Middle Time Points Research Articles

Abstract 82: Single-cell cytokine secretion kinetics probed by Time-lapse flow (TM) cytometry

Abstract Cytokine secretion from immune cells is a central element of the immune response and a major determinant of immunotherapy efficacy. Several methods, including bulk in-solution assays and flow cytometry, have been used to measure cytokine secretion. Population-level analyses have shown that different cytokines can have different rates of secretion, depending upon their differentiation state or the stimulus itself. However, no current method can interrogate cytokine secretion from individual cells over time. Here, we describe a novel flow cytometry-based assay for measuring single-cell cytokine secretion kinetics. Our approach, called “Time-lapse flowTM” uses semiconductor probes called laser particles (LPs) to optically barcode individual cells, providing a means to track single cells across a series of measurements over time. We applied this approach to measure T-cell cytokine secretion kinetics using stimulated primary human T cells. Isolated T cells from cryopreserved human PBMCs were barcoded with LPs and stained with a bispecific antibody that binds to the cell surface on one end and “catches” secreted cytokines (IFNy and IL-2) on the other end. This permits analysis of cytokines from live cells. Next, T cells underwent three sequential stimulations over the course of 3 hours with PMA/Ionomycin. After the first stimulation, cells were stained with a surface marker panel that defined T cell memory phenotypes (CD3, CD4, CD8, CD45RA, CCR7, CD57, CD27, and CD95) and “detection” antibodies that recognized IFNy and IL-2 bound to the cell surface. The detection antibodies were re-added after each stimulation to quantify the accumulating cytokine on the cell surface. In total, kinetic profiles from over 100,000 CD3+ cells were measured. We discovered massive heterogeneity of responding cells, identifying at least three distinct groups of cells defined by their initial time of cytokine secretion (early, middle, and late responders) and varying intensities of cytokine accumulation over time. Cells peaking in cytokine expression at the middle time points were mainly memory types with higher intensity, while later responders skewered towards naïve cells with less cytokine. However, IFNy variation wasn’t solely due to these markers, as diverse kinetics occurred across all subsets. For IL-2, we found that most responses initiated later than those of IFNy, and there was a reduced heterogeneity of both intensity and kinetics at these time points. These results were consistent across multiple donors and replicates (n=3). Laser particle barcoding enables measurement of single-cell function over time. This novel time-lapse data allows interrogation of functional heterogeneity of immune cells at scale, enabling discovery of new relationships between T cell responses, diseases, and immunotherapies. Citation Format: Marissa Fahlberg, Sarah Forward, Emane Rose Assita, Trevor Brown, Pratip Chattopadhyay, Sheldon Kwok. Single-cell cytokine secretion kinetics probed by Time-lapse flow (TM) cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 82.

Physically and Chemically Crosslinked Hyaluronic Acid-Based Hydrogels Differentially Promote Axonal Outgrowth from Neural Tissue Cultures.

Our aim was to investigate axonal outgrowth from different tissue models on soft biomaterials based on hyaluronic acid (HA). We hypothesized that HA-based hydrogels differentially promote axonal outgrowth from different neural tissues. Spinal cord sliced cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) were maintained on a collagen gel, a physically crosslinked HA-based hydrogel (Healon 5®) and a novel chemically crosslinked HA-based hydrogel, with or without the presence of neurotrophic factors (NF). Time-lapse microscopy was performed after two, five and eight days, where axonal outgrowth was assessed by automated image analysis. Neuroprotection was investigated by PCR. Outgrowth was observed in all groups; however, in the collagen group, it was scarce. At the middle timepoint, outgrowth from SCSCs was superior in both HA-based groups compared to collagen, regardless of the presence of NF. In DRGCs, the outgrowth in Healon 5® with NF was significantly higher compared to the rest of the groups. PCR revealed upregulation of NeuN gene expression in the HA-based groups compared to controls after excitotoxic injury. The differences in neurite outgrowth from the two different tissue models suggest that axons differentially respond to the two types of biomaterials.

