In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes

Recently, we reported that 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with >70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 degrees C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1+/-4.0% of doxorubicin over 3 h at 37 degrees C in the presence of serum. The drug was almost completely released within 120s at 42 degrees C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 degrees C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL.     

Toxicogenomics in the assessment of immunotoxicity

Microarray analysis is used for simultaneous measurement of expression of thousands of genes in a given sample and as such extends and deepens our understanding of biological processes. Application of the technique in toxicology is referred to as toxicogenomics. The examples of assessment of immunotoxicity by gene expression profiling presented and discussed here, show that microarray analysis is able to detect known and novel effects of a wide range of immunomodulating agents. Besides the elucidation of mechanisms of action, toxicogenomics is also applied to predict consequences of exposing biological systems to toxic agents. Successful attempts to classify compounds using signature gene expression profiles have been reported. These did, however, not specifically focus on immunotoxicity. Databases containing expression profiles can facilitate the applications of toxicogenomics. Platforms and methodologies for gene expression profiling may vary, however, hampering data compiling across different laboratories. Therefore, attention is paid to standardization of the generation, reporting, and management of microarray data. Obtained gene expression profiles should be anchored to pathological and functional endpoints for correct interpretation of results. These issues are also important when using toxicogenomics in risk assessment. The application of toxicogenomics in evaluation of immunotoxicity is thus not yet without challenges. It already contributes to the understanding of immunotoxic processes and the development of in vitro screening assays, though, and is therefore expected to be of value for mechanistic insight into immunotoxicity and hazard identification of existing and novel compounds.     

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.     

Genetic variation and potential coinfection of Wolbachia among widespread Asian citrus psyllid (Diaphorina citri Kuwayama) populations

Wolbachia can profoundly influence the survival, reproduction, and defenses of insect hosts. These interactions could potentially be harnessed for managing pests or insecttransmitted diseases. Diaphorina citri Kuwayama is a phloem-feeding pest capable of transmitting the putative causal agent of citrus greening, Candidatus Liberibacter asiaticus (CLas). Like many insects, D. citri is also infected with Wolbachia (wDi). Recent studies indicate that the relative abundance of wDi could be associated with the abundance of CLas, and that wDi may contribute to regulating expression of phage lytic cycle genes in CLas, suggesting the need for better understanding of wDi biology in general. This study investigated the genetic diversity of wDi among D. citri in populations spanning eleven countries and two U.S. territories. Six Wolbachia genes, wsp, coxA.fbpA.ftsZ, gatB, and hep A, were sequenced and compared across samples. Two prevalent wDi strains were identified across the samples, and screening of clone libraries revealed possible coinfection of wDi strains in specific populations. D. citri mitochondrial cytochrome oxidase subunit I gene (mtCOI) were more divergent between D. citri populations that were infected with different wDi strains or had different infection statuses (single infection vs. coinfection). While we could not eliminate the possibility that maternal transmission may contribute to such patterns, it is also possible that wDi may induce cytoplasmic incompatibility in their host. These fin dings should contribute to the understanding of wDi population ecology, which may facilitate manipulation of this endosymbiont for management of citrus greening disease worldwide.     

De Novo Mutations in CHAMP1 Cause Intellectual Disability with Severe Speech Impairment

CHAMP1 encodes a protein with a function in kinetochore-microtubule attachment and in the regulation of chromosome segregation, both of which are known to be important for neurodevelopment. By trio whole-exome sequencing, we have identified de novo deleterious mutations in CHAMP1 in five unrelated individuals affected by intellectual disability with severe speech impairment, motor developmental delay, muscular hypotonia, and similar dysmorphic features including short philtrum and a tented upper and everted lover lip. In addition to two frameshift and one nonsense mutations, we found an identical nonsense mutation, c.1192C>T (p.Arg398^∗), in two affected individuals. All mutations, if resulting in a stable protein, are predicted to lead to the loss of the functionally important zinc-finger domains in the C terminus of the protein, which regulate CHAMP1 localization to chromosomes and the mitotic spindle, thereby providing a mechanistic understanding for their pathogenicity. We thus establish deleterious de novo mutations in CHAMP1 as a cause of intellectual disability.     

Whole Exome Sequencing of Distant Relatives in Multiplex Families Implicates Rare Variants in Candidate Genes for Oral Clefts

A dozen genes/regions have been confirmed as genetic risk factors for oral clefts in human association and linkage studies, and animal models argue even more genes may be involved. Genomic sequencing studies should identify specific causal variants and may reveal additional genes as influencing risk to oral clefts, which have a complex and heterogeneous etiology. We conducted a whole exome sequencing (WES) study to search for potentially causal variants using affected relatives drawn from multiplex cleft families. Two or three affected second, third, and higher degree relatives from 55 multiplex families were sequenced. We examined rare single nucleotide variants (SNVs) shared by affected relatives in 348 recognized candidate genes. Exact probabilities that affected relatives would share these rare variants were calculated, given pedigree structures, and corrected for the number of variants tested. Five novel and potentially damaging SNVs shared by affected distant relatives were found and confirmed by Sanger sequencing. One damaging SNV in CDH1, shared by three affected second cousins from a single family, attained statistical significance (P = 0.02 after correcting for multiple tests). Family-based designs such as the one used in this WES study offer important advantages for identifying genes likely to be causing complex and heterogeneous disorders.