Scale-free vertical tracking microscopy.

The behavior and microscale processes associated with freely suspended organisms, along with sinking particles underlie key ecological processes in the ocean. Mechanistically studying such multiscale processes in the laboratory presents a considerable challenge for microscopy: how to measure single cells at microscale resolution, while allowing them to freely move hundreds of meters in the vertical direction? Here we present a solution in the form of a scale-free, vertical tracking microscope, based on a 'hydrodynamic treadmill' with no bounds for motion along the axis of gravity. Using this method to bridge spatial scales, we assembled a multiscale behavioral dataset of nonadherent planktonic cells and organisms. Furthermore, we demonstrate a 'virtual-reality system for single cells', wherein cell behavior directly controls its ambient environmental parameters, enabling quantitative behavioral assays. Our method and results exemplify a new paradigm of multiscale measurement, wherein one can observe and probe macroscale and ecologically relevant phenomena at microscale resolution. Beyond the marine context, we foresee that our method will allow biological measurements of cells and organisms in a suspended state by freeing them from the confines of the coverslip.

Adjuvant hormonal therapy for early breast cancer: an epidemiologic study of medication adherence.

PURPOSE: The aim of this study was to determine the prevalence of adherence to adjuvant hormonal therapy (AHT) and to identify risk factors for medication non-adherence in clinical practice in patients with early-stage hormone receptor (HR)-positive breast cancer (BC) previously treated with chemotherapy. METHODS: We carried out a cross-sectional, observational, prospective, and multicenter survey based on a structured self-report postal questionnaire (35 items investigating six areas). A sample of 474 patients was drawn from 676 patients potentially eligible. The structured and validated Morisky Medication Adherence Scale-4 items was used for measuring medication adherence. An analysis of risk factors for non-adherence to AHT was performed using a two-step approach: univariate, then multivariate analysis. RESULTS: A total of 280 patients out of the 428 analyzed patients participated in the survey, yielding a response rate of 65.4% [60.9-69.9]. The prevalence of adherence to AHT was estimated at 68.6% [63.1-74.0], corresponding to a high level of adherence. Three risk factors for non-adherence to AHT were identified: > 2 medications to treat comorbidities (p-value = 0.003), age less than 65 years (p-value = 0.008), and patient management in a university hospital setting (p-value = 0.014). CONCLUSIONS: Non-adherence is a common, complex, and multidimensional healthcare problem. This better understanding and knowledge of risk factors will allow healthcare providers (such as oncologists, general practitioners, pharmacists) to more easily identify patients at risk for non-adherence and help them provide appropriate information about AHT and its management, thus improving medication adherence in their patients.

Post-mortem determination of insulin using chemiluminescence enzyme immunoassay: preliminary results.

Insulin determination in blood sampled during post-mortem investigation has been repeatedly asserted as being of little diagnostic value due to the rapid occurrence of decompositional changes and blood haemolysis. In this study, we assessed the feasibility of insulin determination in post-mortem serum, vitreous humour, bile, and cerebrospinal and pericardial fluids in one case of fatal insulin self-administration and a series of 40 control cases (diabetics and non-diabetics) using a chemiluminescence enzyme immunoassay. In the case of suicide by insulin self-administration, insulin concentrations in pericardial fluid and bile were higher than blood clinical reference values, though lower than post-mortem serum concentration. Insulin concentrations in vitreous (11.50 mU/L) and cerebrospinal fluid (17.30 mU/L) were lower than blood clinical reference values. Vitreous insulin concentrations in non-diabetic control cases were lower than the estimated detection limit of the method. These preliminary results tend to confirm the usefulness of insulin determination in vitreous humour in situations of suspected fatal insulin administration. Additional findings pertaining to insulin determination in bile, pericardial, and cerebrospinal fluid would suggest that analysis performed in post-mortem serum and injection sites could be complemented, in individual cases, by investigations carried out in alternative biological fluids. Lastly, these results would indicate that analysis with chemiluminescence enzyme immunoassay may provide suitable data, similar to analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoradiometric assay, to support the hypothesis of insulin overdose.

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione.

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) was investigated. RIA overestimated concentrations compared to LC-MS/MS on 59 clinical samples (RIA=1.79×LC-MS/MS+0.94). RIA kit and LC-MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA=1.35×LC-MS/MS-0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC-MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC-MS/MS calibrant material (SHBG, 31±2 and 38±2nmol/l; pH, 8.27±0.18 and 8.66±0.03, respectively). No correlation was observed between androstenedione RIA and LC-MS/MS discrepancy and lipid or protein. LC-MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99-108%). The corresponding RIA results overestimated androstenedione by 52-174% compared to LC-MS/MS. The results here demonstrate that LC-MS/MS is the more accurate method.

Phosphorylation of p27(KIP1) homologs KRP6 and 7 by SNF1-related protein kinase-1 links plant energy homeostasis and cell proliferation.

