The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration. As a result, a defined set of rules has evolved for assembly from standardized units (bricks). The BioBrick system was one of the first attempts to standardize assembly using recombinant DNA technology Recombinant DNA technology was the first technique proposed for targeted manipulation of DNA and has had a wide-ranging and lasting impact on many fields in vitro recombination techniques such as sequence and ligation – independent cloning (SLIC) have been developed in vitro single-strand annealing. They employ relatively short overhangs of 15 to 30 bp, and can be modified into in vitro building brick systems Recently, in vitro cloning. The Invitrogen Gateway system in vitro cloning as they do not have the binary recombination features of the attachment sequence system (such as the attachment sequence recombination: attB + attP  =  attL + attR) but remain the same after recombination. The Integron system has a similar binary mechanism to that of the Lambda phage attachment system and has been applied to the assembly of multiple genes Site-specific recombination methods have been applied widely to in vitro technologies. They rely on both high reaction and high transformation efficiencies. Standardized building brick systems aim to simplify the construction process, both for design and experiments. Since complex designs can be managed via a software platform, the reduction of experimental complexity is an important issue. One possibility is to carry out the assembly in vivo to avoid all in vitro operations. In vivo recombination, such as homologous recombination in Bacillis subtillisin vivo site-specific recombination is more efficient than in vivo homologous recombination, it has rarely been applied to DNA assembly except for some iterative systems E. coli for gene knockout in vivoin vivo recombination methods have potential in parallel DNA assembly.All the above strategies are in vivo recombination and to develop a standard engineering interface. It proves to be an efficient system for assembly of operons into small and medium-sized gene systems. High recombination efficiency was demonstrated by the experimental assembly of five genes.A novel theoretical framework is proposed here in order to both explore the potential of parallel DNA assembly by http://www.synthenome.com/) was applied to simulate and manage all in silico steps in the assembly process The program Vexcutor  for parallel DNA assembly developed here is given in Escherichia coli (E. coli) and helper plasmids that assist with integration and excision of DNA. These tools are shown in A number of tools are required for the experimental demonstration of SRAS. These include the two unit plasmids (pUnitP and pUnitR), the two extraction plasmids (pUnitExP and pUnitExR), the TARGET plasmid within the host cat gene and its promoter) derived from pKD3, the synthesized lambda attL1, attL2, attR1 and attR2, the HK022 attP and attR cloned from CRIM plasmid pAH70 and its integration product, the phi80 attL and attR cloned from the integration product of the CRIM plasmid pAH153, and a cloning site region from pBHR68 in silico (to view download the program from http://www.synthenome.com/ as described above and open the File S1).A series of short DNA components including the kanamycin-resistant operon and R6Kgamma from pKD13, the chloramphenicol-resistant operon  with the codons from the Lambda Int (pAH57), because this hybrid Int gene can specifically catalyze the LxR reaction of HK022 attL and attR Escherichia coli (E. coli) TOP10 strain by the HK022 BxP recombination; the plasmid backbone was then replaced with lambda red recombination with pKD13 to act as a template for polymerase chain reaction (PCR); finally the pKD13 kan gene was removed by pCP20 resulting in the HOST strain with the desired chromosomal sequence shown schematically in The TARGET plasmid was integrated into the A synthesized T7 promoter and terminator region was inserted into the pUnitP and pUnitR plasmids. The details of this procedure and sequences are described in the fourth Vexcutor file in the File S4. Add T7 Promoter and Terminator to the Unit Vectors.vxt. Note that the T7 sequence is omitted from Together, the four plasmids and host chromosome shown in To demonstrate the application of SRAS, the five genes in the lux Operon  in pSB406 To perform the DNA assembly from these initial five pUnit vectors, the LuxC and luxD genes were first sequentially assembled into the E. coli chromosome generating the strain HOSTLuxDLuxC, as shown to the right of The luxA, luxB and luxE genes were next sequentially assembled into the E. coli host chromosome generating strain HOSTLuxELuxBLuxA. These steps, involved the same stages described above and shown in The DNA sequences HOSTLuxDLuxC and HOSTLuxELuxBLuxA were extracted from the host chromosome using recombination between the sites attLPhi80 and attRPhi80, as shown in In the final step, the luxDC genes in pUnitLuxDLuxC were integrated into the chromosome of HOSTLuxELuxBLuxA strain, using the recombination between attP HK022 and attB HK022, resulting in the strain HOSTLuxDLuxCLuxELuxBLuxA . The intDNA fragments. If unrelated sequences are to be excluded from assembly products, each fragment needs two tightly flanking reactive ends (recombination sites). A DNA fragment with two reactive ends is defined as an assembly unit. The assembly process for linear assembly units is simply the reaction of reactive ends. The internal sequence between the two reactive ends will not influence the assembly product. Two units in linear carriers can be assembled by one reaction between their reactive ends  needs an additional recombination site as a ar forms . Two linSelective markers, which are usually constitutively expressed antibiotic resistant genes, can simplify the screening process. When the assembled fragments need to be recovered from host vectors or genomes, extra extraction sites  may also be required. Therefore, DNA fragments, reactive ends, topology breakers and selective markers are the necessary elements in an assembly; extraction sites are optional. In each assembly cycle, positive clones can be screened by the use of selective markers.There are three rules for the assembly strategy proposed here:-active ends must become inactive after the assembly reaction;topology breakers must be reactive throughout the assembly process;assembled products must have the same structure as the assembly units to allow another round of assembly to proceed.Rules 1 and 3 are similar to the concepts developed for iGEM BioBricks attL and attR from the Phi80 or HK022 phage attachment sequence recombination system to satisfy Rule 1. Additional extraction vectors that enable further assembly are also required. The appropriate design of extraction vectors allows the system to obey Rule 2. Briefly, there are two ways to achieve this goal:-Following assembly, the selective marker needs to be removed. The iterative part of this cycle is similar to that of a previous report an assembly unit can be integrated into the host vector or genome by recombination at the reactive ends; the vector backbone is then removed by a second recombination at the topology breakers;an assembly unit can be integrated into the host vector or genome by recombination at the topology breakers; the vector backbone is then removed by a second recombination at the reactive ends.de facto exclusive single-copy selective marker to ensure that all grown colonies are recombinants. As the whole assembly process features integration and excision plus extraction, it is referred to as an In/Out-Extract strategy.The design of the assembly units developed here and schematic diagrams of the assembly steps taken to assemble DNA fragments are shown in in silico using the Vexcutor program, which was designed to manage complex assembly pathways and sequences in processes such as SRAS.The In/Out-Extract SRAS system described in The five genes in the lux Operon  were selected for the experimental demonstration of the In/Out-Extract SRAS system, as they represent a small well characterized set of prokaryotic genes that have been widely applied as a reporter system The luxC and luxD genes were sequentially integrated into the E. coli host chromosome generating the strain HOSTLuxDLuxC, following the scheme shown in CTACATCAATAAAACTTAA for both plasmids. This PCR verification step is suggested for all assembly schemes presented here, as this step confirms DNA assembly has occurred and eliminates the possibility of incorporation of unwanted by-products that could complicate subsequent rounds of assembly.The identity of the assembled DNA sequences was confirmed by PCR using the reactive site ends flanking each lux gene (see File S7.Sequencing Results of the Extracted Plasmids.vxt for sequence details). The sequencing primers were ATF, ATR, BTF, BTR, CTF, CTR, DTF, DTR, ETF and ETR (see Table S2), corresponding to the reactive end sites attB1, attB2, and attBPhi80 flanking the lux D, C, E, B and A genes. The sequence obtained by PCR was found to match that obtained for each gene in each individual pUnit vector . For exaHaving confirmed the successful integration and initial assembly steps of the two and three gene sequences, the smaller of the two DNA sequences containing the luxD and luxC genes in pUnitLuxDLuxC was integrated into the chromosome of HOSTLuxELuxBLuxA strain in a single integration step (using recombination between attP HK022 and attB HK022), producing the final host strain named HOSTLuxDLuxCLuxELuxBLuxA . This fiE.coli-based assembly process.The PCR product successfully demonstrates the assembly of the five individual gene sequences from the five original pUnit vectors. A total of six integration steps were required to assemble the five DNA sequences using a total of six pUnit vectors, with several steps occurring in parallel. This parallel approach will be a particular advantage where larger gene assemblies are required. Notably, hundreds of colonies appeared following integration at each stage of the process indicating the robust nature of this in vivo parallel assembly system. The components required for the In/Out-Extract SRAS approach were developed experimentally and the assembly of a set of five genes from the lux Operon  was realized. The only in vitro operation required, following the construction of the starting materials containing the individual genes to be assembled, is the extraction of plasmids and confirmation of sequence identity by PCR. The experimental demonstration performed here provides a proof of concept of the In/Out-Extract SRAS system using a small well-characterized operon. Theoretically, the absence of in vitro operations permits the assembly of large fragments via this technique with an assembly turnover cycle as short as 1–2 days for 2 fragments ) in terms of time complexity for computer science).A theoretical framework is proposed for the design of an An inherent component of synthetic biology is the synthesis of chromosomes and genomes. Methods such as the Gibson isothermal assembly and yeast recombination have been developed for parallel DNA assembly and chromosome fabrication in vitro operations constitutes a third advantage.The advantage of the building brick method for such optimization is that the built-in adapters used for alignment and combination can be used in any order with no additional subcloning. This potentially results in lower costs for both time and labour when larger assemblies are considered. Furthermore, the native DNA fragments can be of any size, so the building brick systems have the second advantage of low sensitivity to unit size. Lastly, a lower dependency on , a linear plasmid approach to double or triple selective marker systems (linear DRAS and linear TRAS) or a seamless linear Bi-Swap TRAS. These variations on the parallel method of DNA assembly are discussed in turn here.The SRAS approach demonstrated here could be further extended by the use of a Double-Selective Marker Recombination system (DRAS), a Triple-Selective-Marker Recombination Assembly System (TRAS)When the system contains more than one selective marker, the In/Out-Extract strategy can still be implemented with minor modifications. The In/Out steps can be simplified by the swap of the two selective markers. Extraction vectors are also required. A design of this method is shown in When three or more selective markers are available, both the In/Out-Extract and Swap/Extract strategies can be implemented. Moreover, the selective marker swap strategy can be applied between unit plasmids so that integration into the host is not necessary and the extraction step is not required. This is designated a bidirectional swap (Bi-Swap) strategy. A typical design employing the Bi-Swap strategy for the directional assembly of two DNA fragments is shown in E. coli and many other prokaryotes, there are also linear plasmids such as the N15 phage plasmids In A final variation for TRAS, is a seamless model based on the MAGIC A further modification could be the addition of a bacterial conjugation component In vivo recombination based building brick methods presented here have the potential to be logistically simpler than other in vitro methods where the assembly of large numbers of fragments is required, due to the assembly of 2N fragments in N cycles and shorter turnover time, as they require only plasmid transformation steps and fewer in vitro manipulations. The number of steps and theoretical time taken to assemble 32 fragments with different methods including the SRAS method, the Bi-Swap TRAS method (with and without conjugation), the Gibson isothermal assembly method and Recombinant DNA (or BioBrick) method is presented in in vitro steps; the Bi-Swap TRAS with conjugation is performed in vivo, whereas the Bi-Swap TRAS requires plasmid purification. Both TRAS approaches are quicker than the Gibson Isothermal Assembly and BioBrick methods, which rely on in vitro operations. The Gibson isothermal assembly method also assembles 4 fragments in each turnover cycle, while the SRAS, TRAS and Recombinant DNA method assemble 2.in vivo recombination is near 100% for the integration of a single copy of CRIM plasmid into the E. coli genome in vivo integration. In our study, we also found that the number of colonies transformed with the six Lux pUnit vectors for in vivo integration in the SRAS assembly was similar to that observed for the transformation of pUnit into the E. coli S17-1 pir host in which the pUnit plasmids can replicate, i.e., the in vivo integration step did not reduce cell viability, implying that the in vivo integration efficiency is near to 100%. In the theoretical case of Swap or Bi-Swap approaches  Design of a meet-and-cut seamless assembly system M&C Linear-TRAS. Linear-TRAS containing the unit vectors of C-DNA-G, G-DNA-K, K-DNA-C, G-DNA-C, K-DNA-G and C-DNA-K, where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. I-CreI, I-SceI, I-NanI, I-TevI, I-PpoI and I-NitI in yellow rectangles indicate the homing endonuclease sites. I-CreI, I-SceI, I-NanI, I-TevI, I-PpoI and I-NitI in red triangle-ended rectangles indicate the homing endonuclease sites. Lx, La, Lb and Lc stand for the landing pad. (B) Assembly of two DNA fragments DNA1 and DNA2. (TIF)Click here for additional data file.Figure S2Circular Bi-Swap BioBrick plasmid system. (A) Design of a Circular Bi-Swap BioBrick plasmid system. The system contains the unit vectors K-DNA-G, K-DNA-C, C-DNA-K, C-DNA-G, G-DNC-G and G-DNA-K, where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. XbaI, SpeI, NheI and EcoRI in rectangles indicate the endonuclease sites. Rep ori in the round-ended rectangles indicates the replication origin for each plasmid.(B) Assembly of two DNA fragments DNA1 and DNA2. (TIF)Click here for additional data file.Figure S3Linear Bi-Swap BioBrick plasmid system. (A) Design of a Linear Bi-Swap BioBrick plasmid system. The system containing the unit vectors K-DNA-G, K-DNA-C, C-DNA-K, C-DNA-G, G-DNC-G and G-DNA-K, where the chloramphenicol, gentamycin and kanamycin resistance genes are designated cat, gen and kan, respectively. XbaI, SpeI, NheI and EcoRI in rectangles indicate the endonuclease sites. Rep ori in the round-ended rectangles indicate the replication origin for each plasmid. (B) Assembly of two DNA fragments DNA1 and DNA2. About vxt File Format: Please visit: http://www.synthenome.com to download the Vexcutor to view the vxt files. This program is free.(TIF)Click here for additional data file.File S1Construction of the Unit and Extraction Plasmids.vxt. Describes the construction of the Unit vectors, the extraction vectors and the TARGET vector.(VXT)Click here for additional data file.File S2Construction of Host Strain.vxt. Describes the modification of the E. coli TOP10 genome HK022 attB region with the TARGET vector fragment creating the host chromosome ready for integration.(VXT)Click here for additional data file.File S3Construction of the Helper Plasmids.vxt. Describes the construction of helper plasmids from existing CRIM vectors.(VXT)Click here for additional data file.File S4Add T7 Promoter and Terminator to the Unit Vectors.vxt. Describes the construction of the Unit vectors each with a T7 promoter and terminator.(VXT)Click here for additional data file.File S5Construction of Lux Gene Libraries.vxt. escribes the construction of the pUnit vectors, each containing one of the five genes from the lux operon.(VXT)Click here for additional data file.File S6Recombination Process.vxt. Describes the assembly process for the five lux genes cloned in pUnit vectors.(VXT)Click here for additional data file.File S7Sequencing results of the extracted plasmids.vxt. Describes the sequencing results of the extracted plasmids (i.e. pUnitLuxELuxBLuxA and pUnitLuxDLuxC). The sequence of the extracted plasmids was compared to the sequence of the same lux genes individually subcloned into the pUnit vectors. The nodes whose IDs contain “XX_B1012...”are the sequencing results of subcloned individual genes, while the nodes whose IDs contain “XX_B1102...”are the sequencing results of extracted vectors. Sequence alignments can be found in the “NA Compare”nodes.(VXT)Click here for additional data file.File S8Construct DRAS Host.vxt. Describes the construction of the DRAS host E. coli strain by modifying the Phi80 attB region.(VXT)Click here for additional data file.File S9Construct DRAS lux Library.vxt. Describes the process of subcloning the five genes of the lux operon into individual pDRASUnit vectors.(VXT)Click here for additional data file.File S10DRAS Recombination.vxt. Describes the assembly of the five lux genes subcloned in pDRASUnit vectors into the chromosome.(VXT)Click here for additional data file.File S11In Silico Implementation of Bi-Swap TRAS.vxt. Describes a possible design for a Bi-Swap TRAS strategy where five DNA fragments are assembled in order.(VXT)Click here for additional data file.File S12In Silico Implementation of Linear Bi-Swap TRAS.vxt. Describes a possible design for a linear Bi-Swap TRAS strategy where five DNA fragments are assembled in order.(VXT)Click here for additional data file.Table S1Enzymes and Antibiotics.(DOCX)Click here for additional data file.Table S2Primers.(DOCX)Click here for additional data file.


The presence of bone metastases has excluded participation of prostate cancer patients in exercise intervention studies to date and is also a relative contraindication to supervised exercise in the community setting because of concerns of fragility fracture. However, this group of patients often have developed significant muscle atrophy and functional impairments from prior and continuing androgen deprivation that is exacerbated by subsequent and more intensive interventions such as chemotherapy. The aim of this study is to determine the efficacy and safety of a modular multi-modal exercise program in prostate cancer patients with bone metastases.Multi-site randomized controlled trial in Western Australia and New South Wales to examine the efficacy and safety of a modular multi-modal physical exercise program in 90 prostate cancer survivors with bone metastases. Participants will be randomized to (1) modular multi-modal exercise intervention group or (2) usual medical care group. The modular multi-modal exercise group will receive a 3-month supervised exercise program based on bone lesion location/extent. Measurements for primary and secondary endpoints will take place at baseline, 3 months (end of the intervention) and 6 months follow-up.Delaying or preventing skeletal complication and improving physical function for men with bone metastases would provide clinically meaningful benefits to patients. However, exercise programs must be designed and executed with careful consideration of the skeletal complications associated with bone metastatic disease and cumulative toxicities from androgen deprivation such as osteoporosis and increased risk of fractures. The results from this study will form the basis for the development of a specific exercise prescription in this patient group in order to alleviate disease burden, counteract the adverse treatment related side-effects and enhance quality of life.ACTRN12611001158954ACTRN:  Metastases to bone occurs in approximately 80% of men with advanced prostate cancer  and the Long-term androgen deprivation therapy (ADT) remains the first-line treatment for advanced metastatic prostate cancer, however it acts by inducing severe hypogonadism causing a number of cumulative adverse effects ,9. Some Apart from previous ADT, men with bone metastases and castrate-resistant prostate cancer would commonly receive chemotherapy given the results of initial phase III trials indicating modest survival benefit for those with symptomatic disease ,16. A nuClinical trials investigating the efficacy of exercise in prostate cancer survivors including our recent randomized controlled trial excluded those who had bone metastases -24, or bThis is a two-armed prospective randomized controlled trial that will examine the efficacy and safety of a modular multi-modal physical exercise program in prostate cancer survivors with bone metastases. The trial will be a multisite study with clinical sites in Western Australia and New South Wales. Participants randomly assigned to the M3EP exercise intervention group will receive a 3-month supervised exercise intervention program (based on bone lesion location/extent) Table . ParticiSubjects will be recruited by invitation of their specialist (radiation oncologist/urologists). Those entering the study will undertake a series of familiarisation sessions and baseline measurements prior to randomisation.Patients will be randomly allocated in a ratio of 1:1 to the two study arms for the experimental M3EP exercise intervention or usual medical care groups, subject to maintaining approximate balance regarding stratification for current chemotherapy (yes/no). A research methods consultant with no patient contact will be responsible for randomisation. The exercise physiologists and other researchers conducting the study measures will be blinded to a given participant's group allocation. The exercise intervention will be provided by exercise physiologists not in the research team or performing the tests.Ninety men (45 subjects per arm) with prostate cancer and established bone metastases with no regular exercise (undertaking structured aerobic or resistance training two or more times per week) within the past 3 months will be recruited through invitation by their attending specialist in Perth, Western Australia, and the Central Coast region of New South Wales. All participants will require physician consent. Exclusion criteria will include acute illness, significant bone pain, musculoskeletal or cardiovascular or neurological disorders that could inhibit or put them at risk from exercising. The protocol has been approved (ID: 7699 GALVÃO) by the Edith Cowan University Human Research Ethics Committee and all participants will provide written informed consent.Data from our research team in prostate cancer patients indicates that the standard deviation (SD) for change in our primary outcome which is the physical function subscale of the Medical Outcomes Study Short-Form 36 (SF-36) equates to ~12 points following a 3-month intervention. Given the health status of men in the proposed study we anticipate that our 3-month exercise regimen will result in an increase of ~5 points in the physical function subscale of the SF-36 whereas the usual medical care group will result in an overall loss of ~3 points over 3 months. Therefore, we anticipate a difference between the exercise and usual medical care groups of ~8 points in change of the physical function subscale from baseline to 3 months. A priori, 36 subjects per group will be required to achieve 80% power at an alpha level of 0.05 (two-tailed), and to demonstrate a difference between groups at the end of the 3-month intervention. Therefore, to adequately ensure that we have sufficient subject numbers at the end of the intervention (accounting for a drop-out rate of ~20%), 90 subjects will be randomized in a ratio of 1:1 to exercise and usual medical care groups, respectively.Measurements for primary and secondary endpoints will take place at baseline, 3 months (end of the intervention) and 6 months follow-up.The physical function subscale of the Medical Outcomes Study Short-Form 36 (SF-36) questionnaire will be used as an indicator of patient rated physical functioning.A battery of tests will be used to assess functional performance ,23,27. TDynamic muscular strength of the upper and lower body will be assessed using the one repetition maximum (1RM) method . The 1RMA Neurocom Smart Balancemaster  will be used to assess static and dynamic balance. This device measures ground reaction force to track whole body centre of pressure and a tilting visual field and support platform to separate the visual, somatosensory and vestibular balance sense of the patient . Falls sThe safety of the exercise program will be assessed by recording the incidence and severity of any adverse events and skeletal complications throughout the intervention. Skeletal complications include pain at known bone metastases sites and pathological skeletal fractures . Bone paRegional and whole body lean mass  and fat mass will be derived from a whole body dual-energy X-ray absorptiometry scan . Measurement of trunk adiposity is an important indicator of chronic disease risk, and will be assessed from trunk fat mass obtained from the whole body scan and the ratios of trunk fat to limb fat, and trunk fat to total fat.Peripheral Quantitative Computed Tomography  will be used to measure muscle density  and muscle cross-sectional areas of the upper and lower limbs [Health-related quality of life outcomes on general health, pain, vitality, social functioning, emotional role, and mental health will be measured using the SF-36 . The FACThe Brief Symptom Inventory-18 will provide a global measure of current psychological distress with subscale scores for anxiety, depression, and somatisation .The Impact of Events Scale (IES) and the Memorial Anxiety Scale for Prostate Cancer (MAX-PC) will be used to measure cancer specific distress ,36. The Fatigue will be assessed using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) questionnaire. The FACIT-F is a 13-item scale commonly used to assess fatigue in cancer patients  as well Items from the Pittsburgh Sleep Quality Index (PSQI) will be used to measure sleep quality . The PSQThe Theory of Planned Behaviour (TPB) is the most widely utilised behavioural framework when examining physical activity motivation in cancer survivors . TherefoSelf report physical activity level will be assessed by the Godin Leisure-Time Questionnaire. ActiGraph activity monitors  will be used to objectively assess physical activity levels over a 7-day period . A 6-iteTolerance of the exercise program will be evaluated by recording participants' ratings of perceived exertion on a Borg scale after every exercise session. Additionally, a custom designed survey examining exercise tolerance using a 7-point Likert scale will be administered prior to the first exercise session each week. The number of participants completing the 3-month program as well as the number of sessions attended will be recorded.PSA will be assessed at baseline, 3 months (end of the intervention) and 6 months follow-up by an accredited National Association of Testing Authorities laboratory .The modular multi-modal physical exercise intervention program (M3EP) will comprise resistance, aerobic and flexibility exercises undertaken 3 times per week in an exercise clinic setting supervised by an exercise physiologist. Exercise training sessions will take approximately 60 min (this includes the warm-up and cool-down periods) and will be conducted in the Exercise Clinics at Edith Cowan University in Perth, and at the University of Newcastle's Central Coast campus in Ourimbah. The programme will include 6 exercises that target the major trunk, upper and lower body muscle groups, which we have used in a number of previous studies ,27,43-46Data will be analysed using the SPSS statistical software package and an intention-to-treat approach will be applied. Analyses will include standard descriptive statistics, Student's t tests, correlation and regression, and two-way (group × time) repeated measures ANOVA (or ANCOVA as appropriate) to examine differences between groups over time. All tests will be two-tailed and an alpha level of 0.05 will be applied as the criterion for statistical significance.Prostate cancer patients with bone metastases experience considerable morbidity resulting from skeletal complications and often fatigue secondary to chemotherapy ,7. DelayADT: Androgen deprivation therapy; IES: Impact of events scale; MAX-PC: Memorial anxiety scale for prostate cancer; M3EP: Modular multi-modal exercise program; PSA: Prostate specific antigen; SF-36: Medical outcomes study short-form 36; FACIT-F: Functional assessment of chronic illness therapy-fatigue; TPB: Theory of planned behaviour; LL-FI: The late life - function index; PSQI: Pittsburgh sleep quality index.The authors declare that they have no competing interests.DAG, PC, NS, DRT, and RUN developed the study concept and protocols and initiated the project. SKC, CPM, MB, JD, GG and DJ assisted in further development of the protocol. DAG, PC, DRT and RUN drafted the manuscript. NS, JD, and DJ will provide access to patients. DAG, PC, DRT, CPM, MB and RUN will implement the protocol and oversee collection of the data. All authors contributed and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/517/prepub


Previous studies have demonstrated that patients with treatment-resistant depression (TRD) and treatment-sensitive depression (TSD) differed at neural level. However, it remains unclear if these two subtypes of depression differ in the interhemispheric coordination. This study was undertaken for two purposes: (1) to explore the differences in interhemispheric coordination between these two subtypes by using the voxel-mirrored homotopic connectivity (VMHC) method; and (2) to determine if the difference of interhemispheric coordination can be used as a biomarker(s) to differentiate TRD from both TSD and healthy subjects (HS).Twenty-three patients with TRD, 22 with TSD, and 19 HS participated in the study. Data of these participants were analyzed with the VMHC and seed-based functional connectivity (FC) approaches.Compared to the TSD group, the TRD group showed significantly lower VMHC values in the calcarine cortex, fusiform gyrus, hippocampus, superior temporal gyrus, middle cingulum, and precentral gyrus. Lower VMHC values were also observed in the TRD group in the calcarine cortex relative to the HS group. However, the TSD group had no significant change in VMHC value in any brain region compared to the HS group. Receiver operating characteristic curves (ROC) analysis revealed that the VMHC values in the calcarine cortex had discriminatory function distinguishing patients with TRD from patients with TSD as well as those participants in the HS group.Lower VMHC values of patients with TRD relative to those with TSD and those in the HS group in the calcarine cortex appeared to be a unique feature for patients with TRD and it may be used as an imaging biomarker to separate patients with TRD from those with TSD or HS. Despite the rapid progress that has been made in the development of antidepressants over the years, approximately one-third of depressed patients still fail to respond to standard antidepressant treatments As a part of the efforts to understand the pathogenesis of MDD, the structural and functional changes associated with MDD have been explored with imaging technologies. A bulk of positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) studies have accumulated enough evidence to suggest that MDD is related to widespread local changes in many brain areas within the cortico-limbic circuit  studies. For example, several studies revealed that patients with MDD had alterations of orbitofrontal cortex-precuneus coupling Functional homotopy, the synchrony in patterns of spontaneous activity between homotopic regions in each brain hemisphere, is one of the most salient aspects of the brain’s intrinsic functional architecture Patients with TRD and TSD respond to antidepressants differently, suggesting that the two subtypes of depression differ at the neural level. In a seed-based FC study, TRD was related to disrupted FC mainly in thalamo-cortical circuits, while non-treatment refractory depression was associated with more distributed decreased FC in the limbic-striatal-pallidal-thalamic circuits To determine if there is any difference in interhemispheric coordination between patients with two types of depression as well as between depressed patients and healthy subjects, we analyzed an existing dataset Twenty-four patients with TRD, 31 with first-episode, treatment-naive MDD were recruited from the Mental Health Institute of the Second Xiangya Hospital, Key Laboratory of Psychiatry and Mental Health of Hunan Province, Central South University, Hunan, China. The subjects in the present study were from one of our previous studies In order to be eligible for this present study, all patients had to meet the following inclusion criteria: (1) In a current major depressive episode diagnosed with a Structured Clinical Interview for DSM-IV (SCID) All patients with TRD were in a treatment-resistant state and had taken at least two classes of antidepressants before participating in the study. For patients with TRD there was a one week drug-free period prior to the MRI scan. This one week wash-out period was selected based on the fact that the half-life of most antidepressants (except for fluoxetine) is from 12- to 24-hours. Treatment resistance was defined as non-responsiveness to at least two adequate treatments with adequate dosage and duration , compliance, and use of different types of antidepressants The treatment-naive patients were directed to take an antidepressant at a minimum dose of 150 mg/day of imipramine equivalents for 6 weeks after the MRI scan. The antidepressant included one of the three typical classes: tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), and serotonin-norepinephrine reuptake inhibitors (SNRIs). Treatment response was defined as more than a 50% reduction in the HRSD scores after treatment Twenty HS were recruited from the community. They were matched with the patient groups in age, gender, and education level. Patients or HS were excluded if they had any of the following: (1) Any history of neurological diseases, other medical illnesses, or other psychiatric disorders such as schizophrenia, bipolar disorders, or substance-induced mood disorder; (2) Any current Axis I comorbidities such as anxiety disorders, alcohol or drug dependence; (3) Any severe Axis II personality disorders or mental retardation; and (4) Any contraindication for MRI.The study was approved by the Ethics Committee of the Second Xiangya Hospital. All participants were given information about the procedures and signed an informed consent document.Scanning took place on a 1.5T GE scanner  equipped with high-speed gradients at the recruitment day. Participants were instructed to lie motionless, close their eyes and remain awake. The following parameters were used to obtain functional images: repetition time/echo time (TR/TE) = 2000/40 ms, 20 slices, 64×64 matrix, 90° flip angle, 24cm FOV, 5 mm slice thickness, 1 mm gap, and 180 volumes (6 min).http://www.fil.ion.ucl.ac.uk/spm). The first 10 volumes were discarded to ensure a steady state condition. Following steps including slice timing, head motion correction, and spatial normalization (voxel size: 3×3×3 mm3) were performed using Data Processing Assistant for Resting-State fMRI (DPARSF) Resting-state data were preprocessed in Matlab using the statistical parametric mapping software package  between a given voxel and a mirrored voxel in the opposite hemisphere were computed to generate VMHC maps. The details of VMHC obtainment were described in a previous study The VMHC was processed with software REST. Briefly, Pearson correlations (Fisher p<0.005 for multiple comparisons using Gaussian Random Field (GRF) theory . Post-hoc t-tests, restricted to the voxels showing significant differences by ANOVA analyses, were performed to identify differences between each pair of groups. The resulting statistical map was set at p<0.005 (GRF corrected). Since small amount of head motion can influence resting-state FC results Voxel-based comparisons of the entire VMHC maps with ANOVA were conducted in REST with a significance threshold of Brain regions exhibiting significant differences between the TRD and TSD groups and between the TRD and HS groups were designed as masks. Mean VMHC values were extracted within the masks for further receiver operating characteristic curves (ROC) analysis.p<0.005, GRF corrected).To examine the association of VMHC and clinical variables such as HRSD scores and illness duration, voxel-based correlations were implemented to the VMHC values of each depressed patient and clinical variables for each depressed group separately and together . Afterwards, voxel-based comparisons of the entire brain z maps with ANOVA were conducted in REST by using an explicit mask from the union set of the one-sample t-test results . Then post-hoc t-tests were performed to identify differences between each pair of groups . The FD of each subject was used as a covariate at the group level analyses.Seed-based FC was performed using a temporal correlation approach Data from four subjects  were excluded for further analyses due to excessive head motion. Data from 7 treatment-naive patients were discarded due to treatment non-response. Finally, the participants included 23 patients with TRD, 22 patients with TSD and 19 HS. Demographics and clinical characteristics of these participants are presented in As shown in As mentioned above, the calcarine cortex exhibited lower VMHC values in the TRD group compared to both the TSD group and the HS group. We examined whole-brain resting-state FC associated with two ROIs . Most of seed ROIs showed altered FC within the brain regions of the posterior perceptual and motor areas  along with a few subcortical regions  .Since the calcarine cortex exhibited significant VMHC differences between the patient groups and between patients with TRD and the HS group, it might be used as a biomarker to separate the patients with TRD from those with TSD and from HS. To test this possibility, mean VMHC values were extracted from the calcarine cortex  and ROC No correlation was found between VMHC in any brain region and HRSD scores in each patient group and the combined patient group by voxel-based correlation analyses. There was also no correlation between VMHC and age, education level, the overall illness duration, or the current illness duration.To our knowledge, this is the first study of using the VMHC to explore interhemispheric resting-state FC in patients with TRD or TSD and healthy controls. We found that patients with TRD had lower VMHC values in the calcarine cortex compared to both TSD and HS groups. However, there was no significant difference between TSD and HS in VMHC values in the calcarine cortex.The calcarine cortex is where the primary visual cortex is located and plays an important role in “refocusing” of attention Reduced VMHC values in other brain regions in patients with TRD compared to patients with TSD suggest that the disconnection of other brain regions may play a role in the pathogenesis of TRD. The fusiform gyrus is involved in the perception of facial emotion. The impairment of the fusiform gyrus has been observed in MDD p<0.005, postcentral gyrus also exhibited significantly decreased VMHC values , first-episode, and drug-naive MDD. The short duration of the illness may be responsible for the insignificant VMHC change in the TSD group. Second is the sensitivity of scanners. Using a 3T Siemens version scanner, we found that patients with first-episode and drug-naive MDD had reduced VMHC values in the medial prefrontal cortex (MPFC) and the posterior cingulate cortex/precuneus (PCC/PCu) C values . These dThere have been reports that the PFC acts in a key role with both cognition and emotion. Abnormal activity in the PFC has been observed in patients with MDD by using both R-fMRI p value adopted in the present study. When we loosed our statistical power to uncorrected voxel-wise threshold of p<0.005, some PFC areas also exhibited significantly decreases in VMHC values  method in the same group of patients with TRD or TSD did not show any white matter alteration between the mirrored regions with decreased VMHC Second, gray matter dysfunctions might account for the deficits in VMHC. In the intrinsic neural activity, the correlation of fMRI signals of gray matter is an indirect index of synchrony Since epidemiological studies have shown that women have a higher risk for developing MDD than men, gender difference in brain structure and function has attracted researchers’ attention. However, the gender difference in imaging data is far from settled Since clinical variables such as the duration and severity of a depressive episode have been reported as predictors for treatment non-response In addition to the relatively small sample size, the present study has several other limitations. First, patients with TRD were in the current treatment-resistant state and had tried at least two types of antidepressants before the recruitment. Previous studies have shown that antidepressants made brain activity of depressed patients more similar to that of healthy subjects In conclusion, the decreased VMHC values in the calcarine cortex may represent an imaging biomarker for TRD and may be used to separate patients with TRD from those with TSD as well as from HS. The findings also suggest that VMHC may offer an important new avenue of exploring the homotopic FC in both TRD and TSD in order to better understand the nature of the deficits in patients with MDD.Table S1Current treatment details of patients with TRD.(DOC)Click here for additional data file.Table S2The number of episodes of patients with TRD.(DOC)Click here for additional data file.Table S3p<0.005, GRF correction).Significant VMHC differences between pooled patients and HS ((DOC)Click here for additional data file.Table S4p<0.005).Significant VMHC differences between groups (uncorrected voxel-wise threshold of (DOC)Click here for additional data file.


Zirconia was introduced as an alternative to alumina for use in the femoral head. The yttria stabilized zirconia material was improved by adding alumina. We evaluated highly cross-linked polyethylene wear performance of zirconia in total hip arthroplasty. The hypothesis was that alumina stabilized zirconia could decrease highly cross-linked polyethylene wear.Highly cross-linked polyethylene wear was measured with a computerized method (PolyWare) in 91 hips. The steady-state wear rates were measured based on the radiographs from the first year postoperatively to the final follow-up and were compared between hips with yttria stabilized zirconia and alumina stabilized zirconia.The steady-state wear rate of highly cross-linked polyethylene against zirconia was 0.02 mm/year at a mean follow-up of 7 years. No significant difference was observed between groups with yttria stabilized zirconia and alumina stabilized zirconia.Addition of alumina to the zirconia material failed to show further reduction of highly cross-linked polyethylene wear and our hypothesis was not verified. Polyethylene wear debris is a known cause of osteolysis and loosening in total hip arthroplasty (THA). Femoral heads with improved surface characteristics have been introduced to reduce the wear. In an attempt to reduce polyethylene wear, ceramic femoral heads have been used as an alternative bearing surface. Alumina ceramic was first introduced as a ceramic material for femoral heads. Alumina ceramic has excellent geometric form and wettable surface, enhances hardness, thereby maintaining lubrication, and increases resistance to third-body wear . Despite2O3 allows the retention of the tetragonal structure at room temperature 4] reportin vivo and in artificial aging studies, maintaining its smooth surface finish and hardness [Recent clinical studies have reported a low wear rate of highly cross-linked polyethylene against zirconia Table  13,15-1-115-17. hardness .The present study failed to show further reduction of highly cross-linked polyethylene wear using the new zirconia. The mean age of the patients with the new zirconia was 57 years as opposed to 64 years in the patients with PSZ. Since patients are usually more active when they are younger, it is not fair to conclude that the newer-style heads did not contribute to lower wear. Hernigou and Bahrami  showed tThe steady-state wear rate of highly cross-linked polyethylene against PSZ and ASZ heads were very low. No mechanical failures of the polyethylene or zirconia occurred. While our numbers were insufficient to compare degradation of material, addition of alumina to the zirconia material (ASZ) contributed to a reduction in the transformation rate; however, further reduction of highly cross-linked polyethylene wear was not achieved and our hypothesis was not verified.The authors declare that they have no competing interests.MH and AS participated in the design and the coordination of the study. MH drafted the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/154/prepub


Technical details and experimental examples are given for a novel sample environment allowing X-ray investigations of self-assembly phenomena induced by controlled bulk solvent evaporation and nanoscale solvent adsorption/desorption. in situ deposition of NP solutions on solid substrates, (ii) rate-controlled evaporation of the bulk solvent, and (iii) adsorption/desorption of nano-thick solvent films onto preformed NP assemblies. Within this hermetically sealed chamber all the stages of self-assembly, including macroscopic solution evaporation, NP thin-film formation and its subsequent structural transformation induced by nano-thick solvent films, can be characterized in situ by X-ray scattering techniques. Here, technical design and calibration details are provided, as well as three experimental examples highlighting the chamber’s performances and potential. Examples include the controlled adsorption of thin toluene films on flat silicon wafers, the observation of transient accumulation of gold NPs near the toluene–vapour interface, and preliminary data on the structural effects of fast macroscopic solvent evaporation followed by nanoscale solvent adsorption/desorption from a vapour phase. By combining bulk evaporation rate control, fine tuning of the thickness of adsorbed solvent films and in situ X-ray characterization capabilities, this cell enables explorations of both near-to-equilibrium and far-from-equilibrium routes to NP self-assembly.Spontaneous ordering of nanoparticles (NPs) occurring as a consequence of solvent evaporation can yield highly ordered and extended NP superlattices bearing both fundamental scientific interest and potential for technological application. A versatile experimental chamber has been developed allowing (i) controlled  In the subsequent sections the wetting cell performance is demonstrated by three experimental examples: (i) the determination of the effective Hamaker constant for the interaction between toluene and silicon oxide, which was performed as a validation test before applying the chamber to the study of NP-based systems, (ii) the observation of an unexpected transient NP accumulation near the surface of freshly injected dilute suspensions of thiol-stabilized Au NPs in toluene, and (iii) preliminary results concerning the NP self-assembly processes occurring after fast evaporation of the bulk solvent followed by slow nanoscale solvent adsorption and desorption.2 Hamaker constant was determined viain situ XR measurements at the beamline ID15C  at an X-ray energy of 38.7 keV. The NP systems were investigated at the ID15A beamline . In the following the X-ray data sets are presented as a function of the wavevector transfer q, whose Cartesian components are qx = qy = e)k = All the experiments were carried out at the European Synchrotron Radiation Facility in Grenoble, France. The toluene-SiO2.macroscopic solvent evaporation and microscopic solvent adsorption/desorption onto bare or NP-loaded substrates.The sample environment developed for our investigations is composed of an external stainless steel vessel and an internal copper chamber Fig. 1. The exta) and 1(b)dNP dispersions can be injected directly onto a horizontally lying substrate mounted on a sample holder suspended inside the inner chamber  and for 307 K < T < 320 K a)c)The fitted parameters for the second box, describing the adsorbed toluene film, are listed in Table 2et al., 1991b2). The Hamaker constant −1 −1 and, considering the toluene surface tension at et al., 2003et al., 1988The best fit  and the injection shear rate is only ∼10−2 s−1. Weak residual charging of the NPs may play a role; however, further investigations are needed to better understand the origin of this phenomenon.The general scenario that we can infer from these measurements is that, after injection, a part of the particles present in the solution are driven towards the solution–vapour interface, forming a dilute and disordered monolayer. This NP layering effect was reproduced at each new injection of fresh NP solution. Furthermore, the surface NP accumulation is transient, and the NPs slowly re-dissolve into the bulk toluene phase over periods of several hours. We report this totally unexpected finding, although the physical force responsible for the formation of this surface NP layer is not clearly identified, since under normal conditions the particles are expected to be well dissolved in toluene. Shear effects during the injection process cannot account for the particle interfacial accumulation, since the NP dispersion is very dilute et al., 2002During solvent evaporation, the particles are free to diffuse by Brownian motion in the bulk liquid while it progressively evaporates. The Peclet number, Pe, is the parameter used to gauge the dominance of the Brownian velocity relative to the speed of vertical [a)cbversus time in Fig. 7(d)−1, with a maximum value of ∼120 µm min−1, reached during the second half of the process.With the aim of achieving the highest possible Pe number, we heated the sample using an additional power supply driven by one of the temperature controllers. In this way we could reach the temperature ramp plotted in Fig. 7et al., 2006faed)For the calculation of the Pe number attained during this evaporation process, we neglect the fact that  Fig. 7e. We takeg)et al., 2002gThe Pe number resulting from this procedure is plotted in Fig. 7 and the higher-order peaks indicate a well developed in-plane hexagonal arrangement, with lattice constant −1, it is possible to estimate the in-plane correlation length to −1 is the experimental horizontal resolution and As a demonstrative example, we show the 6.in situ X-ray scattering techniques (e.g. XR and GISAXS), from the initial bulk NP dispersion to the resulting dry film after macroscopic solvent evaporation and/or solvent annealing. In principle, this experimental chamber can be used for static and dynamic X-ray investigations of self-assembly processes involving both NPs and a wide range of additional classes of mesoscopic self-assembling building blocks.We have designed and realised an experimental chamber for the study of solvent-mediated NP self-assembly. The chamber adopts a novel approach: the combination of tunable-rate macroscopic solvent evaporation and precisely controlled solvent adsorption/desorption at the nanoscale. The process of NP self-assembly can be characterized throughout all of its phases by


The FAST project (Food Allergy Specific ImmunoTherapy) aims at the development of safe and effective treatment of food allergies. Classical allergen-specific immunotherapy (SIT) for treatment of food allergy using subcutaneous injections with food extracts has proven to be effective but too dangerous due to anaphylactic side-effects. FAST aims at developing a safe alternative by replacing food extracts with hypo-allergenic recombinant major allergens, the active ingredients of SIT. Fish allergy is caused by a single major allergen, parvalbumin. In phase I and II of the study randomized double-blind placebo-controlled trials will be performed. A recipe for fishblinding does not exist and is needed to determine the clinical reactivity when including patients in the trial and to assess efficacy in the Phase II trial.The aim was to develop a recipe that could blind the highest possible amount of freeze-dried cod in the lowest amount of edible low-allergenic vehicle. Several recipes were developed and tried in the attempt to hide the texture and taste of the freeze-dried cod. A burger primarily made of minced chicken meat, onion, rice and spices was able to blind cod successfully. Cumin seed is an important ingredient, that has the ability to mask the taste of cod that would otherwise remain in the mouth after intake.Freeze-dried cod powder Minced chicken meatOnion, choppedOlive oilFlattened rice (rice flakes)ThymeCumin seedsSaltPepperTurmericAll ingredients are thoroughly mixed in a food processor, to a homogeneous paste. Formed to a burger and cooked in a pre-heated oven at 180°C.7 doses are given from 50ug - 4g protein, accumulated dose of 22g of cod fish. Preliminary testing (triangle test) show that it is not possible to tell the active challenge from placebo.


Osteosarcoma is the most common primary bone cancer of children and is established during stages of rapid bone growth. The disease is a consequence of immature osteoblast differentiation, which gives way to a rapidly synthesized incompletely mineralized and disorganized bone matrix. The mechanism of osteosarcoma tumorogenesis is poorly understood, and few proteomic studies have been used to interrogate the disease thus far. Accordingly, these studies have identified proteins that have been known to be associated with other malignancies, rather than being osteosarcoma specific. In this paper, we focus on the growing list of available state-of-the-art proteomic technologies and their specific application to the discovery of novel osteosarcoma diagnostic and therapeutic targets. The current signaling markers/pathways associated with primary and metastatic osteosarcoma that have been identified by early-stage proteomic technologies thus far are also described.  Osteosarcoma (OS) accounts for approximately 3% of all childhood cancers . The majOsteosarcoma affects the skeleton of patients primarily in regions of rapid bone growth such as the distal femur, proximal tibia and proximal humerus .  Figureα-1 (Cbfa1) ) [The majority of OS tumors are heterogeneous, characterized by the various stages of mesenchymal differentiation, each of which demonstrates different levels of gene and protein expression, adding to the complexity of the disease. Despite the heterogeneity within individual tumors and individual patients, mutations in p53 and RB have consistently been identified –12. Thes(Runx2)) –15, and (Runx2)) .In this paper, we focus on the growing list of available state-of-the-art proteomic techniques and their application to the discovery of OS targets and signaling pathways associated with primary and metastatic OS. These new approaches provide the rationale to shift the focus of OS research away from the single-marker/single-pathway paradigm, to a systems biology approach enabling the analysis of multiple signaling pathways that are involved in primary OS and subsequent metastatic disease .Genomic studies provide valuable insight into the regulation of genes involved in the pathogenesis of disease but are limited in their ability to identify multiple protein products derived from individual genes, and it is well established that mRNA levels do not necessarily correlate to the protein level, which is the functional output. Proteomics is a technology for studying protein expression, protein-protein interactions, and posttranslational modifications from complex mixtures of fluids or tissues that reflect the mechanism of disease . ProteomMost proteomic techniques involve a protein separation step followed by identification of proteins. The traditional protein separation technique used to study OS includes two-dimensional gel electrophoresis (2DE) followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for protein identification , 25–29. More recently, proteomic technologies have greatly improved both protein separation and protein identification and can be readily applied to offer deeper insight into OS pathogenesis . One tecThe development of MudPIT gave rise to other shotgun proteomic technologies, which refers to the direct analysis of complex protein mixtures to rapidly generate a global profile of the protein complement within a sample mixture . Anothert-test and other more robust parametric methods [Differentiating proteins between normal bone, OS primary tumors, and metastatic tumors can be accomplished using spectral counting, that is, the number of tandem mass spectra assigned to a given protein . This is methods . We have methods . Protein methods . Another methods –42.An alternative to label-free shotgun proteomics for protein quantitation includes stable isotope labeling approaches. These include isotope-coded affinity tag (ICAT) –45, stabThe proteomic technologies discussed above have been drastically underappreciated in osteosarcoma research with fewer than 70 analyses published in the last decade , many ofThe majority of studies have used differential protein expression to identify new protein markers that can be translated into new diagnostic and therapeutic targets, but they fail to characterize their global contribution to disease pathogenesis and instead focus on single marker/single-pathway identifications leaving out the complexity of multiple pathway interactions. Some examples of proteomics studies which focus mainly on known signaling pathways instead of taking a more global proteomic approach to identify the complexity of protein interactions do exist and are summarized below.Cates et al. compared protein expression profiles between two clonally related murine OS cell lines with low (K12) and high (K7M2) metastatic potential using two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI . 2D-DIGE Y. Li et al. compared primary OS to benign bone tumor samples using 2D gel electrophoresis and the protein spots identified with MALDI-TOF MS . They we Zhang et al. took a subcellular comparative approach . They anEngin et al. studied the role of Notch signaling in tumors of mesenchymal origin . Notch hLi et al. used a proteomic analysis approach to understand the role of E2F1 in p53-negative tumor cells . E2F1 is The c-Jun N-terminal kinase (JNK) signal transduction pathway that is known to play a role in the proliferation, differentiation, and apoptosis of osteoblasts in normal bone has also been studied . These i Recently, a noninvasive approach for the proteomic profiling of blood from osteosarcoma patients was described . Using sOS is the primary bone cancer occurring during the second decade of life and rarely in older adults with Paget's disease. These are times in which there is rapid bone development and turnover. Proteomics has slowly begun to be utilized for the identification of proteins that characterize OS and that may play a role in OS tumorigenesis . The majIn addition to proteomics, new technologies are constantly emerging that provide complementary approaches for the identification of disease-specific biomarkers. Techniques include surface plasmon resonance (SPR) –79 and nNanowires/nanotubes are used to target specific low-molecular-weight proteins. The platform consists of an electrochemical immunosensor, which consists of multiwalled carbon nanotubes for real-time detection of cancer biomarkers in human serum . The tecde novo protein identification. Complementing proteomic-based biomarker platforms with these new approaches may indeed provide the sensitivity and specificity needed for a comprehensive analysis of OS. In sum, we eagerly await the impact of these evolving technologies on the pathophysiology, diagnosis, and treatment of OS.Although exciting, these biomarker discovery approaches are currently in the proof-of-concept stage and have been used for targeted discovery of low-abundant proteins in human serum and not for The Supplemental Material reviews the current state of the proteomic studies performed since 2000 to the present. The table includes references, sample types, proteomic techniques used in the study, the identified proteins that were up- or down-regulated in OS, and the validated biomarkers from each of the proteomic studies.Click here for additional data file.


Several states in the US have passed laws mandating coverage of colorectal cancer (CRC) screening tests by health insurance plans. The impact of these state mandates on the use of colorectal cancer screening has not been evaluated among an age-eligible target population with access to care .We collected information on state mandates implemented by December 31, 2008 and used data on insured adults aged 50 and 64 years from the Behavioral Risk Factor Surveillance System between 2002 and 2008 to classify individual-level exposure to state mandates for at least 1 year. Multivariate logistic regression models  were used to estimate the effect of state mandates on recent endoscopy screening (either flexible sigmoidoscopy or colonoscopy during the past year).From 1999-2008, twenty-two states in the US, including the District of Columbia passed comprehensive laws requiring health insurance coverage of CRC screening including endoscopy tests. Residence in states with CRC screening coverage mandates in place for at least 1 year was associated with a 1.4 percentage point increase in the probability of utilization of recent endoscopy .The findings suggest a positive, albeit small, impact of state mandates on the use of recent CRC screening endoscopy among the target eligible population with health insurance. However, more research is needed to evaluate potential effects of mandates across health insurance types while including controls for other system-level factors (e.g. endoscopy and primary care capacity). National health insurance reform should strive towards a system that expands access to recommended CRC screening tests. Colorectal cancer (CRC) is the third leading cause of cancer-related death in the U.S. among both men and women . CRC scrIn recent years, in an effort to improve CRC screening utilization promulgated by clinical guidelines as well as to expand choice of tests given individual preferences,-15 stateThe Behavioral Risk Factor Surveillance System (BRFSS) is a publicly available state level survey conducted in the United States that measures information on general health and cancer-related risk factors . The BRFFrom the compiled four-year cross-sectional BRFSS survey data, we derived an analytic sample consisting of respondents aged 50 to 64 years who indicated having any health insurance coverage and were residing in 44 states or D.C. (details of state exclusions are explained below).For the purpose of these analyses, states with coverage mandates for CRC screening were those that had implemented comprehensive legislation requiring private insurance plans to cover the full range of colorectal cancer screening tests, including endoscopy procedures, consistent with American Cancer Society guidelines on or beIn the BRFSS, participants were read this script  to enable recall and reporting of endoscopy screening for CRC. Then, participants' information on endoscopy testing was collected by asking these two questions: "Have you ever had either of these exams?" and "How long has it been since you had your last sigmoidoscopy or colonoscopy?" From this information, we defined the outcome as a dichotomous (yes/no) measure of the receipt of an endoscopy CRC screening test in the past year. Though clinical guidelines recommend varying intervals for different types of endoscopy testing for CRC, we used the 'past year' interval in order to minimize potential misclassification of case/control status as a result of respondents' plausible migration between states with and without laws.All multivariate analyses controlled for age , sex , race/ethnicity , education level , household income level , self-reported health status , and marital status . Even though state mandates primarily affect privately insured populations, the BRFSS did not collect information about the type of health plans among those with health insurance coverage. As we could not restrict our analytic sample to the privately insured group, we considered a state level variable measuring the privately insured proportion among 50-64-year-olds  for 2002, 2004, 2006 and 2008 as a covariate in all statistical models in additThe independent effect of state CRC screening coverage mandates on CRC screening was evaluated using state- and year- fixed effects models. Multivariate logistic regression analyses were used to calculate adjusted probabilities  and odds ratios of colorectal cancer screening among respondents with and without state coverage mandates. Models included a state fixed effect that controlled for state factors that do no vary over time (invariant factors) and a year effect that controlled for national secular trends, in addition to individual-level covariates, and state privately insured population rates among those 50-64 years. We present these estimates in comparison with unadjusted probabilities (obtained from unadjusted logistic regression models) in order to understand the influence of other covariates on the relationship between state mandates' exposure and screening probabilities. Further, we performed sensitivity analysis that considered a lag variable in order