Drug delivery systems: polymers and drugs monitored by capillary electromigration methods

In this paper, different electromigration methods used to monitor drugs and polymers released from drug delivery systems are reviewed. First, an introduction to the most typical arrangements used as drug delivery systems (e.g., polymer-drug covalent conjugates, membrane or matrix-based devices) is presented. Next, the principles of different capillary electromigration procedures are discussed, followed by a revision on the different procedures employed to monitor the release of drugs and the degradation or solubilization of the polymeric matrices from drug delivery systems during both in vitro and in vivo assays. A critical comparison between these capillary electrophoretic methods and the more common chromatographic methods employed to analyze drugs and polymers from drug delivery systems is presented. Finally, future outlooks of these electromigration procedures in the controlled release field are discussed.     

Megapixel digital PCR

We present a microfluidic 'megapixel' digital PCR device that uses surface tension-based sample partitioning and dehydration control to enable high-fidelity single DNA molecule amplification in 1,000,000 reactors of picoliter volume with densities up to 440,000 reactors cm(-2). This device achieves a dynamic range of 10(7), single-nucleotide-variant detection below one copy per 100,000 wild-type sequences and the discrimination of a 1% difference in chromosome copy number.     

Molecular type distribution and fluconazole susceptibility of clinical Cryptococcus gattii isolates from South African laboratory-based surveillance, 2005–2013

As is the case globally, Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV (101/146, 70%), followed by VGI (40/146, 27%), VGII (3/146, 2%) and VGIII (2/146, 1%). Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294 (14/146, 10%) and ST155 (10/146, 7%) being most commonly observed. The fluconazole MIC₅₀ and MIC₉₀ values of 105 (of 146) randomly selected C. gattii isolates were 4 μg/ml and 16 μg/ml, respectively. VGIV isolates had a lower MIC₅₀ value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers (99/136; 73% vs. 2/10; 20%), the confidence interval (CI) was wide (95% CI: 1.93–55.31, p = 0.006). Whole genome phylogeny of 98 isolates of South Africa’s most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen.     

Potato-associated arbuscular mycorrhizal fungal communities in the Peruvian Andes

The world's fourth largest food crop, potato, originates in the Andes. Here, the community composition of arbuscular mycorrhizal fungi (AMF) associated with potato in Andean ecosystems is described for the first time. AMF were studied in potato roots and rhizosphere soil at four different altitudes from 2,658 to 4,075 m above mean sea level (mamsl) and in three plant growth stages (emergence, flowering, and senescence). AMF species were distinguished by sequencing an approx. 1,500 bp nuclear rDNA region. Twenty species of AMF were identified, of which 12 came from potato roots and 15 from rhizosphere soil. Seven species were found in both roots and soil. Interestingly, altitude affected species composition with the highest altitude exhibiting the greatest species diversity. The three most common colonizers of potato roots detected were Funneliformis mosseae, an unknown Claroideoglomus sp., and Rhizophagus irregularis. Notably, the potato-associated AMF diversity observed in this Andean region is much higher than that reported for potato in other ecosystems. Potato plants were colonized by diverse species from 8 of the 11 Glomeromycota families. Identification of the AMF species is important for their potential use in sustainable management practices to improve potato production in the Andean region.     

Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene

Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.     

Neurochemical Evidence that Lysine Inhibits Synaptic Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity and Provokes Oxidative Damage in Striatum of Young Rats In vivo