The ctenophore Mnemiopsis leidyi deploys a rapid injury response dating back to the last common animal ancestor

Regenerative potential is widespread but unevenly distributed across animals. However, our understanding of the molecular mechanisms underlying regenerative processes is limited to a handful of model organisms, restricting robust comparative analyses. Here, we conduct a time course of RNA-seq during whole body regeneration in Mnemiopsis leidyi (Ctenophora) to uncover gene expression changes that correspond with key events during the regenerative timeline of this species. We identified several genes highly enriched in this dataset beginning as early as 10 minutes after surgical bisection including transcription factors in the early timepoints, peptidases in the middle timepoints, and cytoskeletal genes in the later timepoints. We validated the expression of early response transcription factors by whole mount in situ hybridization, showing that these genes exhibited high expression in tissues surrounding the wound site. These genes exhibit a pattern of transient upregulation as seen in a variety of other organisms, suggesting that they may be initiators of an ancient gene regulatory network linking wound healing to the initiation of a regenerative response.

Leukemic Driver Clones Can Arise Independently and Outcompete MPN Cells in Post-MPN Secondary AML

The JAK2V617F variant is an acquired somatic mutation recurrently found in myeloproliferative neoplasm (MPN) patients and is used as a diagnostic marker. While considered a strong MPN driver mutation, this variant is sometimes absent in secondary acute myeloid leukemia (sAML) samples which transform from antecedent JAK2-mutant MPNs. The transformative evolution of MPN to sAML is thought to be driven by acquisition of additional genetic mutations affecting epigenetic modifiers (e.g. TET2), tumor suppressor genes (e.g. TP53), and splicing regulation, but the contribution and sequence of onset for these specific genetic events is not well understood. It is currently unknown why or how patients with a JAK2-mutant MPN may develop a JAK2 wild-type sAML. This transformation sequence is coupled with very limited treatment options and a survival of less than 6-months. Elucidating the evolutionary trajectory of secondary leukemic mutation occurrence is paramount for disease monitoring and early detection. To understand this evolutionary trajectory, we employed single cell proteogenomic analysis for three separate paired MPN patient samples (2 MF; 1 PV) who progressed to sAML stage. Clinical sequencing data indicated presence of the JAK2 V617F mutation in all three samples at the MPN stage whereas this mutation was not detected at the sAML stage. We employed the MissionBio Tapestri platform to delineate the clonal trajectory of transformation to determine if sAML and MPN driver clones share a common founder clone or if they arise through independent, non-clonally related founders. Utilization of this single-cell platform enabled us to investigate two potential models of transformation. First, two distinct mutation phases of MPN to sAML occur wherein the JAK2 -mutant clone and sAML driver mutations originate form a shared founder clone. In this model, an MPN phenotype driven by JAK2-mutant clones arises in patients first, followed by the sAML disease stage driven by leukemic mutations. An alternative tested model is that MPN and sAML originate as separate clones wherein the JAK2 mutant hematopoietic stem cells (HSCs) give rise to an MPN phenotype first, while the independent sAML clone(s) grow slowly yet separately in the background of the existing MPN. Both models provide a plausible explanation which results in leukemic blast stage clones harboring leukemic mutations but lacking the MPN driving mutation JAK2V617F. Single-cell analysis detected the JAK2V617F mutation in both the MPN (MF) as well as the sAML stage in one of the paired patient samples in which JAK2 was previously reported as wild-type at the sAML stage. This sample also contained a RUNX1 mutation at the sAML stage as well as a clonally unrelated ATM mutation (Figure 1A). The single-cell data for the remaining two paired patient samples (1MF; 1PV) demonstrated that pre-leukemic driver mutations were not detected within the JAK2-mutant clones at the MPN stage or during a middle time-point stage before AML diagnosis. Single-cell sequencing from the sAML time point for both samples demonstrated loss of the JAK2-mutant clone (verified by ddPCR analysis), amidst presence of leukemic driver mutant clones (Figure 1B). This clonal hierarchy suggests that the leukemic mutations were not present within the JAK2-mutant clones during the MPN stage and arose in the background of the MPN where they outcompeted the MPN driver clone during sAML transformation. Further, this hierarchy suggests that the AML driver mutations do not share a clonal intermediate ancestor with the JAK2 mutant cells which drove the MPN. These data demonstrate that one clonal trajectory of JAK2-mutant MPN to JAK2 wild-type sAML is through independent leukemic clones arising in the background of MPN and outcompeting the JAK2-mutant clones. In these instances, it is more appropriate to view these patients as de novo AML cases arising in a background of pre-existing MPN, which could influence treatment decisions and disease surveillance.