SNF1-related protein kinase-1 (SnRK1), the plant kinase homolog of mammalian AMP-activated protein kinase (AMPK), is a sensor that maintains cellular energy homeostasis via control of anabolism/catabolism balance. AMPK-dependent phosphorylation of p27(KIP1) affects cell-cycle progression, autophagy and apoptosis. Here, we show that SnRK1 phosphorylates the Arabidopsis thaliana cyclin-dependent kinase inhibitor p27(KIP1) homologs AtKRP6 and AtKRP7, thus extending the role of this kinase to regulation of cell-cycle progression. AtKRP6 and 7 were phosphorylated in vitro by a recombinant activated catalytic subunit of SnRK1 (AtSnRK1α1). Tandem mass spectrometry and site-specific mutagenesis identified Thr152 and Thr151 as the phosphorylated residues on AtKRP6- and AtKRP7, respectively. AtSnRK1 physically interacts with AtKRP6 in the nucleus of transformed BY-2 tobacco protoplasts, but, in contrast to mammals, the AtKRP6 Thr152 phosphorylation state alone did not modify its nuclear localization. Using a heterologous yeast system, consisting of a cdc28 yeast mutant complemented by A. thaliana CDKA;1, cell proliferation was shown to be abolished by AtKRP6(WT) and by the non-phosphorylatable form AtKRP6(T152A) , but not by the phosphorylation-mimetic form AtKRP6(T152D). Moreover, A. thaliana SnRK1α1/KRP6 double over-expressor plants showed an attenuated AtKRP6-associated phenotype (strongly serrated leaves and inability to undergo callogenesis). Furthermore, this severe phenotype was not observed in AtKRP6(T152D) over-expressor plants. Overall, these results establish that the energy sensor AtSnRK1 plays a cardinal role in the control of cell proliferation in A. thaliana plants through inhibition of AtKRP6 biological function by phosphorylation.

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Androgen dependence of hirsutism, acne, and alopecia in women: retrospective analysis of 228 patients investigated for hyperandrogenism.

Hirsutism, acne, alopecia, and oligo-amenorrhea are clinical expressions of hyperandrogenism, one of the most frequent endocrine disorders in women of reproductive age. Women referred to our endocrine clinics for skin symptoms of hyperandrogenism underwent a laboratory workup to evaluate hormone measurements and received antiandrogen therapy. We retrospectively analyzed the outcome of 228 consecutive patients investigated over 6 years.Patients with hirsutism had higher levels of androstenedione, dehydroepiandrosterone sulfate (DHEAS), and salivary testosterone; lower levels of sex hormone-binding globulin (SHBG); and a higher prevalence of oligo-amenorrhea than patients with alopecia, while patients with acne showed intermediate values. Hirsutism score correlated positively with androstenedione, DHEAS, and salivary testosterone, and correlated negatively with SHBG; salivary testosterone showed the highest correlation coefficient. Total testosterone was not significantly different among patients with hirsutism, alopecia, or acne, and did not significantly correlate with hirsutism score. Hirsutism and oligo-amenorrhea were the most sensitive symptoms of hyperandrogenism, and no androgenic parameter alone allowed us to identify all cases of hyperandrogenism.Patients of central European origin sought consultation with milder hirsutism scores than patients of southern European origin. There was, however, no difference in the clinical-biological correlation between these groups, arguing against differences in skin sensitivity to androgens.Polycystic ovary syndrome, defined as hyperandrogenism (hirsutism or elevated androgens) and oligo-amenorrhea, was diagnosed in 63 patients (27.6%), an underestimate compared with other reports that include systematic ovarian ultrasound studies. Neither pelvic ultrasound, used in a limited number of cases, nor the luteinizing hormone/follicle-stimulating hormone ratio helped to distinguish patients with polycystic ovary syndrome from the other diagnostic groups. These included hyperandrogenism (hirsutism or elevated androgens) and eumenorrhea (101 patients; 44.3%); normal androgens (acne or alopecia and eumenorrhea) (51 patients; 22.4%); isolated low SHBG (7 patients; 3.1%); nonclassical congenital adrenal hyperplasia (4 patients; 1.8% of total, 4.9% of patients undergoing cosyntropin stimulation tests); and ovarian tumor (2 patients; 0.9%).Ethinylestradiol and high-dose cyproterone acetate treatment lowered the hirsutism score to 53.5% of baseline at 1 year, and was also effective in treating acne and alopecia. The clinical benefit is ascribed to the peripheral antiandrogenic effect of cyproterone acetate as well as the hormone-suppressive effect of this combination. Salivary testosterone showed the most marked proportional decrease of all the androgens under treatment. Cost-effectiveness and tolerance of ethinylestradiol and high-dose cyproterone acetate compared well with other antiandrogenic drug therapies for hirsutism. The less potent therapy with spironolactone only, a peripheral antiandrogen without hormone-suppressive effect, was effective in treating isolated alopecia in patients with normal androgens.