Lysine (Lys) accumulation in tissues and biological fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and other inherited metabolic disorders. In the present study we investigated the effects of acute administration of Lys on relevant parameters of energy metabolism and oxidative stress in striatum of young rats. We verified that Lys in vivo intrastriatal injection did not change the citric acid cycle function and creatine kinase activity, but, in contrast, significantly inhibited synaptic Na⁺,K⁺-ATPase activity in striatum prepared 2 and 12 h after injection. Moreover, Lys induced lipid peroxidation and diminished the concentrations of glutathione 2 h after injection. These effects were prevented by the antioxidant scavengers melatonin and the combination of α-tocopherol and ascorbic acid. Lys also inhibited glutathione peroxidase activity 12 h after injection. Therefore it is assumed that inhibition of synaptic Na⁺,K⁺-ATPase and oxidative damage caused by brain Lys accumulation may possibly contribute to the neurological manifestations of FH and other neurometabolic conditions with high concentrations of this amino acid.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

Genesis and evolution of the <Emphasis Type="Italic">Evx</Emphasis> and <Emphasis Type="Italic">Mox</Emphasis>genes and the extended Hox and ParaHox gene clusters

Background

Hox and ParaHox gene clusters are thought to have resulted from the duplication of a ProtoHox gene cluster early in metazoan evolution. However, the origin and evolution of the other genes belonging to the extended Hox group of homeobox-containing genes, that is, Mox and Evx, remains obscure. We constructed phylogenetic trees with mouse, amphioxus and Drosophila extended Hox and other related Antennapedia-type homeobox gene sequences and analyzed the linkage data available for such genes.

Results

We claim that neither Mox nor Evx is a Hox or ParaHox gene. We propose a scenario that reconciles phylogeny with linkage data, in which an Evx/Mox ancestor gene linked to a ProtoHox cluster was involved in a segmental tandem duplication event that generated an array of all Hox-like genes, referred to as the 'coupled' cluster. A chromosomal breakage within this cluster explains the current composition of the extended Hox cluster (with Evx, Hox and Mox genes) and the ParaHox cluster.

Conclusions

Most studies dealing with the origin and evolution of Hox and ParaHox clusters have not included the Hox-related genes Mox and Evx. Our phylogenetic analyses and the available linkage data in mammalian genomes support an evolutionary scenario in which an ancestor of Evx and Mox was linked to the ProtoHox cluster, and that a tandem duplication of a large genomic region early in metazoan evolution generated the Hox and ParaHox clusters, plus the cluster-neighbors Evx and Mox. The large 'coupled' Hox-like cluster EvxHox/MoxParaHox was subsequently broken, thus grouping the Mox and Evx genes to the Hox clusters, and isolating the ParaHox cluster.

     

Aberrant endometrial steroid receptor expression in in-vitro maturation cycles despite hormonal luteal support: A pilot study

Clinical outcomes of fresh embryo transfer in non-hCG triggered in vitro maturation (IVM) cycles are inferior compared to vitrified-warmed embryo transfer. This is a prospective observational pilot study in a consecutive cohort of 31 polycystic ovary syndrome (PCOS) patients and 37 normo-ovulatory egg donors who underwent IVM without fresh embryo transfer between July 2009 and June 2014. All subjects received 150 IU of highly purified menotropin (HP-hMG) daily for three days. On cycle day 6, all patients started transdermal oestradiol (E2) at a daily dose of 9 mg. There was no human chorionic gonadotropin (hCG) trigger before oocyte retrieval (OR). Vaginal micronized progesterone was commenced on the evening after OR, at a daily dose of 600 mg. Additional luteal phase support (LPS) was administered as follows: Group A: no additional LPS; Group B: 1500 IU of hCG administered 4 h after OR and Group C: 5000 IU of hCG administered 4 h after OR + an additional injection of 5000 IU of hCG 1 day before endometrial biopsy. Endometrial biopsy for histology and immunohistochemistry (IHC) was performed on day 5 or 6 after OR. Instead of being downregulated, both PR-B and ERα in endometrial glands and stroma were moderately to strongly expressed in all three protocols, suggesting that the mid-luteal histological signature of endometrial receptivity is deficient in a non-hCG-triggered IVM cycle. Poor clinical outcomes after fresh embryo transfer following IVM are probably related to inappropriate endometrial development which may be linked to the short follicular phase of IVM cycles.