This representation is inspired by a technique from digital electronic design, termed stochastic logic, in which values are represented by the probability of 1’s in a stream of randomly generated 0’s and 1’s. Research in the electronic realm has shown that a variety of complex functions can be computed with remarkably simple circuitry with this stochastic approach. This paper demonstrates how stochastic electronic designs can be translated to molecular circuits. It presents molecular implementations of mathematical functions that are considerably more complex than any shown to date. All designs are validated using mass-action simulations of the chemical kinetics of DNA strand displacement reactions.This paper discusses the implementation of mathematical functions such as exponentials, trigonometric functions, the sigmoid function and the perceptron function with molecular reactions in general, and DNA strand displacement reactions in particular. The molecular constructs for these functions are predicated on a novel representation for input and output values: a fractional  A particularly promising strategy for molecular computation is based on the mechanism of DNA strand displacement6.Molecular computing holds the promise for transforming research in areas such as disease monitoring and drug delivery. Since early, pioneering work by Adleman14. These circuits have been used as building blocks for both digital signal processing20, and mixed-signal  computation21. Using these simple circuits, complex genetic circuits have been constructed to perform computation in cells22. To automate the design of genetic and DNA circuits, computer-aided design (CAD) systems have been presented23. There has been interest in activating and inhibiting pathways by “filtering” concentrations of molecular types in different frequency bands26.Various computational structures have been proposed for DNA-based systems, as well as in other contexts. Simple logic primitives, such as AND, OR, NAND, NOR, and XOR have been demonstratedcomputer programming framework. Work on CRNs includes programs for computing different sorts of mathematical functions such as polynomials28, and logarithms30.The theory of computing with abstract chemical reactions, termed chemical reaction networks (CRNs), has evolved into a bona fide 31. The hyperbolic regime can be used to realize exponential and logarithm while ultrasensitive regime can be used to realize sigmoid function. The CRNs in31 describe analog behavior of the system while the CRNs described by the proposed approach describe digital behavior. In31, each region of operation is described by a specific input-output characteristic. This implies that the exponential and sigmoid functions are realized for specific ranges of input concentrations. Furthermore, the sigmoid function in29 describes a hard-limit response. On the other hand, the proposed approach realizes digital circuits and the function behavior is not limited to a specific range of input concentrations (only the ratio of two concentrations used to represent a variable is important not their concentrations).In prior work, different approaches to compute complex functions such as exponentials and sigmoids have been presented. For example, it has been shown that CRNs describing covalent modification cycle can realize exponential, logarithm and sigmoid functions32. Thus, our method provides a systematic way to design DNA reactions that compute mathematical functions. These computational constructs are central to the topic of perceptrons, that represent simple machine learning algorithm.This paper presents a method for designing CRNs that compute a wide range of mathematical functions, ranging from simple to complex. The building blocks in the proposed methodology are units composed of four chemical reactions. All chemical reactions in the proposed system have exactly two reactants. Such bimolecular chemical reactions can be implemented as DNA strand-displacement reactions in a robust way34. Molecular implementations of machine learning classifiers could have important applications. One can imagine instances where inference and learning might be an integral part of tasks such as biochemical sensing. For example, genetic logic circuits for cell classification can sense features of mRNAs; they can detect their expression patterns and selectively respond to specific cell types38. Such circuits could enable the production of personalized “smart” drugs that target specific diseases for specific patients39.Machine learning classifiers have become ubiquitous in the computational sciences. Their physical realization using different technologies has been considered44. As early theoretical research on the topic, Hjelmfelt et al. presented chemical reactions that, based on the ordinary differential equations of mass action kinetics model, can emulate so-called McClulloch-Pitts neurons44. These chemical neurons can be coupled together to build chemical neural networks or finite-state machine45. Also in a theoretical vein, Mills et al. described a DNA implementation of a Hopfield neural network as well as a DNA implementation of a multi-layer perceptron47. The authors speculated that networks containing as many as 109 neurons might be feasible.Past work on neural computation with molecular reactions includeset al. performed pattern recognition with chemical (as opposed to biomolecular) reactions, in a continuous flow stirred tank reactors48. Lim et al. implemented pattern recognition with differentially-labeled probe DNA molecules that competitively hybridized to compute the decisions49. Zhang and Seelig described an implementation of a linear classifier using DNA strand displacement50. Design of DNA circuits for supervised learning of a class of linear functions using buffered strand displacement reactions has been presented in51. Finally, Qian et al. demonstrated a complete artificial neural network, implemented experimentally using DNA strand displacement52.Laplante 52 or linear transfer function50 as the activation function.In general, an artificial neural network consists of one or more layers where, in each layer, a neuron computes a weighted sum followed by a nonlinear activation (transfer) function. Typically the activation function corresponds to a sigmoid function. Prior work on molecular implementations of ANNs has considered either a hard-thresholdThis paper discusses the implementation of mathematical functions such as exponentials, trigonometric functions, the sigmoid function and a perceptron function with the limitation that the weighted sum of the inputs is scaled down by the dimension of the input vector.representation for the input and output variables of chemical reaction networks (CRNs) have been considered:The value of each variable corresponds to the concentration of a specific molecular type; this is referred to as a direct representation.53.The value of each variable is represented by the difference between the concentrations of a pair of molecular types; this is referred to as a dual-rail representationIn prior work on molecular computing, two types of fractional representation28. Here a pair of molecular types is assigned to each variable, e.g.,  for a variable x. The value of the variable is determined by the ratio of the concentrations for the assigned pair,X1] and [X0] represent concentrations of molecules X1 and X0, respectively. Note that the value of x is confined to the unit interval, . With the values confined to the unit interval, we refer to the representations as a unipolar fractional encoding.In recent work, we have proposed a new type of representation, referred to as a X0, X1), given by:bipolar fractional encoding.Variables with values in the range  can be represented by a slightly different encoding on the assigned pair b using both the unipolar and bipolar fractional representations. These molecular circuits are, respectively, referred to as Mult, NMult, and MuX. Secondly, this paper demonstrates that unipolar and bipolar fractional coding approaches can be used to design CRNs for computing complex mathematical functions such as ex−, sin(x), and sigmoid (x). The proposed CRNs can readily be implemented using DNA strand displacement.The unipolar fractional coding and the connection that it makes between molecular computation and electronic stochastic logic design have been introduced instochastic logic, in which values are represented by the probability of seeing 1’s in a stream of randomly generated 0’s and 1’s59. Research in the electronic realm has shown that a variety of complex functions can be computed with remarkably simple circuitry with this stochastic approach.The fractional representation is inspired by a technique from digital electronic design, termed 28 and this paper lies in the approach proposed to design and synthesize computing CRNs. The approach in28 uses so-called control generating reactions and the transferring reactions that lead to reactions with m reactants for a polynomial of degree m. In contrast, this paper uses simple molecular units such as Mult and NMult described in the next section. Regardless of the complexity of the target functions, the molecular reactions designed by the new approach are only composed of simple reactions with two reactants and one product. These reactions are more suitable for DNA implementation. The molecular implementations presented in this paper are inspired by the stochastic implementations of functions presented in60.The main difference betweenX0 and X1 molecular types, correspond to the probability of seeing 0’s and 1’s, respectively, in the random streams. This paper demonstrates how stochastic electronic designs can be translated to molecular circuits.The fractional encoding discussed in this paper is analogous to the stochastic representation. The concentrations of the One should notice that the bipolar fractional coding is just a representation of the value of a variable using two molecular types. This means that it is not required to actually calculate Equation . In otheSection 1 introduces molecular reactions for the Mult and NMult units; these perform multiplication in the unipolar fractional representation. Section 2 presents an approach for mapping specific target functions to a cascade of Mult/NMult units. Section 3 introduces a molecular MUX unit that performs scaled addition, as well as Mult/NMult units for multiplication using the bipolar representation. Section 3 also presents an application: CRNs for implementing a single-layered neural network . Section 4 discusses the DNA implementations of the proposed CRNs.c = a × b, and NMult computes c = 1 − a × b where a,b, and c are in the unipolar fractional representation. The units are described below.Based on the fractional coding discussed in the previous section, we propose two simple sets of CRNs for computing multiplication. We refer to these as Mult and NMult. These sets will serve as fundamental units in the construction of other desired functions in Section 2. Mult computes c as the multiplication of two inputs a and b, all in unipolar fractional representation. So if Consider the four reactions shown in Fig. C0 and C1 in the molecular reactions of the Mult unit, we obtain what we call an NMult unit which computes 1 − a × b in the unipolar fractional coding. Figure a × b is very similar to the proof for Mult unit. It can be obtained by switching C0 and C1 in the proof presented for Mult unit.If we switch A0, A1) and , are preserved. The details are presented in Section S.2 of the Supplementary Information.Note that the CRNs in Fig. Figure In this section we propose a framework for designing CRNs to compute different functions. Our method is illustrated