Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199).

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma. In CLL, duvelisib monotherapy is associated with high iwCLL (International Workshop on Chronic Lymphocytic Leukemia) and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene expression studies (RNAseq, Nanostring, Affymetrix array and real-time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex vivo-cultured CLL cells obtained from patients on duvelisib compared with pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

Effectiveness of the photochemical reflectance index to track photosynthetic activity over a range of forest tree species and plant water statuses.

In this study, we investigated the potential of the photochemical resistance index (PRI) to track photosynthetic activity under water stress conditions by measuring PRI, leaf fluorescence, the xanthophyll cycle and photosynthetic activity in different forest tree species subjected to progressive drought. The PRI declined with pre-dawn water potential and a significant relationship between PRI and the xanthophyll de-epoxidation state (DEPS) was observed, although with large interspecific variability in the sensitivity of PRI to changes in DEPS. For single tree species, a strong relationship was observed on either PRI light saturated photosynthesis or PRI maximum photochemical efficiency of PSII (ΔF/Fm'); a larger variability in both relationships was apparent when data from different species were pooled together. However, an improved correlation was shown only in the former relationship by plotting the ΔPRI (dawn PRI minus the midday PRI values). Thus, we conclude that PRI is able to provide a good estimate of maximum CO2 assimilation at saturating light and ΔF/Fm' for single tree species, despite the severe drought conditions applied. PRI should be applied more cautiously when dealing with multispecific forests because of confounding factors such as the strong interspecific differences in the initial value of PRI and in the sensitivity of PRI to changes in DEPS in response to drought.

alpha-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate.

BACKGROUND: A strategy to reduce the secondary effects of anti-cancer agents is to potentiate the therapeutic effect by their combination. A combination of vitamin K3 (VK3) and ascorbic acid (AA) exhibited an anti-cancer synergistic effect, associated with extracellular production of H(2)O(2) that promoted cell death. METHODS: The redox-silent vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) was used in combination with VK3 and AA to evaluate their effect on prostate cancer cells. RESULTS: Prostate cancer cells were sensitive to alpha-TOS and VK3 treatment, but resistant to AA upto 3.2 mM. When combined, a synergistic effect was found for VK3-AA, whereas alpha-TOS-VK3 and alpha-TOS-AA combination showed an antagonist and additive effect, respectively. However, sub-lethal doses of AA-VK3 combination combined with a sub-toxic dose of alpha-TOS showed to induce efficient cell death that resembles autoschizis. Associated with this cell demise, lipid peroxidation, DNA damage, cytoskeleton alteration, lysosomal-mitochondrial perturbation, and release of cytochrome c without caspase activation were observed. Inhibition of lysosomal proteases did not attenuate cell death induced by the combined agents. Furthermore, cell deaths by apoptosis and autoschizis were detected. CONCLUSION: These finding support the emerging idea that synergistic combinations of some agents can overcome toxicity and other side-effects associated with high doses of single drugs creating the opportunity for therapeutically relevant selectivity.

The interactive effect of hunger and impulsivity on food intake and purchase in a virtual supermarket.

OBJECTIVE: It has been shown repeatedly that impulsivity, obesity and food intake are related; obese people are more impulsive than lean people and impulsive people eat more than less impulsive people. The relation between impulsivity and food intake might be state dependent; hunger motivates food seeking behaviour and food consumption, especially of high caloric food. Difficulties to overrule automatic behavioural tendencies might make impulsive people more susceptible to the effects of hunger on food selection. Therefore, they are expected to increase their intake more than low impulsive people when feeling hungry. STUDY 1: Fifty-seven female participants were randomly assigned to a hunger or sated condition. Response inhibition (a measure of impulsivity) and food intake were measured. Results show that impulsive participants ate significantly more, but only when feeling hungry. STUDY 2: Ninety-four undergraduate students participated. Hunger, response inhibition and the purchase of food in a virtual supermarket were measured. The same interaction was found: impulsive participants bought most calories, especially from snack food, but only when feeling hungry. CONCLUSION: Hunger and impulsivity interact in their influence on consumption. These data suggest that reducing hunger during calorie restricting diets is important for successful weight loss, particularly for the impulsive dieters.

Dielectrophoretic trapping in microwells for manipulation of single cells and small aggregates of particles.

