Correlating single cell motility with population growth dynamics for flagellated bacteria

Many bacteria used for biotechnological applications are naturally motile. Their "bio-nanopropeller" driven movement allows searching for better environments in a process called chemotaxis. Since bacteria are extremely small in size compared to the bulk fluid volumes in bioreactors, single cell motility is not considered to influence bioreactor operations. However, with increasing interest in localized fluid flow inside reactors, it is important to ask whether individual motility characteristics of bacteria are important in bioreactor operations. The first step in this direction is to try to correlate single cell measurements with population data of motile bacteria in a bioreactor. Thus, we observed the motility behavior of individual bacterial cells, using video microscopy with 33 ms time resolution, as a function of population growth dynamics of batch cultures in shake flasks. While observing the motility behavior of the most intensively studied bacteria, Escherichia coli, we find that overall bacterial motility decreases with progression of the growth curve. Remarkably, this is due to a decrease in a specific motility behavior called "running". Our results not only have direct implications on biofilm formations, but also provide a new direction in bioprocess design research highlighting the role of individual bacterial cell motility as an important parameter.     

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor

The angiotensin II type I (AT(1)) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn(111) of the AT(1) receptor constitutively activates the receptor leading to Gq-coupled IP(3) production independent of Ang II binding. The Ang II-activated conformation of the AT1(N111G) receptor was proposed to be similar to that of the wild-type AT(1) receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT(1) receptors are different. Upon Ang II binding an activated conformation of the wild-type AT(1) receptor activates G protein and recruits beta-arrestin. In contrast, the agonist-bound AT1(N111G) mutant receptor preferentially couples to Gq and is inadequate in beta-arrestin recruitment.     

Process-induced cell cycle oscillations in CHO cultures: Online monitoring and model-based investigation

The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through the cell cycle causes metabolic regulations with variable productivities in antibody-producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle-derived population dynamics. In this study, process-induced cell cycle synchronization was assessed in repeated-batch and fed-batch cultures. An automated flow cytometry set-up was developed to measure the cell cycle distribution online, using antibody-producing CHO DP-12 cells transduced with the cell cycle-specific fluorescent ubiquitination-based cell cycle indicator (FUCCI) system. On the basis of the population-resolved model, feeding-induced partial self-synchronization was predicted and the results were evaluated experimentally. In the repeated-batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed-batch process with up to cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high-frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle-dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.     

New N-benzhydrylpiperazine/1,3,4-oxadiazoles conjugates inhibit the proliferation,migration, and induce apoptosis in HeLa cancer cells via oxidative stress–mediated mitochondrial pathway

N-benzhydrylpiperazine and 1,3,4-oxadiazoles are pharmacologically active scaffolds which exhibits significant inhibitory growth effects against various cancer cells, however, antiproliferation effects and the underlying mechanism for inducing apoptosis for aforementioned scaffolds addressing HeLa cancer cells remains uncertain. In this study, N-benzhydrylpiperazine clubbed with 1,3,4-oxadiazoles ( 4a–4h ) were synthesized, subsequently characterized using high resolution spectroscopic techniques and eventually evaluated for their antiproliferation potential by inducing apoptosis in HeLa cancer cells. The MTT assay screening results revealed that among all, compound 4d ( N-benzhydryl-4-((5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl)methyl)piperazine) in particular, exhibited IC ₅₀ value of 28.13 ± 0.21 μg/mL and significantly inhibited the proliferation of HeLa cancer cells in concentration-dependent manner. The in vitro anticancer assays for treated HeLa cells resulted in alterations in the cell morphology, reduction in colony formation, and inhibition of cell migration in concentration-dependent treatment. Furthermore, G2/M phase arrest, variations in the nuclear morphology, degradation of chromosomal DNA confirmed the ongoing apoptosis in treated HeLa cells. Increase in the expression of cytochrome C and caspase-3 confirmed the involvement of intrinsic mitochondrial pathway regulating the cell death. Also, elevation in reactive oxygen species level and loss of mitochondrial membrane potential signified that compound 4d induced apoptosis in HeLa cells by generating the oxidative stress. Therefore, compound 4d may act as a potent chemotherapeutic agent against human cervical cancer.     

Interaction of DBC1 with polyoma small T antigen promotes its degradation and negatively regulates tumorigenesis

Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1–DBC1–AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.     

