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The detection of tumor-specific nucleic acids from blood increasingly is being used as a method of liquid biopsy and minimal residual disease detection. However, achieving high sensitivity and high specificity remains a challenge. Here, we perform a direct comparison of two droplet digital PCR (ddPCR)-based detection methods, circulating plasma tumor RNA and circulating plasma tumor DNA (ptDNA), in blood samples from newly diagnosed Ewing sarcoma patients. First, we developed three specific ddPCR-based assays to detect EWS-FLI1 or EWS-ERG fusion transcripts, which naturally showed superior sensitivity to DNA detection on in vitro control samples. Next, we identified the patient-specific EWS-FLI1 or EWS-ERG breakpoint from five patient tumor samples and designed ddPCR-based, patient-specific ptDNA assays for each patient. These patient-specific assays show that although plasma tumor RNA can be detected in select newly diagnosed patients, positive results are low and statistically unreliable compared with ptDNA assays, which reproducibly detect robust positive results across most patients. Furthermore, the unique disease biology of Ewing sarcoma enabled us to show that most cell-free RNA is not tumor-derived, although cell-free-DNA burden is affected strongly by tumor-derived DNA burden. Here, we conclude that, even with optimized highly sensitive and specific assays, tumor DNA detection is superior to RNA detection in Ewing sarcoma patients.

Introduction Hypercoagulability is a common blood alteration in newly diagnosed chemotherapy naïve patients with multiple myeloma. The identification of the procoagulant potential of cancer cells, which is principally related to tissue factor (TF) expression, attracts particular interest. The mechanisms by which myeloma plasma cells (MPCs) activate blood coagulation have been poorly investigated. Aim To identify the principal actors related with MPCs that boost thrombin generation (TG). Methods TF and annexin V expression by MPCs and MPC-derived microparticles (MPC-dMPs) was analyzed by flow cytometry. TF activity (TFa) and TF gene expression were also determined. TG in the presence of MPCs or MPC-dMPs was assessed with the calibrated automated thrombogram assay (CAT) in normal human PPP and in plasma depleted of factor VII or XII. TG was also assessed in plasma spiked with MPCs and MPC-dMPs. Results MPC-dMPs expressed approximately twofold higher levels of TF as compared with MPCs. The TFa expressed by MPC-dMPs was significantly higher compared with that expressed by MPCs. MPCs and MPC-dMPs enhanced TG of human plasma. TG was significantly higher with MPC-dMPs compared with MPCs. Conclusion MPCs indirectly induce blood-borne hypercoagulability through the release of MPC-dMPs rich in TF. Since MPCs, expressing low TFa, represent a weak procoagulant stimulus, the hypercoagulability at the microenvironment could be the resultant of MPC-dMPs rich in TF.

Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking. Here we develop Förster resonance energy transfer (FRET)-based tension sensors to measure the forces experienced by desmoplakin, an obligate desmosomal protein that links the desmosomal plaque to intermediate filaments. Our experiments reveal that desmoplakin does not experience significant tension under most conditions, but instead becomes mechanically loaded when cells are exposed to external mechanical stresses. Stress-induced loading of desmoplakin is transient and sensitive to the magnitude and orientation of the applied tissue deformation, consistent with a stress absorbing function for desmosomes that is distinct from previously analyzed cell adhesion complexes., Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells but direct evidence for their load-bearing nature is lacking. Here the authors develop FRET-based tension sensors to measure the forces experienced by desmoplakin and infer that desmosomes become mechanically loaded when cells are exposed to external mechanical stresses.