A Non-Randomized Trial Investigating the Impact of Brown Rice Consumption on Gut Microbiota, Attention, and Short-Term Working Memory in Thai School-Aged Children.

While dietary fiber has been shown to influence the composition of gut microbiota and cognitive function in adults, much less is known about the fiber-microbiome-cognition association in children. We profiled gut microbiota using quantitative PCR (qPCR) and evaluated cognitive function using the Corsi block-tapping test (CBT) and the psychomotor vigilance test (PVT) before, during, and after the dietary intervention of 127 school-aged children in northern Thailand. While we found that Sinlek rice (SLR) consumption did not significantly alter the abundance of gut microbiota or the cognitive performance of school-aged children, we did find age to be associated with variations in both the gut microbiota profiles and cognitive outcomes. Gammaproteobacteria was significantly lower in the control and SLR groups during the middle time points of both phases (Weeks 4 and 61), and its abundance was associated with age. Cognitive performance using CBT and PVT were also found to be age-sensitive, as older children outperformed younger children on both of these cognitive assessments. Finally, a multiple factor analysis (MFA) revealed that age and cognitive performance best explain individual variation in this study. Collectively, these findings further describe the influence of host variables on the microbial profiles and cognitive outcomes of school-aged children consuming Sinlek rice in Thailand.

Patient reported outcomes for quality of life (QOL) by Expanded Prostate Cancer Index (EPIC) on average 15 years post treatment

Objective/purposePreviously patient reported quality of life (QOL) was reported in men with prostate cancer a mean 2 and 6 years post treatment with open radical prostatectomy (RP), 3D conformal radiation therapy (3D CRT), or 125I low dose rate (LDR) brachytherapy (BT). Herein we update the results 15 years post-treatment QOL. Materials/methodsThe Expanded Prostate Cancer Index (EPIC) domains were scored with differences evaluated at a median 15.8 years follow up based upon mean EPIC summary domains by ANOVA with pairwise post-hoc comparisons adjusted for age. Patient differences of current survey from first cross-section are reported as median change in summary score for each treatment group at median of 2.2 and 6.0, and 15.8 years. ResultsAmong men still alive response rate was 52% in BT, 60% in 3D CRT, and 62% in RP resulting in 30, 41, and 330 QOL questionnaires to evaluate for each corresponding modality at median follow up of 15.8 years. Men were a mean 75.3, 83.6, and 79.3 years of age after RP, 3DCRT, and BT, respectively.At a median of 15.8 years, there were largely persistent differences in EPIC domains without substantial evolution in QoL from middle time points. Persistent worsening in urinary irritative and bowel domain with 3DRT or BT compared to RP. Trend towards worse urinary incontinence with RP were noted without statistical differences within radiotherapy options. ConclusionAs the EPIC patient reported outcomes with the longest follow-up, these data uniquely reveal temporal trends from 2 to 15 years post treatment. However, the treatment modalities of open RP, 3D CRT without image guidance or intensity modulation, and BT without peripheral loading or MRI guidance may not reflect modern techniques.