Saussurea lappa Exhibits Anti-Oncogenic Effect in Hepatocellular Carcinoma,HepG2 Cancer Cell Line by Bcl-2 Mediated Apoptotic Pathway and Mitochondrial Cytochrome C Release

Background and Objectives: Saussurea lappa (S. lappa) is an important species of the Asteraceae family with several purposes in traditional medicine. This study intended to explore the cytotoxic effect of S. lappa on HepG2 cancer cell proliferation. Materials and Methods: The effects of an S. lappa n-butanol extract on the induction of apoptosis were investigated by flow cytometry and mitochondrial cytochrome C-releasing apoptosis assay. Additionally, real-time PCR was employed to confirm apoptosis initiation. Further, qualitative estimation of the active constituent of S. lappa was done by gas chromatography–mass spectroscopy (GC–MS). Results: The cell viability study revealed that the n-butanol extract of S. lappa demonstrated potent cytotoxicity against HepG2 cancer cells, with an IC50 value of 56.76 μg/mL. Cell morphology with dual staining of acridine orange (AO)-ethidium bromide (EB) showed an increase in orange/red nuclei due to cell death by S. lappa n-butanol extract compared to control cells. Apoptosis, as the mode of cell death, was also confirmed by the higher release of cytochrome C from mitochondria, the increased expression of caspase-3 and bax, along with down regulation of Bcl-2. Conclusion: These findings conclude that S. lappa is a cause of hepatic cancer cell death through apoptosis and a potential natural source suggesting furthermore investigation of its active compounds that are responsible for these observed activities.     

Genistein: A Potent Anti-Breast Cancer Agent

Genistein is an isoflavonoid present in high quantities in soybeans. Possessing a wide range of bioactives, it is being studied extensively for its tumoricidal effects. Investigations into mechanisms of the anti-cancer activity have revealed many pathways including induction of cell proliferation, suppression of tyrosine kinases, regulation of Hedgehog-Gli1 signaling, modulation of epigenetic activities, seizing of cell cycle and Akt and MEK signaling pathways, among others via which the cancer cell proliferation can be controlled. Notwithstanding, the observed activities have been time- and dose-dependent. In addition, genistein has also shown varying results in women depending on the physiological parameters, such as the early or post-menopausal states.     

Adult human brain expresses four different molecular forms of neural cell adhesion molecules

Using antibodies to rat neural cell adhesion molecules (NCAM), we analyzed the NCAM of adult human brain. Various regions of the brain were analyzed quantitatively by Western blot. Grey matter showed four bands of NCAM with apparent molecular weights of 180,000, 170,000, 140,000 and 120,000. White matter showed one major band with an apparent M_r of 120,000 and a minor band of 180,000. Cerebellar grey matter contained mainly 170,000, 140,000 and 120,000, white cerebellar white matter had only 180,000 and 120,000 M₁ NCAMS. Spinal cord showed mainly 120,000 M_r NCAM. Deglycosylation using N-glycanase resulted in 170,000, 160,000, 130,000 and 110,000 M_r proteins, suggesting that the four forms of human NCAM are derived from individual polypeptides. The presence of 170,000 M₁ NCAM is unique to human brain.     

Germination response of Arabian desert species to gibberellic acid and potassium nitrate seed treatment

Abstract

Desert plant species commonly use seed dormancy to prevent germination during unfavorable environmental conditions and thus increase the probability of seedling survival. Seed dormancy presents a challenge for restoration ecology, particularly in desert species for which our knowledge of dormancy regulation is limited. In the present study the effect of gibberellic acid (GA₃) and potassium nitrate (KNO₃) on seed dormancy release was investigated on eight Arabian desert species. Both treatments significantly enhanced the germination of most species tested. GA₃ was more effective than KNO₃ in enhancing germination percentage, reducing mean germination time and synchronizing the germination in most of the studied species. Light requirement during germination was species-specific, but in general the presence of light promoted germination more effectively when combined with KNO₃ and GA₃. The wide variation in dormancy and germination requirements among the tested species is indicative of distinct germination niches, which might assist their co-existence in similar habitat/environmental conditions. Seed pre-treatments that optimize germination in this habitat must therefore be assessed for individual species to improve the outcomes of ecological restoration.