in Fig. In the proposed methodology, the functions are approximated by truncating their Maclaurin series expansions. Note that other expansion methods such as Taylor series could also be used. The approximated polynomials are then mapped into equivalent forms that can be readily implemented using Mult and NMult units.f(x) = ex− as an example.The Mult/NMult units are then mapped to CRNs. These are implemented by DNA strand-displacement reactions. We describe these steps using Step 1- Approximate the functionf(x) that is infinitely differentiable at a point a corresponds to the power seriesThe Taylor series of any function a = 0, then the series is called a Maclaurin series. As an example for f(x) = ex− the Maclaurin expansion is given by:If the Taylor series is centered at zero, i.e., n, in order to approximate the desired function. As an example if n = 5, i.e., the first six terms are retained, for f(x) = ex− we obtainThe series is truncated to a polynomial of degree Step 2- Reformat the approximation and map it toMult/NMultunits55 and Gaines56 in the late 1960’s, stochastic processing implements logical computation on random bit streams. Numbers are encoded by the probability of obtaining a one versus a zero in stream of random bits.As the second step, the approximating polynomials obtained in the first step are mapped into equivalent forms can be implemented using Mult and NMult units. The Mult and NMult units are analogous to AND and NAND gates in electronic design paradigm called stochastic processing. First developed by Poppelbaum60 has shown that the form of polynomials that we generate in this step can be changed in a way that they can be mapped to a cascade of AND and NAND logic gates. The approach presented by Parhi and Liu uses the well known Horner’s rule in order to map polynomials with alternating positive and negative coefficients and decreasing magnitudes to AND and NAND gates60. This approach can be used for Maclaurin series of the functions ex−, sin(x), cos(x), log(1 + x), tanh(x), and sigmoid (x). Note that for the trigonometric functions, the operand x is in radians. We use the approach of Parhi and Liu60 to change the form of the desired approximating polynomials and then map them to a cascade of Mult and NMult units. We briefly describe this approach.In this work, the Mult and NMult units perform the same operation on molecular concentrations in the unipolar fractional encoding as AND and NAND gates do, respectively, in stochastic logic. Recent work in stochastic logicP(x) of degree n given in its power form asConsider a polynomial 60, Eq. , cos(x), log(1 + x), tanh(x), and sigmoid (x), listed in equations (41) to (46) of the Supplementary Information, meet these requirements and can be represented using Equation (In order to guarantee 0 ≤ Equation .f(x) = ex−, shown in . The stochastic sigmoid circuit shown in Fig. 60 the reader is referred to60 for details of the derivation. This performs a soft decision of whether the output should be close to 0 or 1. For the perceptron system that we implement, the inputs are binary, that is to say either xi = 0 or xi = 1, and the coefficients, i.e., wi’s, are between −1 and 1. All multiply-add operations are implemented using bipolar Mult units. Since the input of the sigmoid function is between −1 and 1, we implement the sigmoid function using a bipolar fractional coding.This section describes implementation of a single-layered neural network, also called a perceptron, by molecular reactions. As shown in Fig. 52. No prior publication has considered molecular ANNs using a sigmoid activation function. In this section we describe the implementation of the bipolar MUX unit and the bipolar Mult and NMult units.Note that prior biomolecular implementations of artificial neural networks (ANNs) have considered either hard limit or linear activation functionsc as the weighted addition of two inputs a and b as c = a × (1 − s) + b × s, where 0 ≤ s ≤ 1. Here a, b, and c can be in either the unipolar or the bipolar fractional representation while the weight s must be in the unipolar representation. The set of four reactions in Fig. The MUX unit, shown in Fig. c as the multiplication of two inputs a and b, where a, b and c are represented in bipolar fractional representation. So if c = a × b.The bipolar Mult unit, shown in Fig. C0 and C1 in the reactions of the bipolar Mult unit, we obtain the bipolar NMult unit which computes −a × b. Figure c = −a × b in the bipolar fractional coding.Analogous to the way that we obtained NMult from Mult unit in the unipolar fractional coding, if we switch C0 and C1 meaning that in the proof for bipolar Mult instead of C1 − C0 in the numerator we have C0 − C1. This leads to having c = −ab instead of c = ab.The proof is very similar to the bipolar Mult unit. Indeed, for bipolar NMult we just switch x ≤ 1. The output of this function, however, is still in the unit interval  and can be represented by a unipolar fractional representation. In fact, for x ∈  the corresponding output range is . In Parhi and Liu60, it is shown that the sigmoid function using hybrid format, i.e., for bipolar input and unipolar output can be implemented by electronic stochastic logic circuits, namely, XOR and XNOR gates and multiplexers. These electronic circuits perform multiplication and weighted addition for stochastic bit streams analogous to the same operations that bipolar Mult, NMult, and MUX units in Fig. N bipolar Mult units having the same output. Details for the molecular implementation of the inner product are described in Section S.5 of the Supplementary Information.The bipolar fractional representation can be used to implement the sigmoid function, presented in Section 2.1.1 for the unipolar fractional representation. Therefore, the function can be computed for inputs between −1 and 1, i.e., −1 ≤ N = 32 using 32 coefficients. Three perceptrons are simulated. The 32 binary inputs are selected at random such that each bit is equally likely to be 1 or 0. It is important to note that the inputs are not constrained to be binary in the proposed methodology, but are constrained to lie between −1 and 1. For each perceptron, the same 100 input vectors are simulated. The input vectors are illustrated in Fig. state depending on whether it is positive or negative. Then Perceptron B has more molecules that activate the state whereas Perceptron C has more molecules that inhibit the state, whereas Perceptron A has equal number of molecules that either activate or inhibit the state. For equally likely binary inputs, the probabilities of the weighted sum for the Perceptrons A, B, and C, respectively, correspond to 0, 1.5 and −1.5. The expected sigmoid values for the three perceptrons correspond to 0.5, 0.8175, and 0.1825, respectively. Each perceptron output is classified as 1 or 0 using a threshold of 0.5. If very large number of random input vectors are simulated, we would expect the percent of input vectors classified as 1 in these three perceptrons to be 50%, 81.75% and 18.25%, respectively. For the 100 input vectors, the classification results for the three perceptrons are illustrated in Fig. By cascading the inner product part and the sigmoid function, we can implement molecular perceptrons with binary inputs as shown in Fig. xi and wi, respectively, represent the binary value of the ith input and its associated weight. Figure The simulation results in Fig. et al. for the construction of DNA tweezers2. A general method for translating CRNs to DSD reactions was proposed by Soloveichik et al.6 and is illustrated in Supplementary Information Section S.8 and Figure Constructs in the previous sections were presented in terms of abstract CRNs. In this section, we translate our Mult/NMult circuits to DNA strand displacement (DSD) reactions. The idea of DSD reactions based on toehold mediation was first introduced by Yurke et al. showed that bimolecular reactions, such as A + B → C, can be implemented by linear, double-stranded DNA complexes that are compatible with natural DNA32. We note that our computational units are all constructed from bimolecular reactions and so these could be implemented using the framework proposed by Chen et al.32.Recently Chen 6 we simulate the reactions using DSD. Figures x = 0.3 and x = 0.7. Table mean square error (MSE) at the eleven points. The error may be due to several factors: the approximation of the function with a truncated series expansion; the emulation of the related CRNs by DSD reactions; and the limited simulation time (of 50 hours for DSD reactions). As the results show, the error is less than 1 × 10−3. For a visual comparison, Figure Using the software tool provided by Erik Winfree’s group in Caltechy(j) from molecular or DNA simulation, xi[j] represents the ith bit position of input vector j, and wi represents the ith weight. The mean square error values for molecular and DNA simulations are small as the dynamic range of the sigmoid function with scaled weighted sum of binary inputs is small. For example, sigmoid(1.5/32) and sigmoid(−1.5/32), respectively, correspond to 0.5117 and 0.4882. Although the DNA implementation of the perceptron achieves 100% classification accuracy in simulation, we caution that in an actual experiment the DNA perceptron may not achieve perfect classification accuracy.Table systematic way to design molecular systems to compute mathematical functions. This paper presents a systematic methodology for designing CRNs to implement complex mathematical functions robustly. The proposed method is unique in that it relies exclusively on bimolecular reactions, with no requirements on the reaction rates. According to the work of Chen et al., bimolecular reactions are compatible with natural DNA32. This means that, the computational elements we propose here could potentially be used for in vivo applications. A key contribution of this paper is the ability to map any stochastic logic circuit to a molecular circuit based on fractional coding. Numerous prior papers have demonstrated stochastic logic implementations of digital filters, error control coders such as low-density parity check codes and polar codes. The proposed molecular logic gates can be used to design molecular digital filters and molecular error control coders in a straightforward manner.Although there have been numerous examples of CRNs for computing specific functions presented in the literature, as yet there has been no et al.28. In that paper we showed how arbitrary polynomials can be mapped to a CRN. Although that method could be used to compute truncated Maclaurin series of desired functions, it uses a rather complex set of chemical reactions with m reactants and at least m + 1 products, with m≥2, for polynomials of degree m. Implementing reactions with more than two reactants may be biologically infeasible, since this entails large complexes. In contrast, the methodology proposed in this paper requires only bimolecular reactions and so is readily implementable.This paper builds on our prior work. The computation of polynomials was presented in Salehi 66, requires molecules to be bounded between −1 and 1. Thus, complete dynamic range of a function cannot be computed by the proposed method. For example, the proposed method can only compute scaled sine and cosine values. The molecular perceptron cannot compute the sigmoid value of the weighted sum of the binary inputs. This is an inherent limitation of the proposed method as the sigmoid function processes a scaled version of the weighted inputs . Furthermore, the weight values are constrained to lie between −1 and 1. Molecular implementations of general perceptrons with arbitrary weights remains a topic for future research. In addition, further research needs to be directed towards molecular implementations of perceptrons used in inference applications as opposed to binary classification applications.Although molecular and DNA implementations of several mathematical functions using fractional coding have been demonstrated, the proposed method suffers from numerous limitations. Use of fractional coding, inspired by stochastic logicSupplementary Information


