Sequence-Specific and Strand-Specific Cleavage In Oligodeoxyribonucleotides and DNA Containing 3'-Thiothymidine

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-di-isopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.

Political Violence and Child Adjustment: Longitudinal Tests of Sectarian Antisocial Behavior, Family Conflict, and Insecurity as Explanatory Pathways

Understanding the impact of political violence on child maladjustment is a matter of international concern. Recent research has advanced a social ecological explanation for relations between political violence and child adjustment. However, conclusions are qualified by the lack of longitudinal tests. Toward examining pathways longitudinally, mothers and their adolescents (M = 12.33, SD = 1.78, at Time 1) from 2-parent families in Catholic and Protestant working class neighborhoods in Belfast, Northern Ireland, completed measures assessing multiple levels of a social ecological model. Utilizing autoregressive controls, a 3-wave longitudinal model test (T1, n = 299; T2, n = 248; T3, n = 197) supported a specific pathway linking sectarian community violence, family conflict, childrens insecurity about family relationships, and adjustment problems.

Risk factors for developing prolonged grief during bereavement in family carers of cancer patients in palliative care: A longitudinal study.

Context: Family carers of palliative care patients report high levels of psychological distress throughout the caregiving phase and during bereavement. Palliative care providers are required to provide psychosocial support to family carers; however, determining which carers are more likely to develop prolonged grief (PG) is currently unclear.

Objectives: To ascertain whether family carers reporting high levels of PG symptoms and those who develop PG disorder (PGD) by six and 13 months postdeath can be predicted from predeath information.

Methods: A longitudinal study of 301 carers of patients receiving palliative care was conducted across three palliative care services. Data were collected on entry to palliative care (T1) on a variety of sociodemographic variables, carer-related factors, and psychological distress measures. The measures of psychological distress were then readministered at six (T2; n = 167) and 13 months postdeath (T3; n = 143).

Results: The PG symptoms at T1 were a strong predictor of both PG symptoms and PGD at T2 and T3. Greater bereavement dependency, a spousal relationship to the patient, greater impact of caring on schedule, poor family functioning, and low levels of optimism also were risk factors for PG symptoms.

Conclusion: Screening family carers on entry to palliative care seems to be the most effective way of identifying who has a higher risk of developing PG. We recommend screening carers six months after the death of their relative to identify most carers with PG.

Antioxidant Activity of Sesame (Seasamum indicum L.) Cake Extract for the Stabilization of Olein Based Butter

This study investigated the effect of ethanolic sesame cake extract on oxidative stabilization of olein based butter. Fractionation of cream was performed by the dry fractionation technique at 10 °C, ethanolic sesame cake extract (SCE) was incorporated into olein butter at three different concentrations; 50, 100, 150 ppm (T1, T2, T3) and compared with a control. The total phenolic content of SCE was 1.72 (mg gallic acid equivalent g−1 dry weight). The HPLC characterization of ethanolic sesame cake revealed the presence of antioxidant substances viz. sesamol, sesamin and sesamolin in higher extents. The DPPH free radical scavenging activity of SCE was 83 % as compared to 64 and 75 % in BHA and BHT. Fractionation of milk fat at 10 °C significantly (p < 0.05) influenced the fatty acid profile of olein and stearin fractions from the parent milk fat. Concentration of oleic acid and linoleic acid in olein fraction was 29.62 and 33.46 % greater than the parent milk fat. The loss of C18:1 in 90 days stored control and T3 was 24.37 and 3.58 %, respectively, 58 % C18:2 was broken down into oxidation products over 8.55 % loss in T3. The peroxide value of control, T1, T2, BHT and T3 in the Schaal oven test was 8.59, 8.12, 5.34, 4.52 and 2.49 (mequiv O2/kg). The peroxide value and anisidine value of 3 months stored control and T3 were 1.21, 0.42 (mequiv O2/kg) and 27.25, 13.25, respectively. The concentration of conjugated dienes in T3 was substantially less than the control. The induction period of T3 was considerably higher than BHT with no difference in sensory characteristics (p > 0.05). Ethanolic SCE can be used for the long-term preservation of olein butter, with acceptable sensory characteristics.

