Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva

Background

Third-stage larvae (L3) of the canine hookworm, Ancylostoma caninum, undergo arrested development preceding transmission to a host. Many of the mRNAs up-regulated at this stage are likely to encode proteins that facilitate the transition from a free-living to a parasitic larva. The initial phase of mammalian host invasion by A. caninum L3 (herein termed “activation”) can be mimicked in vitro by culturing L3 in serum-containing medium.

Methodology/Principal Findings

The mRNAs differentially transcribed between activated and non-activated L3 were identified by suppression subtractive hybridisation (SSH). The analysis of these mRNAs on a custom oligonucleotide microarray printed with the SSH expressed sequence tags (ESTs) and publicly available A. caninum ESTs (non-subtracted) yielded 602 differentially expressed mRNAs, of which the most highly represented sequences encoded members of the pathogenesis-related protein (PRP) superfamily and proteases. Comparison of these A. caninum mRNAs with those of Caenorhabditis elegans larvae exiting from developmental (dauer) arrest demonstrated unexpectedly large differences in gene ontology profiles. C. elegans dauer exiting L3 up-regulated expression of mostly intracellular molecules involved in growth and development. Such mRNAs are virtually absent from activated hookworm larvae, and instead are over-represented by mRNAs encoding extracellular proteins with putative roles in host-parasite interactions.

Conclusions/Significance

Although this should not invalidate C. elegans dauer exit as a model for hookworm activation, it highlights the limitations of this free-living nematode as a model organism for the transition of nematode larvae from a free-living to a parasitic state.     

Role of phenolic acids and enzymes of phenylpropanoid pathway in resistance of chayote fruit (Sechium edule) against infestation by melon fly,Bactrocera cucurbitae

Melon fly is a serious pest of cucurbits all over the world causing huge losses to yield. However, the only exception is the chayote fruit (Sechium edule) that shows resistance to melon fly infestation. Studies on culture of melon fly indicated the absence of plant traits resisting oviposition on chayote fruit. However, the melon fly was unable to complete its life cycle successfully on chayote showing that factors inhibiting larval development in melon fly could be attributed to biochemical constituents. Studies were, therefore, carried out to compare the biochemical responses of chayote, a melon fly resistant species and bitter gourd, a susceptible species to melon fly infestation with regard to the levels of phenolic acids and activities of the enzymes of phenylpropanoid pathway (PPP) leading to synthesis of lignin. The resistant chayote exhibited significantly higher accumulation of lignin associated with higher activities of phenylalanine ammonia‐lyase (PAL), tyrosine ammonia‐lyase (TAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD). On the contrary, the susceptible bitter gourd recorded lower activities of PAL, CAD and POD and a decreasing trend of TAL during infestation associated with a lower lignin content. The monomer composition of lignin in the resistant chayote showed twofold higher level of guaiacyl (G) and syringyl (S) units compared to susceptible bitter gourd and the G/S ratio during infestation increased in chayote while decreasing in bitter gourd. The levels of PPP intermediates, p‐coumaric acid was higher in chayote while p‐hydroxy benzoic acid, a chemo‐attractant, was higher in bitter gourd. Incorporation of p‐coumaric acid in the larval diet strongly inhibited larval growth even as p‐hydroxy benzoic acid promoted growth confirming the direct role of p‐coumaric acid in conferring resistance to chayote. The level of salicylic acid, a signal molecule involved in induction of defence response, was higher in chayote compared to bitter gourd. Chayote also exhibited higher level of activity of POD in the phloem exudates compared to bitter gourd. The higher concentration of sugars in exudates of chayote might act like signalling molecules causing activation of plant genes, especially of the phenylpropanoid biosynthesis pathway and possibly produce an osmotic effect inducing resistance against the melon fly. Thus, the study revealed that the resistance in chayote to melon fly infestation is a complex, multi‐layered process in which the activities of PPP enzymes generating phenolic intermediates leading to lignin biosynthesis and the composition of exudates appear to play significant roles. Besides, the study also indicated that different forms of lignin might play a role in the resistance of chayote against melon fly infestation.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Weekly vs. tri-weekly cisplatin based chemoradiation in carcinoma cervix: a prospective randomized study of toxicity and compliance