Chemical cell lysis is an interesting topic in the research to Lab-on-a-Disc (LOD) platforms on account of its perfect compatibility with the centrifugal spin column format. However, standard procedures followed in chemical cell lysis require sophisticated non-contact temperature control as well as the use of pressure resistant valves. These requirements pose a significant challenge thereby making the automation of chemical cell lysis on an LOD extremely difficult to achieve. In this study, an LOD capable of performing fully automated chemical cell lysis is proposed, where a combination of chemical and thermal methods has been used. It comprises a sample inlet, phase change material sheet (PCMS)-based temperature sensor, heating chamber, and pressure resistant valves. The PCMS melts and solidifies at a certain temperature and thus is capable of indicating whether the heating chamber has reached a specific temperature. Compared to conventional cell lysis systems, the proposed system offers advantages of reduced manual labor and a compact structure that can be readily integrated onto an LOD. Experiments using Salmonella typhimurium strains were conducted to confirm the performance of the proposed cell lysis system. The experimental results demonstrate that the proposed system has great potential in realizing chemical cell lysis on an LOD whilst achieving higher throughput in terms of purity and yield of DNA thereby providing a good alternative to conventional cell lysis systems. Over the last decade, development of integrated and inexpensive point-of-care (POC) devices for rapid diagnostics has been on the rise owing to miniaturization and automation of analytical protocols based on microfluidics. The obvious advantages of the use of microfluidic devices include reduced sample consumption, enhanced efficiency, and fast reaction times ,2. The sSpecific to POC applications, improvements to present systems are required to ensure proper integration of microfluidic functions and reduction of fabrication costs; this is in addition to the necessary adaptation of bio-assays to a microfluidic format. In bio-assays, finding and analyzing information regarding genetic and/or disease characteristics is essential ,4. Cell Recently, a variety of microfluidic devices have been proposed and demonstrated to perform cell lysis. Cell lysis techniques, based on mechanical, biological, and chemical methods, applied to microfluidic devices have been widely investigated ,10,11,12Amongst numerous microfluidic technologies available, this study proposes the design of a centrifugal microfluidic chip, also known as Lab-on-a-Disc (LOD), which is regarded as one of the most outstanding platforms in microfluidics ,17,18,19Although many integrated LOD platforms have already been demonstrated for use in biochemistry and whole-blood immunoassay processing ,30,31,32Through complete integration of a molecular assay for Salmonella species Identification, the objective of this study is to demonstrate the implementation of robust microfluidic functions. Accordingly, the materials, fabrication means, and assembly strategies have been selected to meet requirements for further realistic applications of the device. Phase change material sheet (PCMS) and an illuminance sensor have been used in the proposed system, performing fully automated cell lysis with a combination of chemical and thermal methods. Use these materials enable ease of fabrication, automation of the heating process, and accurate sensing of temperature changes. This proposed platform is expected to be equally applicable to other LOD automation platforms that require wireless heating parts to be fully integrated.The proposed design of the LOD system is depicted in The thermally controlled disc platform, capable of performing three parallel sample processes, was fabricated by laminating together five layers comprising two double-sided adhesive films  and three polycarbonate layers. The top and bottom polycarbonate layers were each 1.2-mm thick, and the middle polycarbonate layer measured 4 mm in thickness. The CAD model of the disc was designed using ProEngineer software. Layers of polycarbonate were carved using a computer numeric control (CNC) machine. The top layer combines the PCM structure with the black EVA valve and consists of vent holes for the lysate chamber. The lysis and lysate chambers are located in the middle layer. The lysis chamber (volume = 840 µL) was sufficiently large to handle typical sample solution volumes of 470 µL. The lysate chamber (volume = 750 µL) was positioned away from the center of the disc on account of the sequential progression of the liquid under the action of the centrifugal force.® Genomic DNA extraction kit, K-3032, Bioneer, Korea). The solution in the column tube was first washed at 13,000 rpm for 1 min. The column tube was then changed, 500 μL of washing buffer 1 was added, and the solution was centrifuged the same way as in the earlier step. The above step was then repeated using washing buffer 2, and the solution was centrifuged once more to reduce any reagents on the wet membrane. 200 μL of the elution buffer was subsequently added to the solution in the column tube, which was then incubated for 5 min at room temperature. Finally, the column tube was changed, and the solution was centrifuged for 1 min at 13,000 rpm to complete the purification stage. The purified solution was then analyzed by checking the ratio of optical density at DNA and protein absorbance wavelengths of 260 and 280 nm, respectively.Salmonella typhimurium  was used as the target strain to demonstrate the cell lysis operation and liquid control performance of the proposed system. The salmonella sample was inoculated in 25-mL tryptic soy broth and incubated overnight. Subsequently, the sample was subcultured once with the same medium grown to an OD600 value of 1.0, and made aliquoted into cultured salmonella samples measuring 1 mL each in volume. Simultaneously, PBS was washed twice with harvesting and reducing supernatants of the aliquots. To obtain the lysis sample, the amount of supernatant used in the third harvesting was reduced to 250 µL. 200 µL of binding buffer and 20 µL of proteinase K were then added to comprise a total sample volume of 470 µL. Here, the enzyme proteinase K, which is usually activated at 60 °C, will free the DNA from its histones around which DNA is tightly wrapped during the cell lysis. The lysis operation performed on the disc at 60 °C lasted for 10 min, and the lysate was delivered to the lysate chamber. 400 µL of the lysate was pipetted out and passed into a spin column tube that is used to purify DNA from cell debris and chaotropic agents present in the lysis reagent. The purifying procedure was performed following the manual enclosed in the kit , PCM-60  was used in this study. Automation of the heating process for cell lysis was one of the objectives of the proposed study. Since the lysis process is performed at 60 °C, the AccuprepPhase change material (PCM) is a substance with a high heat of fusion which, melting and solidifying at a certain temperature, is capable of storing and releasing large amounts of energy . When thPolycarbonate was used as the main material in the fabrication of the disc in order to withstand the heat generated during the lysis heating process. Inlet and vent holes were drilled at innermost locations on the disc. The inlet and vent holes form essential parts for sample liquid injection. The EVA valve, Y-channel, lysis chamber, vinyl valve, and lysate chamber were arranged on the disc as depicted in The lysis chamber was heated up to 60 °C by the laser module. When the liquid inside the chamber was heated up, the internal pressure increased, which caused a leak. Liquid leakage can occur at the inlet hole and vent hole, and it must be blocked. Therefore, the EVA valve was designed for each of the inlet holes and vent holes. The EVA valve, as shown in The process of increasing the temperature of the sample liquid to 60 °C depends on the situation—the uniformity of liquid distribution, ambient humidity, and temperature—in the lysis chamber. As such, it was impossible to automate the system by merely specifying the heating time. In order to achieve temperature changes in real time, the authors designed a system that could directly detect the temperature, and PCMS was used as the material of choice for this system on account of its temperature-dependent properties. In the proposed study, a temperature of 60 °C—since the target temperature of lysis is 60 °C—was maintained using PCM-60, which undergoes a phase change at 60 °C.As the temperature in the lysis chamber increased, the heat generated was transferred to the PCMS causing it to gradually melt. At 60 °C, the PCMS was found to have completely liquefied with its phase changing to that of a transparent liquid. The black floor of the plate beneath the PCMS layer, which also served as a light absorber, was, thus, exposed. As a result, the amount of reflected light entering the illuminance sensor had gradually diminished. To demonstrate performance of the proposed lysis disc, the lysis experiment inside the disc and the conventional method—spin column—were carried out concurrently. To see reproducibility, 10 replicate experiments were performed . The avePCR was used as the amplification method in this study. In order to perform PCR, 10 μL of NobleZyme™ PCR Plus Premix 2×  was used and mixed with an upstream primer, 10 μM (2 μL); downstream primer, 10 μM (2 μL); DNA template (3 μL); and nuclease-free water (3 μL) as reaction components. The PCR protocol was based on the thermal cycling profile—denaturation at 95 °C for 2 min, 30 cycles at 95 °C for 45 s, at 52 °C for 45 s, at 72 °C for 60 s, and a final extension at 72 °C for 5 min. Electrophoresis procedures—using 2% agarose gel  with 0.5X TBE buffer  at 100 V for 30 min—were used to obtain results of the PCR process.—were used to obtain results of the PCR process. The DNA size Marker  was loaded with 5 μL on the first lane, and resultant solutions obtained from the conventional and proposed methods were loaded with 5 μL on 5 lanes each. 3X green gel stain (10 mL) was then placed on agarose in a dark room for 30 min, and stained DNA were finally observed using a fluorescence treatment .A new heating system that serves to dissolve bacterial cells in DNA on an LOD platform is proposed in this paper. In order to prevent liquid leakage under increased pressure, an EVA valve with sufficient thermal durability has been designed. A dedicated laser module is used to heat the lysis chamber and a PCM array is used to maintain optimum chamber temperature. Additionally, a dedicated LED and illuminance sensor have been added to automate the entire heating process. Effectiveness of the proposed design has been demonstrated by measuring the purity and yield of the extracted DNA templates and their subsequent amplification using PCR. The results demonstrate that the lysis process was successfully performed and was fully automated. The authors expect that use of the proposed device would lead to user-friendly DNA analysis procedures in the near future. On the top of that, these experimental results tell us how stable the closed-loop system under PCM based temperature measurement is.