Glioblastoma is the most aggressive brain tumor with poor overall survival bellow 2 years. The natural compounds with anti-cancer properties, are thus gaining attention for possible adjuvant GBM treatment. In various cancer models Enterolobium contortisiliquum Trypsin Inhibitor (EcTI) proved to have anti-cancer effects. Here, we investigated the EcTI effects on GBM U87 cells and on mesenchymal stem cells (MSC) compared to their direct coculture (MSC/U87). MSC are present in tumor stroma, modulating GBM cells phenotype, and also represent potential drug delivery vehicle due to their tumor tropism. We showed that in p53-wild type U87 cells, metabolic activity was less affected by EcTI as in MSC monocuture, but the metabolic rate of mixed coculture was significantly reduced at lower EcTI concentration. Under coculture condition, EcTI potentiated MSC induced cell cycle arrest, possible due to highly increased p53, p21 and lower D1 expression, but there was no effect on apoptosis. Accordingly, in the coculture EcTI also enhanced Ca2+ signalling mediated via bradykinin receptor 2, being associated with nitric oxide release that highly impaired proliferation and invasion. The mechanism did not seem to involve changes in cell adhesion but rather it down-regulated the β1 integrin signaling with associated p-FAK in U87 cells, both supporting inhibition of invasion. Finally, some cytokines were down-regulated, indicating that EcTI inhibition of signalling might be mediated by cytokines. In conclusion, these results indicate that in cocultured MSC/U87 cells EcTI impairs the metabolic activity, proliferation, and reduced invasion, possibly associated with observed cytokines secretion. In this context, we confirmed that the plant derived protein potentiated the anticancer effects, induced by MSC, as represented by GBM U87 cell line.

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

In this work, we have investigated a series of aniline-based squaraines, with varying solubilizing alkyl chains, as donor materials in bulk heterojunction (BHJ) solar cells. Although these squaraine molecules exhibit similar absorbance spectra and crystal structures, the difference in properties that drive the OPV performance becomes apparent when each squaraine is blended with PCBM. Thin film X-ray diffraction results demonstrate a disruption of squaraine crystallization in the presence of PCBM, more so for shorter side chain squaraines. As a result, the hole mobilities of BHJ films of shorter side chain squaraines show the largest drop when compared to their neat films, whereas the mobility decrease for the longer side chain counterparts is small. However, morphological studies have shown that the phase separation rapidly happens during the spin-casting process for longer side chain squaraines. Ultimately, it is the extent of phase separation that dominates the final device efficiency. Therefore, ration...

To determine the association between cow's milk-fat and non-high-density lipoprotein (non-HDL) cholesterol, a marker of cardiovascular disease (CVD) risk in young children, and whether this association is mediated by the typical volume of cow's milk consumed.A longitudinal study in 2- to 8-year-old children (n = 2890) was conducted through The Applied Research Group for Kids (TARGet Kids!), a practice-based research network in Toronto, Canada. Generalized estimating equations were used to examine the relationship between parent-reported cow's milk-fat percentage intake and serum non-HDL cholesterol concentrations as well as having high non-HDL cholesterol (≥3.75 mmol/L [145 mg/dL]), adjusting for covariates including age, sex, body mass index z score, breastfeeding duration, mother's ethnicity, and parental history of CVD. Bootstrap resampling (10 000 repetitions) was used to assess whether typical volume consumed mediated the association between cow's milk-fat percentage and non-HDL cholesterol.In total, 156 (5.4%) had high non-HDL cholesterol. Each percent increase in cow's milk-fat was associated with a 0.035 mmol/L (1.35 mg/dL) (P .001) and 0.024 mmol/L (0.92 mg/dL) (P = .01) increase in non-HDL cholesterol, unadjusted and adjusted for covariates respectively. Cow's milk-fat percentage was not associated with greater odds of having high non-HDL cholesterol. Volume of cow's milk partially mediated the association between cow's milk-fat percentage and non-HDL cholesterol, accounting for 28% of the relationship (P .001).Consumption of higher-fat cow's milk was associated with a small increase in non-HDL cholesterol but not greater odds of having high non-HDL cholesterol. Further research is needed to assess this relationship with other CVD risk factors in young children.ClinicalTrials.gov: NCT01869530.

