Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography

In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.     

Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.     

Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds

Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.     

Effect of a diabetic state on myometrial ultrastructure and isolated uterine contractions in the rat

Alterations in rat myometrial ultrastructure and in vivo uterine contractile responses to oxytocin were examined in estradiol-treated (40 micrograms/kg) euglycemic and streptozotocin-induced (85 mg/kg) diabetic rats. Myometrial morphology was examined 18, 24, and 36 hr after estradiol administration. At the time points examined, nuclei of myometrial cells from euglycemic and diabetic rats were pleomorphic and contained large areas of heterochromatin dispersed throughout the nuclei. Mitochondria were round to oval in shape and contained a dense matrix with cristae that extended across the mitochondria. Myofilaments were found in both euglycemic and diabetic cells but the relative number of myofilaments in diabetic cells appeared to be less than the number found in myometrial cells removed from euglycemic animals. The number of free cytoplasmic ribosomes in diabetic cells also appeared to be less than those found in euglycemic cells. In order to determine if apparent differences in the number of myofilament found in diabetic myometrial cells could be correlated with changes in uterine contractile responses to hormones, oxytocin dose-response curves (10(-8) to 10(-5) M) were examined in isolated uteri removed from saline-injected and estradiol-injected (24-hr pretreatments) euglycemic and diabetic rats. The maximal contractile responses (milligrams tension developed per milligrams tissue) in saline-injected euglycemic and diabetic rats were 49 +/- 5 and 36 +/- 4, respectively, while maximal contractile responses in estradiol-injected euglycemic and diabetic rats were 68 +/- 7 and 45 +/- 5, respectively. Maximal contractile responses induced by oxytocin in estradiol-treated diabetic uteri were significantly smaller than the contractile responses measured in euglycemic estradiol-treated uteri. This study demonstrates that estradiol-induced changes in both myometrial cell morphology and in vitro uterine contractile responses to oxytocin are altered in diabetic rats.     

Accessibility of antigenic sites recognized by AUA1, HMFG1 and HMFG2 monoclonal antibodies: its influence on antibody binding of live cells

We assessed the immunoreactivity of live and alcohol-fixed monolayers of HRA-19, a rectal adenocarcinoma cell line, to the monoclonal antibodies AUA1, HMFG1 and HMFG2. Differences in staining patterns between live and alcohol-fixed colonies were found. The well-polarized cells forming the centers of the monolayer colonies showed strong membrane staining when the cells were alcohol-fixed prior to AUA1 incubation, but showed no staining when the cells were alive during the incubation. When AUA1 incubation was done both before and after alcohol fixation, membrane staining was again seen, ruling out the possibility of antigenic modulation. Incubation of live cells with AUA1 together with EDTA showed strong staining of dissociating cells. It is concluded that AUA1 antigenic sites, which on polarized cells are basolateral in location, are inaccessible to the antibody-containing culture fluid, which bathes the apical aspects of the cells, but they become accessible after alcohol fixation, or treatment with EDTA. HMFG1 antigenic sites are located on the apical cell membrane, and accordingly, no differences were seen between incubation of live and alcohol-fixed cells when incubated with HMFG1. The antigenic sites of HMFG2 are partly intracellular, and in our monolayer model, the staining of live cells was weaker and more scarce than on alcohol-fixed cells. It is concluded that immunostaining of cytological and histological material of tumours may not adequately predict antibody binding on live cells, and thus, these findings are of importance in the context of selection of monoclonal antibodies for clinical radio-immunotargeting.     

Probable Relationships between the Lower Jurassic Crocodilomorph Trackways Batrachopus and Selenichnus: Evidence and Implications Based on New Finds from the St. George Area Southwestern Utah

Some small and medium-sized crocodylomorph footprints are described from the Late Jurassic (Kimmeridgian) coastal and deltaic units of the northern Spain (Asturias). There are at least four footprint morphotypes. Three of them, with well preserved trackways, are included in the ichnogenus Crocodylopodus (Crocodylopodus isp. and Crocodylopodus meijidei); the fourth one, documented by some isolated large footprints, is referable to the ichnogenus Hatcherichnus. This ichnoassociation confirms the presence of small crocodilians in palaeoenvironments apparently dominated by dinosaurs. The presence of Hatcherichnus seems to confirm the affinity between the Iberian and North American ichnofaunas.     

Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

Hydrophobins represent a class of unique fungal proteins that have low molecular mass, are cysteine rich, and can self-assemble into two-dimensional arrays at water/air interfaces. These highly surface-active proteins are able to decrease the surface tension of water, thus allowing fungal structures to penetrate hydrophobic–hydrophilic barriers. Due to their unusual biophysical properties, hydrophobins have been suggested for use in a wide range of biotechnological applications. Here we describe a simple method for producing a functionally active class I hydrophobin derived from the entomopathogenic fungus, Beauveria bassiana, in an E. coli host. N-terminal modifications were required for proper expression and purification, and the hydrophobin was expressed as a fusion partner to a cleavable N-terminus chitin-binding domain-intein construct. The protein was purified and reconstituted from E. coli inclusion bodies. Self-assembly of the recombinant hydrophobin was followed kinetically using a thioflavin T fluorescence binding assay, and contact angle measurements of purified recombinant hydrophobin protein (mHyd2) films on a variety of substrata demonstrated its surface modification ability, which remained stable for at least 4 months. Filament or fibril-like structures were imaged using atomic force and transmission electron microscopy. These data confirmed the functional properties of the purified protein and indicate amino acid flexibility at the N-terminus, which can be exploited for various applications of these proteins.     

Antimicrobials and in vitro systems: antibiotics and antimycotics alter the proteome of MCF-7 cells in culture

Cell culture is widely used to study gene or protein changes in response to experimental conditions. The value of such experiments depends on stringent control and understanding of the in vitro environment. Despite well-documented evidence describing toxic effects in the clinical setting, antibiotics and antimycotics are routinely used in cell culture without regard for their potential toxicity. We cultured MCF-7 breast cancer cells in the presence/absence of antibiotics (penicillin/streptomycin) and/or the antimycotic amphotericin B. Differential protein expression was assessed using 2D-DIGE and MALDI-MS/MS. Antibiotics caused 8/488 spots (1.3% of the protein) to be generally down-regulated. The affected proteins were principally chaperones and cytoskeletal. In marked contrast, amphotericin B induced a more dramatic response, with 33/488 spots (9.5% of the total protein) generally up-regulated. The proteins were mostly involved in chaperoning and protein turnover. Combining antibiotics and amphotericin B had little overall effect, with only one (unidentified) protein being up-regulated. As this study identifies differential protein expression attributable to antibiotics/antimycotics, we urge caution when comparing and interpreting proteomic results from different laboratories where antibiotics/antimycotics have been used. We conclude that as antibiotics and antimycotics alter the proteome of cultured cells in markedly different ways their use should be avoided where possible.