Eating Earlier and More Frequently Is Associated With Better Diet Quality in Female Brazilian Breast Cancer Survivors Using Tamoxifen

BackgroundEmerging literature demonstrates that eating time and frequency are associated with quality and quantity of food consumption and anthropometric measurements. Considering that unhealthy dietary choices and obesity are important modifiable risk factors for breast cancer incidence and recurrence, this subject is relevant and has not been studied sufficiently in breast cancer survivors. ObjectiveThis study’s aim was to examine the association of eating time and frequency with diet quality, quantity of food consumption, anthropometric measurements, and body composition parameters in female breast cancer survivors using tamoxifen. DesignThis was a cross-sectional study. Participants/settingThis study was conducted from March 2015 to March 2016 at a Brazilian university hospital (Clinic’s Hospital, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil) and included an assessment of 84 female breast cancer survivors using tamoxifen (mean [SD] age was 53.1 [8.7] years). Main outcome measuresQuantitative dietary assessment consisted of three 24-hour dietary recalls. The Brazilian Healthy Eating Index Revised was used for the qualitative diet analysis. Participants were classified by median eating time (early or late eaters of breakfast, lunch, and dinner), as well as by considering the middle time point between the first and last meal of the day (early or late midpoint eaters). Participants were also classified by median eating frequency (<5 or ≥5 eating episodes per day). Anthropometric measurements and body fat percentage by bioelectrical impedance were obtained. Statistical analysisGeneralized linear models and generalized mixed models were used to assess the associations between variables. ResultsEarly breakfast and dinner eating and early midpoint eating were associated with better scores for specific Brazilian Healthy Eating Index Revised components (P < .05). Early breakfast and dinner eating were also associated with better scores for the total index (P = .035 and P = .017, respectively). Early dinner eaters and early midpoint eaters had significantly lower daily energy consumption (P = .007 and P = .002, respectively). Eating ≥5 episodes per day was also associated with better scores of specific Brazilian Healthy Eating Index Revised components and the total index (P < .05). No significant associations between eating time and frequency with anthropometric measurements and body composition parameters were found (P > .05). However, women in the healthy body mass index category vs women in the overweight/obesity category had higher energy consumption at breakfast (P = .046). ConclusionsEarlier food intake time was associated with better diet quality and lower daily energy consumption. Higher frequency of eating was also associated with better diet quality. Future studies, such as randomized controlled trials, are needed to evaluate interventions addressing the timing and frequency of meals and their effect on diet quality and quantity in breast cancer survivors.

Water-Based Training Programs Improve Functional Capacity, Cognitive and Hemodynamic Outcomes? The ACTIVE Randomized Clinical Trial

ABSTRACT Purpose: To compare the effects of 16 weeks of aerobic training (AT) to 8 weeks of AT followed by 8 weeks of combined training (CT), as well as to a control group (CG), on the functional, cognitive and hemodynamic responses of older women exercising in the aquatic environment. Materials and Methods: The study is a three-armed parallel randomized controlled clinical trial. For this, 52 older women were first randomized in an aerobic training (AT; n = 35) or control group (CG; n = 17). After 8 weeks of intervention, participants from AT group were again randomized into another 8-week period of either AT (n = 17) or combined training (CT; n = 18). AT was performed at the percentage of the heart rate corresponding to the anaerobic threshold, resistance training was performed with sets at maximal effort and the control group performed low-intensity therapeutic exercises in water. All outcomes were assessed before (week 0) and after the intervention (week 17). In addition, some outcomes were also investigated in the middle timepoint (week 9). Generalized Estimating Equations (GEE) and Bonferroni post-hoc tests (α = 0.05) were used to compare timepoints and groups. Results: All groups similarly improved functional capacity (3–11%) and blood pressure (−4 to −10%), whereas cognitive function was not modified for any group. Conclusion: Water-based training programs and therapeutic exercises (i.e., CG) improve functional capacity and blood pressure responses in older women. Clinical Trials NCT03892278.

