Folate-targeted PEG as a potential carrier for carboplatin analogs. Synthesis and in vitro studies

Like most low molecular weight drugs, carboplatin has a short blood circulation time, which reduces tumor uptake and intracellular DNA binding. Drugs conjugated to PEG carriers benefit from prolonged blood circulation, but suffer from reduced cell permeability. In this work we attempted to develop long-circulating PEGylated carboplatin analogues with improved cell permeation abilities, by conjugating the platinum moiety to folate-targeted PEG carriers capable of utilizing the folate receptor-mediated endocytosis (FRME). Two bifunctional FA-PEG conjugates, FA-PEG-Pt and FA-PEG-FITC, were prepared, and their cell uptake, DNA binding, and cytotoxicity were studied by fluorescent microscopy, FACS, and platinum analysis. Folate-targeted PEG conjugates enter the cells efficiently by the FRME pathway but form relatively few DNA adducts and have higher IC(50) values than carboplatin and their nontargeted analogues. Nontargeted PEG-Pt conjugates have a lower cellular uptake but produce higher levels of DNA binding and improved cytotoxicity. Carboplatin, used as a control, has the fastest cellular uptake, but after 16 h of postincubation a large percentage of the drug is excreted from the cells. The findings of this study suggest that folate-targeted conjugates such as FA-PEG-Pt, may not be an optimal prodrug for the carboplatin family compounds, because the conjugates or the active moieties are neutralized or blocked during the FRME process and do not manage to effectively reach the nuclear DNA.     

Erasure of kinase phosphorylation in astrocytes during oxygen-glucose deprivation is controlled by ATP levels and activation of phosphatases

We have examined the relationship between adenosine triphosphate (ATP) concentration and loss of maintenance of kinase-signalling cascades in primary cortical astrocytes during oxygen-glucose deprivation (OGD) as this may constitute an irreversible step that commits astrocytes to cell death. We report that the phosphorylation of Akt, ERK, JNK and p38 kinases, whose activities depend on serine, threonine and tyrosine phosphorylation, were all increased during OGD. All these phosphorylations were reduced to below detection limits when ATP levels were less than 10% of normal levels. Using ERK and Akt as representative examples, we show that this erasure is not irreversible as both ERK and Akt phosphorylations can be partially restored by addition of glucose under anoxic conditions. We further investigated whether OGD caused any change in phosphatase activity. The PP1/PP2A phosphatase inhibitors okadaic acid and caliculyn A, but not cyclosporine A, delayed the removal of ERK and Akt phosphorylation under OGD. By comparing the extent of phosphorylation increase under OGD and normoxic conditions, we calculate that phosphatase activity was increased by approximately 3.6-fold during OGD. These data show that ATP levels control an important checkpoint in kinase function, and that ATP levels may need to be considered when studies of kinase function in relation to OGD are conducted.     

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates

The potential of RNAs and RNA-protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA (trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.     

Structure-activity relationships of aminoglycoside-arginine conjugates that bind HIV-1 RNAs as determined by fluorescence and NMR spectroscopy

We present here a new set of aminoglycoside-arginine conjugates (AACs) that are either site-specific or per-arginine conjugates of paromomycin, neamine, and neomycin B as well as their structure-activity relationships. Their binding constants (KD) for TAR and RRE RNAs, measured by fluorescence anisotropy, revealed dependence on the number and location of arginines in the different aminoglycoside conjugates. The binding affinity of the per-arginine aminoglycosides to TAR is higher than to RRE, and hexa-arginine neomycin B is the most potent binder (KD=5 and 23 nM, respectively). The 2D TOCSY NMR spectrum of the TAR monoarginine-neomycin complex reveals binding at the bulge region of TAR.     

The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes

Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging autophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow autophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDa)-traced by immunoblotting and verified by [(3)H]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.     

Isolation of a dehydrin cDNA from orange and grapefruit citrus fruit that is specifically induced by the combination of heat followed by chilling temperatures

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K-segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a 'Navel' orange 202-bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm-type K-segment consensus sequence, and of which the expression was down-regulated by exposure to low oxygen levels. The full-length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5'-and 3'-RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202-bp 'Navel' dehydrin PCR fragment was used to screen a 'Star Ruby' grapefruit flavedo cDNA library, and its full-length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N-terminus but differed in their C-terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S-segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K-segment consensus sequence, whereas cpDHN contains two dehydrin K-segments. Both csDHN and cpDHN represent single copy genes, in 'Navel' orange and 'Star Ruby' grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre-storage hot water treatment, given to enhance fruit-chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2 degrees C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2 degrees C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non-chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.     

FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.     

Regulation of spindle pole function by an intermediary metabolite

Spore formation in the yeast Saccharomyces cerevisiae depends on a modification of spindle pole bodies (SPBs) at the onset of meiosis II that allows them to promote de novo membrane formation. Depletion of the environmental carbon source during sporulation results in modification of only one SPB from each meiosis II spindle and formation of a two-spored ascus, called a nonsister dyad (NSD). We have found that mutants impaired in the glyoxylate pathway, which is required for the conversion of acetate to glucose, make NSDs when acetate is the primary carbon source. Wild-type cells make NSDs when the carbon source is glycerol, which is converted to glucose independently of the glyoxylate pathway. During NSD formation in glycerol, only the two SPBs created at the meiosis I/II transition ("daughters") are modified. In these conditions, the SPB components Mpc70p and Spo74p are not recruited to mother SPBs. Moreover, cooverexpression of Mpc70p and Spo74p suppresses NSD formation in glycerol. Our findings indicate that flux through the glyoxylate pathway during sporulation regulates modification of mother SPBs via recruitment of Mpc70p and Spo74p. These results define a cellular response in which the accumulation of an intermediary metabolite serves as a measure of biosynthetic capacity to regulate the number of daughter cells formed.     

A Typical Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Clone Is Widespread in the Community in the Gaza Strip

Epidemiological data on community acquired methicillin-resistant-Staphylococcus aureus (CA-MRSA) carriage and infection in the Middle-East region is scarce with only few reports in the Israeli and Palestinian populations. As part of a Palestinian-Israeli collaborative research, we have conducted a cross-sectional survey of nasal S. aureus carriage in healthy children and their parents throughout the Gaza strip. Isolates were characterized for antibiotic susceptibility, mec gene presence, PFGE, spa type, SCCmec-type, presence of PVL genes and multi-locus-sequence-type (MLST). S. aureus was carried by 28.4% of the 379 screened children-parents pairs. MRSA was detected in 45% of S. aureus isolates, that is, in 12% of the study population. A single ST22-MRSA-IVa, spa t223, PVL-gene negative strain was detected in 64% of MRSA isolates. This strain is typically susceptible to all non-β-lactam antibiotics tested. The only predictor for MRSA carriage in children was having an MRSA carrier-parent (OR = 25.5, P = 0.0004). Carriage of the Gaza strain was not associated with prior hospitalization. The Gaza strain was closely related genetically to a local MSSA spa t223 strain and less so to EMRSA15, one of the pandemic hospital-acquired-MRSA clones, scarcely reported in the community. The rapid spread in the community may be due to population determinants or due to yet unknown advantageous features of this particular strain.     

High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging

Background

Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.

Results

Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.

Conclusions

The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-864) contains supplementary material, which is available to authorized users.