Molecular analysis of blood group Rh transcripts from a rGr variant

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the rGr phenotype. Nucleotide sequence analysis revealed that the rG allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid rG gene (c-C-e or c-D-e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.     

Crack propagation from a pre-existing flaw at a notch root – II: Detailed form of the stress intensity factors at the initial crack tip and conclusion.

This paper pursues the study of crack kinking from a pre-existing crack emanating from some notch root. It was shown in Part I that the stress intensity factors at the tip of the small initial crack are given by universal (that is, applicable in all situations, whatever the geometry of the body and the loading) formulae; they depend only on the `stress intensity factor of the notch' (the multiplicative coefficient of the singular stress field near the apex of the notch in the absence of the crack), the length of the crack, the aperture angle of the notch and the angle between its bisecting line and the direction of the crack. Here we identify the universal functions of the two angles just mentioned which appear in these formulae, by considering the model problem of an infinite body endowed with a notch with straight boundaries and a straight crack of unit length. The treatment uses Muskhelishvili's complex potentials formalism combined with some conformal mapping. The solution is expressed in the form of an infinite series involving an integral operator, which is evaluated numerically. Application of Goldstein and Salganik's principle of local symmetry then leads to prediction of the kink angle of the crack extension. It is found that although the direction of the crack is closer to that of the bisecting line of the notch after kinking than before it, the kink angle is not large enough for the crack tip to get closer to this line after kinking, except perhaps in some special situations.     

Crack propagation from a pre-existing flaw at a notch root. I. Introduction and general form of the stress intensity factors at the initial crack tip

This paper and its companion are devoted to the study of crack kinking from some small pre-existing crack originating from a notch root (the notch root radius being zero). Both the notch boundaries and the initial crack are allowed to be curved; also, the geometry of the body and the loading are totally arbitrary. The ingredients required are knowledge of the stress intensity factors at the initial crack tip and use of a suitable mixed mode propagation criterion. This paper is devoted to the first point, and more specifically to establishing the general (that is, not yet fully explicit) form of the formulae giving these stress intensity factors. The method used is based on changes of scale (homogeneity properties of the equations of elasticity) on the one hand, and on continuity of the displacement and stresses at a given, fixed point with respect to the crack length on the other hand. The formulae derived for the stress intensity factors at the tip of the small crack are of universal value: they apply to any situation, whatever the geometry of the body, the notch and the crack and whatever the loading, the stress intensity factors depending always only upon the `stress intensity factor of the notch' (the multiplicative coefficient of the singular stress field near the notch root in the absence of the crack), the length of the crack, the aperture angle of the notch and the angle between its bisecting line and the direction of the crack.     

Decrease in ventilation time with a standardized weaning process

OBJECTIVE: To test the hypothesis that standardizing the process of weaning from mechanical ventilation would decrease ventilation times and length of stay in a surgical intensive care unit. DESIGN: Comparison of historic ventilation times with physician-directed weaning with those obtained with protocol-guided weaning by respiratory therapists. SETTING: Urban, teaching surgical intensive care unit with open admission policy and no dominant diagnosis related group. RESULTS: From January 1, 1995, through December 31, 1995, 378 patients who underwent physician-directed weaning from a ventilator had 64488 hours of ventilation, compared with 57796 ventilation hours in 515 patients with protocol-guided weaning (April 1, 1996, through May 31, 1997). The mean hours of ventilation decreased by 58 hours, a 46% decrease (P<.001). The length of hospital stay decreased by 1.77 days (29% change), while the Acute Physiology and Chronic Health Evaluation III score remained at 50 to 51. The number of reintubations did not change. The marginal cost savings was $603580. CONCLUSION: Protocol-guided weaning from mechanical ventilation leads to more rapid extubation than physician-directed weaning and has great potential for cost savings.     

Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I-III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10-50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D(IVb) variant), method III (D(VI) and DFR variants) and method IV (D(VI) variants), but not method I. Weak D (D(u)) was correctly detected as D-positive by all methods, but most cases of Rh(null) appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C- or E-allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.     

Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O₂^•−) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O₂^•− levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma.     

Antimicrobial functionalization of wool: assessment of the effect of Cecropin-B and [Ala5]-Tritrp7 antimicrobial peptides

This investigation provides a new strategy to impart antimicrobial properties into wool-based materials using Cecropin-B and [Ala5]-Tritrp7 antimicrobial peptides (AMPs). The process was conducted using exhaustion method at 40 °C for 1–3 h. The presence of the AMPs in the modified-wool samples was confirmed by colorimetric assay of Bradford reagent and possible changes in the morphology of the fibers and damage to its surface were analyzed by scanning electron microscopy. Results showed that 1 h were long enough for the functionalization to occur effectively and that the morphology of the fibers was not influenced by the functionalization process. Furthermore, the antimicrobial activity of the AMPs applied on wool was assessment by JIS L 1902-2002 against Staphylococcus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352). The results showed that both AMPs have a high reduction in bacterial growth (Cecropin-B resulting in 71.67% reduction against S. aureus and 85.95% against K. pneumoniae. While [Ala5]-Tritrp7 resulting in 66.74% reduction against S. aureus and 88.65% against K. pneumoniae).     

Comparison of the antibacterial activity of modified‐cotton with magainin I and LL‐37 with potential as wound‐dressings

Wounds are the ideal setting for the development of micro‐organisms, so it is often necessary to apply a dressing to control bacterial colonization. Cotton is commonly used in dressings, as it exhibits important hydrophilic characteristics such as high moisture and fluid retention properties, but it may provide a sustainable media for the development of micro‐organisms. In this way, the development of new strategies to provide cotton materials with lasting and effective antimicrobial properties is of the utmost importance. Consequently, here we described two processes to develop cotton‐dressings functionalized with antimicrobial peptides (AMPs) magainin I (MagI) and LL‐37, in order to give cotton‐dressings an antibacterial effect. The AMPs showed no cytotoxic effect against human fibroblasts so they are safe to contact with skin. In addition, the functionalized materials with either LL‐37 or MagI present an antimicrobial effect exhibiting inhibition ratios of 89% against Klebsiella pneumoniae and 58% against Staphylococcus aureus, respectively. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40997.