Activation of the alternative complement cascade has been implicated in the pathogenesis of age related macular degeneration (AMD) and Alzheimer’s disease (AD). Amyloid β (Aβ), a component of drusen, may promote complement activation by inhibiting CFI bioactivity. We determined whether Aβ reduced CFI bioactivity and whether antibodies against Aβ including a monoclonal antibody, GSK933776 could restore CFI bioactivity. We also measured CFI bioactivity in plasma of subjects with AMD and AD. In support of the GSK933776 development program in AMD (geographic atrophy), we developed a quantitative assay to measure CFI bioactivity based on its ability to cleave C3b to iC3b, and repeated it in presence or absence of Aβ and anti-Aβ antibodies. Using this assay, we measured CFI bioactivity in plasma of 194 subjects with AMD, and in samples from subjects with AD that had been treated with GSK933776 as part of the GSK933776 development program in AD. Aβ reduced the CFI bioactivity by 5-fold and pre-incubation with GSK933776 restored CFI bioactivity. In subjects with AMD, plasma CFI levels and bioactivity were not significantly different from non-AMD controls. However, we detected a positive linear trend, suggesting increasing activity with disease severity. In subjects with AD, we observed a 10% and 27% increase in overall CFI bioactivity after treatment with GSK933776 during the second and third dose. Our studies indicate that CFI enzymatic activity can be inhibited by Aβ and be altered in proinflammatory diseases such as AMD and AD, in which deposition of Aβ and activation of the alternative complement cascade are believed to play a key role in the disease process. Activation of the alternative complement cascade is believed to be involved in the pathogenesis of age-related macular degeneration (AMD), a common cause of central vision loss among individuals over 55 years of age , 2. Earlin vitro and mouse models, Wang et al (2008)). It has been shown that while both factors co-precipitate with Aβ, the inhibitory effect of Aβ is related to CFI and not to CFH [in vitro.CFI plays a key role in the regulation of all complement cascades, alternative, classical and lectin, by channeling C3-derived C3b toward production of iC3b in the breakdown pathway . CFI bioSK933776 , resultet to CFH . Wang ett to CFH  used wesAPOE4 knock in mice fed a high fat and cholesterol-enriched diet, Ding et al [The inhibitory effect of Aβ on CFI is an indication for the potential role of Aβ as in indirect activator of the complement pathway. It is well documented that Aβ is a key component of drusen associated with AMD , 31. Drung et al  showed din vitro assays to quantify CFI bioactivity. We detected no statistical significant differences in CFI bioactivity across these groups. However, we observed a statistically significant linear trend across the disease severity continuum, suggesting progressive increases in systemic CFI bioactivity with disease progression. Recently, in a 2 independent cohort study in patients with AMD [in vitro data recapitulate the role of Aβ in inhibiting CFI bioactivity, and therefore induce a push towards activation of the complement system and formation of MAC locally in the eye.Since these studies were designed to support the development program of GSK933776 in geographic atrophy, we also used the CFI bioactivity assay to assess whether concentrations and, more importantly, CFI bioactivity changes throughout the AMD disease continuum using a cohort of subjects with various stages of AMD. Subjects included normal (no-AMD) and AMD, based on AREDS criteria . These iwith AMD , we havewith AMD . Althougwith AMD , 36. Folin vivo.Using the same methodology, we also studied the effects of anti-Aβ mAb, GSK933776, on CFI bioactivity in a few achieved samples from subjects from phase 1 trial of anti-Aβ systemic therapy for AD. Plasma samples were collected around the second and third infusion periods see . Interesin vitro assays described in this study have allowed efficient and accurate quantification of CFI bioactivity. The methodology could be applied to RPE cells and to determine whether or not RPE-generated CFI is adversely affected by local Aβ deposits. Interestingly, our clinical data show that in AMD, systemic CFI may not robustly engage in pro-inflammatory signaling of the alternative complement pathway, and perhaps these pathways are more robust directly at the ocular tissue level where Aβ concentrations are higher. Recent reports indicating that CFI polymorphisms are associated with AMD [The with AMD , 11, 37


Leontowicz identifies the topic of argumentation as a critical competency for medical students and practicing physicians, and suggests that the topic be included as part of medical education programs.The word argumentation, as typically defined, involves the integration of two components: the first is critical/creative thinking, and the second is effective communication and messaging. Since these two components are both typically included within existing medical curricula, the topic of argumentation could involve enriched learning activities which build upon and integrate these two components.I have one suggestion regarding definitions. The word argumentation is often equated with debate. In fact however, debate represents one important form of argumentation, but is by no means the only one. Argumentation can also be employed in less adversarial forms of discourse such as dialogue, discussion, dialectics, devil’s advocate, and others. Each of these forms of discourse have their own specific tone, purpose, and orientation, and each may be appropriate, or not, in a given social context. For example, the adversarial tone which characterizes a true debate might be appropriate in a detached academic scholarly setting, but may be completely inappropriate conversation between a doctor and a patient.Argumentation can also be employed in constructing proposals, and in other forms of persuasive communication such as policy advocacy. So I would suggest focusing on a broad conception of argumentation, and identify debate as simply one context within which argumentation competencies can be learned and applied. Related competencies might include the ability to define each of the above forms of discourse  to explain how they differ, and to identify situations under which each might be appropriate.The topic of argumentation is also related to other curriculum topics such as shared decision-making, evidence-based practice, and clinical reasoning. It may thus be possible to integrate argumentation into existing learning activities involving these closely related topics as well.Second, the topic of argumentation as broadly conceived has a potential place in each of the seven CanMEDS roles. It may thus be useful for the purpose of comprehensive curriculum planning, to look at each of the CanMEDS roles, and to identify where and how the topic of argumentation might fit.constructive criticism and socially appropriate argumentation must be respected. Implications for curriculum design could include the distinction between being right and being helpful. Curriculum design should also embody the principle that the greater the degree of cognitive/sociopolitical conflict in a given social context, the greater the need for sensitivity, compassion, and psychological safety.Third, as the author indicates, communication and messaging represent potentially powerful psychosocial interventions. A well formulated, logically correct, and accurate statement – the essence of critical thinking – no matter how well intended and how accurately delivered, always carries with it the potential to do more harm than good. The potential for argumentation to have unintended negative consequences thus needs to be recognized, and the importance of Fourth, personality variables play a significant role in the way argumentation is delivered and received. Implications for medical schools could include discussions of variables such as tolerance for conflict, tolerance for ambiguity, sensitivity to criticism, and how attention to these personality traits can help ensure positive learning outcomes.Finally, in an era where the need for deep, meaningful, transformational change in healthcare and medical systems is becoming more and more urgent, the role of argumentation in change leadership should be underscored. This could involve more deeply integrating the topic of argumentation into the collaborator, leader-manager, and advocate roles. This might be effectively accomplished through various forms of experiential learning such as participatory educational theatre, which have been shown to be effective for cultivating competencies pertaining to capacity in conversational change leadership.Please note that these suggestions are meant to be neither definitive nor exhaustive, but rather simply to illustrate a few ways in which the topic of argumentation could be developed and effectively integrated across the full spectrum of medical and allied health professions education.I therefore hope that the author’s letter on the lost art of argumentation will serve to start a rich and productive conversation, and thus help to give this topic the attention that it deserves, thereby supporting the development of effective clinicians, and physician-leaders, for addressing the many complex health-related challenges of the next decade.Detailed references addressing the themes discussed in this commentary are available from the author.For an excellent review addressing the topics of strategic messaging, interpersonal influence and conversational change leadership, the following work is recommended:I’m right and you’re an idiot: the toxic state of public discourse and how to clean it up. Gabriola Island, BC, Canada: New Society Publishers, 2016.Hoggan J, Litwan G.


