Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. A great effort has been devoted, during the past decade, to the development of new devices for specific DNA sequences detection, due to the relevance of nucleic acid analysis in many areas, including clinical diagnostics , environi.e., single-molecule PCR) [It is for this reason that a key step in nucleic acid detection protocols is the amplification of the target sequence. The amplification generates a large number of target copies, greatly enhancing the assay sensitivity . The mosule PCR) ,9 due toule PCR) ,10. AmonThis review will first discuss the use of microfluidic-based systems for nucleic acid amplification by underlining advantages coming from the rapidity of the process and the low sample consumption and discussing drawbacks caused by surface-dependent processes. Miniaturized isothermal amplification systems will be then illustrated including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA).Since its advent, microfluidic has drawn great attention for the capability of miniaturizing typical laboratory operations by using a fraction of the volume of reagents in significantly less time . CompareA microfluidic device may include several components such as valves, mixers and pumps capable of controlling the fluid flow within the device and other components useful for sample pre-treatments or detection. Liquids confined into the microfluidic environment experience an interplay of multiple physical effects that lead to variants of physical properties of fluids moving in large channels. Microfluidics has been able to exploit differences existing between fluids flowing at the macroscale or travelling through microchannels, allowing one to perform techniques and experiments which are impossible at the macroscale . In the typical low Reynolds number regime of microfluidics, viscous forces overwhelm inertial forces and fluids adopt a laminar flow ,14. ThisPassive mixing principles are very attractive because they rely on diffusion or chaotic advection through specially-designed microchannels. They can also operated by avoiding the need of any external energy source. Active mixing schemes, on the other hand, improve the mixing performance by applying external forces to perturb sample flows and consequently to accelerate the diffusion processes. The latter configuration often leads to a complex device design .Enhanced sensitivity can sometimes be achieved by increasing the reaction time before the detection as concerned in the stopped-flow technology . Using aDifferent microfluidic-coupled PCR amplification approaches have been developed over the last few years ,21. ThesImprovement of the PCR efficiencies can be accomplished through passivation procedures ,29, eithThe introduction of a droplet-based technology provides a convenient way to prevent most of the above-mentioned issues since amplification reactions occur within droplets. Any interaction between the PCR sample and the surface of the microfluidic device is eliminated. Droplet-based microfluidics, unlike continuous flow systems, creates discrete fraction of water solutions by using immiscible fluids that are driven into separate microchannels via an independently controlled flow . Most ofThe ability to rapidly create highly uniform aqueous droplets with controlled contents enables the rapid analysis of very small quantities of reagents in a portable, automated and inexpensive format. Reagents are confined in droplets and each droplet is isolated from channel walls by the immiscible liquid. A similar configuration greatly reduce the reagent dispersion and sample contaminations thus offering fundamental advantages for successful single-molecule DNA amplification. Moreover, each droplet can be considered as an isolated microreactor and each microreactor can be potentially individually controlled and analyzed ,35 by alDroplets help in achieving a rapid mixing of reagents: the rate of DNA hybridization in a homogeneous solution is about 40-fold faster than the hybridization rate in solid-liquid interfaces such as those of microarrays. The typical experimental configuration of devices performing droplet microfluidic PCR amplification includes an oil stream that cyclically drives droplets through alternating temperature zones for denaturation and annealing thus obtaining a rapid and efficient PCR thermocycling . The impA number of different isothermal amplification procedures have been introduced over the last 10 years. These include nucleic acid sequence-based amplification (NASBA), loop-mediated amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA) . IsotherMany miniaturized isothermal amplification systems exploit the strand-displacement activity of a DNA polymerase to cyclically amplify a target in less than an hour. Reactions take place in a microchamber or in a microchannel; reservoirs can be included to inject reagents necessary for reactions and to mix them with the sample. Primers, enzymes and reagents can also be stored in the chip and, in order to obtain multiplex analyses, different primers for several targets can be employed.et al. in 2000 [Loop-mediated isothermal amplification (LAMP), described for the first time by Notomi  in 2000 , utilize in 2000 , severe  in 2000 , Staphyl in 2000 , Salmone in 2000 .LAMP reaction is initiated by a forward inner primer (FIP), containing sequences of the sense strand of the target DNA, which hybridizes to F2c in the target and initiates complementary strand synthesis . Then, tThe subsequent cycles, comprising elongation and recycling steps, lead to final products constituted by a mixture of stem-loop DNAs having various stem lengths and cauliflower-like structures with multiple loops formed after the annealing between alternately inverted repeats of the target sequence in the same strand. It is to underline that only inner primers are used for strand displacement DNA synthesis during cycling reactions while all four primers are used during initial steps of the LAMP reaction. The initial use of four primers that enables the recognition of six distinct sequences, followed by the use of two primers, ensures the high selectivity for target amplification.The LAMP method does not require thermocycling since the amplification is performed at a constant temperature between 60 °C and 65 °C. Another important advantage of the LAMP method is its simplicity for detection of the amplification reaction. In fact, pyrophosphate byproducts are produced during the reaction. These byproducts form a white precipitate that increases the turbidity of the solution. The detection of amplification products can be obtained by using a variety of methods including electrophoretic , turbidiet al. have developed an integrated isothermal device for both amplification and real-time detection of hepatitis B virus (HBV) DNA via the LAMP amplification method [Although most analytical tools rely on bulk measurements of LAMP amplification, LAMP has also been down-scaled by integrating it in microfluidic devices. Lee n method . The devn method ,51. It set al. presented an on-chip LAMP carried out with a total reaction volume of 10 μL for visual determination by naked eye with SYBR Green I [et al., who integrated the LAMP method in a microfluidic chip called microLAMP (μLAMP) to quantitatively detect target nucleic acids [Zhang  Green I . The reaic acids . In partPotential for further miniaturization has been demonstrated by the successful analysis of single DNA templates encapsulated in a polyacrylamide (PAA) gel based microchamber . A microE. coli bacterial target without any pre-treatment.The electrochemical detection of microfluidic integrated LAMP amplicons has been applied for the detection and the quantification of Escherichia coli . Among mEscherichia coli in 1976 [Progresses in understanding properties of helicases, discovered in  in 1976 ,59, have in 1976 . It mimiA scheme of HDA mechanism is shown in vs. DNA) [Helicases are motor proteins that bind nucleic acids and move directionally along them. They separate dsDNA by using the energy generated by the nucleoside 5'-triphosphate (NTP) hydrolysis. Helicases can be classified according to their directionality , their source , their oligomeric or monomeric nature and their substrate specificity  ,63,64. Svs. DNA) . Conservvs. DNA) .Escherichia coli UvrD helicase (~82 kDa) [The first HDA system was developed by using ~82 kDa)  that can~82 kDa) . UvrD be~82 kDa) ,68.Bacillus stearothermophilus polymerase I at 60–65 °C [The limited capability of UvrD HDA system in efficiently amplifying long target sequences ,70 was i60–65 °C .N. gonorrhoeae and S. aureus directly on surface bound primers has been reported [Multiple reports on the use of HDA for the detection of bacterial and viral target DNA and RNA in standard microtube protocols have been described ,73. Recereported . It combreported .et al. [Ramalingam et al.  describeet al. .It has been reported that the speed and the robustness of HDA are highly dependent on the synchronization of helicase, single-stranded DNA binding protein and large fragment of Bst DNA polymerase . By incrThe integration in a microfluidic chip of isothermal amplification by HDA and micro solid phase extraction (μSPE) column for DNA isolation has been also described . In thisThe rolling circle amplification (RCA) method exploits the continuous amplification of a circular DNA template by a strand displacing DNA polymerase. The DNA polymerase displaces the synthesized strand and ‘rolls’ on with DNA synthesis. This method operates at a constant temperature and produces a long single-stranded DNA molecule with tandem repeats of the circular template . Both liEfficient amplification by RCA requires small and circular single-stranded DNAs acting as the