TFE/translocation renal cell carcinoma (tRCC) was formally recognized by the WHO in 2004 as a distinct, typically translocation-associated, RCC with characteristic morphology and immunohistochemical expression of TFE3 or TFEb. Cytogenetic translocations may include TFE3-ASPS, TFE3-PRCC, TFEb-alpha, or other variants; mechanisms for TFE upregulation may be heterogenous. TFE3 and TFEB are members of the MiTF/TFE family of basic helix-loop-helix-leucine zipper transcription factors [1–3].

CBA mouse macrophages control Leishmania major infection yet are permissive to Leishmania amazonensis. Few studies have been conducted to assess the role played by autophagy in Leishmania infection. Therefore, we assessed whether the autophagic response of infected macrophages may account for the differential behavior of these two parasite strains. After 24 h of infection, the LC3-II/Act ratio increased in both L. amazonensis- and L. major-infected macrophages compared to uninfected controls, but less than in chloroquine-treated cells. This suggests that L. amazonensis and L. major activate autophagy in infected macrophages, without altering the autophagic flux. Furthermore, L. major-infected cells exhibited higher percentages of DQ-BSA-labeled parasitophorous vacuoles (50%) than those infected by L. amazonensis (25%). However, L. major- and L. amazonensis-induced parasitophorous vacuoles accumulated LysoTracker similarly, indicating that the acidity in both compartment was equivalent. At as early as 30 min, endogenous LC3 was recruited to both L. amazonensis- and L. major-induced parasitophorous vacuoles, while after 24 h a greater percentage of LC3 positive vacuoles was observed in L. amazonensis-infected cells (42.36%) compared to those infected by L. major (18.10%). Noteworthy, principal component analysis (PCA) and an hierarchical cluster analysis completely discriminated L. major-infected macrophages from L. amazonensis-infected cells accordingly to infection intensity and autophagic features of parasite-induced vacuoles. Then, we evaluated whether the modulation of autophagy exerted an influence on parasite infection in macrophages. No significant changes were observed in both infection rate or parasite load in macrophages treated with the autophagic inhibitors wortmannin, chloroquine or VPS34-IN1, as well as with the autophagic inducers rapamycin or physiological starvation, in comparison to untreated control cells. Interestingly, both autophagic inducers enhanced intracellular L. amazonensis and L. major viability, while the pharmacological inhibition of autophagy exerted no effects on intracellular parasite viability. We also demonstrated that autophagy induction reduced NO production by L. amazonensis- and L. major-infected macrophages but not alters arginase activity. These findings provide evidence that although L. amazonensis-induced parasitophorous vacuoles recruit LC3 more markedly, L. amazonensis and L. major similarly activate the autophagic pathway in CBA macrophages. Interestingly, the exogenous induction of autophagy favors L. major intracellular viability to a greater extent than L. amazonensis related to a reduction in the levels of NO. Fil: Dias, Beatriz R.S.. Gonçalo Moniz Institute; Fil: de Souza, Carina S.. Gonçalo Moniz Institute; Fil: Almeida, Niara de Jesus. Gonçalo Moniz Institute; Fil: Lima, José G.B.. Gonçalo Moniz Institute; Fil: Fukutani, Kiyoshi F.. Gonçalo Moniz Institute; Fil: dos Santos, Thiale B.S.. Gonçalo Moniz Institute; Fil: França-Cost, Jaqueline. Universidade Federal da Bahia; Brasil. Gonçalo Moniz Institute; Fil: Brodskyn, Claudia I.. Gonçalo Moniz Institute; Fil: de Menezes, Juliana P.B.. Gonçalo Moniz Institute; Fil: Colombo, Maria Isabel. Universidad Nacional de Cuyo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Veras, Patricia S.T.. Gonçalo Moniz Institute