Local recurrence in patients with large and locally advanced breast cancer treated with primary chemotherapy

Background: Primary chemotherapy is being given in the treatment of large and locally advanced breast cancers, but a major concern is local relapse after therapy. This paper has examined patients treated with primary chemotherapy and surgery (either breast-conserving surgery or mastectomy) and has examined the role of factors which may indicate those patients who are subsequently more likely to experience local recurrence of,disease.

Methods: A consecutive series of 173 women, with data available for 166 of these, presenting with large and locally advanced breast cancer (T2 >4 cm, T3, T4, or N2) were treated with primary chemotherapy comprising cyclophosphamide, vincristine, doxorubicin, and prednisolone and then surgery (either conservation or mastectomy with axillary surgery) followed by radiotherapy were examined.

Results: The clinical response rate of these patients was 75% (21% complete and 54% partial), with a complete pathological response rate of 15%. A total of 10 patients (6%) experienced local disease relapse, and the median time to relapse was 14 months (ranging from 3 to 40). The median survival in this group was 27 months (ranging from 13 to 78). In patients having breast conservation surgery, local recurrence occurred in 2%, and in those undergoing mastectomy 7% experience local relapse of disease. Factors predicting patients most likely to experience local recurrence were poor clinical response and residual axillary nodal disease after chemotherapy.

Conclusions: Excellent local control of disease can be achieved in patients with large and locally advanced breast cancers using a combination of primary chemotherapy, surgery and radiotherapy. However, the presence of residual tumor in the axillary lymph nodes after chemotherapy is a predictor of local recurrence and patients with a better clinical response were also less likely to experience local disease recurrence. The size and degree of pathological response did not predict patients most likely to experience recurrence of disease. (C) 2003 Excerpta Medica, Inc. All rights reserved.

A Novel Dried Blood Spot-LCMS Method for the Quantification of Methotrexate Polyglutamates as a Potential Marker for Methotrexate Use in Children

Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS).

Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 μl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 μm, 2.1x150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization.

Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique.

Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology. © 2014 Hawwa et al.

The value of adjuvant radiotherapy on survival and recurrence in triple-negative breast cancer: a systematic review and meta-analysis of 5,507 patients

BACKGROUND: The value of adjuvant radiotherapy in triple negative breast cancer (TNBC) remains unclear. A systematic review and meta-analysis was conducted in TNBC patients to assess survival and recurrence outcomes associated with radiotherapy following either breast conserving therapy (BCT) or post-mastectomy radiotherapy (PMRT). METHODS: Four electronic databases were searched from January 2000 to November 2015 (PubMed, MEDLINE, EMBASE and Web of Science). Studies investigating overall survival and/or recurrence in TNBC patients according to radiotherapy administration were included. A random effects meta-analysis was conducted using mastectomy only patients as the reference.  RESULTS: Twelve studies were included. The pooled hazard ratio (HR) for locoregional recurrence comparing BCT and PMRT to mastectomy only was 0.61 (95% confidence interval [CI] 0.41-0.90) and 0.62 (95% CI 0.44-0.86), respectively. Adjuvant radiotherapy was not significantly associated with distant recurrence. The pooled HR for overall survival comparing BCT and PMRT to mastectomy only was 0.57 (95% CI 0.36-0.88) and HR 1.12 (95% CI 0.75, 1.69). Comparing PMRT to mastectomy only, tests for interaction were not significant for stage (p=0.98) or age at diagnosis (p=0.85). However, overall survival was improved in patients with late-stage disease (T3-4, N2-3) pooled HR 0.53 (95% CI 0.32-0.86), and women <40 years, pooled HR 0.30 (95% CI 0.11-0.82). CONCLUSIONS: Adjuvant radiotherapy was associated with a significantly lower risk of locoregional recurrence in TNBC patients, irrespective of the type of surgery. While radiotherapy was not consistently associated with an overall survival gain, benefits may be obtained in women with late-stage disease and younger patients.