We demonstrate that the population of the molecular rotational state through a stimulated impulsive Raman excitation can be controlled by tailoring the femtosecond laser pulse with a V-style phase modulation. The results show that, by precisely manipulating the modulation parameters, both the odd and even populations of the molecular rotational state can be completely suppressed or reconstructed. Meanwhile, the relative excitation between the odd and even populations can be obtained. Finally, we show that field-free molecular alignment can be controlled due to the modulation of the molecular rotational state populations. The bPulse shaping techniques have been notably applied to exercise control on the ground state molecular populations. It features major interest related to the involvement of molecular vibrational or rotational wave packets in a wide area of quantum dynamics. The reason is that the temporal evolution of the rotational wave packets can lead to the macroscopic molecular alignment or orientation along the laser polarization –20. Ther2.E(t), as shown in figure 1a, the molecular transition probability from the rotational states E(t); JE is the energy of the Jth rotational state with h is Planck's constant, B is the molecular rotational constant, c is the speed of light in vacuum, J is the rotational quantum number and Trot is the molecular rotational period with P can be written as:JW is the Boltzmann weight factor with Q is the rotational partition function, k is the Boltzmann constant and T is the molecular rotational temperature.When a molecule is exposed to a weak femtosecond laser field heory as 2.1PJ→J+P is correlated with the laser spectral phase b shows the modulated laser spectrum by the V-style phase modulation with the defined function of Φ(ω) = τ|ω − ω0 − δω|, here τ and δω represent, respectively, the modulation depth and the modulation position, and ω0 is the laser central frequency. With the advent of the ultra-fast pulse shaping technique, the programmable 4f-configuration zero-dispersion pulse shaper combined with a one-dimensional liquid-crystal spatial light modulator has shown to be a well-established arrangement to shape the femtosecond laser pulse in the laboratory. By applying this V-style phase modulation on the liquid-crystal modulator of the femtosecond pulse shaping system, the shaped femtosecond laser pulse can be easily obtained.As can be seen in equation (2.2), the Raman transition probability B = 1.93 cm–1. Thus, the rotational period of the CO molecule can be calculated as Trot = 1/(2Bc) ≈ 8.64 ps. The central frequency of the laser pulse is assumed as ω0 = 12 500 cm–1, corresponding to the central wavelength of 800 nm, and the spectral bandwidth  is 200 cm–1. In the calculation, total 50 rotational states are considered to ensure covering all the thermal and dynamic rotational levels distribution.In our theoretical simulation, the CO molecule is used as the example, and the molecular rotational constant is 3.Podd (a), Peven (b) and P (c) as a function of the modulation depth τ and the modulation position δω at the rotational temperature T = 300 K. Here, Podd, Peven and P represent, respectively, the contributions of the odd, even and total rotational states to the population. One can see that both Podd and Peven and then P are strongly modulated by varying the parameters of the modulation depth τ and the modulation position δω. The transition probability Podd is suppressed at τ = 1140 fs (or 3180 fs) and reconstructed at τ = 1020 fs (or 3300 fs) while Peven is suppressed at τ = 1020 fs (or 3300 fs) and reconstructed at τ = 1140 fs (or 3180 fs). This means relative excitation between the populations of the odd and even rotational state can be obtained by scanning the modulation depth τ. Both the Podd and Peven are fully suppressed at τ = 2160 fs and reconstructed at τ = 4320 fs and so is the total transition probability P. But the transition probability at τ = 4320 fs cannot be fully reconstructed as that induced by the transform-limited laser pulse (i.e. τ = 0). Meanwhile, one can find that the modulation of both the positive and negative δω has the same effect on the transition probabilities. When the modulation position δω is δω = 0, the control effect is most obvious. To further clearly observe the manipulations of the spectral phase modulation to the transition probabilities, we present Podd (red dashed line), Peven (blue dotted line) and P (green solid line) as a function of the modulation depth τ with the modulation position δω = 0, and the result is shown in Podd, Peven and P can be relatively excited (i.e. around τ = 1080 fs and 3240 fs), suppressed (i.e. τ = 2160 fs) or reconstructed (i.e. τ = 4320 fs) by precisely manipulating the modulation depth τ and the modulation position δω. Therefore, we can conclude that the V-style spectral phase modulation provides an effective method to control the transition probability, i.e. the population of the molecular rotational state.τ determines the time separation Δt of the two sub-pulses with Δt = 2τ, and the absolute value of the modulation position τ is 1080, 2160, 3240 and 4320 fs, the time separation between the two sub-pulses is Δt = 2τ = 2.16, 4.32, 6.48 and 8.64 ps, corresponding to the quarter, half, three-quarters and full molecular rotational period . This means the relative excitation between the Podd and Peven can be obtained when the separation of the two sub-pulses is around a quarter or three-quarters of the molecular rotational period (i.e. Trot/4 or 3Trot/4), and both the Podd and Peven can be completely suppressed or reconstructed when the separation of the two sub-pulses is half or full of the molecular rotational period (i.e. Trot/2 or Trot). The transition probability cannot be fully reconstructed as that induced by the transform-limited laser pulse due to the spectral phase shaping induced intensity reduction. On the other hand, because the positive and negative δω with the same absolute value δω. The control effect is most obvious for δω = 0 due to the fact that two sub-pulses have equal intensity, i.e. the relative intensity ratio is 1 : 1 in this case.In order to explore the physical control process of the manipulation of the transition probability by the V-style spectral phase modulation, next we analyse the temporal intensity profile of the shaped laser pulse. As described in detail elsewhere ,22, two 13 W cm−2, and the molecular rotational temperature is 300 K.When a molecule is excited with a laser pulse, a coherent rotational wave packet is created from the contributions of the populated molecular rotational states by the interaction between the anisotropic polarizability of the molecule and the polarized laser field. The evolution of the coherent rotational wave packet then leads to the macroscopic molecular alignment along the direction of the laser polarization ,24. As s2θ〉 (green solid lines) induced by V-shaped laser pulse with the modulation position δω = 0 and the modulation depth τ = 1020 fs (a), 1140 fs (b), 2160 fs (c) and 4320 fs (d), together with the contributions of the odd (red dashed lines) and even (blue dotted lines) rotational state and the temporal intensity profile of the V-shaped laser pulse (olive dash-dotted lines). As can be seen in figure 4a,b, when τ = 1020 fs (or 1140 fs), corresponding to the separation of the shaped two sub-pulses 2.04 ps (or 2.28 ps), the molecular alignment of the τ = 2160 fs, corresponding to the separation of the shaped two sub-pulses 4.32 ps (i.e. Trot/2), both of the molecular alignment τ = 4320 fs, corresponding to the separation of the shaped two sub-pulses 8.64 ps (i.e. Trot), both of the molecular alignment 4.In conclusion, we demonstrate in this paper the control of the molecular rotational state populations and, further, the rotational dynamics by the pulse shaping technique-based coherent control. The excitation femtosecond laser pulse is tailored by the well-defined V-style spectral phase function. It is shown that, by precisely manipulating the phase modulation parameters, both the odd and even populations of the molecular rotational state can be completely suppressed or reconstructed. The relative excitation between the odd and even rotational components of the wave packet can be obtained. Finally, we show that field-free molecular alignment can be controlled together due to the modulation of the molecular rotational state populations.