Background Chemotherapy is used preoperatively for children with bilateral Wilms tumor (BWT) or unilateral high-risk Wilms tumor (UHRWT) to promote tumor regression to facilitate renal preservation with nephron-sparing surgery (NSS). In adults, various surgical techniques have been described to preserve renal tissue. Few studies have examined the use of surgical adjuncts in NSS in children with renal tumors. Methods We performed a multi-institutional retrospective review of patients with BWT or UHRWT. Patient demographics, tumor size at diagnosis, following neoadjuvant chemotherapy, utilization of surgical adjuncts including intraoperative ultrasound (IOUS), margin status, complications, renal function, and follow-up were recorded. Results The cohort comprised 23 patients: 18 BWT, 3 UHRWT, and 2 patients with solitary kidney. Twenty-two of the 23 patients had successful NSS. IOUS was used 19 times, and seven had positive margins after surgery. Cooling/vascular isolation was used six times. At a median follow-up of 18 mo, median estimated glomerular filtration rate Schwartz was 126 mL/min/1.73 m2 and median serum creatinine 0.39 mg/dL in the 22 patients who had successful NSS. There have been no tumor recurrences. Conclusions In patients with BWT and UHRWT, surgical adjuncts such as cooling/vascular isolation are uncommonly performed. IOUS may be helpful but does not guarantee negative microscopic margins. Level of evidence Level 4, Case series with no comparison group.

Objective To better understand the impact of nephron-sparing surgery (NSS) on renal function in patients with nonsyndromic unilateral Wilms tumor (uWT), a group of such patients treated with NSS were compared with a stage-matched cohort managed with radical nephrectomy (RN). The recommended management of nonsyndromic uWT is RN. However, NSS may decrease the risk of long-term renal disease and associated comorbidities. Materials and Methods An international, multi-institutional review was conducted of nonsyndromic uWT cases managed with NSS and compared with a stage-matched RN cohort. Data were collected on demographics, oncologic characteristics and outcomes, serum creatinine, and estimated glomerular filtration rate (eGFR) calculated via Schwartz formula. Results Fifteen patients who underwent NSS (6 females and 9 males) met study criteria and were diagnosed at a median age of 2.5 years (range, 0.2-8.2 years) and followed for a median of 8.4 years (range, 0.5-31.8 years). The stage-matched RN cohort consisted of 15 patients (8 females and 7 males) diagnosed at a median age of 3.7 years (0.3-7.4) and followed for a median of 2.1 years (0.6-10.5 years). The median preoperative eGFR was 91.7 (39.4-237.7) and 149.9 (93.8-215.9) for NSS and RN, respectively, P = .026. The median eGFR at last follow-up was 135.3 (57.5-185.8) and 131.0 (98.6-161.2) for NSS and RN, respectively, P = .95. The median change in eGFR during the study period was a gain of 28.6 (−51.9 to 83.0) for the NSS cohort vs a loss of 19.1 (−54.7 to 25.2) for the RN cohort, P = .007. Conclusion In a highly selected patient population with nonsyndromic uWT, NSS provides excellent renal function preservation when compared with RN. These data require validation via prospective investigation on a larger scale.

Ewing sarcoma is a small round blue cell malignancy arising from bone or soft tissue and most commonly affects adolescents and young adults. Metastatic and relapsed Ewing sarcoma have poor outcomes and recurrences remain common. Owing to the poor outcomes associated with advanced disease and the need for a clear research strategy, the Children’s Oncology Group Bone Tumor Committee formed the New Agents for Ewing Sarcoma Task Force to bring together experts in the field to evaluate and prioritize new agents for incorporation into clinical trials. This group’s mission was to evaluate scientific and clinical challenges in moving new agents forward and to recommend agents and trial designs to the Bone Tumor Committee. The task force generated a framework for vetting prospective agents that included critical evaluation of each drug by using both clinical and non-clinical parameters. Representative appraisal of agents of highest priority, including eribulin, dinutuximab, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, anti-angiogenic tyrosine kinase inhibitors, and poly-ADP-ribose polymerase (PARP) inhibitors, is described. The task force continues to analyze new compounds by using the paradigm established.

