogen are constrained by an indeterminate pool of detrital material and intracellular storage compounds. The ATP‐biomass indicator is sensitive, economical, and can be readily standardized among laboratories and across environments.

Molecular Mechanisms of Renal Progenitor Regulation: How Many Pieces in the Puzzle?

Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/β-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-like receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single-cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic.

Long-term cell culture and electrically in situ monitoring of living cells based on a polyaniline hydrogel sensor.

Accurate, in situ and long-term electrically monitoring of cell development plays an important role in cell study, which brings in challenges in terms of biocompatibility, processability, and sensing capability of electrochemical sensors. Based on biocompatible conductive polyaniline (PAni) hydrogels, we constructed a flexible sensor with flexible carbon cloth for electrical analysis of living cells. The carbon fiber substrate modified with conductive PAni hydrogels was selected as the electrode to promote the current collection of the sensor. The three dimensional nanostructured mesoporous matrix of PAni hydrogels is favorable for in situ generation of catalytic Pt nanoparticles and cell growth. With these hierarchically nanostructured features, the hydrogel electrochemical sensor was endowed with high sensitivity and selectivity in the detection of H2O2 (with a low detection limit of 1.6 μM in 0.01 M PBS and a wide linear range from 10 μM to 10 mM), and good biocompatibility for cell growth as long as 5 days. The accurate detection of H2O2 released from cells enabled us to differentiate the physiological states of cells and imitate the different stimuli-responsive behavior, which can provide real-time information on cell biological events. With outstanding biocompatibility, operability and repeatability, this strategy can be expanded to the fields of other biosensor fabrication and cell-related biomarker monitoring, which exhibits a broad application potential in bioanalysis catering to new generation sensors.