template. Unfortunately, a large number of relevant diagnostic DNA targets are composed of double stranded linear DNA molecules thus making it difficult the amplification process to be established. For this reason, oligonucleotides called padlock probes have been designed . PadlockSample pre-treatments are required before the padlock probe circularization in order to allow the transformation of duplex genomic DNA into accessible single-stranded DNA targets. For this reason genomic DNA is first digested with restriction enzymes that produce small sized dsDNA, then subjected to degradation by 5'-3' exonucleolysis. Even mRNA can be amplified through the RCA approach but, in this case, an mRNA pre-treatment is performed which consists of cDNA synthesis by a reverse-transcription followed by mRNA degradation by RNase H digestion . In bothAlthough small circular DNA molecules are uncommon in higher organisms, they are widespread in several microorganisms such as viruses, bacteriophages and bacteria. RCA has also been used to amplify circular genomes of DNA viruses, to identify phage genomes carrying lethal mutations and bacteria by using primers that include the sequence of the target DNA ,86. The et al. introduced an integrated platform for RCA and circle-to-circle amplification (C2CA) of padlock probes on a poly(methyl methacrylate) (PMMA) microchip [Mahmoudian icrochip . C2CA isicrochip . After eicrochip . In the in vitro translation (IVT) system supplemented with fluorescein-di-β-D-galactopyranoside (FDG) that is transformed into fluorescein by β-galactosidase.A droplet of microfluidics-based hyperbranched rolling circle amplification (HRCA) for single-molecule DNA amplification and analysis has been also described . The micet al. [Another RCA-based system that benefits from the typical low sample consumption and short processing time advantages of microfluidics is the rolling-circle enhanced enzyme activity detection (REEAD). REEAD was combined with a droplet microfluidic lab-on-a-chip platform by Juul et al. .The principle of REEAD approach is the circularization of RCA substrate by the Plasmodium enzyme topoisomerase I (pTopI) together with the conversion of single DNA cleavage-ligation events to micrometer-sized products detectable at the single-molecule level. The REEAD-on-a-chip procedure appears attractive since it allows the specific, quantitative and highly sensitive detection of the human-malaria-causing Plasmodium parasites in small volumes (pL droplets) of unprocessed and crude clinical samples with a detection limit of less than one parasite/μL. −6 mutations/nucleotide) [−4 mutations/nucleotide) [MDA is an isothermal method for whole genome amplification (WGA) capable of producing a large number of amplification products from a few DNA molecules . MDA expleotide)  in compaleotide) ,96. The leotide) . MDA does not require the temperature-modulated denaturation of the genomic DNA to facilitate the annealing of random primers. In fact, the random priming on the double-stranded DNA target is efficient enough to obtain an initial priming step that is followed by the strand-displacement process that produces the amplification. It has been also demonstrated that the omission of the denaturation step results in a reduced template degradation and in an improved specificity of the amplification . MDA hasIt is important to recognize that MDA tends to be a self-limiting reaction. The amplification yield is independent of the input amount of DNA template and reaches a plateau at a 0.7–1.0 µg/µL concentration of the amplified DNA .et al. in 2006 in combination with a novel probe-based detection approach [RPA is a low temperature (about 37 °C) isothermal amplification method that couples the isothermal recombinase-driven primer targeting of the template material with the strand-displacement DNA synthesis. The method, introduced by Piepenburg approach , amplifiRPA uses nucleoprotein complexes constituted by oligonucleotide primers and recombinase proteins to facilitate the primer binding to the template DNA. In particular, recombinase-primer complexes scan the double-stranded DNA by promoting the primer binding at the target sequence of dsDNA and the displacement of the non-template strand. The displaced strand is stabilized by ssDNA binding proteins while the recombinase disassembly leaves 3'-end of the primer accessible to DNA polymerase. An exponential amplification of the target sequence is obtained after the cyclic replication of the process.RPA isothermal amplification has been integrated in a centrifugal lab-on-a-chip device . The fluet al. developed a passive microfluidic batch mixing chip for RPA fabricated through a simple and low-cost method which combines dry film resist technology and direct wafer bonding [Hakenberg  bonding . In this bonding , fluoresDigital microfluidic isothermal amplification methods that take place at temperatures close to the room temperature are convenient because the polymerization reaction is chemistry rather that temperature actuated. However, these methods suffer from the unwanted characteristic that the amplification reaction is expected to proceed at room temperature if the nucleic acid sample is premixed with initiation reagents prior to compartmentalization. Conversely, in digital PCR the amplification reaction is controlled by temperature and any low-temperature non-specific pre-amplification process can be eliminated by using specific procedures . As a coRPA has been implemented in a droplet microfluidic chip for the simultaneous chemical initiation of over one thousand sequence-specific RPA in parallel . The ampNASBA is an isothermal, transcription-based amplification method specifically designed for the amplification of single-stranded RNA or DNA sequence. It was first introduced by Compton in 1991  and is pi.e., avian myeloblastosis virus reverse transcriptase, T7 DNA-dependent RNA polymerase (DdRp) and RNase H). NASBA utilizes two RNA target-specific primers and three enzymes   which aret al. [Real-time NASBA technology has been integrated in a microfluidic system by Gulliksen et al. . In thisAn improvement in the real-time NASBA integration in microfluidic environment was introduced by detecting artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples in cyclic olefin copolymer (COC) microchips . In thiset al. presented the development of a “proof of principle” self-contained and hands-free diagnostic platform for NASBA which performs sample preconcentration, nucleic acid extraction, amplification and real-time fluorescent detection with minimal user interfacing [More recently Gulliksen erfacing . The auterfacing . The micet al. [A lab-on-a-chip (LOC) device based on the real-time immuno nucleic acid sequence-based amplification (immuno-NASBA) for quantitative detection of waterborne pathogens has been recently designed by Zhao et al. . The immIsothermal DNA amplification is rapid, cost effective, easy-to-use and more tolerant to inhibitory components from a crude sample compared with PCR, which greatly simplifies point-of-care diagnosis, but at the same time showing equivalent or higher sensitivity and reliability in clinical diagnosis. The use of fully enclosed microstructured devices into which performing the isothermal amplification reduces the risk of sample contamination and allows integration and portable device realization. Furthermore, the implementation of isothermal DNA amplification in microfluidic systems has the added benefit of reduced reaction volumes that result in a decreased reagent and sample consumption. The possibility of lowering sample requirement constitutes an important usefulness, especially when limited template material is available. Reactions take place in a microchamber or in a microchannel; reservoirs can be included to inject reagents necessary for reactions and to mix them with the sample. Primers, enzymes and reagents can also be stored in the chip and, in order to obtain multiplex DNA analyses, different primers for several targets can be employed.i.e., LAMP and HDA). The described approaches differ from each other in terms of operating temperature, diagnostic application, mechanism, strengths and weaknesses. From this perspective, it is worthy of noting that NASBA, RCA, and RPA reactions allow nucleic acid amplification at low temperature, thus requiring less energy to operate than higher temperature isothermal amplification  to cyclically amplify a target in less than an hour. Target application can vary from single stranded DNA or RNA molecules to double stranded DNA samples. DNA is often preferred, where appropriate, because it is more stable than RNA and will be likely associated with a higher sensitivity in samples stored or transported in suboptimal conditions.A summary of isothermal nucleic acid amplification methods here described is provided in It should be noted that the choice of the method is strictly related to the target. NASBA for example is especially suited for RNAs such as mRNA, rRNA, tmRNA or genomic RNA, while it cannot amplify double-stranded DNAs unless subjected to a denaturation step. On the other hand, NASBA is not free from drawbacks, since it is prone to ribonuclease (RNase) contamination and non-specific adsorption that can affect the amplification signal and the signal curve of NASBA assay.Contrary to NASBA, LAMP enables duplex DNA amplification because, in the condition of dynamic equilibrium at 65 °C, one of the LAMP primers can anneal to the complimentary sequence of double-stranded DNA without any initial denaturation step. LAMP can also be applied to the detection of RNA with the addition of reverse transcriptase. Another important advantage of the LAMP method is its simplicity for detection of the amplification reaction, due to the insoluble pyrophosphate byproduct that can be easily quantified turbimetrically.In a different manner, the HDA reaction exploits the activity of a DNA helicase in combination with other accessory proteins to separate complementary strands of ds DNAs, thus mimicking the denaturation mechanism in living organisms. However HDA suffers from the limited speed and processivity of UvrD helicase that is able to amplify only short DNA sequences (from 70 to 120 bp). As an attempt to circumvent this drawback, the limited capability of UvrD HDA system in efficiently amplifying long target sequences was improved by using a thermostable UvrD helicase (Tte-UvrD).The target applications of the RCA method are wide, ranging from small and circular single-stranded DNAs to double stranded linear DNA molecules and even mRNA. Sample pre-treatments are required before padlock probe circularization in order to allow the transformation of duplex genomic DNA into accessible single-stranded DNA targets; this additional heating step is responsible for increased power demand and more complicated control mechanisms. In mRNA detection, instead, the pre-treatment step consists of cDNA synthesis by a reverse-transcription followed by mRNA degradation by RNase H digestion. On the other hand, ss the circularizing step of a padlock probe is strictly target-dependent, high enough specificity is ensured to allow SNP analysis. Another relevant advantage of RCA is its capability to detect miRNA samples, which arises from its inherent nature for specific detection of short sequences. In RPA, an initial denaturation step is not required thanks to the activity of recombinase-primer complexes that scan the double-stranded DNA by promoting the primer binding at the target sequence and the displacement of the non-template strand. The RPA reaction has the ability to proceed at a variety of temperatures since it is tolerant to fluctuations of the incubation temperature ranging from 37 °C to 42 °C. This remarkable property of RPA is of great appeal for field applications where precise temperature control is often technically challenging.MDA exploits random exonuclease-resistant primers and the activity of φ29 DNA polymerase, already used in RCA, but to amplify linear genomic DNA rather than for circular DNA amplification. The random priming on the double-stranded DNA target is efficient enough to obtain an initial priming step that is followed by the strand-displacement process. The MDA method is capable of producing a large number of amplification products from a few DNA molecules regardless of the type of clinical sample used including buccal swabs, whole blood, finger stick blood and Guthrie cards. In addition, the use of a high-fidelity proofreading DNA polymerase ensures a significantly lower error rate during the amplification reaction and preserves DNA sequence information, an advantage to consider when an accurate genotyping is required as in whole genome amplification (WGA).The microfluidic lab-on-a-chip technology for the amplification of nucleic acids offers great potential for lower cost, higher speed, smaller sample consumption and automation of all processes from sample preparation to signal detection. The integration of multiple functional fluidic modules enables the analysis of smaller sample volumes with increased reproducibility, faster analysis and more accurate quantification.In this review, the use of microfluidic-based methods for isothermal nucleic acid amplification has been described. In particular, the integration in microfluidic device of NASBA, LAMP, HDA, RCA, MDA and RPA isothermal amplification methods have been discussed. Isothermal reactions allow nucleic acid amplification at both constant and low temperature. Although PCR is the most widespread technology for DNA amplification, the need for an electrically powered thermal cycler with a precise temperature control and for an optimized experimental setup makes the use of PCR at point-of-care settings more complex and increases the cost of PCR-based devices. Conversely, isothermal amplification processes offer multiple advantages if compared with thermal cycling requiring methods, such as reduced needs for instrumental complexity, thermal control and power consumption, thus rendering their integration in microfluidic platforms significantly easier. In addition, the use of isothermal amplification techniques coupled with miniaturized devices for analysis ensure a higher percentage of specific amplification product obtained from the targeted DNA template and reduced amplification bias, enabling also accurate and high fidelity single-cell genome amplification in nanoliter volumes.


Previous studies have shown different clinical and imaging pattern in tremordominant and akinetic-rigid Parkinson’s disease (PD) subtypes. The association between dopaminergic and glucose metabolism has in contrast not been investigated yet. Therefore, this study compared PD subtypes with respect to clinical and imaging findings with the aim of establishing a relationship between clinical subtypes, dopamine and glucose metabolism.18F]-fluoro-L-dopa (F-dopa) and [18F]-fluorodeoxyglucose (FDG)-PET.Two groups of a total of 64 idiopathic PD patients  were analysed: akinetic-rigid  and tremor-dominant  patients. Both were compared with respect to differential involvement of local striatal dopamine and glucose metabolism using [The analysis of PD subgroups showed significant differences in the F-dopa uptake in the anterior putamen. Using the results of the local striatal dopamine difference as a volume of interest for the FDG-analysis, analysis of AR patients revealed a significantly lower normalised cerebral metabolic rate of glucose (nCMRGlc) within the ventral striatum.The dual tracer study illlustrates clear differences between TD and AR subtypes in the ventral striatum. In accordance with previous FP-CIT-SPECT studies, it discloses congruent results for the presynaptic dopaminergic system and extends the knowledge about an additional involvement of local metabolic activity in the caudate and anterior putamen. The findings corroborate the specific role of distinct PD subtypes within the cerebello-thalamo-cortical-circuits. Multitracer PET imaging may thus enhance the knowledge about the clinical segregation into subtypes. Parkinson’s disease (PD) patients differ concerning the clinical progress, symptom severity and symptom onset Previously, our group demonstrated distinct differences in the dopaminergic system in classical PD subtypes both at baseline In this context and regarding the glucose metabolism, the group of Eidelberg developed a distinct pattern for tremor and rigidity in PD, the Parkinson’s Diseases Related Pattern (PDRP) 18F]-fluorodeoxyglucose (FDG) and [18F]-fluoro-L-dopa (F-dopa) PET scans in identical patients with specific subtypes of PD. We hypothesized subtype-specific binding patterns with reduced F-dopa and FDG uptake particularly in akinetic-rigid patients. The multimodal aspect of this study considered the local uptake of F-dopa and FDG in the same striatal regions identified as subtype-specific key structures.Nevertheless, to our knowledge, little is known about the relationship between local dopaminergic uptake and the local neuronal function represented through the glucose metabolism in identical regions for specific PD subtypes. In order to refine the knowledge and the association between dopaminergic and glucose metabolism and about specific PD forms, we used -fluorodeoxyglucose (FDG): After the injection of 370 MBq of FDG, cerebral glucose metabolism was measured representing the regional metabolic activity. Arterialized venous blood sampling allowed absolute quantification for all subjects.[18F]-fluoro-L-dopa (F-dopa): To avoid peripheral decarboxylation of F-dopa, 100 mg carbidopa was administered orally to the patients before the intravenous injection of 370 MBq F-dopa. Patients were scanned during 90 minutes, recording a dynamic series of nine 10-minute frames in a 3-dimensional mode.[Due to the applied techniques of using quantified FDG-PET data and semi-quantified, dynamic F-dopa PET-scans, this dataset achieves a high standard of data acquisition in order to reveal in-vivo processes.To define local differences of dopaminergic uptake between subgroups, we used a voxel-based approach (SPM). The consecutive regional difference between these subtypes was used as a three dimensional volume of interest (VOI) to identify the local glucose metabolism in the respective individual FDG-PET scans. For an overview of the methodology see To compare the PET-scans with differing sizes and proportions in voxel-wise statistics, scans were preprocessed in SPM8 . Images were smoothed by a 10 mm full width at half maximum (FWHM) Gaussian filter and subsequently spatially normalized to a standard stereotactic space using an in-house created FP-CIT-template for the F-dopa scans ” function in SPM (see www.nf.mpg.de/vinci3). This produced individual values for the rCMRGlc in this specific volume  was calculated as the averaged voxel activity in an elliptic region of interest encompassing all visible brain structures on a transaxial PET image plane transecting the striatum. The gCMRGlc between subgroups was compared using an independent samples t-test as distribution was parametric . In order to reveal the regional CMRGlc (rCMRGlc) in the region for the dopaminergic difference between subtypes, the three-dimensional SPM mask was used on all individual normalized FDG-scans in an in-house created imaging tool  of the patients were divided by the occipital uptake values and “1” was subtracted. This resulted in striatal-to-occiptial-ratios (SOR) -1 values for each subject. Due to a extremely low extrastriatal signal in KOverall, 32 pairs of matched AR patients  