Vibrio vulnificus is a Gram-negative halophilic bacterium commonly found in warm coastal waters. The bacterium can cause severe gastroenteritis from consumption of raw seafood as well as wound infections and necrotizing fasciitis, with mortality rates for sepsis and wound infection at 50% and 17%, respectively [V. vulnificus is responsible for the most deaths caused by Vibrios [V. vulnificus is expanding due to global warming and rising sea temperatures, such that the incidence of infection has risen worldwide [V. vulnificus in previously unaffected regions, suggesting there may be future increase of infections [ Vibrios . Additioorldwide . Even mofections .V. vulnificus are relatively rare in food and the environment.Risk factors for infection include advanced age, male gender, and underlying disease, particularly liver cirrhosis, immunodeficiency, diabetes, and hemochromatosis . HoweverV. vulnificus is a diverse bacterial species [V. vulnificus pathogenesis. Discovery and characterization of these factors may then facilitate identifying potentially deadly strains. However, this requires researchers to address critical questions regarding V. vulnificus human pathogenesis in the context of its natural environment.As  species , the speV. vulnificus is naturally competent and frequently exchanges DNA via horizontal gene transfer (HGT), complicating classification systems [V. vulnificus into biotypes, with Biotype 1 (BT1) originally composed of human clinical and related environmental strains, Biotype 2 (BT2) containing mostly eel pathogens, and Biotype 3 (BT3) representing a recent outbreak of systemic hybrid strains geographically limited to Israel [vcg into Clinical (C) and Environmental (E) types [For years, researchers have attempted to identify genotypic or phenotypic markers to classify this species into “pathogenic” or “non-pathogenic” strains. However, this has proven difficult due to the nature of the bacterium:  systems . One meto Israel , 6. BT1 E) types .V. vulnificus is in linkage disequilibrium, meaning there is a nonrandom association of alleles at multiple loci [This split of BT1 into two lineages was apparent also by multilocus sequence typing (MLST) , with mople loci . AdditioV. vulnificus strains suggested two ecotypes, A and B [V. vulnificus pathogenicity.A separate whole-genome SNP analysis of hundreds of clinical and environmental  A and B . Unexpec A and B  and whol A and B , most of A and B  and thatV. vulnificus infections. There have been several proposed answers to this question.Because of the abundance of strains and classification systems in addition to an ability to rampantly undergo recombination, development of a rapid identification method, for example in commercial seafood, would be a useful strategy to combat Vv toxin (RtxA1). Due to HGT, the rtxA1 gene and its encoded toxin can be re-arranged into at least seven different combinations of effector domains, with each effector conferring a distinct cytotoxic activity [A widely accepted virulence factor is the large composite MARTXactivity . Thus, iactivity . TherefoV. vulnificus is known to vary expression of CPS via phenotypic conversion, switching from a virulent opaque (Op) colony phenotype to a translucent (Tr) phenotype at a frequency of 10−3 to 10−4 [V. vulnificus pathogenesis, all strains can switch, except that C-type strains exhibit a significantly lower rate of conversion from Op to Tr phenotypes than E-type strains. This suggests phase variation is more related to environmental survival than to human pathogenicity [The capsular polysaccharide (CPS) is a virulence factor that has differential expression between strains. Unencapsulated strains are less virulent than their encapsulated counterparts, which are more invasive in subcutaneous tissue infections than nonencapsulated strains . Further to 10−4 . Howevergenicity  and is nV. vulnificus also variably produces sialic acid-like molecules presented as modifications to the lipopolysaccharide, which likely evolved to facilitate a marine environmental lifestyle. However, these modifications show significant benefit to survival during bloodstream infections and dissemination in vivo [V. cholerae was shown to uptake host-derived sugars, specifically sialic acid, giving this pathogen a competitive advantage in intestinal colonization. This is an important aspect of pathogenicity to be investigated further, especially in pathogens capable of gastrointestinal infection, including V. vulnificus [V. vulnificus, since these host-like molecules are required for full motility and biofilm formation, providing a benefit for bacterial survival in the bloodstream [ in vivo . In addilnificus . On the odstream .V. vulnificus virulence factor to human pathogenesis can be postulated as a useful typing scheme to identify virulent versus nonvirulent strains.Overall, in comparison to other pathogens, very few factors have been tested for a role in pathogenesis across a broad distribution of strain isolates. Thus, given the genomic diversity and disequilibrium, more research is needed before direct linkage of any single V. vulnificus produces an N-acetylglucosamine (GlcNAc)-binding protein A (GbpA), which mediates bacterial adhesion to chitin [V. vulnificus and other bacteria [V. cholerae has also been shown to facilitate binding to mucin, which has similar carbohydrate subunits to chitin [V. vulnificus colonization. In fact, V. vulnificus GbpA is also a mucin-binding protein and is essential for pathogenesis in a mouse model [V. vulnificus strains integrate into marine aggregates (chitin) more efficiently than C-type strains. This study also found that expression of gbpA was significantly higher in C-type strains [V. vulnificus human pathogenesis. Further understanding of selective forces that drive environmental pathogenesis versus commensal colonization of aquatic host could also be useful in discerning the basis of pathogenic emergence (Alternatively, one might consider how this bacterium exists in the environment, as an environmental host is likely the selective force for recurrent emergence of pathogenic strains through HGT. o chitin . This chbacteria . The Gbpo chitin . Mucin ise model . Interes strains . While mmergence .V. vulnificus expanding due to global warming, this bacterium is becoming more important as a human pathogen. While previous classification systems are helpful in classifying strains to biotype, they have not been predictive of pathogenic potential. Overall, an overarching question is how to track human pathogenic strains rapidly enough to detect harmful variants within estuarine waters and seafood? The answer may lie in the continued identification of more virulence factors beyond those already known (With the geographical area impacted by dy known . This co


The rapidly expanding corpus of medical research literature presents major challenges in the understanding of previous work, the extraction of maximum information from collected data, and the identification of promising research directions. We present a case for the use of advanced machine learning techniques as an aide in this task and introduce a novel methodology that is shown to be capable of extracting meaningful information from large longitudinal corpora and of tracking complex temporal changes within it. Our framework is based on (i) the discretization of time into epochs, (ii) epoch-wise topic discovery using a hierarchical Dirichlet process-based model, and (iii) a temporal similarity graph which allows for the modelling of complex topic changes. More specifically, this is the first work that discusses and distinguishes between two groups of particularly challenging topic evolution phenomena: topic splitting and speciation and topic convergence and merging, in addition to the more widely recognized emergence and disappearance and gradual evolution. The proposed framework is evaluated on a public medical literature corpus. Recent years have witnessed a remarkable convergence of two broad trends. The first of these concerns information, i.e. data, rapid technological advances coupled with an increased presence of computing in nearly every aspect of daily life, have for the first time made it possible to acquire and store massive amounts of highly diverse types of information. Concurrently and in no small part propelled by the environment just described, research in artificial intelligence—in machine learning –5, 8, daLarge amounts of highly heterogeneous data types are pervasive in medicine. Usually the concept of so-called big data in medicine is associated with the analysis of Electronic Health Records , 41, 43,temporally locally static.A limitation of most models described in the existing literature lies in their assumption that the data corpus is static. Here the term ‘static’ is used to describe the lack of any associated temporal information associated with the documents in a corpus—the documents are said to be exchangeable . HoweverIn this section, we introduce our main technical contributions. We begin by reviewing the relevant theory underlying Bayesian mixture models and then explain how the proposed framework employs these for the extraction of information from temporally varying document corpora.Mixture models are appropriate choices for the modelling of the so-called heterogeneous data whereby heterogeneity is taken to mean that observable data is generated by more than one process (source). The key challenges lie in the lack of observability of the correspondence between specific data points and their sources and the lack of a priori information on the number of sources .Bayesian non-parametric methods place priors on the infinite-dimensional space of probability distributions and provide an elegant solution to the aforementioned modelling problems. Dirichlet Process (DP) in particular allows for the model to accommodate a potentially infinite number of mixture components : 1\docuγ,H) is defined as a distribution of a random probability measure G over a measurable space A1,A2,…,Ar) of Θ the random vector (G(A1),…,G(Ar)) is a Dirichlet distribution with parameters (γH(A1),…,γH(Ar)). A Dirichlet process mixture model (DPM) is obtained by associating different mixture components with atoms ϕk and assuming f(.) is the kernel of the mixing components  the CDF operating point, we prune the edge between topics ϕj,t and ϕk,t+1 iff Our methodology comprises two stages. Firstly, we consider all inter-topic connections present in the initial fully connected graph and extract the empirical estimate of the corresponding cumulative density function (CDF). Then, we prune the graph based on the operating point on the relevant CDF. In other words, if We now analyse the performance of the proposed framework empirically on a large real-world data set.We used the PubMed interface to access the US National Library of Medicine and retrieve from it scholarly articles. We searched for publication on the autism spectrum disorder (ASD) and the metabolic syndrome (MetS) using respectively the keyword ’autism’ and the keyphrase ‘metabolic syndrome’ and collected only those achieved papers which were written in English. The earliest publications found were that by Kanner  on ASD aThe raw data collected from PubMed is in the form of free text. To prepare it for automatic analysis a series of ‘pre-processing’ steps are required. The goal is to remove words which are largely uninformative, reduce dispersal of semantically equivalent terms, and thereafter select terms which are included in the vocabulary over which topics are learnt.We firstly applied soft lemmatization using the WordNet®; lexicon  to normaζ=0.9.We stared evaluation by examining whether the two topic relatedness measures (BHD and KLD) are capturing different aspects of relatedness. To obtain a quantitative measure, we looked at the number of inter-topic connections formed in respective graphs both when the BHD is used as well as when the KLD is applied instead. The results were normalized by the total number of connections formed between two epochs, to account for changes in the total number of topics across time. Our results for the MetS corpus are summarized in Fig. x coordinate of a point corresponding to a pair of topics and the latter as its y coordinate. The scatter of data in the plot corroborates our previous observation that the two similarity measures indeed do capture different aspects of topic behaviour.To get an even deeper insight into the contribution of the two relatedness measures, we examined the corresponding topic graphs before edge pruning. The plot in Fig. We also performed extensive qualitative analysis which is necessitated by the nature of the problem at hand and the so-called semantic gap that underlies it. In all cases, we found that our algorithm revealed meaningful and useful information as verified by medical experts on the ASD and MetS. This observation is consistent with previous reports in the literature using an algorithm with a similar structure . The keyOur final contribution comprises a web application which allows users to upload and analyse their data sets using the proposed framework. A screenshot of the initial window of the application when a data set is loaded is shown in Fig. The application allows a range of powerful tasks to be performed quickly and in an intuitive manner. For example, the user can search for a given topic using keywords (and obtain a list of topics ranked by the degree to which they match the query), trace the origin of a specific topic backwards in time, or follow its development in the forward direction, examine word clouds associated with topics, display a range of statistical analyses, or navigate the temporal relatedness graph freely. Some of these capabilities are showcased in Fig. In this work, we presented a case for the importance of the use of advanced machine learning techniques in the analysis and interpretation of large longitudinal text corpora such as specialized medical literature. We described a novel framework based on non-parametric Bayesian techniques which is able to extract and track complex, semantically meaningful changes to the topic structure of a longitudinal document corpus. Moreover, this work is the first to describe and present a method for differentiating between two types of topic structure changes, namely topic splitting and what we termed topic speciation. Experiments on a large corpus of medical literature concerned with the metabolic syndrome was used to illustrate the performance of our method. Lastly, we developed a web application which allows users such as medical researchers to apply our method for their analysis.The present work illuminates several promising avenues for future work. Firstly, considering the important difference between topic interaction phenomena such as topic splitting and speciation, and topic merging and convergence, we intend to focus on developing an information-theoretic framework which is capable of handing all of the aforementioned interactions in a unified manner. Such a framework would confer the additional benefit of removing the need for the free parameter necessitated by our method—the proportion of edges retained in our topic relatedness graph. The second direction of research we intend to pursue concerns the free parameters used to handle the temporal dimension. Although it is prima fasciae clear that the duration of the epoch length used to discretize time, as well as the degree of overlap between consecutive epochs, should be dependent on the dynamics of a particular longitudinal corpus , currently, there are no principled methods for choosing the values of these parameters. Lastly, we believe that the nature of the problem at hand, that of temporal topic modelling, offers opportunities for a more objective manner of performance assessment, in contrast to the usual post hoc, qualitative approaches used in the existing literature on topic modelling.


