Rapid nested-PCR for tyrosinase gene detection on chip

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.     

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.     

Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16^INK4A, p18^INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16^INK4A, p18^INK4C, CDK4 and CDK6 expressions in physiological term villous explants.

PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16^INK4A, p18^INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay.

We reported significantly increased p16^INK4A and p18^INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16^INK4 and p18^INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast.

We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.     

Effects of Pleiotrophin Overexpression on Mouse Skeletal Muscles in Normal Loading and in Actual and Simulated Microgravity

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca²⁺ concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.     

Suitability of Solanum lycopersicum L. ‘Microtom’ for growth in Bioregenerative Life Support Systems: exploring the effect of high‐LET ionising radiation on photosynthesis,leaf structure and fruit traits

• The realisation of manned space exploration requires the development of Bioregenerative Life Support Systems (BLSS). In such self‐sufficient closed habitats, higher plants have a fundamental role in air regeneration, water recovery, food production and waste recycling. In the space environment, ionising radiation represents one of the main constraints to plant growth.
• In this study, we explore whether low doses of heavy ions, namely Ca 25 Gy, delivered at the seed stage, may induce positive outcomes on growth and functional traits in plants of Solanum lycopersicum L. ‘Microtom’. After irradiation of seed, plant growth was monitored during the whole plant life cycle, from germination to fruit ripening. Morphological parameters, photosynthetic efficiency, leaf anatomical functional traits and antioxidant production in leaves and fruits were analysed.
• Our data demonstrate that irradiation of seeds with 25 Gy Ca ions does not prevent achievement of the seed‐to‐seed cycle in ‘Microtom’, and induces a more compact plant size compared to the control. Plants germinated from irradiated seeds show better photochemical efficiency than controls, likely due to the higher amount of D1 protein and photosynthetic pigment content. Leaves of these plants also had smaller cells with a lower number of chloroplasts. The dose of 25 Gy Ca ions is also responsible for positive outcomes in fruits: although developing a lower number of berries, plants germinated from irradiated seeds produce larger berries, richer in carotenoids, ascorbic acid and anthocyanins than controls.
• These specific traits may be useful for ‘Microtom’ cultivation in BLSS in space, in so far as the crew members could benefit from fresh food richer in functional compounds that can be directly produced on board.

     

Cadmium-Resistance Plasmid Affected Cd+2 Uptake More Than Cd+2 Adsorption in Klebsiella oxytoca

A bacterial strain was isolated from Petra City Wastewater Treatment Plant. This isolate was identified as Klebsiella oxytoca based on 16S rDNA analysis. A single plasmid (> 23 kb) was detected in this strain and transformed into Esherichia coli JM83. The transformed E. coli cells exhibited elevated resistance to cadmium as compared to parental plasmid-free cells. The sodium dodecyl sulfate (SDS)-treated cells showed higher efficiency in plasmid curing than the ethidium bromide–treated cells. The ethidium bromide–cured cells grew only in a 10 μ g/ml Cd^{+ 2} minimal tolerable concentration, whereas the SDS-treated cells had no growth in any of the Cd concentrations tested (2, 5, 10, 20, 30, 40, and 50 ppm). Contrary to the Freundlich model, the Langmuir model gave a good fit to the Cd biosorption data by K. oxytoca cells. Plasmid curing caused 80%, 82%, and 70% inhibition in the Cd biosorption, adsorption, and uptake, respectively. Furthermore, the absence of lysine decarboxylase (LDC) activity in the cured strain strongly implies that the structural gene-encoding LDC in this bacterium is plasmid encoded. After curing of the plasmid, 100% of the antibiotic-resistant loci were observed as chromosomal encoded. All of the results shown above indicated that the Cd resistance is plasmid mediated.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

Interaction of nitroaromatic radiosensitizers with irradiated polyadenylic acid as measured by an indirect immunochemical assay with specificity for the 8,5'-cycloadenosine moiety

The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'-cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity.