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Background and Objectives: Cellulite and lipodystrophy are often found together, especially in areas of the buttocks and thighs, causing skin surface irregularities. Each of these conditions is currently treated independently as two separate surgical procedures. In our practice, we developed a novel combined approach for the simultaneous treatment of cellulite and lipodystrophy, as a single stage procedure in the same anatomic area. For the treatment of cellulite, we used the Nd:YAG laser at a wavelength of 1,440-nm, along with an innovative 1,000-micron directional side-firing fiber optic laser system. For the treatment of lipodystrophy, the Nd:YAG laser with a 1,440 nm wavelength, along with a fiber optic laser system was used. The objective of this study is to determine the efficacy and safety of a combined approach for the simultaneous treatment of cellulite and lipodystrophy., Study Design, Patients and Methods: In 2012, 16 subjects with noticeable cellulite, Grade II and Grade III, accompanied by mild-to-moderate lipodystrophy of the lower body received single treatments of the Nd:YAG laser at a wavelength of 1,440-nm along with the 1,000-micron side-firing fiber optic laser system for simultaneous treatments of both cellulite and lipodystrophy. Patients were assessed at baseline and 3-6 months post-treatment by a modified Nurnberger-Muller scale utilized to quantify the cellulite severity. Additionally, patient satisfaction and a global aesthetic improvement scale were used to measure the improvement in lipodystrophy., Results: Blinded reviewers identified the correct baseline photographs 97% of the time when presented with a set of photographs. The median modified Nurnberger-Muller scale score at baseline was 4.75 ± 1.2 and the average improvement was 2.0 ± 1.2. Global aesthetic improvement scores ranged from 1 to 3 with an average of 1.58 indicating a much-improved overall appearance. Satisfaction was high for both physicians and patients with scores corresponding to extremely satisfied/satisfied., Conclusion: Precise, effective delivery of laser energy to the dermal-adipose tissue, as well as the deep adipose lipodystrophy is feasible as a safe modality for the simultaneous treatment of cellulite and lipodystrophy in the buttocks and thighs, as a single stage procedure., (© 2015 Wiley Periodicals, Inc.)

Extensive re-organisation of the actin cytoskeleton and changes in the expression of its binding proteins is a characteristic feature of cancer cells. Previously we have shown that the tropomyosin isoform Tpm3.1, an integral component of the actin cytoskeleton in tumor cells, is required for tumor cell survival. Our objective was to determine whether cancer cells devoid of Tpm3.1 would evade the tumorgenic effects induced by H-Ras transformation. The tropomyosin isoform (Tpm) expression profile of a range of cancer cell lines (21) demonstrates that Tpm3.1 is one of the most broadly expressed Tpm isoform. Consequently, the contribution of Tpm3.1 to the transformation process was functionally evaluated. Primary embryonic fibroblasts isolated from wild type (WT) and Tpm3.1 knockout (KO) mice were transduced with retroviral vectors expressing SV40 large T antigen and an oncogenic allele of the H-Ras gene, H-RasV12, to generate immortalized and transformed WT and KO MEFs respectively. We show that Tpm3.1 is required for growth factor-independent proliferation in the SV40 large T antigen immortalized MEFs, but this requirement is overcome by H-Ras transformation. Consistent with those findings, we found that Tpm3.1 was not required for anchorage independent growth or growth of H-Ras-driven tumors in a mouse model. Finally, we show that pERK and Importin 7 protein interactions are significantly decreased in the SV40 large T antigen immortalized KO MEFs but not in the H-Ras transformed KO cells, relative to control MEFs. The data demonstrate that H-Ras transformation overrides a requirement for Tpm3.1 in growth factor-independent proliferation of immortalized MEFs. We propose that in the SV40 large T antigen immortalized MEFs, Tpm3.1 is partly responsible for the efficient interaction between pERK and Imp7 resulting in cell proliferation, but this is overidden by Ras transformation.

Colorectal cancer (CRC) transcriptional subtypes have been recently identified by gene expression profiling. Here we describe an analytical pipeline, microRNA master regulator analysis (MMRA), developed to search for microRNAs potentially driving CRC subtypes. Starting from a microRNA-mRNA tumour expression data set, MMRA identifies candidate regulator microRNAs by assessing their subtype-specific expression, target enrichment in subtype mRNA signatures and network analysis-based contribution to subtype gene expression. When applied to a CRC data set of 450 samples, assigned to subtypes by 3 different transcriptional classifiers, MMRA identifies 24 candidate microRNAs, in most cases downregulated in the stem/serrated/mesenchymal (SSM) poor prognosis subtype. Functional validation in CRC cell lines confirms downregulation of the SSM subtype by miR-194, miR-200b, miR-203 and miR-429, which share target genes and pathways mediating this effect. These results show that, by combining statistical tests, target prediction and network analysis, MMRA effectively identifies microRNAs functionally associated to cancer subtypes.

We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

Here, we show a mechanism for expansion regulation through mutations in the green revolution gene gibberellin20 (GA20)-oxidase and show that GAs control biosynthesis of the plants main structural polymer cellulose. Within a 12,000 mutagenized Sorghum bicolor plant population, we identified a single cellulose-deficient and male gametophyte-dysfunctional mutant named dwarf1-1 (dwf1-1). Through the Sorghum propinquum male/dwf1-1 female F2 population, we mapped dwf1-1 to a frameshift in GA20-oxidase. Assessment of GAs in dwf1-1 revealed ablation of GA. GA ablation was antagonistic to the expression of three specific cellulose synthase genes resulting in cellulose deficiency and growth dwarfism, which were complemented by exogenous bioactive gibberellic acid application. Using quantitative polymerase chain reaction, we found that GA was positively regulating the expression of a subset of specific cellulose synthase genes. To cross reference data from our mapped Sorghum sp. allele with another monocotyledonous plant, a series of rice (Oryza sativa) mutants involved in GA biosynthesis and signaling were isolated, and these too displayed cellulose deficit. Taken together, data support a model whereby suppressed expansion in green revolution GA genes involves regulation of cellulose biosynthesis., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.

Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying MET amplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naïve GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other MET-addicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.

Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques.

In a screen for root hair morphogenesis mutants in Arabidopsis thaliana L. we identified a T-DNA insertion within a type III J-protein AtDjC17 caused altered root hair development and reduced hair length. Root hairs were observed to develop from trichoblast and atrichoblast cell files in both Atdjc17 and 35S::AtDJC17. Localization of gene expression in the root using transgenic plants expressing proAtDjC17::GUS revealed constitutive expression in stele cells. No AtDJC17 expression was observed in epidermal, endodermal, or cortical layers. To explore the contrast between gene expression in the stele and epidermal phenotype, hand cut transverse sections of Atdjc17 roots were examined showing that the endodermal and cortical cell layers displayed increased anticlinal cell divisions. Aberrant cortical cell division in Atdjc17 is proposed as causal in ectopic root hair formation via the positional cue requirement that exists between cortical and epidermal cell in hair cell fate determination. Results indicate a requirement for AtDJC17 in position-dependent cell fate determination and illustrate an intriguing requirement for molecular co-chaperone activity during root development.

Natural food colorants with functional properties are of increasing interest. Prior papers indicate the chemical suitability of sorghum leaf 3-deoxyanthocyanidins as natural food colorants. Via mutagenesis-assisted breeding, a sorghum variety that greatly overaccumulates 3-deoxyanthocyanidins of leaf tissue, named REDforGREEN (RG), has been isolated and characterized. Interestingly, RG not only caused increased 3-deoxyanthocyanidins but also caused increased tannins, chlorogenic acid, and total phenolics in the leaf tissue. Chemical composition of pigments was established through high-performance liquid chromatography (HPLC) that identified luteolinidin (LUT) and apigeninidin (APG) as the main 3-deoxyanthocianidin species. Specifically, 3-deoxyanthocianidin levels were 1768 μg g⁻¹ LUT and 421 μg g⁻¹ APG in RG leaves compared with trace amounts in wild type, representing 1000-fold greater levels in the mutant leaves. Thus, RG represents a useful sorghum mutagenesis variant to develop as a functionalized food colorant.

Background: Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock., Results: It was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure., Conclusion: Antithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum.

Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus×giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.

Objective: Upon colon cancer metastasis resection in liver, disease outcome is heterogeneous, ranging from indolent to very aggressive, with early recurrence. The aim of this study is to investigate the capability of metastasis associated in colon cancer 1 (MACC1) levels measured in liver metastasis specimens to predict further recurrence of the disease., Methods: Gene expression and gene dosage of MACC1, hepatocyte growth factor (HGF), and hepatocyte growth factor receptor (MET) were assessed using quantitative realtime polymerase chain reaction on a cohort of 64 liver metastasis samples from patients with complete follow-up of 36 months and detailed clinical annotation. The most relevant mutations associated to prognosis in colorectal cancer, KRAS, and PIK3CA were assessed on the same specimens with Sanger sequencing., Results: Receiver operating characteristic (ROC) analysis revealed that MACC1 mRNA abundance is a good indicator of metastatic recurrence (AUC = 0.65, P < 0.05), whereas no such results were obtained with MET and HGF, nor with gene dosage. Generation of MACC1-based risk classes was capable of successfully separating patients into poor and good prognosis subgroups [hazard ratio (HR) = 5.236, 95% confidence interval (CI) = 1.2068-22.715, P < 0.05]. Also KRAS mutation was significantly associated with higher risk of recurrence (HR = 2.07, 95% CI = 1.048-4.09, P < 0.05). Cox regression multivariate analysis supported the independence of MACC1, but not KRAS, from known prognostic clinical information (Node Size HR = 3.155, 95% CI = 1.4418-6.905, P < 0.001, Preoperative carcinoembryonic antigen HR = 2.359, 95% CI = 1.0203-5.452, P < 0.05, MACC1 HR = 7.2739, 95% CI = 1.6584-31.905, P < 0.01)., Conclusions: MACC1, a new easily detectable biomarker in cancer, is an independent prognostic factor of recurrence after liver resection of colorectal cancer metastasis.

Background: Historically, treatments for cellulite have not been able to address all of its physiological components and require multiple sessions., Objective: The authors evaluate the safety and efficacy of a single, subdermal procedure to treat the underlying structure of cellulite., Methods: Fifty-seven patients underwent a 3-step cellulite treatment with a 1440-nm Nd:YAG laser with a side-firing fiber and temperature-sensing cannula. Efficacy was measured by the ability of blinded evaluators to distinguish baseline photos from those taken at 3 and 6 months posttreatment, as well as their rating of the results on a 5-point, 2-category ordinal photonumeric scale when comparing baseline photos to those taken at 2, 3, and 6 months posttreatment. Patient and physician satisfaction was assessed based on completion of a satisfaction survey at 2, 3, and 6 months posttreatment. Adverse events (AE) were recorded throughout the study., Results: At 6 months posttreatment, blinded evaluators rated at least a 1-point improvement in the appearance of cellulite in 96% of treated sites. Blinded evaluators were also able to correctly identify baseline versus posttreatment photos in 95% of cases. At least 90% of patients and physicians reported satisfaction with the results of treatment throughout 6 months. AE were mild in intensity and transient to treatment., Conclusions: A single, 3-step, minimally invasive laser treatment using a 1440-nm Nd:YAG laser, side-firing fiber, and temperature-sensing cannula to treat the underlying structure of cellulite proved to be safe and maintained effectiveness at least 6 months posttreatment., Level of Evidence: 2.

Survivin is an essential component of the chromosomal passenger complex and a member of the inhibitor of apoptosis family. It is expressed at high levels in a large variety of malignancies, where it has been implicated in drug resistance. It was also shown previously that survivin is up-regulated during telomerase-mediated immortalization, which occurs at a relatively early stage during carcinogenesis. This study shows that up-regulation of survivin during immortalization of human myofibroblasts is an indirect consequence of the repression of p16(INK4a). Survivin and p16(INK4a) were functionally linked by assays that showed that either the up-regulation of survivin or repression of p16(INK4a) rendered telomerase-transduced MRC-5 myofibroblasts resistant to oxidative stress. Conversely, siRNA-mediated down-regulation of survivin activated caspases and enhanced the sensitivity of immortal MRC-5 cells to oxidative stress. The E2F1 transcription factor, which is negatively regulated by the pRB/p16(INK4a) tumor suppressor pathway, was implicated in the up-regulation of survivin. Using the ChIP assay, it was shown that E2F1 directly interacted with the survivin gene (BIRC5) promoter in cells that spontaneously silenced p16(INK4a) during telomerase-mediated immortalization. E2F1 binding to the BIRC5 was also enhanced in telomerase-transduced cells subjected to shRNA-mediated repression of p16(INK4a). Together, these data show that repression of p16(INK4a) contributes to the up-regulation of survivin and thereby provides a survival advantage to cells exposed to oxidative stress during immortalization. The up-regulation of survivin during immortalization likely contributes to the vulnerability of immortal cells to transformation by oncogenes that alter intracellular redox state.

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Unlabelled: Only a fraction of patients with metastatic colorectal cancer receive clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) antibodies, which calls for the identification of novel biomarkers for better personalized medicine. We produced large xenograft cohorts from 85 patient-derived, genetically characterized metastatic colorectal cancer samples ("xenopatients") to discover novel determinants of therapeutic response and new oncoprotein targets. Serially passaged tumors retained the morphologic and genomic features of their original counterparts. A validation trial confirmed the robustness of this approach: xenopatients responded to the anti-EGFR antibody cetuximab with rates and extents analogous to those observed in the clinic and could be prospectively stratified as responders or nonresponders on the basis of several predictive biomarkers. Genotype-response correlations indicated HER2 amplification specifically in a subset of cetuximab-resistant, KRAS/NRAS/BRAF/PIK3CA wild-type cases. Importantly, HER2 amplification was also enriched in clinically nonresponsive KRAS wild-type patients. A proof-of-concept, multiarm study in HER2-amplified xenopatients revealed that the combined inhibition of HER2 and EGFR induced overt, long-lasting tumor regression. Our results suggest promising therapeutic opportunities in cetuximab-resistant patients with metastatic colorectal cancer, whose medical treatment in the chemorefractory setting remains an unmet clinical need., Significance: Direct transfer xenografts of tumor surgical specimens conserve the interindividual diversity and the genetic heterogeneity typical of the tumors of origin, combining the flexibility of preclinical analysis with the informative value of population-based studies. Our suite of patient-derived xenografts from metastatic colorectal carcinomas reliably mimicked disease response in humans, prospectively recapitulated biomarker-based case stratification, and identified HER2 as a predictor of resistance to anti-epidermal growth factor receptor antibodies and of response to combination therapies against HER2 and epidermal growth factor receptor in this tumor setting., (© 2011 AACR.)

Strains of non-pathogenic pseudomonad bacteria, can elicit host defence responses against pathogenic microorganisms. Pseudomonas fluorescens strain MKB158 can protect cereals from pathogenesis by Fusarium fungi, including Fusarium head blight which is an economically important disease due to its association with both yield loss and mycotoxin contamination of grain. Using the 22 K barley Affymetrix chip, trancriptome studies were undertaken to determine the local effect of P. fluorescens strain MKB158 on the transcriptome of barley head tissue, and to discriminate transcripts primed by the bacterium to respond to challenge by Fusarium culmorum, a causal agent of the economically important Fusarium head blight disease of cereals. The bacterium significantly affected the accumulation of 1203 transcripts and primed 74 to positively, and 14 to negatively, respond to the pathogen (P = 0.05). This is the first study to give insights into bacterium priming in the Triticeae tribe of grasses and associated transcripts were classified into 13 functional classes, associated with diverse functions, including detoxification, cell wall biosynthesis and the amplification of host defence responses. In silico analysis of Arabidopsis homologs of bacterium-primed barley genes indicated that, as is the case in dicots, jasmonic acid plays a role in pseudomonad priming of host responses. Additionally, the transcriptome studies described herein also reveal new insights into bacterium-mediated priming of host defences against necrotrophs, including the positive effects on grain filling, lignin deposition, oxidative stress responses, and the inhibition of protease inhibitors and proteins that play a key role in programmed cell death.

The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when packaged with chemotherapeutics. When targeted via antibodies to tumor-cell-surface receptors, minicells can specifically and sequentially deliver to tumor xenografts first siRNAs or short hairpin RNA (shRNA)-encoding plasmids to compromise drug resistance by knocking down a multidrug resistance protein. Subsequent administration of targeted minicells containing cytotoxic drugs eliminate formerly drug-resistant tumors. The two waves of treatment, involving minicells loaded with both types of payload, enable complete survival without toxicity in mice with tumor xenografts, while involving several thousandfold less drug, siRNA and antibody than needed for conventional systemic administration of cancer therapies.

In this study the effect of Agrobacterium tumefaciens genotype of two strains AGL1 and LBA4404 was investigated in regard to the propensity for backbone integration during the transformation of potato for blight tolerance conferred by the resistant to blight (RB) gene carried by the vector pCLDO4541. A PCR based walking approach was employed to identify left and right backbone sequences as well as for selected genes carried on the plasmid backbone. It was found that adjacent to the left border insertion site, the integration of backbone sequence was greater for AGL1 than for LBA4404; however, the opposite was observed with regards to the right border T-DNA junction. Considering both T-DNA borders LBA4404 was found to have a two fold greater integration potential for backbone than the AGL1. The possibility of only backbone integration in T-DNA negative plants was also investigated with the average rate of integration between the two strains calculated at 4.2% with LBA4404 recording a three fold greater occurrence of backbone integration than AGL1. In summary, evidence of Agrobacterium genotype dependency showed that LBA4404 has greater potential to integrate non-T-DNA vector sequence than AGL1 and this should be taken into account when utilising the listed A. tumefaciens genotypes in generating transgenic potato. Additionally, the application of a PCR and primer walking system proved to be reliable and allows for fine detailed studies of backbone sequence integration of transgenic plant.

BRAF(V600E) mutation has been frequently reported in different types of melanocytic lesions, but its role in melanomagenesis is poorly understood, having been associated with either the proliferative-induced MAPK pathway activation or the acquisition of oncogene-driven senescence. The presence of BRAF alterations has been related to sun exposure, although the molecular mechanisms underlying this event are only partly known. To elucidate the relationships among BRAF/NRAS alterations, MAPK pathway activation, and sun exposure, we examined 22 acquired nevi and 18 cutaneus melanomas from 38 patients. Microdissected tissues from each lesion were subjected to BRAF/NRAS mutation analysis by sequencing, allele-specific PCR and pyrosequencing assay. The same lesions were also examined for the expression of phosphorylated ERK1/2. Phototype and an accurate history of sun exposure were evaluated for each patient. BRAF(V600E) mutation was detected in 50% of the acquired nevi and in 70% of the cutaneus melanomas in the absence of NRAS alterations. The fraction of alleles carrying BRAF(V600E) substitution was variable but strongly associated with sun exposure. In contrast, no relationship was evidenced between the presence of this mutation and patients' phototype, phosphorylated ERK1/2 expression, or Clark's level. Our findings indicate that in melanocytic lesions, BRAF(V600E) mutation can affect a subset of the cells and is associated with the type and quantity of sun exposure. This mutation is independent of the nevo-melanoma progression and unrelated to ERK phosphorylation, suggesting that alternative mechanisms to the MAPK activation are also involved in this type of transformation.

The characteristics of sebaceous gland hyperplasia (SGH) consist of yellowish or skin-colored papules and nodules. Chronic sun exposure and immunosuppressed conditions are the main environmental risk factors, whereas chronological aging regulated by hormones and molecular changes are the intrinsic risk factors. We have evaluated the contribution of BRAF, K-Ras, and N-Ras mutations to the pathogenesis of SGHs in four patients belonging to three MYH-associated polyposis (MAP) pedigrees. MAP is an autosomal-recessive disease characterized by multiple colorectal adenomas and cancer. Immunohistochemistry of mismatch repair and APC proteins was performed. DNA isolated from blood lymphocytes and formalin-fixed or paraffin-embedded SGHs was PCR amplified and sequenced. In the SGH patients, we detected T1796A heterozygous substitution (V600E) in the BRAF gene. Compound biallelic germline MYH mutations (Y165C/G382D, R168H/379delC, and Y90X/delGGA464) were detected in the MAP patients. In contrast to the majority of melanocytic lesions, activating hotspot mutations in BRAF have not been involved so far in the pathogenesis of SGH. BRAF mutation is not a specific marker of melanocytic cancerogenesis, and it can also be involved in SGHs. In both melanocytic and non-melanocytic skin tumors, BRAF mutation is linked to early tumorigenesis events.

Most patients with herpes simplex virus (HSV) central nervous system (CNS) infection have abnormal cerebrospinal fluid (CSF) indices. Therefore, we implemented screening criteria based on CSF values and host immune status to guide testing. All CSF samples submitted for HSV PCR analysis from January 1999 through December 2004 were included in the study. Specimens from patients with human immunodeficiency virus, a history of transplants, an age of <2 years, a CSF white blood cell count of >5 cells/mm(3), or a protein level of >50 mg/dl were tested upon request. All other samples were rejected and frozen. To validate our screening criteria, rejected specimens were pooled and tested retrospectively. Electronic medical records were also reviewed. A total of 1,659 HSV PCR requests from 1,458 patients were screened. Of the 1,296 specimens (78.1%) accepted for testing, 1,213 were negative, 7 were positive for HSV type 1 (HSV-1), 26 were positive for HSV-2, and 50 had unavailable results. Sixteen requests were rejected because an alternative microbiologic diagnosis had been established. Of the 347 samples rejected based on criteria, 222 (64.0%) remained available for pooled testing. No HSV-1-positive samples were identified in the rejected specimens. Two rejected specimens tested positive for HSV-2 DNA, but both met acceptance criteria which had not been communicated to the laboratory. Few patients (7.8%) with rejected specimens were treated with acyclovir, which suggests a low clinical concern for HSV encephalitis. Acceptance criteria based on CSF parameters and host immune status saved time and cost and did not miss patients with HSV CNS infection. Communication between the clinician and the laboratory is imperative for a successful screening program.

Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS(Q61R) oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF(V600E) oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS(Q61R) transfectant displayed progressive accumulation in G(0)-G(1) phase of the cell cycle and stained for the senescence-associated beta-galactosidase activity (SA-beta-Gal). Inducible expression of NRAS(Q61R), by the Tet-Off system, in clone 21 cells (21NRAS(61ON)) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-beta-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21(waf1/Cip1). These effects were detected neither in 21 cells with silenced NRAS(Q61R) (21NRAS(61OFF)) nor in cells transfected with an inducible wild-type NRAS gene (21NRAS(WTON)). In addition, when compared with parental 21 cells, or with 21NRAS(61OFF), 21NRAS(61ON) and constitutive NRAS(Q61R) transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen-restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against "double-mutant" cells.

Biotechnology-based tools are now widely used to enhance and expand the traditional remit of potato in food production. By modifying its functionality, the capacity of the potato to produce, for example, therapeutic or industrial compounds is now a reality, and its ability to resist disease can also be radically improved. Two developments have been crucial to expanding the role of potato: the recent advances in the fields of structural and functional potato genomics and the ability to integrate genes of interest into the potato genome. In this review we discuss how both developments have diversified the remit of this crop.