to capture the effect of mandates at different times after implementation. We tested for significant differences in the adjusted rates among residents in states with mandates for different time periods after implementation .We tested for potential two- and three-way interactions between the mandates and demographic (race and ethnicity) and SES variables (education- and income- level), as it is plausible that the effect of these mandates would be more concentrated among certain groups than others, given that some groups may have had lower access to these tests prior to the passage of these mandates and may thus benefit more after these laws are passed. SAS-callable-SUDAAN Version 10 was used to apply the sampling weights in the calculation of weighted estimates and standard errors (using Taylor Series Linearization Method) that taOf the 45 states included in the study, 22 states and DC had passed mandates requiring private insurance plans to cover the full range of colorectal cancer screening tests consistent with ACS guidelines on or before December 31, 2008  . However, linear contrasts of the adjusted probabilities indicated that the effect of mandates was significant and stronger in higher-educated respondents  but not in lower-educated respondents . A three-way interaction variable between state mandates, a collapsed two-level version of the household income , and education level  was statistically significant (p < 0.005). Linear contrasts of the adjusted probabilities from the interaction term indicated that among low- and middle-income individuals, the effect of state mandates was positive and significant only among higher-educated individuals  but not among lower-educated individuals . Conversely, state mandates resulted in marginally higher CRC utilization rates among lower educated-high income individuals , but not among high educated-high income individuals .In this study, we provide the first assessment of the utilization effects of state mandates requiring private insurers to cover recommended CRC screening. The main findings suggest that state mandates are positively associated with utilization of CRC endoscopic screening (1.4% absolute increase in probability of past-year endoscopy test use) in adults aged 50 to 64 years with access to care. In addition, our results suggest that among low- and middle- income groups, mandates seemed to have benefited higher-educated groups more than lower-educated, whereas education-related disparities in high income groups appear to have been reduced as a result of mandates. Even though we know of no prior study that has examined this association in a non-Medicare insured population of adults aged 50 to 64 years, our study findings are generally consistent with previous studies conducted among the elderly Medicare beneficiaries, showing positive effects of policy changes -24.In reflecting on these study's finding suggesting a small effect of state CRC screening mandates (in absolute percentage differences), we have to consider several contextual issues and challenges that may stimulate additional health policy research endeavors. First, there is the issue that state-mandated benefits for CRC screening are limited in their reach because of certain exemptions. State mandates only apply to certain types of insurance coverage in the commercial private marketplace. It is the case that, the federal Employee Retirement Income Security Act (ERISA) exempts states from regulating self-funded employer-based health plans from state laws that govern health insurance ,26. We wScreening for colorectal cancer is a beneficial, cost-effective way to advance the public's health by reducing the incidence and mortality of CRC ,5. ColorThe authors declare that they have no competing interests.VC initiated the research idea, designed the study, and wrote the manuscript. PB contributed to the study design, conducted the analysis and wrote parts of the manuscript. MS provided valuable support in the compilation of state mandates information as well as review/editing of the manuscript. KV provided valuable comments on the analysis and editorial suggestions on the manuscript. EW provided valuable comments and editorial suggestion on the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/19/prepub


Recently, important efforts have been dedicated to the realization of a fascinating class of new photonic materials or metamaterials, known as photonic quasicrystals (PQCs), in which the lack of the translational symmetry is compensated by rotational symmetries not achievable by the conventional periodic crystals. As ever, more advanced functionality is demanded and one strategy is the introduction of non-linear and/or active functionality in photonic materials. In this view, core/shell nanorods (NRs) are a promising active material for light-emitting applications. In this article a two-dimensional (2D) hybrid a 2D octagonal PQC which consists of air rods in an organic/inorganic nanocomposite is proposed and experimentally demonstrated. The nanocomposite was prepared by incorporating CdSe/CdS core/shell NRs into a polymer matrix. The PQC was realized by electron beam lithography (EBL) technique. Scanning electron microscopy, far field diffraction and spectra measurements are used to characterize the experimental structure. The vertical extraction of the light, by the coupling of the modes guided by the PQC slab to the free radiation via Bragg scattering, consists of a narrow red emissions band at 690 nm with a full width at half-maximum (FWHM) of 21.5 nm. The original characteristics of hybrid materials based on polymers and colloidal NRs, able to combine the unique optical properties of the inorganic moiety with the processability of the host matrix, are extremely appealing in view of their technological impact on the development of new high performing optical devices such as organic light-emitting diodes, ultra-low threshold lasers, and non-linear devices.PACS: 81.07.Pr Organic-inorganic hybrid nanostructures, 81.16.-c Methods of nanofabrication and processing, 42.70.Qs Photonic band-gap materials. Quasiperiodic crystals are a new class of materials that exhibit long-range aperiodic translational order and high rotational symmetries ,2. UnlikPQCs possess photonic band-gaps (PBGs) with very interesting properties of light transmission ,16, waveAdvances in 2D photonic structures are expected in the introduction of non-linear and/or active functionality into a 2D PQC. 1D semiconductor nanostructures are likewise promising materials both in fundamental research and in practical applications -33. As aThese characteristics open the way to a new class of hybrid devices based on polymers and colloidal NRs in which the unique optical properties of the inorganic moiety are combined with the processability of the host matrix to develop new high performing optical devices such as organic light-emitting diodes, ultra-low threshold lasers, and non-linear devices. One of the challenges of these applications is the incorporation of inorganic nanoparticles into organic polymer matrices, since this is usually accompanied by phase separation, aggregation of nanoparticles, loss of transparency, and luminescence quenching due to exciton energy transfer ,39.In this paper, for the first time to our knowledge, a 2D hybrid eightfold symmetric aperiodically ordered PQC which consists of air rods in a nanocomposite prepared by incorporating CdSe/CdS core/shell nanorods (NRs) in a polymer is proposed and experimentally demonstrated. The semiconductor nanocrystal based 2D PQC was fabricated by directly patterning and releasing a thin film of a functional material composed by NRs dispersed in a positive electronic resist.Scanning electron microscopy (SEM) and optical measurements are used to characterize the structures. The vertical extraction of the light, by the coupling of the modes guided by the PQCs slab to the free radiation via Bragg scattering, consists of a narrow red emissions band at 690 nm with a FWHM of 21.5 nm.The dot/rod core/shell CdSe/CdS nanocrystals were synthesized by a "seeded growth" approach  which yiA morphological analysis of the stock solution of CdSe/CdS NRs dissolved in toluene was carried out by means of transmission electron microscopy (TEM) and is reported in Figure 3 on an indium tin oxide (ITO) coated glass were obtained.We firstly fabricated CdSe/CdS NRs-PMMA polymer composite film by combining the CdSe/CdS NRs with PMMA of high optical transparency in the visible region and spin-coating the composite solution. Since PMMA is transparent in the visible spectral range and it is an electron-sensitive material, it was chosen as the embedding matrix for NRs. Clear nanocomposite films without bubbles and smooth surfaces with sizes up to 1.5 × 2.5 × 0.0000600 cmOur experimental structure was fabricated by using a high-resolution electron beam lithography (EBL) technique. The EBL facility employed consisted of a Raith 150 system. Such system enables the writing of patterns of arbitrary geometries with a spatial resolution up to 10 nm. The samples were obtained by exposing a layer of our mixture of CdSe/CdS NRs doped PMMA deposited on an ITO coated glass. The e-beam is locally focused on the sample to expose selected regions of material homogenously along the depth of the substrate according to the calculated desired pattern. Exposed areas were dissolved away leaving a 2D eightfold octagonal quasiperiodic structure made of air-filled cylinders, located at the vertices of the octagonal lattice, lying in the polymer matrix.2. The air rods arrays were fabricated using EBL on quartz substrates with a 15 nm layer ITO for conduction. A 600-nm-thick layer of colloidal doped PMMA was spin-coated on top of the cleaned substrate. Subsequently, the nanopatterning was defined using the Raith 150 system with current and area dosage of 27.8 pA at 20 keV. The resist was developed in a 1:3 solution of methyl isobutyl ketone (MIBK) and isopropanol (IPA).The resulting 2D QC is made of air rods embedded into a hybrid organic/inorganic matrix of CdSe/CdS NRs doped PMMA. The size of the patterned area is about 650 μmRecently, the importance of tiling geometry in the arrangement of the octagonal quasicrystal was discussed. The fabricated octagonal structure was determined by simulating the quasiperiodic transverse irradiance distribution given by 8-beam interference process and was theoretically analyzed through finite difference time domain (FDTD) simulations of the transmittance spectra . The octA SEM image of the eightfold rotational symmetry PQC realized in the hybrid nanocomposite film with a hole diameter of 535 nm is reported in Figure + laser operating on the TEM00 mode at the wavelength λ = 514.5 nm and having a beam waist w0 = 2 mm. After having spatially filtered the laser beam through a lens-pinhole-lens system, the source is incident on the sample. Through the spatial filter, the incident light is diffracted creating an airy pattern: the central zero-order has an excellent spatial coherence and represents a good approximation of a plane wave. Higher diffraction orders have highly divergent wave vectors with respect to the optical axis. The second pinhole filters only the central spot of the airy pattern. An optical system consisting of two confocal lenses is placed between the two pinholes providing a magnification ratio of 3.47 that ensures a uniform illumination of the experimental sample having a square surface of ~700 μm2. A charge-coupled device (CCD) array coupled with a focusing lens system is finally used to acquire the Fourier spectra of the structure.The experimental octagonal quasicrystal realized has been characterized in the direct space through SEM metrological measurements, whereas in the reciprocal space interesting properties of the aperiodic octagonal array can be provided through the experimental determination of the Fourier spectra of the samples. Diffraction patterns are produced as a result of the regional periodicity. Figure The diffraction pattern possesses eightfold rotational symmetry, and contains a series of spots of different intensity, surrounding the central undiffracted beam. These spots can be associated with vectors in reciprocal space. The observed peaks are sharp and symmetrically distributed. Each order has rings of spots at different cone angles around the zero-order central spot. The picture clearly