In this work we present a novel concept of active microwells based on cylindrical wells able to vertically trap and control single particles by means of negative dielectrophoresis. The device is fabricated by drilling through holes on a polyimide substrate with copper-gold or aluminum metals, forming three annular electrodes within the well. A channel under the device provides a fluid flow filling the microwell by capillarity. Particles are delivered from the top by a microdispenser and applying sinusoidal signals to the electrodes at frequencies ranging from 100kHz to 1.5MHz and amplitudes between 2V and 7V they are successfully trapped and levitated at the level of the central electrode in the middle of microwells with a diameter of 125mum. By changing signal phases, other configurations are also enabled to load particles in the well or eject them from the bottom. The extension to an array of microwells is presented and design rules are described for routing electrode connections and setting signal parameters. K562 cells cultured with Ara-C 1000nM were successfully trapped and controlled in physiological media. Polystyrene beads were also levitated in water and were used for experimental measurements on minimum amplitudes and phase differences in the signals required to levitate beads, confirming the results obtained by simulation.

The interaction between impulsivity and a varied food environment: its influence on food intake and overweight.

OBJECTIVE: The current study tests the influence of two factors, the obesogenic environment and impulsivity, on food intake in primary school children. Our current food environment offers a large variety of cheap and easily available sweet and fatty foods. This obesogenic environment is believed to be a cause of the recent obesity epidemic. Impulsive people are generally less successful at inhibiting prepotent responses and they are reward sensitive. We investigate whether the interaction between an obesogenic environment and an impulsive person leads to overeating. DESIGN: A quasi-experimental 2 (reward sensitive versus not reward sensitive) by 2 (successful response inhibitors versus unsuccessful response inhibitors) by 2 (monotonous versus varied food environment) between-subjects design with caloric intake during a taste test as the main dependent variable. The link between impulsivity and overweight was also examined. SUBJECTS: 78 healthy primary school children (age: 8-10 years). MEASUREMENTS: We measured two aspects of impulsivity: reward sensitivity and deficient response inhibition. Subsequently, one aspect of the obesogenic environment was manipulated; half of the participants received monotonous food during a bogus taste test whereas the other half tasted food that was varied in colour, form, taste and texture. RESULTS: As expected, reward sensitivity interacted with variety. In the monotony group there was no difference in food intake between the less and more reward-sensitive children (183 kcal+/-23 s.d. versus 180 kcal+/-21 s.d.). However, in the variety group the more reward-sensitive children ingested significantly more calories than the less reward-sensitive children (237 kcal+/-30 s.d. versus 141 kcal+/-19 s.d.). Reward sensitivity was not linked to overweight. Deficient response inhibition did not interact with variety, but it was linked to overweight. CONCLUSION: It is suggested that reward sensitivity could be a causal mechanism for overeating in an obesogenic environment whereas prepotent response inhibition may be a maintaining factor of the problem of overeating.

Microfluidic channel fabrication in dry film resist for production and prototyping of hybrid chips.

Microfluidic networks are patterned in a dry film resist (Ordyl SY300/550) that is sandwiched in between two substrates. The technique enables fabrication of complex biochips with active elements both in the bottom and the top substrate (hybrid chips). The resist can be double bonded at relatively low temperatures without the use of extra adhesives. A postbake transfers the resist into a rigid structure. The resist is qualified in terms of resolution, biocompatibility and fluidic sealing. Fabrication in both a fully equipped cleanroom setting as well as a minimally equipped laboratory is described. The technique is applied for dielectrophoresis-based cell separation systems and a fuel cell reaction chamber with micropillars. The dry film resist can be considered a cheap and fast alternative to SU-8.

Levitation and movement of human tumor cells using a printed circuit board device based on software-controlled dielectrophoresis.

In this study we describe an original, efficient, and innovative printed circuit board (PCB) device able to generate dielectrophoresis-based, software-controlled cages that can be moved to any place inside a microchamber. Depending on their dielectrophoretic properties, eukaryotic cells can be "entrapped" in cages and moved under software control. The main conclusion gathered from the experimental data reported is that the PCB device based on dielectrophoresis permits levitation and movement of different tumor cells at different dielectrophoresis conditions. The results presented herein are therefore the basis for experiments aimed at forced interactions or separation of eukaryotic cells using "lab-on-a-chip." In fact, because many cages can be controlled at the same time, and two or more cages can be forced to share the same or a different location, it is possible, in principle, either to bring in contact cells of a differing histotype or to separate them.

