Malnutrition and cachexia in patients with head and neck cancer treated with (chemo)radiotherapy

AimTo highlight the problems associated with nutrition that occur in patients with squamous cell carcinoma of the head and neck (SCCHN).BackgroundSCCHN is associated with weight loss before, during and after radiotherapy or concurrent chemoradiotherapy. Because of serious consequences of malnutrition and cachexia on treatment outcome, mortality, morbidity, and quality of life, it is important to identify SCCHN patients with increased risk for the development of malnutrition and cachexia.Materials and methodsCritical review of the literature.ResultsThis review describes pathogenesis, diagnosis and treatment of malnutrition and cancer cachexia. Treatment of malnutrition and cancer cachexia includes nutritional interventions and pharmacological therapy. Advantages and disadvantages of different nutritional interventions and their effect on the nutritional status, quality of life and specific oncological treatment are presented.ConclusionsNutritional management is an essential part of care of these patients, including early screening, assessment of nutritional status and appropriate intervention.     

Force-dependent stepping kinetics of myosin-V

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.     

New statistical software for intralaboratory and interlaboratory quality control in clinical cytology. Validation in a simulation study on clinical samples

OBJECTIVE: To design a statistical software package to provide automated calculations of normal and weighted and 3 indices. STUDY DESIGN: Prompted by the lack of commonly available software to compute weighted kappa and the nonproportionate workload needed to calculate our 3 variability indices manually, the new statistical software package was designed. To demonstrate the performance of the new CONQUISTADOR software, a simulation study (both intralaboratory and interlaboratory) was designed using 5,000 clinical samples randomly selected from a data file of > or = 200,000 conventional Pap smears and programmed to become "analyzed" by 12 cytologists in 5 imaginary laboratories. RESULTS: A representative set of both complete and partial outputs provided by the software, in Excel format (Microsoft, Redmond, Washington, U.S.A.) are shown to illustrate the different functions of the program. In the interlaboratory mode, the software calculates accuracy indicators (sensitivity, specificity, positive and negative predictive value, and their 95% CI), which are not common features of regular statistical packages; kappa and weighted kappa; and their 95% CI (comparison of single laboratories to all laboratories and pairwise comparisons between single laboratories). The 3 diagnostic variability indices can be computed separately for all samples or for only the positive samples. In the intralaboratory mode, the software calculates the same indices for individual cytologists. CONCLUSION: The CONQUISTADOR statistical package has properties that are useful in monitoring cytologic laboratory quality in both intralaboratory and interlaboratory settings. The software will be distributed by the National Institute of Health, Rome, for the delivery costs only.     

Conventional Pap smear and liquid-based cytology as screening tools in low-resource settings in Latin America: experience of the Latin American screening study

OBJECTIVE: To evaluate the performance of the conventional Pap test and liquid-based cytology (LBC) in an ongoing multicenter trial testing optional screening tools (cytology, screening colposcopy, visual inspection with acetic acid, visual inspection with Lugol's Iodine, cervicography and Hybrid Capture II [HCII] (Digene Brazil, S?o Paulo, Brazil) conventional and self-sampling), for cervical cancer in Brazil and Argentina. STUDY DESIGN: A cohort of 12,107 women attending four clinics (Campinas, S?o Paulo, Porto Alegre, Buenos Aires) were randomized into the 8 diagnostic arms. Women testing positive with any of the tests were referred for colposcopy, and cervical biopsies were used as the gold standard to assess performance characteristics of the diagnostic tests. Conventional Pap smears were sampled by all clinics (n = 10,240), and LBC (Autocyte PREP, [TriPath Imaging, Burlington, North Carolina, U.S.A.], n=320, and DNA-Citoliq [Digene Brazil], n =1,346) was performed by 1 of the clinics. RESULTS: Conventional Pap smears showed no squamous intraepithelial lesions (normal) in 8,946 (87.4%) and LBC in 1,373 (82.4%). Using high grade squamous intraepithelial lesions (HSIL) as the cutoff, Pap smears predicted high grade (cervical intraepithelial neoplasia [CIN] 3) with OR 63.0 (95% CI, 36.90-107.70), standard error (SE) 59%, SP 97.8%, positive predictive value (PPV) 68.1% and negative predictive value (NPV) 96.7%. The same figures for Autocyte PREP were: OR 9.0 (95% CI, 2.43-33.24), sensitivity (SE) 33.3%, specificity (SP) 100%, PPV 100% and negative PV (NPV) 88.8%. DNA-Citoliq detected CIN 3 as follows: OR 11.8 (95% CI 2.60-53.26), SE 40.0%, SP 94.6%, PPV 40.0% and NPV 94.6%. Lowering the cutoff to low grade squamous intraepithelial lesions increased SE and NPV but compromised SP and PPV. The detection rates for high grade lesions after an atypical squamous cells of undetermined significance diagnosis were similar with the 3 techniques. In our settings, the 3 methods of cervical cytology were slightly different in performance. The conventional Pap smear had the highest SE, while Autocyte PREP had 100% SP and PPV in detecting CIN3 with the HSIL cutoff. All 3 tests had lower SE but higher SP as compared to HCII.     