DNA translocases are molecular motors that move rapidly along DNA using adenosine triphosphate as the source of energy. We directly observed the movement of purified FtsK, an Escherichia coli translocase, on single DNA molecules. The protein moves at 5 kilobases per second and against forces up to 60 piconewtons, and locally reverses direction without dissociation. On three natural substrates, independent of its initial binding position, FtsK efficiently translocates over long distances to the terminal region of the E. coli chromosome, as it does in vivo. Our results imply that FtsK is a bidirectional motor that changes direction in response to short, asymmetric directing DNA sequences.

To assess the pre- and post-therapy glomerular filtration rate in patients with testicular germ cell tumors to determine its effect on the natural history of renal function.We reviewed an institutional database of patients with testicular germ cell tumor, with pre- and post-therapy serum creatinine levels available. The renal function was estimated using a calculated glomerular filtration rate. We compared the patients treated without chemotherapy (orchiectomy with or without radiotherapy or retroperitoneal lymph node dissection) with those who received systemic chemotherapy. We analyzed the data for the outcome of new-onset chronic kidney disease (CKD) stage 3 between these groups. Kaplan-Meier curves were constructed and compared using a log-rank test.A total of 144 patients were reviewed. The testicular germ cell tumor stage distribution was stage I in 78 (54.2%), stage II in 28 (19.4%), and stage III in 38 (26.4%). Overall, the median creatinine and estimated glomerular filtration rate at diagnosis was 0.9 mg/dL (range 0.5-1.5) and 104.0 mL/min/1.73 m(2) (range 58.7-235), respectively. Of the 144 patients, 102 (70.8%) had CKD stage 0-1, 41 (28.5%) stage 2, and 1 (0.7%) stage 3. The median creatinine and estimated glomerular filtration rate at the last follow-up visit was 1.0 mg/dL (range 0.6-2.6) and 95.5 mL/min/1.73 m(2) (range 31.5-167.6), respectively. This difference between the pre- and post-therapy estimated glomerular filtration rate was significant (P.01). A total of 81 patients (56.3%) received chemotherapy (median 4 cycles, range 1-12), and 63 (43.7%) were treated without chemotherapy. Of the 81 patients who received chemotherapy, 8 (9.9%) developed new-onset CKD 3 compared with none in the nonchemotherapy group (P = .01).Patients with testicular germ cell tumor receiving chemotherapy experienced a significant decrease in the estimated glomerular filtration rate and had a significantly increased risk of developing CKD stage 3 compared with those treated without chemotherapy. These findings offer insight into the long-term risks of testicular germ cell tumor survivorship and will be useful in counseling patients.

Aroma is one of the first sensorial characteristics affecting the perception of quality of salted dried codfish. In this work, the volatile profile of different commercially available salted dried codfishes was characterized by HS-SPME/GC-IT-MS. Samples of three species (Gadus morhua, Gadus macrocephalus and Theragra calchogramma), subjected to two curing processes (Traditional and Yellow curing) for different durations caught in distinct pools, were studied.Thirty compounds were fully characterized and 25 were tentatively identified and comprising several chemical classes (amines, hydrocarbons, esters, chlorinated compounds, aldehydes, aromatic, alcohols, acids, sulphur compounds, ethers, ketones and terpenes), with 26 of them being reported for the first time in codfish. Principal component analysis (PCA) showed major differences for the yellow cured sample and for T. chalcogramma species. Yellow cured codfish presented a higher content of compounds of almost all classes. T. chalcogramma exhibited a hig...