and TD patients  were identified. Patients of both subtypes were comparable regarding age, age at onset, Hoehn & Yahr stage, disease duration, LEDD and gender distribution (p>0.05 Wilcoxon-Mann-Whitney). Patients were matched as one aim of this study was to present different pathophysiologic aspects of PD subtypes. Therefore, it was from our point of view crucial to match patients which are per se comparable. This means to compare patients of the same disease duration, disease severity or LEDD. As far as are aware, there is no scaling factor or a non-linear approach to compare the progress of different PD-subtypes. Thus, we think that our approach of matching subtypes for multiple factors gives the best opportunity to relate these diverse disease entities. The clinical details are summarized in As expected, the SPM analysis for the F-dopa-PET using an independent samples t-test revealed a significant difference  in the caudate and anterior putamen indicating a reduced dopaminergic uptake for AR compared to TD patients see  top.The global FDG-metabolism was not significantly different between subgroups . The nCMRGlc in the striatal volume of interest (VOI) using the SPM mask for the dopaminergic subtype-difference showed a statistically significant difference (p = 0.013) between AR (1.1359 µmol/100 g/min) and TD patients (1.1802 µmol/100 g/min) see  bottom, r = 0.439, p = 0.005 for the overall group). This relationship was more pronounced for the AR patients, indicating a lower dopaminergic uptake is going along with a reduced glucose metabolism in this subtype of PD patients  neuron loss in the substantia nigra pars compacta. This results in DA denervation of the striatum, which in turn induces a demodulation of complex motor circuits comprising the motor and premotor cortices, the putamen, the globus pallidum, the subthalamic nucleus, and the thalamus Structural changes with respect of white matter alterations in MRI scans offer conflicting results, suggesting no relevant overall impact of white matter hyperintensities e.g. . Yet, ceGenerally speaking, there is some evidence that PD patients show elevated levels of glucose metabolism in striatal regions compared to healthy controls as repeatedly demonstrated by different PET studies using partly no absolute quantification The increase in striatopallidal synaptic terminal activity was suspected as a result of nigrostriatal DA denervation Considering metabolic substrates of bradykinesia and tremor, Lozza et al. reported in a FDG-PET study the severity of bradykinesia being correlated with higher CMRGlc in the putamen and globus pallidus whereas tremor scores related to lower CMRGlc in the putamen and cerebellar vermis. Both regions showed an extensive zone of overlap in the putamen bilaterally. This overlap was computed for each clinical variable independently and did not reflect colinearity.The prominent circumscribed reduced metabolism in AR patients was found in the ventral striatum, namely the caudate and the anterior putamen. The anterior putamen was already defined as a subtype-discriminating region in an earlier FP-CIT study of our own group The main strength of this manuscript is the dual tracer approach in a large group of PD patients using quantified & dynamic sets of PET-scans allowing a distinct differentiation of individual glucose/dopaminergic uptakes. The large number of individually characterized patients gives the unique opportunity to detect subtype-specific differences on different pathophysiological levels (glucose and presynaptic dopaminergic metabolism).Previous dual tracer studies employed the combination of dopaminergic and metabolic imaging Beside the weaknesses of a retrospective analysis, the missing UPDRS part III is a main flaw of this study. However, the rigorous exclusion of patients in any doubt of correct assignment to one subtype reduces the risk of an incorrect classification. More than 70% of the patients were excluded due to this procedure. We are rather confident that both the strong experience in this movement disorders center and the strict inclusion criteria based on the charts – as described above - supports an assignment of patients to the correct subtype. However, the lack of UPDRS subscores comprises the risk of an inaccurate classification of patients. As the results for the dopaminergic metabolism are in line with previous findings We had no structural data (e.g. MRI scans) available in these patients. Thus, we could not compare the regional hyper- or hypometabolism with structural gray matter atrophy or white matter hyperintensities as shown before The present findings indicate a different involvement of both presynaptic dopaminergic uptake and glucose metabolism in the ventral striatum for AR and TD subtypes of PD. These results suggest a differential involvement within distinct regions of the basalganglia-cerebello-thalamocortical circuit in these subgroups. Future research is needed to clarify these observations and achieve the longitudinal progress of subgroups.File S1Methodological considerations.(DOCX)Click here for additional data file.


Bone is the most common site for metastasis in human prostate cancer patients. Skeletal metastases are a significant cause of morbidity and mortality and overall greatly affect the quality of life of prostate cancer patients. Despite advances in our understanding of the biology of primary prostate tumours, our knowledge of how and why secondary tumours derived from prostate cancer cells preferentially localise in bone remains limited. Examining the impact of these facets of bone metastasis in vivo remains a significant challenge, as animal models that closely mimic the natural history and malignant progression of clinical prostate cancer are not available.To develop an animal model of human metastatic prostate cancer. Once a model has been developed and optimised, it was this to test efficacy of immunotherapy using T-cells that have been genetically targeted against prostate-specific membrane antigen (PSMA).vivo bioluminescent imaging (BLI) was used to detect metastases.Using PCR, western blot, flow cytometry and ELISA, we performed functional anaylsis of fucosyltransferase 3(FT3) in PC3LN3(PL)and PC3LN3-PSMA (PLP) tumour cell lines. In in vitro. Bioluminescent animal model of metastasised prostate cancer is established to determine the effect of this upon their pattern of metastatic spread in SCID Beige mice.In preliminary studies, we have observed that delivery of a FT3-encoding retroviral vector to PL and PLP enables them to express sialyl Lewis X and to acquire E-selectin binding activity. We also showed that FT3 promotes increased PLP motility and invasiveness in-vivo model of PSMA-expressing prostate cancer. This will serve as a platform to test immunotherapy using P28z+ T-cells.We have established an


MIR10B gene sequence and about 1 kb upstream of the HOXD4 gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the  Atherosclerosis underlies the development of a majority of cardiovascular diseases, which are among the leading causes of mortality worldwide. Different vascular beds vary in their susceptibility to the development of atherosclerosis. Coronary artery has the highest prevalence of atherosclerotic plaques in comparison with other arteries. It has been established that internal mammary arteries as well as great saphenous veins are resistant to the development of atherosclerosis . HoweverAlthough atherosclerosis is caused by the interaction of multiple genetic and environmental factors, these explain only a portion of the total disease risk. Epigenetic mechanisms that underly this pathology have become a promising area of research , 3. CompThe most widely studied and best-characterized epigenetic marker in human genome is DNA methylation. DNA methylation in tissues usually occurs within the context of CpG-dinucleotide sequences (CpG-sites). In somatic mammalian cells, the majority of CpG-sites are methylated. However, CpG-sites located in regions of increased CG-density, known as CpG-islands, generally have low levels of methylation. DNA methylation at gene promoters is important for transcriptional regulation, with dense promoter hypermethylation around the transcription start sites being associated with repressed expression of genes. Outside of CpG-islands, intragenic DNA methylation has been linked to transcriptional and splicing activities .ALOX15), estrogen receptors (ESR1 and ESR2), monocarboxylate transporter 3 (MCT3), and tissue factor pathway inhibitor 2 (TFPI2) genes have been reported using a candidate gene approach - CACTTTAATCTCTAACTATTCC-3’) primers in 50 μl reaction volume including 0.2 mM dNTPs and 2 mM MgClThe results presented in our study were obtained using the log2 ratio between unmethylated and methylated probes  and the beta-statistics [methylated/(unmethylated + methylated)]. Illumina’s GenomeStudio software was used to analyze BeadArray data to assign site-specific DNA methylation beta-values to each CpG-site. The beta-value represents a quantitative measure of the DNA methylation level of specific CpG-sites and ranges from 0 (completely unmethylated) to 1 (completely methylated).http://www.R-project.org) and analyzed using Bioconductor lumi package [The raw data were imported into R environment for statistical computing v3.0.1 . Multi-dimensional scaling was used to display the distance between individual samples based on their methylation status. All CpG-sites were used for scaling.limma package [Differentially methylated CpG-sites  between groups were identified using  package . BenjamiFunctional enrichment analysis of genes containing differentially methylated CpG-sites was performed using the web-based GEne SeT AnaLysis Toolkit . The refStatistically significant differences in DNA methylation level between groups were identified by ANOVA with post-hoc test. An enrichment of hyper- and hypomethylated CpG-sites versus all CpG-sites was calculated using Fisher’s exact test. Correlations between variables were estimated using Pearson’s correlation coefficient. The significance level of p<0.05 was applied. These analyses were carried out using GraphPad software (InStat).The pipeline of the experiment is given in The analysis of DNA methylation profiles in four matched sets of tissues from 6 individuals was performed using Infinium HumanMethylation27 BeadChip assay (Illumina), which contains 27578 CpG targets covering 14495 genes. After quality control, methylation of 27378 CpG-sites (14391 genes) was analyzed.DNA methylation age per tissue was moderately correlated with corresponding chronological age . However, GSV showed evidence of significantly higher mean DNA methylation age than IMA   and 8 hypomethylated  genes were identified as significantly highly differentially methylated genes in the GSV .HOXD4 promoter region were also confirmed as hypomethylated in the independent dataset of CAP in comparison with IMA and GSV.Methylation levels in CAP tissues were significantly different from the average methylation of other tissues . Four CpAberrant DNA methylation has been recognized as an important factor underlying the development and progression of in various common diseases. A majority of studies conducted so far have focused on cancer and only a few have investigated the role of epigenetic mechanisms, such as DNA methylation, in the development of cardiovascular disease . To the We analyzed quantitative single-base resolution DNA methylation patterns on a genome-wide level in vascular tissues obtained from patients with atherosclerosis. We showed that DNA methylation age of vascular tissues was moderately related to chronological age. The great saphenous veins had increased DNA methylation age in comparison to the internal mammary arteries. It has been established elsewhere, that GSV is a more differentiated tissue with lower rates of cell proliferation and apoptosis as compared to IMA . It is pWe observed a high heterogeneity of DNA methylation profiles of the CAP tissues. A plaque constitutes many cell types and other components and, therefore, our finding was not surprising. The average methylation level for all the CpG-sites as well as inside the CpG-islands was significantly higher in the CAP tissues, mainly due to lower frequency of the hypomethylated CpG-sites, as compared with the IMA. Our results contradict to those published by Castillo-Diaz et al. , who repThere is a body of evidences of a fundamental role of inflammation in the development and progression of atherosclerotic plaque . To dateIntriguingly, a high percentage of differentially methylated genes between vascular tissues related to regulation of developmental process were revealed. One possible interpretation of our findings is that these genes are involved in vascular tissue remodeling. Reactivation of the developmental pathways is also needed during organ injury repair. Possibly, these genes are upstream effectors that modulate other pathways that are involved in atherosclerosis. It has been identified that the most highly variably DNA-methylated regions in several tissues were significantly enriched by the development-related genes . AccordiHOX cluster . Differential methylation region comparisons between athero-susceptible and protected sites in swine arterial endothelial methylome also revealed a sharp distinction in the HOX locus [We also identified site-specific DNA methylation changes in comparison between the CAP and IMA as well as GSV and IMA tissues. In our study the majority of differentially methylated CpG-sites were located in developmental genes, including OX locus . AberranOX locus , thus suHOXD4 gene was found to be associated with differentially methylated CpG-island both in the study of Castillo-Diaz et al. [HOXD4 promoter region in CAP tissues as compared to IMA and GSV. This result may be partly attributable to differences in the microarray platforms and the study subjects.The z et al.  and in oz et al.  found thHOXD4 in atherosclerosis remains unclear, a previous study identified differential expression of this gene in the samples of human aorta with varying degrees of atherosclerosis [MIR10B gene sequence. MiR-10b is a microRNA associated with metastasis and/or invasiveness of various cancer types [MIR10B gene sequence in atherosclerosis.Although the functional significance of clerosis . On the er types . It was er types . This mier types . FurtherHoxa2 and Hoxa5 have been cloned from an adult rat vascular smooth muscle cDNA library, although the functions of these proteins were not determined in this study [HoxA5 is regulated by flow in a DNA methylation-dependent manner and that Hoxa5 regulates endothelial inflammation [HoxA5 and downregulation of gene expression in arterial endothelium [HOXA5 is hypermethylated in great saphenous veins as compared with the internal mammary arteries that may contribute to aberrant remodeling of veins after coronary artery bypass grafting surgery.is study . Hoxa5 iis study . Recentlammation . Inductiothelium . Our stuS100A10 gene was hypomethylated in right coronary artery in the area of advanced atherosclerotic plaques compared to atherosclerotic-resistant internal mammary arteries. S100A10 is highly expressed in endothelial cells, macrophages and foam cells of complicated carotid plaque segments [2+-binding protein involved in the plasmin/plasminogen system regulating proteolytic activity and degradation of extracellular matrix, angiogenesis and macrophage invasion. Second, we observed changes in the methylation level of GLRX gene in both comparisons CAP vs. IMA and GSV vs. IMA. Expression of GLRX gene is enhanced in endothelial cells, smooth muscle cells and macrophages of human nonatherosclerotic and atherosclerotic coronary arteries [Some of the highly differentially methylated genes are also very interesting. First, in our study segments . This Caarteries . Glutarearteries  glutaredALOX15, ESR1, ESR2, MCT3 and TFPI2 genes in patients with atherosclerosis [Previous studies of vascular tissue samples revealed alterations in methylation of clerosis . These gF13A1 and CD163) were found in common with our list of differentially methylation genes.We have not tested functional implications of changes in DNA methylation, although this is a subject of pivotal importance for the future studies. It should be noted, however, that Archacki et al.  identifiA limitation of our study is an inability to attribute the methylation patterns to specific cell types. The methylation patterns identified, consequently, may represent the gene set of methylation effect contributed from the different cells. Therefore differences in DNA methylation can result from changes in the proportions of cell types in atherosclerosis-affected and healthy vascular tissue samples. To assess cell type-specific DNA methylation of complex tissues, laser capture microdissection must be used. DNA yields from such samples, however, are too small to use with the current methods of microarray-based genome-wide DNA methylation analysis. In our approach, since the tissues were taken directly from patients with advance disease, the data can be biased due to possible effects of unrecognized factors and drugs used for the treatment of the patients. Unbiased results for vascular tissues can be achieved by sampling prior to the development of the disease, but this is not feasible in humans. The major strength of our study is the use of quantitative methods for the analysis of DNA methylation at single-base resolution. We also studied matched vascular tissues in order to avoid the effect of interindividual variation of DNA methylation patterns. Chen et al.  describeMIR10B gene sequence and about 1 kb upstream of the HOXD4 gene in right coronary artery in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. Further studies need to be carried out to investigate the genes and biological pathways that are involved in the development of atherosclerosis and may serve as targets for prevention and treating vascular diseases.We performed a genome-wide DNA methylation profiling study on the right coronary arteries compared with internal mammary arteries and great saphenous veins from the same patients with coronary heart disease. Modest, but consistent and significant, DNA methylation changes in vascular tissues indicate the importance of epigenetic mechanisms in atherosclerosis. We revealed that a high percentage of differentially methylated genes between vascular tissues related to the regulation of the developmental process. This study identified hypomethylation of four CpG-sites located within the S1 TableFDR-adjusted p<0.05 and delta beta ≥0.2 or delta beta ≤-0.2.(DOCX)Click here for additional data file.S2 TableThe results for each enriched GO category  are listed in the table. C—number of reference genes in the category; O—number of genes in the gene set and also in the category, E—expected number in the category, R—Ratio of enrichment, rawP—p value from hypergeometric test, and adjP—p value adjusted by the multiple test adjustment.(DOCX)Click here for additional data file.S3 TableFDR-adjusted p<0.05 and delta beta ≥0.2 or delta beta ≤-0.2.(DOCX)Click here for additional data file.S4 TableThe results for each enriched GO category  are listed in the table. C—number of reference genes in the category; O—number of genes in the gene set and also in the category, E—expected number in the category, R—Ratio of enrichment, rawP—p value from hypergeometric test, and adjP—p value adjusted by the multiple test adjustment.(DOCX)Click here for additional data file.


Ulcerative Colitis (UC) is a type of inflammatory bowel disease, considered as an important disease of gastrointestinal tract having a huge impact on the health of the patient. Prolonged inflammation of colon in UC patients increases the risk of developing colorectal cancer. MiRNA are reported as a connecting link between inflammation and cancer. Differential miRNA expression is reported in Crohn’s disease (CD) patients involving various regions of the gastrointestinal tract. The current study was performed to dissect out the site specific miRNA expression in the colon biopsy samples of UC patients from Northern India.Biopsy samples were collected from UC patients and healthy controls from Rectosigmoid Area (RS) and Ascending Colon (AC). MiRNA expression was compared between patients with RS and AC using a microarray platform. Differential expression was further validated by Real Time PCR analysis. Demographic and pathological data of UC -associated CRC patients was collected from the hospital database and analyzed for assessing the site of cancer.Upon analysis of data generated on a microarray platform and qRT PCR revealed that the expression of six miRNAs hsa-miR-146b-5p, hsa-miR-335-3p, hsa-miR-342-3p, hsa-miR-644b-3p, hsa-miR-491-3p, hsa-miR-4732-3p were downregulated in patients where RS was involved as compared to AC. The expression of hsa-miR-141-3p was upregulated in patients where RS region was involved as compared to AC. Analysis of the registered UC patient’s database from the hospital revealed that the site of CRC was predomimnantly the rectosigmoid region of the colon in most of the cases.This is the first study to show the differential expression of miRNA involving different sites of colon in UC patients. Taking our data and previous reports into consideration, we propose that differential miRNA expression during UC perhaps contribute in the development of UC-associated CRC at the rectosigmoid area. Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by severe inflammation of the small bowel and/or the colon leading to recurrent diarrhea and abdominal pain. Crohn`s Disease (CD) and Ulcerative Colitis are two major forms of IBD . ChronicMicroRNAs (miRNA) are small endogenous RNA molecules approximately 22 nucleotides long. miRNA can interact with mRNA by binding to 3`UTR and can regulate gene expression at post transcriptional level. It can regulate gene expression either by translational repression or mRNA degradation –8. Each MicroRNAs are reported as a link between chronic inflammation and cancer in various inflammatory diseases , 6. CurrBiopsy samples from UC patients from ascending colon and from recto-sigmoid area were collected to evaluate the differential miRNA expression. Microarray analysis was done for evaluating miRNA expression and validated the results using qRT PCR. We found altered expression of some important miRNA which are already reported in various inflammatory diseases and cancer and few which are not reported so far. A retrospective study was performed on the patient’s history from the hospital registry of UC-associated CRC patients for analyzing the site of carcinoma. Our data shows that differences observed in specific miRNA expression could be one of the factors leading to increased risk of CRC at rectosigmoidal area.This study included 30 UC patients and 20 non-IBD controls. Colonic pinch biopsies from the ascending colon (AC) and rectosigmoid (RS) area of the colon were collected from UC and control individuals. Out of 30 UC patients, 20 colonic pinch biopsy samples were collected from both ascending colon and Rectosigmoid area of Pancolitis patients. Ten biopsy samples from patients were collected only from rectosigmoid area of Left sided colitis patients. Demographic features of participants are given in We collected details of UC associated CRC patients from the patient’s registry maintained by the Gastroenterology department of All India Institute of Medical Sciences (AIIMS), New Delhi, India. Demographic and pathobiological features were analyzed to assess the site of CRC in patients who are suffering from UC.Total RNA was isolated using mirVana miRNA isolation kit . RNA was stored at -80°C until further analysis. Quality of RNA was analysed by running on 1.5% agarose gel for three bands of 28SrRNA, 18SrRNA and 5SrRNA. Bioanalyser  was used to calculate the RIN value for RNA. Samples with RIN value more than 7 were included for microarray analysis.™ microRNA Array (7th gen). The array data has been submitted in NCBI Gene Expression Omnibus (GEO) under the entry series GSE66932. 1.7 fold changes were used as cut off for upregulation and 0.6 fold for downregulation of miRNA expression change.MiRNA microarray was performed by Exiqon A/S, Denmark. Samples were divided in four categories: UC patients Ascending colon and rectosigmoid area and biopsy samples from controlsfrom the corresponding area. Five RNA samples were pooled in each category in equal concentration . RNA samR Green universal PCR Master Mix , 1 μl of each forward and reverse primer (4 picomole/μl), 1 ul cDNA. Relative expression of a miRNAs was calculated using Ct values.Reverse transcription of miRNA was done using gene specific looped primers  by reverThe relative expression was calculated using the equation 2-ΔΔCT where ΔΔCT = Δ CT target miRNA- ΔCT U6. Statistical analysis for qRTPCR was done using unpaired, two way student`s t-test and a probability level of p < 0.05 was considered statistically significant.The site of CRC development in UC is rectosigmoid region in most of the cases. Given the critical role of miRNAs in IBD and its association with CRC incidence, we performed microarray analysis on pinch biopsies from patients where either ascending colon or rectosigmoid area was involved. Here we focused on miRNAs that were showing region specific differences in UC only. Microarray data was validated by qRTPCR in biopsy samples (20 from ascending colon and 30 from rectosigmoid area) of UC patients and 20 samples from non IBD controls. Expression analysis results using qRTPCR showed a similar pattern as observed in microarray analysis with variation in fold changes . Hsa-miRAs per the hospital record, during last 10 years total 20 UC patients were reported to have developed UC associated CRC. We observed that in 15 patients (75%) of the cases, the site of CRC was rectosigmoid area (60% in the rectum and 15% in the sigmoid colon) . There wUC patients are considered to be at increased risk of developing CRC. The prevalence of CRC in UC patients in the Asia-Pacific region has been estimated to range from 0.3%-1.8% . FurtherSite of CRC in UC patients is reported to be the RS area in most of the UC-associated CRC patients. As per the hospital record of the UC patients, we observed that the site of colorectal cancer in 73.68% of UC associated CRC patients was in RS area of colon where none was recorded from ascending colon region of the affected individual. This supported earlier observations seen in IBD patients turning into CRC .Alteration in miRNA expression has been reported in UC compared to healthy controls. Feng Wu et al, 2008 reported differential expression of 11 miRNAs in UC compared to healthy controls . Ueda etUsing microarray analysis and qRTPCR, our results showed that seven miRNA were differentially expressed in active UC patients involving either rectosigmoid area or ascending colon. Our data further confirmed that except hsa-miR-141-3p, six other miRNA were downregulated in patients with rectosigmoid area compared to ascending colon.Among other miRNA that were downregulated in UC patients involving RS, one important miRNA was miR-146b-5p. MiR-146b has been reported to play important role in linking inflammation and cancer. Tumor suppressive role of this miRNA in carcinogenesis has been confirmed through a study conducted by Kanan et al 2012) while tracking the changes in expression of miRNA in CD patients from non neoplastic tissue to dysplasia and eventually to CRC where the expression miR-146 significantly decreased during transition to CRC . It has 12 while miR-335 has been reported to play role in metabolic pathway shared by UC and CRC. Inflammatory cytokines serve as important mediators of continuous crosstalk between the immune system and tumor cells and function at multistage of inflammation- associated tumor process. MiR-335 is known to mediate crosstalk between cytokine-cytokine receptor interaction and chemokine signaling pathway through the regulation of chemokine families . It actsDecreased expression of miR-342-3p observed by us in UC patients involving rectosigmoid area supports the study carried out in an integrated miRNA and mRNA expression profiling of inflammatory breast cancer sub type . MiR-342High levels of miR-644b-3p were found to be associated with shorter overall survival of acute myeloid leukemia (AML) . HoweverIncreased expression of miR-141-3p observed in microarray data was validated by qRTPCR in biopsy samples of individual UC patients with RS was compared with AC. We found eight fold increased expression of only one miRNA, miR-141-3p in tumor prone RS area of colon. This context dependent expression pattern observed in the present study supported earlier study carried out in tumour samples of stage III CRC patients where increased expression was observed . Cheng eMiRNA are reported as an important link between inflammatory diseases and cancer . In thisBased on our data and the important role of dysregulated miRNAs in inflammation and the development of cancer, we propose that site -specific change in miRNA expression may be one of the contributing factor for increased incidence of UC associated CRC at RS region of colon.Our study indicated that site specific miRNA expression may be one of the contributors in CRC development in rectosigmoid colon in UC patients. Based on our analysis we propose that increase in expression of tumor promoting miRNA and decrease in expression of tumor suppressive miRNA may be one of the reasons for increased occurrence of CRC in the rectosigmoid area during ulcerative colitis . FunctioS1 FigRelative miRNA expression in Rectosigmoid region compared to Ascending colon of UC patients by microarray analysis.(TIF)Click here for additional data file.


Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs) to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1) BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2) An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3) Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways. Bacterial secondary metabolites mediate a broad range of microbe-microbe and microbe-host interactions, and are widely used in human medicine, agriculture and manufacturing. Despite recent advances in synthetic biology, efforts to engineer their biosynthetic genes for the production of unnatural variants are frustrated by a high failure rate. In an effort to better understand what types of genetic changes are most likely to lead to successful improvements, we systematically analyzed the ways in which biosynthetic genes naturally evolve to generate new compounds. We show that large gene clusters appear to evolve through the merger of sub-clusters, which function independently, and are promising units for cluster engineering. Moreover, a subset of gene clusters evolve by concerted evolution, which generates sets of interoperable domains that may enable predictable domain swapping. Finally, many biosynthetic gene clusters evolve in family-specific modes that differ greatly from each other. Overall, this quantitative perspective on the ways in which gene clusters naturally evolve suggests novel strategies for using synthetic biology to engineer the production of unnatural metabolites. Bacterial secondary metabolites are widely used as pharmaceutical, agricultural, and dietary agents. They consist of many classes of compounds including polyketides (PKs), nonribosomal peptides (NRPs), ribosomally synthesized and post-translationally modified peptides (RiPPs), terpenoids, saccharides, and a plethora of hybrids. The genetic basis for this rich molecular diversity can be found in biosynthetic gene clusters (BGCs), physically clustered groups of genes that encode the enzymatic pathways necessary to construct specific chemicals The diversity of extant natural products and BGCs raises important questions about their evolutionary origin. These include the basic question of how Nature invents new molecules, and a series of applied questions relevant to biotechnology: for example, the evolutionary modularity of NRP and PK BGCs has long been seen as a feature that might allow large libraries of new compounds to be generated by mixing and matching their constituent domains and modules Regardless of these apparent constraints to genetic change, Nature appears to have been quite successful at engineering biosynthetic pathways through the process of gene cluster evolution: even a conservative estimate suggests that the number of broad biosynthetic gene cluster families that have evolved exceeds 6,000 So far, insights into the key principles underlying the evolution of BGC architectures and repertoires have been derived from limited case studies Table S1), we found not only that they may have been transferred horizontally at high frequency , but also display exceptionally high rates of insertions, deletions, duplications and rearrangements . Phylogenetic profiling P<0.001), based on the χ2 test. These motifs comprise 591 different Pfam domains and have an average length of 5.3 domains (Table S2). As expected, they include many well-known and widely conserved motifs that appear to be linked to specific sub-functionalities of gene clusters, such as precursor biosynthesis, transport or synthesis of a specific chemical moiety, and motifs belonging to modular BGC architectures of NRPSs and PKSs , indicating that the macrolactam- and AHBA-producing sub-clusters have been co-evolving for a long time (instead of multiple independent AHBA sub-cluster acquisitions having occurred in different macrolactam-producing polyketide BGCs). Hence, the multi-hybrid rubradirin gene cluster might have arisen from a rifamycin-like ancestor  that already harbored the combination of a modular type I PKS sub-cluster and an AHBA biosynthesis sub-cluster, and which then acquired new sub-clusters for the biosynthesis of the aminocoumarin, 3,4-dihydroxydipicolinate and nitrosugar moieties (which are not found in any other closely related ansamycins). Contrary to the shared evolutionary histories of AHBA and ansamycin type I PKS sub-clusters, a clear example of sub-cluster transfer between BGCs of different types can be seen for 6-methylsalicylic acid (MSAS)/orsellinic acid (OSAS) sub-clusters, as inferred from a maximum-likelihood phylogenetic tree of MSAS/OSAS-producing iterative PKSs . The topology of this tree strongly indicates that MSAS/OSAS sub-clusters have largely evolved independent of the scaffold types of their parent gene clusters , and that they have been transferred between multiple types of BGCs during their evolutionary past. In conclusion, in the context of the bricks-and-mortar analogy, some bricks move around between different structures more often than others. Finally, we should note that there are also BGC families which evolve over long periods of time without major changes to the gene cluster architecture or the scaffold of the core molecule made: for example, the large family of over >1,000 aryl polyene BGCs that we described recently Although the complex patterns of sub-cluster sharing, in which various sub-clusters are shared between otherwise completely different gene clusters Fig. 2, Many chemical scaffold types of secondary metabolite classes are quite distinct, which raises the question of how BGC families encoding the synthesis of distinct scaffolds are related. To assess this question, we calculated the proportion and similarity of Pfam domains shared between all pairs of BGCs within our data set of 732 known gene clusters using multiple sequence alignments for each Pfam domain Fig. 3 aStreptomyces gene cluster encoding the lipopeptide antibiotic daptomycin Mycobacterium glycopeptidolipid (GPL) gene clusters Figure S5). When we performed a more in-depth analysis through a phylogenetic analysis of condensation domains, we indeed found that GPL domains consistently cluster together with domains from the NRPSs that synthesize daptomycin . Although both daptomycin and the GPLs are lipopeptides, the Mycobacterium GPLs are shorter (tetrapeptide vs. tridecapeptide), cell-wall-associated rather than diffusible, linear rather than cyclic, and originate from an actinomycete genus that is not closely related to Streptomyces.For example, the Likewise, one of the strongest matches for the gene cluster encoding the immunosuppressant rapamycin Figure S8 which shows that relevant bootstrap values are almost all above 90). Even more remarkably, 14 out of 16 domains responsible for the biosynthesis of the sub-structure shared between these two molecules , showing widespread sequence variability at almost every position in the AT domains, except for the residues near the substrate binding site . Mutations in KS domains, on the other hand, are mostly restricted to the regions in vicinity (around the core) of the substrate-binding site and the dimerization interface , suggesting their importance in influencing substrate selectivity.The phylogenetic trees of KS and AT domains from our data set of known BGCs revealed another unexpected finding: in spite of the structural similarity of rapamycin and FK520, 63% of the constituent domains of their polyketide synthases (PKSs) cluster into entirely separate clades . For the gene clusters encoding the biosynthesis of the mutually closely related macrolides erythromycin Concerted evolution is not peculiar to the rapamycin family . Firstly, gene clusters encoding glycopeptides, calcium-dependent lipopeptides and macrolides/polyethers appear to be most repetitive, pointing to a history of module duplications and/or a prominent influence of concerted evolution. The syringopeptin NRPS p-values from the N- to C-termini of large synthases, suggesting that some gene clusters evolve to encode the synthesis of larger molecules by iterative duplication of their most N-terminal module, would have the effect of extending an intermediate NRP or PK by the addition of a new starter unit. Thirdly, a group of BGCs including the ones that encode the polyketides psymberin To understand more generally how PKS and NRPS BGCs evolve, we set out to measure the contributions of concerted evolution, duplication, and divergence to the evolution of all multimodular PKS and NRPS BGCs in both our known and predicted BGC data sets. We first collected and quantified 25 different features describing the nature of gene cluster sequences and the relationships among their constituent domains  is largely governed by the evolutionary population dynamics of the species. Many bacteria live in large effective population sizes and have relatively short generation times, leading to very strong purifying selection and, consequently, rigorous genome streamlining Concerning the birth of new gene cluster architectures, large effective population sizes and short generation times also suggest that BGC modifications should immediately confer an evolutionary advantage in order to be maintained; on the other hand, frequent changes in population size may affect the probability of mutations to be fixated in the population Our analysis of BGC evolution will enable new approaches to BGC engineering informed by the mechanisms by which BGCs evolve naturally. Our results suggest that efforts to engineer the biosynthesis of unnatural natural products could be more successful by observing the modes by which specific BGC classes evolve in nature.For example, conglomerate molecules consisting of multiple different chemical moieties could be designed by engineering BGCs consisting of novel combinations of sub-clusters. Such an effort could be guided by information taken from evolutionary comparisons, which would offer suggestions about which sub-clusters are most likely to function together, based on how often evolution has successfully forged combinations between them.Furthermore, our evolutionary analysis of NRPS and PKS gene clusters suggests that concerted evolution has created sets of domains within gene clusters that are highly homologous. These domain sets are more likely to be mutually interoperable than domain sets chosen at random, and might therefore be of great utility in future engineering efforts.Also, evolutionary strategies towards generating larger and more complex compounds could be mimicked by N-terminally extending certain types of NRPS/PKS gene clusters by duplicating and then carefully modifying the first assembly-line module.Overall, in combination with new synthetic biology techniques that may soon enable the rapid assembly of thousands of clusters from a common set of parts To remove highly similar genomes from these analyses, we used the AMPHORA To search for histidine and tryptophan biosynthetic operons, we modified ClusterFinder Best matching sequence homologs of a query protein domain from a biosynthetic or primary metabolic gene cluster were obtained using MUSCLE 2 test with Bonferroni correction was applied to test for statistical significance, with the null hypothesis stating that the two values are equal.For each BGC, a two-dimensional array of the size corresponding to the numbers of consecutive protein domains that are in Pfam database (rows) and 