Lidocaine is an integral part of most wetting solutions used in liposuction. Although the Physician's Desk Reference states that the permissible dose of lidocaine is 7 mg/kg, doses as high as 75 mg/kg have been used in liposuction. Lidocaine is used in the wetting solution even when the procedure is performed under epidural or general anesthesia. The justification for this is a reduction in postoperative pain. This study compared the pain between paired, mirrored sides of 10 patients when lidocaine was used on only one side. There was no statistically significant difference between the postoperative pain at 5, 30, 60, and 120 minutes and on the first postoperative day. Because there was no difference in pain whether or not lidocaine was used, and because lidocaine is potentially toxic and lethal, this study concludes that lidocaine is not necessary in liposuction.

A case of reversible encephalopathy during treatment with Amphotericin-B (AMB) is described. The comparison of the clinical course of AMB encephalopathy with total dose of AMB, cranial radiotherapy and MRI data available in previously reported cases, shows that this complication is characterized by a progressive, dose-dependent course, possibly influenced by cranial irradiation.

We studied the efficacy of color Doppler sonography in the transcutaneous assessment of acute changes in microvascular flow. Arterial and venous occlusion studies were performed in rabbits after simple isolation of the common femoral artery and vein and after raising an epigastric island flap. Vessel diameters were measured through an operating microscope and compared to color Doppler determinations of the diameters. Vessel patency and blood flow in the major vessels were monitored before and after occlusion. The correlation of vessel diameters measured under the operating microscope and with color Doppler sonography was .91 for the arteries and .39 for the veins. A strong linear correlation existed between Doppler identification of arterial occlusion, but not of venous occlusion. This study demonstrated that color Doppler sonography can be used transcutaneously to reliably assess patency and blood flow in the small vessels. Time-dependent, reproducible patterns in the spectral waveform analysis were indicative of the onset of arterial or venous occlusion.

The clinical course of patients with hematological disease, especially after treatment, is often complicated by gastrointestinal infections. Between 1986 and 1990 a total of 18 patients affected with hematologic disease and presenting with an acute abdomen were admitted to the surgery department at the University of Rome "La Sapienza". Most patients were affected with acute or chronic myeloid leukemia (61%) and lymphoma. Five patients with acute appendicitis, three with necrotizing enterocolitis, three with spontaneous hemoperitoneum, three with cholecystitis, two splenic infarctions and two intestinal occlusions were diagnosed. Symptoms were often vague and non specific and blood counts revealed neutropenia in all but two patients, while anemia was characteristic in spontaneous hemoperitoneum and in neutropenic enterocolitis. Fungemia occurred in only two cases while bacteremia was present in seven. The most critical patients were those affected by neutropenic enterocolitis and acute cholecystitis. Sonography was meaningful in the diagnosis of hemoperitoneum, splenic infarct and acute cholecystitis. All patients underwent surgical procedures within 48 hours of admission to the department. In all cases peritoneal washing was performed and at least one peritoneal drainage was left. In all cases of necrotizing enterocolitis, intestinal resections, either ileal or colonic, were followed by an immediate anastomosis in two layers. Intensive hematological and antibiotic post surgical care was performed in all patients. Seven patients presented minor complications (38.8%), and only one died (5.5%). Emergency surgical treatment may be safely carried out in patients with hematological diseases presenting with an acute abdomen. Intensive postsurgical care is mandatory for the recovery of patients and the patient's critical condition should not be a deterrent to surgical intervention.

The radial forearm flap has become a versatile flap for upper extremity reconstruction. The use of the forearm flap for hand reconstruction in the patient with previously burned forearms has not been widely appreciated. In those patients whose forearms have been previously split-thickness skin-grafted on fascia, we have employed the reverse radial forearm flap as a skin graft-fascial flap for hand reconstruction and have obtained excellent functional results. Three patients at various intervals postburn are presented to demonstrate use of this flap for wrist contracture release, coverage of arthroplasties, first web space contracture release, and acute salvage of phalanges and tendons. Assessment of the hand's vascular anatomy and careful treatment of the donor area have contributed to no added morbidity and an excellent aesthetic result at the donor site.