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is a potent and promising anti-HIV molecule. It is currently being investigated for use as a vaginal microbicide in two-dosage forms, a semi-solid gel and a silicone elastomer ring. Quick-dissolving films are promising and attractive dosage forms that may provide an alternative platform for the vaginal delivery of microbicide drug candidates. Vaginal films may provide advantages such as discreet use, no product leakage during use, lack of requirement for an applicator for insertion, rapid drug release, and minimal packaging and reduced wastage. Within this study, the in vitro bioactivity of dapivirine as compared with the non-nucleoside reverse transcriptase inhibitor UC781 was further established, and a quick dissolve film was developed for vaginal application of dapivirine for prevention of HIV infection. The developed film was characterized with respect to its physical and chemical attributes including water content, mechanical strength, drug release profile, permeability, compatibility with lactobacilli, and bioactivity. The anti-HIV activity of the formulated dapivirine film was confirmed in in vitro and ex vivo models. Importantly, the physical and chemical properties of the film as well as its bioactivity were maintained for a period of 18 months. In conclusion, a vaginal film containing dapivirine was developed and characterized. The film was shown to prevent HIV-1 infection in vitro and ex vivo and have acceptable characteristics which make this film a promising candidate for testing as vaginal microbicide.

Background Pediatric oncology and hematopoietic stem cell transplantation (HSCT) patients are at high risk for influenza infection and its associated complications. Little is known about infection with novel 2009 influenza A (H1N1) in this population. Procedure Prospective laboratory surveillance identified all children with positive influenza test results from 4/27/09–12/5/09. 2009 H1N1 infection was confirmed by PCR subtyping; cases in which subtyping was not performed were considered probable. Medical records of all pediatric oncology and HSCT cases were reviewed. Results Thirty children with cancer or HSCT had laboratory-confirmed influenza A. Patients with ALL (18), CNS tumors (4), CML (1), Ewing sarcoma (1), Hodgkin lymphoma (1), LCH (1), severe aplastic anemia (1), and HSCT (3), had confirmed (5) and probable (25) H1N1 by rapid (22; 73%), DFA (4; 13%), or RVP (4; 13%) assays. Most frequent presenting signs and symptoms were fever (93%; median 38.6°C), cough (97%), and rhinorrhea (83%). Ten patients required hospitalization for a median of 5 days, most commonly for fever and neutropenia (8). Imaging demonstrated lower respiratory tract involvement in three patients. There were no concomitant bacteremias; one patient had rhinovirus co-infection. Three patients required ICU care; 1 developed ARDS, multi-organ failure, and died after 5 days. Chemotherapy was delayed in five patients. Oseltamivir was administered to 28 patients; 1 patient developed an oseltamivir-resistant strain and was treated with zanamivir. Conclusions 2009 influenza A H1N1 infection in children with cancer and HSCT is mild in most patients, but can lead to serious complications. Pediatr Blood Cancer. 2010;56:127–133. © 2010 Wiley-Liss, Inc.

One of the major limitations in scaffold-based bone tissue engineering has been the inability to increase the loading of biologically active inorganic mineral. The present study introduces a novel two step strategy to increase overall mineral content of electrospun scaffolds and employs multiple factor interaction as a statistic to identify the combination of factors that yields maximal scaffold mineralization. Different amounts of nHA (0, 10, 25 and 50% by wt. of polymer) were electrospun in combination with polydioxanone (PDO) or poly(glycolide: lactide) to generate composite scaffolds. Successful incorporation of nHA within, on and in between nanofibers was confirmed by transmission and scanning electron microscopy. These scaffolds were immersed in different types (conventional, revised, ionic and modified) of simulated body fluid (SBF), prepared at 1× and 4× concentrations and the incubation was carried out either in static or dynamic setting at biomimetic conditions. At 2 weeks, the total amount of mineral within the scaffold was quantified using a modified Alizarin Red-based assay. Each of the five independent factors was analyzed independently and tested for interaction using random effects ANOVA. Statistics revealed significant higher order interactions among factors and the combination of PDO containing 50% nHA incubated in 1× revised SBF resulted in maximum mineralization.