reveals the presence of quasiperiodicity within the sample.The light propagating in the glass substrate which is extracted by diffraction was measured at room temperature using a multimode fiber, a CCD imaging telescope (OL610), and a CCD-based spectroradiometer (OL770-LED).A white light was introduced from an edge of the glass and was propagating inside the glass. The QPC area was seen as a red area because of the wavelength-selective diffraction by the hybrid QPC. Figure It has been studied that when the period of a two-dimensional photonic crystal is equal to the cavity wavelength of the guided mode, the guided waves propagating to several in-plane directions are coupled to the radiation mode in the direction normal to the device surface since the Bragg diffraction condition is satisfied. The vertical extraction of the light, by the coupling of the modes guided by the PC slab to the free radiation via Bragg scattering, is ruled by a phasematching condition, namely by the conservation of the in-plane component of the momentum at the air-dielectric interface .In this work we patterned, for the first time to our knowledge, a nanocomposite containing colloidal semiconductor quantum rods of nanometer size scale to fabricate a novel 2D hybrid eightfold symmetric aperiodically ordered PQC. Our hybrid organic/inorganic nanocomposite is formed by using inorganic NRs, core/shell CdSe/CdS quantum rods, as inclusions in an organic polymer matrix (PMMA). Our nanocomposite has been prepared as a film in which each domain/inclusion can perform a specific photonic or optoelectronic (combined electronic and photonic) function. This permits introducing multifunctionality, and each of these functions can independently be optimized.2. The diffraction effect of our sample has been confirmed and consists of a narrow red emission with a FWHM of 21.5 nm. The measured resonances peaks are due to the QPC feedback effect.A semiconductor nanocrystals based 2D QPC pattern with rods with a diameter of 500 nm and a depth of 700 nm has been uniformly formed by EBL in a large area of 650 × 650 μmThe possibility to pattern hybrid materials open the route to the development of new high performing optical devices such as organic light-emitting diodes, ultra-low threshold lasers, and non-linear devices. In a recent work of some of the authors  CdSe/CdSCCD: charge-coupled device; EBL: electron beam lithography; FDTD: finite difference time domain; FWHM: full width at half-maximum; IPA: isopropanol; ITO, indium tin oxide; MIBK, methyl isobutyl ketone; NRs: nanorods; 1D: one-dimensional; PQCs: photonic quasicrystals; PMMA: polymethylmethacrylate; PBGs: photonic band-gaps; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TEM: transverse electromagnetic; 2D: two-dimensional.The authors declare that they have no competing interests.LP conceived of the study, carried out the nanocomposite preparation and the fabrication of the samples, the SEM characterizations and drafted the manuscript. MR carried out the far field diffraction characterization and the spectra measurements. LM supervised the synthesis of colloidal nanorods and provided the TEM characterization. MZ prepared NR solutions. JZ participated in the design of the study. PM participated in the design of the study and its coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells. Nuclear receptor TLX plays an important role in vertebrate brain functions Neural stem cells in adult brains reside in the subgranular cell layer of the hippocampus and the SVZ 3H) thymidine to mark cells undergoing DNA synthesis Classical studies of neurogenesis used tritiated . Co-staining of TLX and BrdU revealed that 66±17% (n = 3) of the BrdU label-retaining cells were also TLX-positive  showed a dramatic reduction in the number of rapidly dividing neural progenitor cells in the SVZ of TLX−/− brains, indicated by a decrease in 6 hr pulsed BrdU-labeling cells , which lThe total cell population in the SVZ, as revealed by nuclear Dapi staining, reduced dramatically in TLX−/− brains, compared to that in wild type brains . Only onConfocal images taken at the surface of whole mounts of the lateral wall of the lateral ventricles revealed intensive GFAP staining in the SVZ of TLX−/− brains . The GFAThis study provided the first evidence of endogenous TLX expression in the SVZ of adult mouse brains by immunostaining with a TLX-specific antibody. TLX immunostaining was performed to determine the cellular identity of the TLX-expressing cells in the SVZ regions of adult mouse brains. Different strategies of thymidine analog incorporation in the wild type and TLX-homozygous mice were used to determine the proliferative status of the TLX-expressing cells.The adult SVZ contains the slowly dividing or relatively quiescent type B neural stem cells and the rapidly dividing progenitors, including the transit-amplifying type C cells and type A neuroblasts T2 reporter under the control of TLX gene regulatory sequences T2 reporter was used to determine TLX expression in the previous study. Secondly, we used Mash1 to label type C cells in this study, while epidermal growth factor receptor (EGFR) was used to label type C cells in the previous study high transit amplifying cells A previous study has shown that TLX is expressed exclusively in type B cells but not in type C cells in the SVZ of adult mouse brains, using a CreERIn this study, we also took advantage of a double tymidine analog labeling approach to investigate the proliferative state of the TLX-expressing cells in the SVZ of adult brains. The thymidine analogs IdU and CIdU have advantages over BrdU labeling alone. Both IdU and CIdU can be injected into the same animal, therefore cells produced at different time points can be simultaneously assessed Interestingly, a large number of TLX-expressing cells that were not labeled by either IdU or CIdU were detected in the SVZ of adult brains. Similarly, a big portion of the TLX-expressing cells did not label with either short-term or long-tem BrdU labeling in the SVZ. These observations suggest the presence of a population of TLX-positive cells that are relatively quiescent and arrested in the cell cycle. Quiescence has been shown to be important for the maintenance of the regenerative potential of adult neurogenic regions, presumably by functioning as a protective mechanism to counter stem cell exhaustion It has been shown that the slowly dividing, BrdU label-retaining cells correspond to neural stem cells in the adult brain All research animals are maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and used in compliance with federal and institutional regulations. Brain tissues were harvested from wild type, TLX-heterozygous, and TLX-homozygous mice under the IACUC protocol 03038 approved by the City of Hope Institutional Animal Care and Use Committee.TLX-heterozygous and homozygous mice were described in our previous study Primary antibodies used include rabbit anti-TLX  To label proliferative cells, we treated mice with BrdU at 50 µg/g body weight in three strategies. For short-term BrdU labeling, animals were injected with BrdU and sacrificed 6 hr later to chase the rapidly dividing cells (6 hr BrdU chase). Alternatively, mice were injected with BrdU once daily for one week and sacrificed the next day after the last dose of BrdU to chase most of the dividing cells. For long-term BrdU labeling, mice were injected with BrdU once daily for one week and sacrificed 4 weeks later to chase the slowly dividing cells (BrdU label-retaining cells). For double IdU and CIdU labeling to chase the continuously slowly dividing cells, mice were treated with IdU at 0.1% in drinking water for 2 weeks. Ten days later, mice were injected with CldU at 50 µg/g body weight once daily for 5 days. The treated animals were allowed to survive for 2 weeks and then sacrificed and processed for immunostaining analysis.Fig. S1Expression of TLX in GFAP-positive type B neural stem cells in the SVZ of mouse brains.A. A single optical scanning image of TLX-GFAP staining. B. A merged image from a z-series scanning of the same staining. Nuclei DAPI staining was shown in blue. LV stands for lateral ventricles.(TIF)Click here for additional data file.Fig. S2Orthogonal images and multiple single plane images of co-staining of TLX, Mash1, and BrdU.A. Orthogonal images of TLX, Mash1, and BrdU co-staining. The 1st, 2nd, 3rd, and 4th cells (from the top) in B. Multiple single plane images of TLX, Mash1, and BrdU co-staining. LV stands for lateral ventricles.(TIF)Click here for additional data file.Fig. S3Orthogonal images of TLX-BrdU co-staining. An example of the TLX-BrdU double-stained cells in long-term BrdU labeling (1) and short-term BrdU labeling (2) is shown in orthogonal planes. LV stands for lateral ventricles.(TIF)Click here for additional data file.


Insulin is one of the major metabolic hormones regulating glucose homeostasis in the organism and a key growth factor for normal and neoplastic cells. Work conducted primarily over the past 3 decades has unravelled the presence of insulin in human breast cancer tissues and, more recently, in human non-small cell lung carcinomas (NSCLC). These findings have suggested that intracellular insulin is involved in the development of these highly prevalent human tumors. A potential mechanism for such involvement is insulin's binding and inactivation of the retinoblastoma tumor suppressor protein (RB) which in turn is likely controlled by insulin-degrading enzyme (IDE). This model and its supporting data are collectively covered in this survey in order to provide further insight into insulin-driven oncogenesis and its reversal through future anticancer therapeutics. It has meanwhile been nearly a century since insulin's stimulatory effects on cell proliferation and tissue growth have been studied -3. Thereintracellular- insulin effects at the level of the cell nucleus [However, historically preceding and coinciding with this model on an extracellular insulin activity, there have also been reports of direct - i.e. (non-insulin receptor-mediated and)  nucleus  and, mor nucleus  as well  nucleus .In the early 1990s, this conceptual framework on an intracellular localization and action of insulin was expanded by a novel proposal according to which insulin may physically interact with the (mainly nuclear) retinoblastoma tumor suppressor protein (RB) and thereby, similar to RB-binding viral oncoproteins, inactivate RB and thus promote cell proliferation -13, primAs a result of an insulin-insulin receptor interaction in the presence of low nanomolar insulin concentrations, a second messenger cascade is activated among which the intracellular enzyme phosphatidylinositol 3-kinase (PI 3-kinase) is a major intermediary molecule . FurtherIn addition to this signalling cascade initiated by insulin at the level of the cell membrane, it has become increasingly apparent over the past three and a half decades that insulin could also act as its own messenger (i.e. without the mediation of other molecules) in order to directly promote cell growth, specifically insulin molecules that are located intracellularly.The main support for such a possibility comes from studies conducted on human cancer tissue specimens and revealing the presence of intratumoral insulin -19. IntrA potential mechanism for such intracellular insulin-driven tumor growth is the insulin-RB complex formation that, so far, has been experimentally demonstrated in several human carcinoma-derived cell lines -13. ThisFurthermore, the probability for this interaction should be higher in neoplastic cells which equally display a dysfunction of insulin-degrading enzyme (IDE) or, respectively, insulysin in the light of previous data showing that an inactivation of IDE leads to an increase in the nuclear localization of insulin . In thisTherefore, the resulting concept on IDE as a potential tumor suppressor protecting RB from inactivation by insulin, as elaborated through structural -24 and pIn case the envisaged insulin-RB complexes and IDE dysfunction will be validated in human cancer specimens, this would then suggest as an antineoplastic treatment strategy the interference with such intracellular carcinogenesis by means of cell-penetrating peptides that bind and thereby neutralize insulin such as those peptides derived from RB and termed MCR peptides ,13,27-30The author declares having no competing interests.