Analysis and Comparison of Electrical Pulse Parameters for Gene Electrotransfer of Two Different Cell Lines

Knowledge of the parameters which influence the efficiency of gene electrotransfer has importance for practical implementation of electrotransfection for gene therapy as well as for better understanding of the underlying mechanism. The focus of this study was to analyze the differences in gene electrotransfer and membrane electropermeabilization between plated cells and cells in a suspension in two different cell lines (CHO and B16F1). Furthermore, we determined the viability and critical induced transmembrane voltage (ITV_c) for both cell lines. In plated cells we obtained relatively little difference in electropermeabilization and gene electrotransfection between CHO and B16F1 cells. However, significant differences between the two cell lines were observed in a suspension. CHO cells exhibited a much higher gene electrotransfection rate compared to B16F1 cells, whereas B16F1 cells reached maximum electropermeabilization at lower electric fields than CHO cells. Both in a suspension and on plated cells, CHO cells had a slightly better survival rate at higher electric fields than B16F1 cells. Calculation of ITV_c in a suspension showed that, for both electropermeabilization and gene electrotransfection, CHO cells have lower ITV_c than B16F1 cells. In all cases, ITV_c for electropermeabilization was lower than ITV_c for gene electrotransfer, which is in agreement with other studies. Our results show that there is a marked difference in the efficiency of gene electrotransfer between suspended and plated cells.     

The dynamics of melittin-induced membrane permeability

The transport of co-encapsulated solutes through the melittin-induced pores in the membrane of giant phospholipid vesicles was studied, and the characteristics of the pore formation process were modeled. Molecules of two different sizes (dextran and the smaller, fluorescent marker Alexa Fluor) were encapsulated inside the vesicles. The chosen individual vesicles were then transferred by micromanipulation from the stock suspension to the environment with the melittin (MLT). The vesicles were observed optically with a phase-contrast microscope and by monitoring the fluorescence signal. Such an experimental setup enabled an analysis of a single vesicle’s response to the MLT on the basis of simultaneous, separate measurements of the outflow of both types of encapsulated molecules through the MLT-induced pores in the membrane. The mechanisms of the MLT’s action were suggested in a model for MLT pore formation, with oligomeric pores continuously assembling and dissociating in the membrane. Based on the model, the results of the experiments were explained as a consequence of the membrane’s permeability dynamics, with a continuously changing distribution of pores in the membrane with regard to their size and number. The relatively stable “average MLT pore” characteristics can be deduced from the proposed model.     

Insights into subtle conformational differences in the substrate-binding loop of fungal 17β-hydroxysteroid dehydrogenase: a combined structural and kinetic approach

The 17β-HSD (17β-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17β-hydroxy counterparts. 17β-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17β-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17β-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17β-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17β-HSDcl [IC50 2.8?μM; at 100?μM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π-π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme-coenzyme-steroid ternary complexes.     

A neurotoxic phospholipase a(2) impairs yeast amphiphysin activity and reduces endocytosis

Background

Presynaptically neurotoxic phospholipases A₂ inhibit synaptic vesicle recycling through endocytosis.

Principal Findings

Here we provide insight into the action of a presynaptically neurotoxic phospholipase A₂ ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin.

Conclusions

We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein–protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A₂ work can open new ways to regulate endocytosis.