Efficient diffeomorphic metric image registration via stationary velocity

Diffeomorphic image registration is a fundamental tool for MRI analysis, and fast growing image data demand highly efficient registration methods. Stationary velocity based method has much faster speed than non-stationary one in producing diffeomorphism, but how to achieve validity for large deformation while keeping its efficiency remains to be solved. To this end, we have proposed a new simplified model for optimal stationary velocity by representing temporal integration form of transformation variation via a single point model, e.g. the middle time point, according to mean value theorem and smoothness of transformation. This model can maintain the same registration accuracy as integral model but reduce the time cost a lot. It also shows as a better strategy than first order approximation model for large deformation mapping. Comparative study has also been conducted between this method and non-stationary approach on both synthesized and real brain images, and this approach demonstrated comparable whole brain mapping accuracy with much faster speed. Results showed efficacy of this approach in reducing complexity of stationary velocity based diffeomorphic registration while achieving validity for large deformation.

Global coherence of GPS-measured high-frequency surface tremor motions

The traditional approach of GPS investigations is determining trends which are connected with the motion of tectonic plates. At the same time, a global GPS network provides the possibility of investigating statistical properties of high-frequency earth surface tremor in different parts of the world. Based on the results of coherence and correlation analysis of noise components of daily three-component GPS time series, representing measurements of earth surface displacements at 1097 stations, we have found that, during 2010–2011, there was a significant increase in the average level of noise coherence or correlation with dominant periods 7–9 days of surface tremor in nine regions of the earth, and in some of these regions, the average level of coherence or correlation is still high and does not return to the previous level. The increase of the average level of coherence and correlation could be detected on the graphs purely visually, while the middle time point of the time interval in which the ascending occurred is detected more precisely by a formal method based on the use of the Fisher’s ratio.

Intrafractional Motion of Target Volumes and Organs at Risk Due to Bladder Filling: Implications for MR-Only Prostate Radiation Therapy

As magnetic resonance simulation (MR-SIM) emerges as a primary treatment planning modality for prostate cancer, it becomes critical to quantify the uncertainties introduced in an MR-only workflow. Given the long image acquisition times encountered in MRI, we sought to characterize the temporal and spatial intra-fractional changes between the prostate, seminal vesicles (SVs), and other organs at risk in response to bladder filling conditions. Serial T2-weighted MR-SIM data (3-6 timepoints/subject at 1.0T, 1.5T, or 3.0T, 38 evaluable timepoints) were acquired in 10 immobilized subjects using a fixed bladder filling protocol (bladder void, 20 oz water consumed, 10 oz consumed mid-session). Delineations were performed by one physician including prostate, SVs, and organs at risk (bladder, rectum, penile bulb) to yield 370 evaluable contours. Bony rigid registration (3 degrees of freedom) was performed for serial T2 data to isolate local organ effects. To quantify the impact of systematic bladder filling, temporal assessments of the volumes, center of mass, and Dice similarity coefficients (DSCs) were performed. To assess serial target displacements, centroid differences between the prostate and proximal SVs were compared across timepoints. Subjects had an average bladder volume difference of 62 ± 15% between initial and final timepoints. When compared to the initial prostate volume, mean DSCs of the prostate went from 0.81 ± 0.07 at the middle timepoint to 0.75 ± 0.07 at the final. A large reduction in DSC was observed for the proximal SVs (0.44 ± 0.22 at the middle time point vs. 0.32 ± 0.17 for the final). Overall, the median vector displacement of the penile bulb was 2.2 mm. After excluding a subject with an extreme case of rectal gas, the average rectum volume difference from the initial timepoint was 5.9 ± 10.3%. The prostate showed <1 mm lateral displacement and > 3mm in centroid shift in 6/10 cases (4 posteriorly and 2 anteriorly due to changes in rectal status). The SVs shifted posteriorly with bladder filling (7/10 cases were > 3mm). The centroid differences between the prostate and proximal SVs were >2mm in 4/10, 6/10, and 7/10 of subjects in the left-right, anterior-posterior, and superior-inferior directions, respectively, suggesting that they move independently with bladder status. As bladder filling increased, DSC decreased for the prostate, SVs, and OARs. In addition, large, systematic shifts were observed in the prostate and SVs (particularly anterior/posterior), although these were subject dependent due to confounders such as rectal status. This suggests that care must be taken during patient preparation to minimize these effects. Future work will incorporate dosimetric consequences using MR-only treatment planning in response to non-uniform organ displacement.