Validation studies for the genetic typing of the D1S80 locus for implementation into forensic casework.

A series of validation experiments were designed to evaluate, according to the Technical Working Group on DNA Analysis Methods (TWGDAM) guidelines, the analysis of the D1S80 locus for casework implementation. Approximately 400 samples from three different populations (Minnesota Caucasian, Minnesota African Americans, and Minnesota Native Americans) were typed to determine allele frequencies. Simulated forensic type specimens (blood, saliva, hair and semen, or vaginal secretions) were typed to demonstrate that deoxyribonucleic acid (DNA) extracted from various tissues of an individual yield the same D1S80 type. Dilution studies were performed and it was determined that a wide range of input DNA (0.5 ng to 40.0 ng) will consistently yield typeable results. The evaluation of DNA from various animals showed that the D1S80 locus is specific to human DNA within the limits of the parameters tested. The reproducibility of the system was tested by duplicate analysis of approximately 200 population samples. Duplicate samples were analyzed on both horizontal and vertical gel systems. In addition, simulated forensic specimens were analyzed by two independent laboratories: the Minnesota Forensic Science Laboratory (MFSL) and the Roche Biomedical Laboratories (RBL). All analyses, including extraction, quantitation, amplification and typing, were performed independently. All typing results for both laboratories were in agreement. By the analysis of mixtures from various simulated casework type mixtures, it was demonstrated that the D1S80 typing system is suitable for analyzing mixtures. In addition to the simulated casework, evidentiary samples from several adjudicated cases previously analyzed by restriction fragment length polymorphism (RFLP) analysis and/or DQA1 were typed at the D1S80 locus. The D1S80 results were consistent with previous RFLP and/or DQA1 results regarding inclusions/exclusions.

HLA DQA1 and Polymarker validations for forensic casework: standard specimens, reproducibility, and mixed specimens.

This study describes the testing performed by the Minnesota Forensic Science Laboratory (MFSL) to validate the Amplitype DQA1 and Amplitype Polymarker (PM) PCR Amplification and Typing Kits before implementation for casework. All studies were based on the analysis of mock forensic case samples, which were assembled from various biological samples from individuals at the MFSL. To address the validation of standard specimens, DNA was isolated from semen, vaginal secretions, saliva, urine, and blood samples. Typing results from all tissues from a particular individual yielded the same typing results using both the DQA1 and PM systems. Reproducibility between laboratories was evaluated by having duplicate samples analyzed by a second laboratory. The Roche Biomedical Laboratories (RBL) were sent a duplicate set of mock cases and all analyses including extraction, quantitation, amplification, and typing were performed at the RBL using their established testing procedures. All typing results for both laboratories, from the approximate 30 single source samples analyzed, were in agreement. Mixed specimens were evaluated by examining the results obtained from semen/vaginal, semen/saliva, semen/blood, semen/ urine, and semen/vaginal/blood mixtures. All typing results of these mixtures for both laboratories were in agreement. It was determined that by incorporating a wash step of the sperm cell pellet, a complete separation of the nonsperm cell fraction was more likely to be attained. After completing the above studies, as well as population studies, environmental insult studies, and proficiency testing, the MFSL determined that both kits were suitable for use on forensic casework.

Isolated left ventricular filling abnormalities may predict interleukin-2-induced cardiovascular toxicity.

Interleukin-2 (IL-2) is a cytokine with proven activity against metastatic renal cell carcinoma (RCC) and malignant melanoma (MM). The intravenous administration of high-dose IL-2 is limited by important cardiovascular side effects such as hypotension, fluid retention, arrhythmias, and myocardial ischemia, which often cause dose reduction and/or treatment withdrawal. The occurrence of these toxic events is not predicted by routine pretreatment examinations. The aim of the present study was to test the reliability of serial echocardiography in predicting subsequent cardiac adverse effects in patients undergoing IL-2 administration. In 19 patients (15 men, 4 women; median age: 51 years, range 27-71 years; 10 affected by metastatic RCC and 9 affected by MM) we performed two-dimensional and Doppler echocardiography before and immediately after 28 continuous intravenous infusions (CIVI) of IL-2 at the dose of 18 MIU/m2/day for 4 days. Left ventricular systolic function and the diastolic transmitral flow pattern were assessed before and after IL-2 administration. Significant changes of two indexes of left ventricular filling were noted: a decrease of the ratio of maximal flow velocity in early diastole to that in late diastole (E/A) (basal: 1.12 +/- 0.46, mean +/- SD; posttreatment: 0.83 +/- 0.27; p < 0.01) and an increase of the percentage of the atrial contribution to left ventricular filling (basal: 37.75 +/- 11.58%; posttreatment: 49.43 +/- 16.48%; p < 0.01). Eight major cardiovascular events causing IL-2 infusion withdrawal were observed (two ischemic electrocardiographic modifications, three grade III-IV hypotension, one atrial fibrillation, one pericardial effusion, one acute heart failure). These major cardiovascular events were observed more often when an abnormal basal E/A ratio < 1.0 (p < 0.05) was found. We conclude that Doppler transmitral flow pattern analysis before and subsequent to IL-2 infusion is a useful and easily available procedure for the monitoring of cardiac modifications during CIVI IL-2 administration. It might also predict a major cardiovascular event during IL-2 administration. Patients with basal E/A ratio < 1.0 should be more carefully monitored during treatment and/or should be treated with lower IL-2 doses to avoid cardiovascular toxicity.