Mikania micrantha H.B.K. (Asteraceae) is one of the world’s most invasive weeds which has been rapidly expanding in tropical Asia, including China, while its close relative M. cordata, the only Mikania species native to China, shows no harm to the local ecosystems. These two species are very similar in morphology but differ remarkably in several ecological and physiological traits, representing an ideal system for comparative analysis to investigate the genetic basis underlying invasion success. In this study, we performed RNA-sequencing on the invader M. micrantha and its native congener M. cordata in China, to unravel the genetic basis underlying the strong invasiveness of M. micrantha. For a more robust comparison, another non-invasive congener M. cordifolia was also sequenced and compared.M. micrantha, M. cordata, and M. cordifolia, respectively. Phylogenetic analyses and divergence time dating revealed a relatively recent split between M. micrantha and M. cordata, i.e., approximately 4.81 million years ago (MYA), after their divergence with M. cordifolia (8.70 MYA). Gene ontology classifications, pathway assignments and differential expression analysis revealed higher representation or significant up-regulation of genes associated with photosynthesis, energy metabolism, protein modification and stress response in M. micrantha than in M. cordata or M. cordifolia. Analysis of accelerated evolution and positive selection also suggested the importance of these related genes and processes to the adaptability and invasiveness of M. micrantha. Particularly, most  positively selected genes found in M. micrantha could be classified into four groups, i.e., energy acquisition and utilization (10 genes), growth and reproduction (13 genes), protection and repair (34 genes), and signal transduction and expression regulation (20 genes), which may have contributed to the high adaptability of M. micrantha to various new environments and the capability to occupy a wider niche, reflected in its high invasiveness.A total of 52,179, 55,835, and 52,983 unigenes were obtained for M. micrantha and its non-invasive congeners, M. cordata and M. cordifolia. A comparison of their transcriptomes provided insights into the genetic basis of the high invasiveness of M. micrantha.We characterized the transcriptomes of the invasive species The online version of this article (10.1186/s12864-018-4784-9) contains supplementary material, which is available to authorized users. When a species is introduced into a new environment, it either does not adapt and quickly goes extinct or persists and establishes in the new environment. Species that live on may become competitive and colonize new areas and niches at high rates, eventually becoming successful invaders. Since many invasive species pose serious threat to native biodiversity and cause severe economic loss in the affected regions –5, bioloMikania micrantha H.B.K. is a perennial vine that belongs to the family Asteraceae. This species is native to tropical America, and has been recognized as one of the world’s most notorious invaders [M. micrantha can rapidly colonize disturbed habitats, while competing with the native vegetation and retarding their growth [Mikania species, most of which native to tropical America, M. micrantha is the only species that has spread from the New World to the Old World, and now coexists with its native congener in the Old World, M. cordata (Burm. f.) B. L. Robinson [M. micrantha in the Old World could be traced back to 1884 in Hong Kong, and after then, M. micrantha has expanded in southern China, covering Hong Kong and the Guangdong and Taiwan provinces [M. cordata is the only Mikania species native in South China, distributed in Yunnan, Hainan, and Taiwan provinces [M. micrantha and M. cordata appear to be quite similar to each other in morphology and life style, they show considerable differences in many ecological traits, such as niche requirements, eco-adaptability, and most importantly, invasiveness. As observed in Taiwan where they co-exist, for example, while M. micrantha run rampant and becomes a major pest of crops and forests [M. cordata grows slowly and exhibits no harm to other native species and the local ecosystems [invaders , 21. Witr growth , 22–24. r growth –29. AmonRobinson –33. The rovinces , 34, 35. forests , 37, M. osystems .M. micrantha were mainly performed on the species alone and/or focused on its ecological impacts and physiological traits [M. micrantha and its non-invasive congeners that share morphological and life-history traits is lacking [M. micrantha compared to M. cordata [M. micrantha. Studies on habitat preferences have revealed that M. micrantha can invade a relatively wider light niche, while M. cordata can only tolerate shady environments in tropical and subtropical China [M. micrantha has also been found to be able to achieve extremely fast growth and reproduction in new environments [M. micrantha to different habitats with differential expression of genes involved in high light intensity stress response, protein folding, and oxidative processes by comparing native and introduced M. micrantha populations [M. micrantha transcriptome, constrained by the sequencing technology and bioinformatics tools developed then, the size and quality of data obtained at that time was relatively limited for a thorough study [M. micrantha from the physiological and ecological perspectives , the underlying molecular mechanisms remains barely explored.Previous studies on the invasion of l traits , 39–49.  lacking , 46, 47. cordata , 28, 47,al China , 51. M. ronments , hintingronments , 54 follgh study . AlthougM. micrantha and M. cordata thus offers an ideal system for comparative analysis. To further exclude possible biases due to stochastic factors or phylogenetic effects from comparing only two species, the inclusion of a third, closely related, non-invasive congener in such a study would aid in the reduction of these stochastic effects to more accurately identify the genomic/transcriptomic features that is unique to the invasive species. In this study, we performed RNA-sequencing (RNA-seq) and assembled the transcriptomes of the invasive plant M. micrantha and its coexisting non-invasive congener M. cordata from China, and another allopatric non-invasive congener M. cordifolia [M. micrantha. The objective of this study was to use large-scale datasets to identify the genetic basis of invasiveness observed in M. micrantha, which would be valuable for further unraveling the molecular mechanisms of invasion success and contribute to the control efforts of the species. In particular, we explored: (1) the degree of sequence differentiation among M. micrantha, M. cordata, and M. cordifolia, and the divergence times of these three species; (2) the differences in gene components and expression patterns between the transcriptomes of M. micrantha and M. cordata/M. cordifolia and their functional roles; and (3) the evolutionary signatures of M. micrantha genes, i.e., the types of genes with putatively accelerated nonsynonymous divergence and genes that show signals of positive selection, and their potential correlations with the adaptability and invasiveness of M. micrantha.Different from intraspecific analysis on gene expression or other physiological characteristics, which has been a common method to investigate the adaptive strategies of invasive species , 49, 56,rdifolia , 58. In M. micrantha, M. cordata, and M. cordifolia leaf transcriptomes, respectively  of 97.51, 92.34 and 97.37%, respectively. Based on these clean reads, 75,179, 76,344, and 93,872 contigs were de novo assembled for M. micrantha, M. cordata, and M. cordifolia, respectively, which further resulted in 62,145, 56,409, and 53,330 non-redundant sequences with N50 values of 975, 1399, and 1561 bp, respectively, suggesting good integrity of the three assemblies. All sequencing reads generated in this study have been deposited in NCBI Sequence Read Archive (SRA) under the accession numbers SRX3520663- SRX3520665.Using RNA-seq, a total of 41.26, 48.76, and 46.76 million paired-end reads were generated from the M. micrantha, M. cordata, and M. cordifolia, 39,511, 30,380, and 39,893, respectively, of the non-redundant sequences had hits in NR databases. As expected, most sequences  of the three assemblies were of plant origins. A considerable amount of fungus-origin sequences was identified from the M. micrantha assembly  while only some were found in the M. cordata  and M. cordifolia  assemblies  of each non-redundant sequence based on its top-hit BLAST result against the NCBI non-redundant protein (NR) database. The BLAST results showed that for M. micrantha, M. cordata, and M. cordifolia, 99.94, 99.24, and 98.03% sites in total were covered by at least one high-quality base , equivalent to an accuracy of ≥99.9% at these sites; and 77.56, 61.03, and 76.04% sites were covered at least ten times by high-quality bases, equivalent to an accuracy of ≥99.99%  and six other species from the Heliantheae alliance, including two species from Eupatorieae (Chromolaena odorata and Stevia rebaudiana), two species from the sister tribe Heliantheae (Ambrosia artemisiifolia and Helianthus annuus) and one species for each of the sister tribe Madieae (Arnica montana) and Helenieae , using Tragopogon dubius (Cichorioideae) as an outgroup. As observed from the phylogenetic tree , and that M. micrantha and M. cordata split approximately 4.81 MYA (95% CI = 4.0–5.7 MYA).To investigate the phylogenetic relationships and divergence times for ree Fig. , M. micrMikania species, their differences in sequences were also measured. Direct comparison on protein sequences of orthologous gene pairs showed an overall similarity of approximately 94.44% for M. micrantha-M. cordata, slightly higher than that for M. micrantha-M. cordifolia (93.67%) and M. cordata-M. cordifolia (93.65%)  