Implementation of intensity modulated radiotherapy for prostate cancer in a private radiotherapy service in Mexico

Intensity modulated radiation therapy (IMRT) allows physicians to deliver higher conformal doses to the tumour, while avoiding adjacent structures. As a result the probability of tumour control is higher and toxicity may be reduced. However, implementation of IMRT is highly complex and requires a rigorous quality assurance (QA) program both before and during treatment. The present article describes the process of implementing IMRT for localized prostate cancer in a radiation therapy department. In our experience, IMRT implementation requires careful planning due to the need to simultaneously implement specialized software, multifaceted QA programs, and training of the multidisciplinary team. Establishing standardized protocols and ensuring close collaboration between a multidisciplinary team is challenging but essential.     

Synthesis and characterisation of a new pH-sensitive amphotericin B--poly(ethylene glycol)-b-poly(L-lysine) conjugate

This paper reports on the synthesis, characterisation, and efficiency of a new intravenous conjugate of amphotericin B (AMB). Twelve molecules of AMB were attached to block copolymer poly(ethylene glycol)-b-poly(L-lysine) via pH-sensitive imine linkages. In vitro drug release studies demonstrated the conjugate (M(w)=26,700) to be relatively stable in human plasma and in phosphate buffer (pH 7.4, 37 degrees C). Controlled release of AMB was observed in acidic phosphate buffer (pH 5.5, 37 degrees C) with the half-life of 2 min. The LD(50) value determined in vivo (mouse) is 45 mg/kg.     

Assimilation dynamics of soil carbon and nitrogen by wheat roots and Collembola

It has been demonstrated that plant roots can take up small amounts of low-molecular weight (LMW) compounds from the surrounding soil. Root uptake of LMW compounds have been investigated by applying isotopically labelled sugars or amino acids but not labelled organic matter. We tested whether wheat roots took up LMW compounds released from dual-labelled (¹³C and ¹⁵N) green manure by analysing for excess ¹³C in roots. To estimate the fraction of green manure C that potentially was available for root uptake, excess ¹³C and ¹⁵N in the primary decomposers was estimated by analysing soil dwelling Collembola that primarily feed on fungi or microfauna. The experimental setup consisted of soil microcosm with wheat and dual-labelled green manure additions. Plant growth, plant N and recoveries of ¹³C and ¹⁵N in soil, roots, shoots and Collembola were measured at 27, 56 and 84 days. We found a small (<1%) but significant uptake of green manure derived ¹³C in roots at the first but not the two last samplings. About 50% of green manure C was not recovered from the soil-plant system at 27 days and additional 8% was not recovered at 84 days. Up to 23% of C in collembolans derived from the green manure at 56 days (the 27 days sampling was lost). Using a linear mixing model we estimated that roots or root effluxes provided the main C source for collembolans (54−79%). We conclude that there is no solid support for claiming that roots assimilated green manure derived C due to very small or no recoveries of excess ¹³C in wheat roots. During the incubation the pool of green manure derived C available for root uptake decreased due to decomposition. However, the isotopic composition in Collembola indicated that there was a considerable fraction of green manure derived C in the decomposer system at 56 days thus supporting the premise that LMW compounds containing C from the green manure was released throughout the incubation. Responsible Editor: A. C. Borstlap.     

Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli

The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS-PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.     

Cellular uptake of ODNs in HIV-1 human-infected cells: a role for viral particles in DNA delivery?

We have previously described how a 16 nucleotides ODN (termed 93del) is capable of inhibiting the activity of recombinant integrase in a cell-free system as well as HIV-1 replication in human-infected cells with IC(50) in the low nanomolar range. Intracellular HIV-1 replication was inhibited when the ODN was added at the onset of infection. These results raise several questions. Is a naked ODN able to enter the cell? Does the virus play a role in ODN entry? The uptake of several ODNs (93del, 60del(sc), TBA, T30923) was evaluated and then tracked by labeling the ODN with a fluorescent dye and assessing its intracellular localization by confocal microscopy. A significant level of cellular uptake of free ODN was observed in several cell lines: HeLa epithelial cells, Huh7 hepatic cells, and H9 lymphocytes, and was detected for all ODNs tested except for TBA. Striking differences were observed when naked ODNs were added to cell in the presence or absence of the virus. When HIV-1 virions were present a sharp increase in cellular fluorescence was observed. These results strongly suggest a role for HIV-1 virions in the uptake of certain ODNs.     

Detection of transient protein-protein interactions by bimolecular fluorescence complementation: the Abl-SH3 case

Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of bimolecular fluorescence complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.     

Caenorhabditis elegans mitochondrial mutants as an investigative tool to study human neurodegenerative diseases associated with mitochondrial dysfunction

In humans, well over one hundred diseases have been linked to mitochondrial dysfunction and many of these are associated with neurodegeneration. At the root of most of these diseases lay ineffectual energy production, caused either by direct or indirect disruption to components of the mitochondrial electron transport chain. It is surprising then to learn that, in the nematode Caenorhabditis elegans, a collection of mutants which share disruptions in some of the same genes that cause mitochondrial pathogenesis in humans are in fact long-lived. Recently, we resolved this paradox by showing that the C. elegans "Mit mutants" only exhibit life extension in a defined window of mitochondrial dysfunction. Similar to humans, when mitochondrial dysfunction becomes too severe these mutants also exhibit pathogenic life reduction. We have proposed that life extension in the Mit mutants occurs as a by-product of compensatory processes specifically activated to maintain mitochondrial function. We have also proposed that similar kinds of processes may act to delay the symptomatic appearance in many human mitochondrial-associated disorders. In the present report, we describe our progress in using the Mit mutants as an investigative tool to study some of the processes potentially employed by human cells to offset pathological mitochondrial dysfunction.