This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique. DNA fragments in ligation reactions are capable of combining to produce numerous products. The ELAN method uses judicious choice of restriction enzyme sites coupled with simultaneous digestion and ligation reactions to create just one product, by converting off-pathway products back into substrate. The experimental parameters critical for a successful ELAN reaction are discussed, and the ordered, one-tube assembly of four DNA fragments in the presence of eight restriction enzymes is demonstrated. This technique will be useful to those performing gene construction, DNA computing, biophysics and even standard molecular cloning. Starting with reactant fragments, the protocol takes 4-16 h to produce nanogram to microgram yields, depending on the complexity of the reaction.

DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of subsequent manipulations (e.g., selection via bacterial transformation) often obviates the need for a high yield of correctly ligated products. However, the ability to perform high-yield, preparative-scale DNA ligations would benefit a number of downstream applications ranging form standard molecular cloning to biophysics and DNA computing. We describe here a ligation technique that specifically converts off-pathway ligation products back into substrate. We term this second-chance strategy enzymatic ligation assisted by nucleases (ELAN) and demonstrate the ordered assembly of four DNA fragments via simultaneous ligation and digestion in the presence of eight restriction enzymes. Use of ELAN increased the yield of the desired product by more than 30-fold.

starting from N-(4-hydroxyphenyl)acetamide (Paracetamol, convergent approach) or from cyanuric chloride in reaction with 4-aminophenol (divergent approach), two synthetic routes towards novel tripodal N-substituted melamines as s-triazine derivatives of (4-aminophenoxy)acetic acid or of 4-(4-aminophenoxy)butyric acid are comparatively defined. The key steps consist of Williamson etherification of N-masked forms of 4-aminophenol and acidic hydrolysis of the N- and / or O-protected (4-aminophenoxy)alkanoic segments.

As genetic material DNA is wonderful, but as a macromolecule it is unruly, voluminous and fragile. Without the action of DNA replicases, topoisomerases, helicases, translocases and recombinases, the genome would collapse into a topologically entangled random coil that would be useless to the cell. We discuss the organization, movement and energetics of these proteins that are crucial to the preservation of a molecule that has such beautiful biological but challenging physical properties.

The increased incidence of human immunodeficiency virus infection in women has identified an urgent need to develop a female-controlled method to prevent acquisition of human immunodeficiency virus and other sexually transmitted diseases. Women would apply the product intravaginally before intercourse. Development of such a product requires a better understanding of the permeability characteristics of the tissues with which such products would come into contact. However, limited studies have been performed in this area. In the present study, water permeability of fresh human cervical and vaginal tissue was evaluated. The average apparent permeability coefficient was found to be 8 × 10 −5 cm/s for fresh human cervical tissue and 7 × 10 −5 cm/s for fresh human vaginal tissue. Considering the lack of regularity in obtaining cervical and vaginal tissue from surgical specimens, additional tests were performed to evaluate the effect of freezing on tritiated water permeability. No statistically significant differences were observed in the permeability values obtained when comparing fresh versus frozen tissues.

The use of advanced oxidation processes (AOPs) to remove pollutants in various water treatment applications has been the subject of study for around 30 years. Most of the available processes (Fenton reagent, O3 under basic conditions, O3/H2O2, O3/UV, O3/solid catalyst, H2O2/Mn+, H2O2/UV, photo-assisted Fenton, H2O2/solid catalyst, H2O2/NaClO, TiO2/UV etc.) have been investigated in depth and a considerable body of knowledge has been built up about the reactivity of many pollutants. Various industrial applications have been developed, including ones for ground remediation (TCE, PCE), the removal of pesticides from drinking water, the removal of formaldehyde and phenol from industrial waste water and a reduction in COD from industrial waste water. The development of such AOP applications has been stimulated by increasingly stringent regulations, the pollution of water resources through agricultural and industrial activities and the requirement that industry meet effluent discharge standards. Nevertheless, it is difficult to obtain an accurate picture of the use of AOPs and its exact position in the range of water treatment processes has not been determined to date. The purpose of this overview is to discuss those processes and provide an indication of future trends.