Particulate emissions from modern and old technology wood combustion induce distinct time-dependent patterns of toxicological responses in vitro

Toxicological characterisation of combustion emissions in vitro are often conducted with macrophage cell lines, and the majority of these experiments are based on responses measured at 24h after the exposure. The aim of this study was to investigate how significant role time course plays on toxicological endpoints that are commonly measured in vitro. The RAW264.7 macrophage cell line was exposed to PM1 samples (150μg/ml) from biomass combustion devices representing old and modern combustion technologies for 2, 4, 8, 12, 24 and 32h. After the exposure, cellular metabolic activity, cell membrane integrity, cellular DNA content, DNA damage and production of inflammatory markers were assessed. The present study revealed major differences in the time courses of the responses, statistical differences between the studied samples mostly limiting to differences between modern and old technology samples. Early stage responses consisted of disturbances in metabolic activity and cell membrane integrity. Middle time points revealed increases in chemokine production, whereas late-phase responses exhibited mostly increased DNA-damage, decreased membrane integrity and apoptotic activity. Altogether, these results implicate that the time point of measurement has to be considered carefully, when the toxicity of emission particles is characterised in in vitro study set-ups.

Cellular and Proteomic Characterization of the Innate Immune Response in Bat Stars (Patiria miniata)

Bat star populations on the west coast have been declining due to the Sea Star Wasting Syndrome. This tissue‐decaying epidemic involves a viral infection and skin lesions leading to disintegration in sea stars. The mechanisms causing decay in the symptomatic individuals are still unknown. We considered the immune response in bat stars after the appearance of skin lesions to determine how immune cells react and which proteins are expressed throughout the viral infection and tissue disintegration. We hypothesized that the viral infection is met with an increase in immune cell concentration and phagocytic activity, and an increase in the secretion of immune‐ and stress‐response related proteins into the coelomic fluid in activated defensive pathways. The morphology, behavior, and concentration of the immune cells per mL of coelomic fluid in healthy and wasting bat stars were compared by extracting coelomic fluid and staining nuclei, photographing the cells, and counting them. Immune cells were extracted at healthy and middle time points with no visible lesions, and at an endpoint once visible lesions were present; the cells were counted and incubated with fluorescent bacteria. Phagocytes, cells that engulf invading pathogenic cells, were found in higher cell concentrations after viral infection and the appearance of lesions, showing higher levels of activity and consuming more fluorescent bacteria. The reactions of the cells to these pathogens were observed in coelomic fluid from healthy and wasting sea stars. From the coelomic fluid samples taken throughout the progression of the disease in several individual sea stars, LC/MS/MS analysis and NCBI BLAST characterization of the proteins present showed dynamic abundance patterns in secreted proteins related to cell signaling, stress, and immune response. We are attempting a more thorough characterization of the proteins involved in the immune reaction to better define the defense mechanisms occurring in the bat stars during the response to the viral challenge. The changes in abundance of certain proteins may give clues about the patterns of disintegration occurring at the cellular level and the pathways involved in the tissue disintegration. We may also be able to identify novel antimicrobial or anti‐viral molecules of pathways, with potential for future use in tissue preservation.Support or Funding InformationCOAST Grant, CSULB 2015 Graduate Summer Research Fellowship.

Trisomy 12 CLLs progress through NOTCH1 mutations

Trisomy 12 CLLs progress through NOTCH1 mutations

Early Evaluation of Cancer Response by a New Functional Biomarker: Apparent Diffusion Coefficient