408 selected organisms (columns) (see “Comparison of HGT with primary metabolism”) was created. The cells in the array consisted of sequence identities between a given domain from a BGC and the most homologous domain  from a given organism. Next, we calculated a Pearson product-moment correlation coefficient (correlation coefficient) for each possible pair of protein domains (rows), resulting into a new matrix, a correlation matrix, of the size corresponding to the number of protein domains  in both dimensions. To take rearrangements into account, we reordered rows and columns of the correlation matrix based on hierarchical clustering of the correlation matrix in both dimensions. We then parsed linear motifs that are likely to evolve in a correlated fashion by selecting consecutive pairs of domains in this reordered correlation matrix  with correlation coefficient >0.5. The analysis was repeated by setting the correlation coefficient cutoff to >0.65 and >0.8. Each motif was divided into all possible sub-motifs of sizes between 2 domains and the total number of domains in a motif. To determine the significance of a (sub)motif occurrence, we next compared the number of (sub)motif occurrences to the number of all possible (sub)motif occurrences in all BGCs that did not pass the correlation coefficient cutoff. Pearson's χData S1) comparing each alignment of two-gene clusters having at least three matching blocks with >70% identity. Rearrangements were defined as an identified difference in the order of 1-kb blocks in an otherwise conserved (piece of) gene cluster, such as when A1-A2-A3-A4-A5 matches to B1-B4-B3-B2-B5 in an alignment of two BGCs A and B. Indels were defined as 1-kb blocks present in one gene cluster but not in the other gene cluster, such as when A1-A2-A3-A4-A5-A6 matches to B1-B2-B5-B6 in an alignment. To make these inferences more reliable, a constraint was used that the flanking regions (of size > = 2 kb) of each indel breakpoint must be homologous between query and hit gene cluster, and the block order must be conserved between them. Finally, duplications were defined as 1-kb blocks that had the best hit towards another block in its own gene cluster, and having a higher copy number in one gene cluster than in the other, such as when A1-A2-A3-A2-A3-A4-A5 aligns to B1-B2-B3-B4-B5, while the mutual sequence identity between the A2 and A3 pairs is higher than between any of the A2/A3 blocks and B2 or B3.We performed an all-versus-all alignment of nucleotide sequences of known and predicted BGCs using the blastn algorithm. Gene cluster sequences were divided into blocks of 1 kb, and then mapped to the most homologous blocks from other gene clusters, as well as from the same gene cluster (to test for genomic duplications). 56% of the blocks  did not map to any homologous regions in the same or other BGCs with >70% identity. Evolutionary events  were detected by a custom-made Python script . The final sub-cluster sharing network was drawn with Cytoscape Sub-clusters with known functions from experimentally characterized gene clusters were manually collected from the literature. Sub-cluster sharing between gene clusters from the training set was calculated using blastp p-value of its closest blastp match inside the gene cluster, given the distribution of the percent identities of all within-gene-cluster blastp hits of all domains of that domain type in the complete set of gene clusters. The internal similarity index was then calculated from these numbers as the mean of all inverse p-values. The same inverse p-values were used for plotting the internal domain similarity across gene clusters. The vertical evolution index of a gene cluster was calculated as the average difference between the p-value of the top 10 percent identities of a domain's blastp hits to all domains from other gene clusters with the p-values of the Lin distances of the gene clusters to the host gene clusters of each of the top 10 hit domains. Consequently, gene clusters with domains with highly similar closest hits to domains in dissimilar gene clusters get a low value, while gene clusters with domains with dissimilar closest hits to domains in similar gene clusters get a high value.To study patterns of evolution in multimodular NRPS and PKS gene clusters, a range of features was calculated describing key characteristics of these gene clusters. The first set of features was based on the topologies of intra-BGC domain similarity networks  and consisted of the average clustering coefficient, average sequence similarity, graph transitivity, number of 2–4 node cliques, number of connected components in a graph with sequence similarity >50%, and average neighbor degree. We also included as features the number of different Pfam domain types in a BGC, the total number of domains in a BGC, the average number of domains per gene, and the averages and standard errors of best-matching pair sequence identities and internal BGC similarity indices. Two evolutionary indices were also added: the internal similarity index and the vertical evolution index. To obtain the internal similarity index of a gene cluster, we calculated for each of its NRPS/PKS domains the PCA analysis was performed with the aforementioned features as an input. Compound types were assigned using the classifications taken from the primary literature.Figure S1The rapid and dynamic evolution of BGCs differs from the evolution of tryptophan operons. Distributions of the best matching sequence homologs with respect to organism similarity (based on 16S rRNA) for predicted BGCs and tryptophan operons suggest significant differences in the ways they evolve, The distribution of all organism-organism similarities (background organism similarities) is trimodal, which may explain why, similarly, the distributions of the best-matching sequence homologs for predicted BGCs and tryptophan operons are also trimodal.(PDF)Click here for additional data file.Figure S2Examples of insertions/deletions in BGCs. Three gene cluster alignments of highly similar BGCs (>70% at the nucleotide level) are shown that are likely to represent relatively recent insertions/deletions in BGCs with functional consequences. In the upper panel, genes that putatively encode one or more sugar moieties have been inserted/deleted from a saccharide biosynthesis gene cluster. In the middle panel, a germacradienol synthase has been replaced by another type of terpene synthase, a pentalenene synthase, as well as an AMP-dependent synthetase. In the lower panel, a gene cluster related to the well-known coelibactin gene cluster from Saccharopolyspora spinosa is shown, which has acquired a MSAS polyketide synthase, a cytochrome P450, a carboxamide synthase and a 3-oxoacyl-(ACP) synthase compared to the coelibactin gene cluster from Streptomyces coelicolor. These genes are predicted to encode a polyketide moiety that might be attached to the NRP siderophore synthesized by the coelibactin NRPS machinery.(PDF)Click here for additional data file.Figure S3Phylogeny of ansamycin KS domains. A FastTree (PDF)Click here for additional data file.Figure S4Phylogenetic tree of MSA/OSA iterative PKSs. Maximum likelihood phylogenetic tree (constructed with RAxML (PDF)Click here for additional data file.Figure S5Similarity between daptomycin and its BGC and other BGCs and their small molecule products. Node sizes correspond to the number of Pfam domains with sequence identity to one of the daptomycin genes higher than the top 10th percentile of the background Pfam sequence identity distribution, and node colors denote the average sequence identity for such Pfam domain pairs.(PDF)Click here for additional data file.Figure S6GPL condensation domains clade with daptomycin condensation domains in a phylogenetic tree. The tree shown was reconstructed using the maximum likelihood method in MEGA (PDF)Click here for additional data file.Figure S7Mutations in AT and KS domains mapped onto their crystal structures.a, We aligned sequences of AT and KS domains from 4 BGCs  and at the homodimer interface. The entropy was not calculated in the regions that fall outside of the Pfam-annotated domains, nor in the indel-rich regions (marked black). The figures were generated using UCSF Chimera (PDF)Click here for additional data file.Figure S8Evidence for concerted evolution in various PKS and NRPS gene clusters. Phylogenetic trees of KS/AT and C/A domains, respectively, involved in the biosynthesis of several families of related polyketide or nonribosomal peptide molecules show various degrees of concerted evolution. For example, trees of the AT and KS domains of macrolide biosynthesis enzymes show a high rate of BGC-specific branching (suggestive of concerted evolution), while hardly any such branching is observed in trees of the C and A domains of glycopeptide biosynthetic enzymes. Phylogenetic trees were constructed in MEGA5 (PDF)Click here for additional data file.Figure S9Clustered heat map of features based on protein sequence alignments and domain-similarity network topologies. Features include the average number of Pfam domains per gene, means and standard deviations of the clustering coefficient and the network transitivity (see (PDF)Click here for additional data file.Figure S10Domain architectures of all 658 BGCs encoding multimodular PKS and NRPS enzymes. The domains are colored by the p-value of the homology to their nearest neighbor within the same gene cluster. BGCs that are mostly red contain domains that are highly similar to other domains in the same gene cluster, whereas BGCs that are mostly blue contain domains that are dissimilar from other domains within the same gene cluster.(PDF)Click here for additional data file.Figure S11Detailed phylogenetic trees of KS and AT domains of polyketide synthases from the rapamycin family. The tree was reconstructed using the neighbor-joining method in MEGA (PDF)Click here for additional data file.Table S1Overview of evolutionary events detected between alignments of gene cluster pairs sharing at least three matching 1 kb-sized blocks in alignments with thresholds of >70% identity (top) or >80% identity (bottom). The numbers of observed indels, duplications and rearrangements are given for BGCs of several sizes classes: 1–10 kb, 11–20 kb, 21–30 kb, 31–40 kb and 40+ kb, or in cumulative combinations of these size classes .(XLSX)Click here for additional data file.Table S2Results from the phylogenetic profiling analysis at three different cross-correlation cutoffs. The first and second column of each table show a number of co-evolving and non-coevolving motifs, followed by p-values from a Chi2-test , a string of Pfam IDs that constitute a motif, and their description.(XLSX)Click here for additional data file.Table S3Sub-cluster type-specific cut-offs to determine sub-cluster sharing between BGCs. Suitable cut-offs were determined by a manual comparison of known sub-clusters to their corresponding known chemical moieties, and cut-offs were set at those sequence identities where the chemistry produced by the enzymes encoded by the sub-clusters was (nearly) identical.(XLSX)Click here for additional data file.Data S1Python script and associated data files that were used to identify insertions/deletions, duplications and rearrangements in homologous gene clusters.(ZIP)Click here for additional data file.