BackgroundAddition of chemotherapy to radiation has improved 5-year survival by 6%. However, the optimal dose and schedule of concurrent cisplatin is not well defined, though widely accepted practice is the weekly schedule of 40 mg/m² for 5 weeks. Repeated admissions for weekly cisplatin drain the limited resources in high volume centres. We intended to study the compliance and toxicity of two cisplatin schedules in our patients diagnosed with carcinoma cervix.Materials and methodsBetween 2007–2011, 212 patients, histologically proven squamous cell carcinoma with stages IIB to IIIB were randomized into two arms. All patients were planned for external beam radiotherapy 45 Gy/25 frs over 5 weeks followed by Intracavitary or Interstitial brachytherapy to a total BED dose of 75–85 Gy. Single agent cisplatin given concomitantly, was scheduled weekly (40 mg/m²/cycle, 5 cycles) in an arm A and three weekly (100 mg/m²/cycle, 2 cycles) in an arm B. Toxicity and compliance were evaluated weekly according to the RTOG guidelines. Analysis of the compiled data was done using SSPS version 20.ResultsOf the evaluable 212, 109 patients received weekly cisplatin chemotherapy and 103 patients received three weekly cisplatin. The most common acute toxicity observed was grade I–II leucopoenia. The upper and lower gastrointestinal reactions were high in three weekly arms, which was statistically significant (57% and 42.7%, p < 0.05). Proctitis was observed in 10% of patients in both of the arms and only two patients had Gr1 Cystitis after 6 months of treatment.ConclusionsTri-weekly cisplatin based concurrent chemoradiation can be adopted in high volume centres with manageable haematological and gastrointestinal acute toxicities.     

Model of T-Cell Nuclear Deformation by the Cortical Actin Layer

Deformations of cell nuclei accompany a number of essential intracellular processes. Although evidence is being accumulated on the primary role actin structures play in controlling the shape of the nucleus, the mechanisms behind this phenomenon remain unknown. Here, we consider theoretically a specific paradigm of nuclear deformation, and a related actin rearrangement, in T cells stimulated by contact with a bead covered by surrogate antigens. We suggest that the nucleus is deformed by the elastic forces developed within a cylindrical layer of polymerized actin, which is generated as a result of the receptor-mediated T-cell activation. We substantiate this proposal with a theoretical analysis of mutual deformations in the actin layer and the nucleus, which recovers the experimentally observed nuclear shapes. Furthermore, we evaluate the forces developed by the actin polymerization that drives the nuclear deformation. The model predicts the mode of actin polymerization generated by the surrogate antigens covering a bead and the values of the elastic moduli of the nuclear shell. We provide a qualitative experimental support for the model assumptions by visualizing the stages of nuclear shape change and the corresponding evolution of the cortical actin.     

Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop

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EGFP-tagged core and linker histones diffuse via distinct mechanisms within living cells

The effect of chromatin organization on EGFP-tagged histone protein dynamics within the cell nucleus has been probed using fluorescence correlation and recovery measurements on single living HeLa cells. Our studies reveal that free fraction of core-particle histones exist as multimers within the cell nucleus whereas the linker histones exist in monomeric forms. The multimeric state of core histones is found to be invariant across mammalian and polytene chromosomes and this is ATP dependent. In contrast, the dynamics of the linker histones exhibits two distinct diffusion timescales corresponding to its transient binding and unbinding to chromatin governed by the tail domain residues. Under conditions of chromatin condensation induced by apoptosis, the free multimeric fraction of core histones is found to become immobile, while the monomeric linker histone mobility is partially reduced. In addition, we observe differences in nuclear colocalization of linker and core particle histones. These results are validated through Brownian dynamics simulation of core and linker histone mobility. Our findings provide a framework to understand the coupling between the state of chromatin assembly and histone protein dynamics that is central to accessing regulatory sites on the genome.     

Chromatin structure exhibits spatio-temporal heterogeneity within the cell nucleus

Local chromatin compaction undergoes dynamic perturbations to regulate genetic processes. To address this, the direct measurement of the fluidity of chromatin structure is carried out in single live cells using steady-state anisotropy imaging and polarization modulation microscopy. Fluorescently tagged core and linker histones are used to probe different structural aspects of chromatin compaction. A graded spatial heterogeneity in compaction is observed for the chromatin besides the distinct positional ordering of core and linker histones. These spatio-temporal features are maintained by active processes and perturbed during death. With cell cycle, the distribution in compaction heterogeneity continually changes maximizing during M-G1 transition where it displays bimodal behavior. Such measurements of spatio-temporal chromatin fluidity could have broader implications in understanding chromatin remodeling within living cells.