The purpose of this article is to investigate whether apparent diffusion coefficient (ADC) might be used as a universal biomarker for response evaluation in different tumors. Twenty-one patients with lung cancer, 12 patients with esophageal cancer, 19 patients with liver metastases, 24 patients with gastric cancer, and 26 patients with rectal cancer were recruited to the study. Percentage changes in the ADC and changes in the size of responding and nonresponding lesions of different tumors after treatment were analyzed using repeated measures analysis of variance. There was no significant difference among the percentage ADC changes of different tumors (F = 1.57; p = 0.192). Clear differences were seen in the percentage ADC changes between responding and nonresponding tumors (F = 21.62; p < 0.001), which were significant at every time point after the start of treatment (early time point, F = 19.75 and p < 0.001; middle time point, F = 11.23 and p = 0.001; and later time point, F = 15.98 and p < 0.001). The percentage size changes after treatment between responding and nonresponding tumors were significantly different (F = 19.38; p < 0.001). However, at the early time point after treatment, the difference was not statistically significant (F = 0.02; p = 0.894). The ADC changes correlated with treatment response in five types of body tumor but were independent of the tumor's location. Early increases in ADC during treatment indicate good response to treatment. ADC change is a promising biomarker for detecting therapeutic responses at an early stage that could be widely used.

Differential expression of genes associated with cell proliferation and apoptosis induced by okadaic acid during the transformation process of BALB/c 3T3 cells

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8–31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

Direct Observation of the Assembly of RecA/DNA Complexes by Atomic Force Microscopy

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATP γS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 × 10 −5 RecA min −1 bp −1. The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min −1. These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.

Steroids modulate transepithelial resistance and Na(+) absorption across cultured porcine vas deferens epithelia.

Epithelial cells were isolated from adult porcine vas deferens and grown in the absence or presence of steroid hormones. Transepithelial resistance (R(te)), basal short circuit current (I(sc)), and the effects of selected ion transport modulators on these parameters were evaluated in modified Ussing chambers at three time points (5-8, 11-14, and 18-22 days postseeding). At the earliest time point, no significant differences were observed. At the middle time point, when compared with R(te) in untreated control monolayers, R(te) in monolayers exposed to 17beta-estradiol, aldosterone, cortisol, cortisone, prednisolone, prednisone, and dexamethasone was significantly lower; in contrast, R(te) in monolayers exposed to testosterone, dihydrotestosterone, or progesterone did not differ from that in control monolayers. Treatments with cortisol, prednisolone, and dexamethasone were associated with an elevated basal I(sc) that was amiloride sensitive, indicating ongoing Na(+) absorption by these monolayers. R(te) was increased by amiloride treatment in glucocorticoid-treated monolayers but remained significantly less than that of control monolayers. At the third time point, the postamiloride R(te) of glucocorticoid-treated monolayers was not different from that of control monolayers. Responses to ATP, forskolin, bumetanide, and DASU-02 were not affected by steroid treatment at any time point. Taken together, these results suggest that estrogens and corticosteroids can modulate epithelial function in the distal excurrent duct of the adult male reproductive system. At physiological or pharmacological concentrations, these hormones would be expected to modify the luminal environment (both the ionic composition and pH) to which sperm are exposed and thus affect male fertility.

Neonatal damage to the rat's infraorbital nerve upregulates both galanin and neuropeptide Y in individual vibrissae-related primary afferent axons.

Previous studies in adult animals have suggested that the peptides galanin and neuropeptide Y (NPY) may be upregulated in the same primary afferent neurons after peripheral axotomy. The present study was undertaken to determine whether such upregulation occurred in vibrissae-related primary afferent neurons and their axons after damage to the infraorbital nerve [ION; the trigeminal (V) branch that innervates the vibrissae follicles]. Double-labelling experiments demonstrated that approximately 75% of axotomized V ganglion cells and the central arbors of vibrissae-related primary afferents expressed both galanin and NPY after perinatal, but not adult, nerve damage. However, additional experiments demonstrated that the sensitive periods for lesion-induced upregulation of the two peptides and the period over which they were expressed after neonatal ION transection differed substantially. Staining for both peptides was increased after ION damage on P-0 through P-14, but only galanin staining was increased in vibrissae-related primary afferents after lesions on P-21. Galanin expression was elevated in vibrissae-related primary afferents in rats killed 3, 8, and 15 days after neonatal ION transection, while increased NPY was observed at only the middle time point. The lesion-induced increases in galanin and NPY in vibrissae-related ION primary afferents suggest that these peptides may modulate central V reorganization after such damage.