Importance of intracellular pH in determining the uptake and efficacy of the weakly basic chemotherapeutic drug, doxorubicin

Low extracellular pH (pH(e)), that is characteristic of many tumours, tends to reduce the uptake of weakly basic drugs, such as doxorubicin, thereby conferring a degree of physiological resistance to chemotherapy. It has been assumed, from pH-partition theory, that the effect of intracellular pH (pH(i)) is symmetrically opposite, although this has not been tested experimentally. Doxorubicin uptake into colon HCT116 cells was measured using the drug's intrinsic fluorescence under conditions that alter pH(i) and pH(e) or pH(i) alone. Acutely, doxorubicin influx across the cell-membrane correlates with the trans-membrane pH-gradient (facilitated at alkaline pH(e) and acidic pH(i)). However, the protonated molecule is not completely membrane-impermeant and, therefore, overall drug uptake is less pH(e)-sensitive than expected from pH-partitioning. Once inside cells, doxorubicin associates with slowly-releasing nuclear binding sites. The occupancy of these sites increases with pH(i), such that steady-state drug uptake can be greater with alkaline cytoplasm, in contradiction to pH-partition theory. Measurements of cell proliferation demonstrate that doxorubicin efficacy is enhanced at alkaline pH(i) and that pH-partition theory is inadequate to account for this. The limitations in the predictive power of pH-partition theory arise because it only accounts for the pH(i)/pH(e)-sensitivity of drug entry into cells but not the drug's subsequent interactions that, independently, show pH(i)-dependence. In summary, doxorubicin uptake into cells is favoured by high pH(e) and high pH(i). This modified formalism should be taken into account when designing manoeuvres aimed at increasing doxorubicin efficacy.     

Heterogeneity in White Blood Cells Has Potential to Confound DNA Methylation Measurements

Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50–179) and in two white blood cell fractions (n = 20), isolated using density gradient centrifugation, in four CGIs (CpG Islands) located in genes HHEX (10 CpG sites assayed), KCNJ11 (8 CpGs), KCNQ1 (4 CpGs) and PM20D1 (7 CpGs). Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter) explained up to 40% (p<0.0001) of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4–15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.     

The CUPID (Cultural and Psychosocial Influences on Disability) study: methods of data collection and characteristics of study sample

Background

The CUPID (Cultural and Psychosocial Influences on Disability) study was established to explore the hypothesis that common musculoskeletal disorders (MSDs) and associated disability are importantly influenced by culturally determined health beliefs and expectations. This paper describes the methods of data collection and various characteristics of the study sample.

Methods/Principal Findings

A standardised questionnaire covering musculoskeletal symptoms, disability and potential risk factors, was used to collect information from 47 samples of nurses, office workers, and other (mostly manual) workers in 18 countries from six continents. In addition, local investigators provided data on economic aspects of employment for each occupational group. Participation exceeded 80% in 33 of the 47 occupational groups, and after pre-specified exclusions, analysis was based on 12,426 subjects (92 to 1018 per occupational group). As expected, there was high usage of computer keyboards by office workers, while nurses had the highest prevalence of heavy manual lifting in all but one country. There was substantial heterogeneity between occupational groups in economic and psychosocial aspects of work; three- to five-fold variation in awareness of someone outside work with musculoskeletal pain; and more than ten-fold variation in the prevalence of adverse health beliefs about back and arm pain, and in awareness of terms such as “repetitive strain injury” (RSI).

Conclusions/Significance

The large differences in psychosocial risk factors (including knowledge and beliefs about MSDs) between occupational groups should allow the study hypothesis to be addressed effectively.     

Cell-based therapy for epithelial wounds

Background aimsBone marrow-derived cells (BMDC) form a significant portion of regenerating epithelial tissue. The purpose of this study was to determine whether exogenous BMDC (containing stroma, stem and progenitor cells), introduced systemically or within the injury site, could enhance the injury repair response.MethodsExcisional wounds (10-mm diameter) were treated by systemic (intravenous; i.v.) and local (subcutaneous; s.c.) administration of BMDC (10–20 × 10⁶/100 μL phosphate-buffered saline). Young and aged BMDC and recipients were studied.ResultsYoung BMDC (2 months old) increased the healing rate compared with older BMDC (1 year old), as measured by the rate of healing and the percentage of healed tissue. Young recipients had statistically better healing efficiency than older recipients. When old BMDC were used, young recipients had a better healing ability than older recipients. In addition, when the size of the healed tissue, the area of repigmentation and hair growth at the injury site were compared, young BMDC and young recipients had superior effects compared with old BMDC and old recipients.ConclusionsThese results demonstrate that cellular therapy is important for wound healing in older recipients that do not heal significantly without intervention. BMDC injections result in normal healing, indistinguishable from young recipients. Significantly, a single injection into the wound margin is sufficient to reverse the wounding process and promote normal wound healing. Although younger recipients eventually healed without therapy, BMDC injections accelerated the process, reduced scarring and increased hair regrowth. These findings provide insight into the treatment of non-healing epithelial tissue with BMDC.     

Extreme genetic diversity in the lizard Atlantolacerta andreanskyi (Werner, 1929): A montane cryptic species complex

ABSTRACT: BACKGROUND: Atlantolacerta andreanskyi is an enigmatic lacertid lizard that, according to the most recent molecular analyses, belongs to the tribe Eremiadini, family Lacertidae. It is a mountain specialist, restricted to areas above 2400 m of the High Atlas Mountains of Morocco with apparently no connection between the different populations. In order to investigate its phylogeography, 92 specimens of A. andreanskyi were analyzed from eight different populations across the distribution range of the species for up to 1108 base pairs of mitochondrial DNA (12S, ND4 and flanking tRNA-His) and 2585 base pairs of nuclear DNA including five loci (PDC, ACM4, C-MOS, RAG1, MC1R). RESULTS: The results obtained with both concatenated and coalescent approaches and clustering methods, clearly show that all the populations analyzed present a very high level of genetic differentiation for the mitochondrial markers used and are also generally differentiated at the nuclear level. CONCLUSIONS: These results indicate that A. andreanskyi is an additional example of a montane species complex.     

Optical Absorption Enhancement in Freestanding GaAs Thin Film Nanopyramid Arrays

Although III–V compound semiconductor multi‐junction cells show the highest efficiency among all types of solar cells, their cost is quite high due to expensive substrates, long epitaxial growth and complex balance of system components. To reduce the cost, ultra‐thin films with advanced light management are desired. Here effective light trapping in freestanding thin film nanopyramid arrays is demonstrated and multiple‐times light path enhancement is realized, where only 160 nm thick GaAs with nanopyramid structures is equivalent to a 1 μm thick planar film. The GaAs nanopyramids are fabricated using a combination of nanosphere lithography, nanopyramid metal organic chemical vapor deposition (MOCVD) growth, and gas‐phase substrate removal processes. Excellent optical absorption is demonstrated over a broad range of wavelengths, at various incident angles and at large‐curvature bending. Compared to an equally thick planar control film, the overall number of photons absorbed is increased by about 100% at various incident angles due to significant antireflection and light trapping effects. By implementing these nanopyramid structures, III–V material usage and deposition time can be significantly reduced to produce high‐efficiency, low‐cost thin film III–V solar cells.