Highly constrained neural networks for industrial quality control.

In this work we investigate techniques for embedding domain-specific spatial invariances into highly-constrained neural networks. This information is used to drastically reduce the number of weights which have to be determined during the learning phase, thus allowing us to apply artificial neural networks to problems characterized by a relatively small number of available examples. As an application of the proposed methodology, we study the problem of optical inspection of machined parts. More specifically, we have characterized the performance of a network created according to this strategy, which accepts images of parts under inspection at its input and issues a flag at its output which states whether the part is defective. The results obtained so far show that the proposed methodology provides a potentially relevant approach for the quality control of industrial parts, as it offers both accuracy and short software development time, when compared with a classifier implemented using a standard approach.

The feasibility of repetitive courses of high-dose continuous intravenous infusion interleukin-2 and subcutaneous alpha-interferon with polychemotherapy in advanced malignant melanoma.

AIMS AND BACKGROUND: A Phase I study of repetitive courses of chemotherapy (carmustine, cis-platinum, dacarbazine) and immunotherapy (continuous intravenous infusion recombinant interleukin-2 (rIL-2) and subcutaneous (sc) alpha-interferon 2b) plus tamoxifen was performed in order to establish a more efficacious way to sequence this kind of treatment for advanced malignant melanoma. STUDY DESIGN: Patients who had measurable metastatic melanoma, a Karnofsky performance status > or = 80, and no clinically significant hematologic or cardiac disfunction were considered eligible. Treatment consisted of BCNU, 150 mg/m2 i.v. day 1 in alternating cycles; DTIC, 220 mg/m2 i.v. days 1, 2 and 3; CDDP, 25 mg/m2 i.v. days 1, 2 and 3; tamoxifen 10 mg twice/day per os continuously; rIL-2, 18 x 10(6) IU/m2/day continuous i.v. infusion days 5-8 (96 h) and days 19-22 (96 h); alpha-interferon (IFN) s.c. 3 x 10(6) U day 12, 6 x 10(6) U day 14, 9 x 10(6) U days 16, 19, 21, 23, 26, and 28 (from cycle 2, 9 x 10(6) U days 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26, and 28). Two consecutive cycles were planned until response evaluation. RESULTS: Three patients were treated according with the protocol; none of them was able to respect the planned dose-intensity schedule. The given dose intensity/planned dose intensity ratios were as follows: DTIC, 0.74 (range, 0.70-0.80); CDDP, 0.77 (0.72-0.80); BCNU, 0.77 (0.72-0.80); rIL-2, 0.65 (0.36-0.80); alpha-IFN, 0.01 (0-0.03); tamoxifen, 1.0. Systemic side effects of rIL-2 and myelotoxicity were the main reasons for treatment delay and/or dose-reduction, and for the long period of hospital care. CONCLUSIONS: We conclude that the treatment schedule is not feasible. However, since we believe that combined chemo-immunotherapy is a potentially active treatment in metastatic malignant melanoma, we have modified it in order to make it more feasible and consequently efficacious.

[Incidence and clinical aspects of Meckel's diverticulum and its complications]. / Frequenza ed aspetti clinici del diverticolo di Meckel e sue complicanze.

Meckel's diverticulum is usually discovered at laparatomy and the AA. ful that it should be removed regardless of its pathology because of the difficulty in making a diagnosis and the significant mortality rate associated with its complications.