or between M. cordata and M. cordifolia (Ka = 0.0195). As for the silent site divergence, the median synonymous substitution rate (Ks) was 0.0560 between M. micrantha and M. cordata, 0.0976 between M. micrantha and M. cordifolia, and 0.0980 between M. cordata and M. cordifolia , 31,944 (57.21%), and 40,991 (77.37%) of M. micrantha, M. cordata, and M. cordifolia unigenes had matches in at least one database , 29,829 (53.42%), and 39,576 (75.00%) unigenes with matches in the NR database, 15,377 (51.37%), 24,624 (82.55%), and 33,820 (85.46%) unigenes matched sequences from their respective top ten species, respectively , 27,561 (49.36%), and 31,178 (58.85%) unigenes had GO terms assigned in the three main categories, including 14,388, 22,144, and 24,909 unigenes with terms from “Biological Process”, 9226, 20,092, and 26,490 from “Cellular Component”, and 17,045, 23,334, and 23,811 from “Molecular Function”, respectively. While the gene distribution patterns (at level two) were generally similar among the three transcriptomes , P < 0.05), with most of them (213 out of 235) being shared , “photosystem II oxygen evolving complex” , “transcription factor activity, protein binding” , “protein binding involved in protein folding” , wound healing , and “metaphase/anaphase transition of cell cycle” .Based on the above BLAST search results, GO annotations were then retrieved. For mes Fig. , detaile 19,945 3.22%, 27, 19,945 3.22%, 27,Mikania species, KEGG pathway assignments and detailed statistical analysis were performed. For M. micrantha, M. cordata, and M. cordifolia, 10,787 (20.67%), 9164 (16.41%), and 23,381 (44.13%) unigenes were assigned 43 KEGG pathway classes comprising 349, 339, and 350 subclass pathways, respectively . Despite the more similar gene distribution patterns between M. micrantha and M. cordata than between these two species with M. cordifolia, which was in accordance to their phylogenetic relationships as mentioned above, it was worth noting that M. micrantha pathway-involving genes showed significantly higher representation than M. cordata and M. cordifolia genes in photosynthesis , a subclass pathway involved in the energy metabolic pathway.To investigate the active biological pathways of the three Mikania species were subjected to a search against the KOG database. For M. micrantha, M. cordata, and M. cordifolia, 11,127 (21.32%), 11,497 (20.59%) and 29,854 (56.35%) unigenes were classified into 26 KOG categories, respectively , followed by “Translation, ribosomal structure and biogenesis”  and “Signal transduction mechanisms” , while that for M. cordifolia was “Signal transduction mechanisms” , followed by “Posttranslational modification, protein turnover, chaperones”  and “Transcription” .For more comprehensive annotation, all unigenes of the three ly Table . SimilarMikania species, the RNA-seq data of M. micrantha, M. cordata, and M. cordifolia were separately mapped to their reference transcriptomes , and genes differentially expressed between M. micrantha and M. cordata/M. cordifolia were identified. Under a priori replicate variance value of 0.2 and an FDR cut off of 0.01  and one Madieae  species was identified. These genes were functionally grouped into 642 different categories according to their GO annotations and used to detect the types of genes showed accelerated evolution. To infer average rates of protein evolution for different GO categories between invasive and non-invasive Mikania species, both M. micrantha-M. cordata and M. micrantha-M. cordifolia ortholog pairs were compared. For those between non-invasive Mikania species, M. cordata-M. cordifolia ortholog pairs were compared. Preliminary analysis on Ka/Ks ratios of nonsynonymous-to-synonymous substitutions for each group of genes revealed elevated Mikania pairwise Ka/Ks values in diverse functional categories  while being absent in those between non-invasive Mikania .To examine the type of genes that showed accelerated evolution and also identify genes under positive selection pressure for the three M. micrantha-M. cordata than in M. cordata-M. cordifolia , arguing that for these categories the higher average Ka/Ks in between invasive and non-invasive Mikania is mainly contributed by certain genes instead of the combined effects of a large fraction of all genes involved. This implication was further supported by the statistical tests on Ka/Ks distributions, which revealed that the M. micrantha-M. cordata/M. cordifolia distributions were not significantly different from the M. cordata-M. cordifolia distributions (P > 0.05 by the Wilcoxon signed-rank test).Further statistical comparisons showed that the average Ka/Ks in several categories, e.g., carbon fixation (0.61 vs. 0.37), chlorophyll biosynthetic process (0.49 vs. 0.26), cellular response to stress (2.77 vs. 1.66), DNA repair (11.38 vs. 5.55), chromosome organization (0.23 vs. 0.19), and transcription cofactor activity (0.52 vs. 0.43), was significantly higher in M. micrantha genes than those of M. cordata and M. cordifolia, as revealed by their median Ka/Ks values of all orthologous genes calculated using the free-ratio model , which can also be observed through the distributions of these Ka/Ks values that showed relatively more M. micrantha genes in the very low Ka/Ks range  and of special significance for the species. To provide full lists of candidate genes that have been under positive selection in the invasive M. micrantha and understand their functional importance, positively selected genes (PSGs) were identified along each Mikania branch using the improved branch-site likelihood ratio test. In total, 213, 225, and 178 PSGs were identified, with 112, 114, and 65 genes left after correcting for multiple tests  for M. micrantha, M. cordata, and M. cordifolia, respectively  in categories including chlorophyll biosynthesis, ATPase activity, response to stimulus, protein folding, and chromosome segregation  were found to be mainly involved in processes of i) energy acquisition and utilization, ii) growth and reproduction, iii) protection and repair, and iv) signal transduction and biological regulation, which might have contributed to the specific traits, e.g., wide eco-adaptability and intense invasiveness, of M. micrantha , with M. cordifolia diverging first , followed by the split of M. micrantha and M. cordata   derived from the late Miocene environmental changes might have driven the subsequent divergence of their ancestral populations and promoted the accumulation of genetic differences that are of benefit for adaptation to their respective habitats. Although the present-day morphological features of M. micrantha and M. cordata are still so similar that it is difficult to distinguish them with the naked eye [M. micrantha and M. cordata, the detected genetic divergence could be the underlying cause for the higher eco-adaptability of M. micrantha, giving it the potential to invade various habitats .For aked eye , the sevM. micrantha and its indigenous sister species M. cordata revealed noteworthy differences between these two species. M. micrantha was found to harbor far more overrepresented GO categories than M. cordata (235 vs. 13). Notably, most of these M. micrantha-enriched functional categories  also had higher representation in M. micrantha when compared to M. cordifolia , and several of them were found to be involved in photosynthesis, energy metabolism, wound healing, protein modification, asexual reproduction, and biological regulation process  , which was, again, identified for M. micrantha in its comparison to M. cordifolia  was much larger than that of down-regulated (751 and 620) in M. micrantha compared with M. cordata or M. cordifolia  for genes in carbon fixation, chlorophyll biosynthesis, cellular response to stress, DNA repair, and transcription regulation  and the distinction of Ka/Ks distribution between species pairs. This could be plausible considering the relatively recent divergence of these Mikania species, especially between M. micrantha and M. cordata.Besides the differences in genetic components as discussed above, characterization of the patterns of sequence divergence across the genomes could further help to clarify the evolutionary processes that gave rise to the distinct features between species, and even the genetic basis and mechanisms of adaptive evolution and speciation –68. RapiM. micrantha (median Ka/Ks = 0.0981) than those in M. cordata and M. cordifolia , these results, combined with the pairwise analysis discussed above, suggested that pronounced molecular evolutionary changes may preferentially imprint on certain genes in M. micrantha while the majority of genes are less likely to be changed. Since genes performing basic cellular functions conserved across different species should have evolved predominantly under constraint [M. micrantha would be very helpful for the better understanding of the molecular correlates for its strong invasiveness. To this end, signatures of positive selection were tested for all orthologous genes along each Mikania branch using the improved branch-site model [P < 0.05), 112 of them showed a significant signature of positive selection after correcting for multiple tests  , 13 genes in growth and reproduction (group II), 34 genes in protection and repair (group III), and 20 genes in signal transduction and biological regulation (group IV)  Fig. , as discM. micrantha, physiological and biochemical studies have demonstrated its greater photosynthesis capability than M. cordata as well as several other associated species [M. micrantha were found to be associated with energy acquisition and utilization  , which in turn provides ample carbon for growth and reproduction [As an energy source and regulatory signal throughout the plant life cycle, light is among the most important environmental factors affecting the optimal growth and development of plants, especially vines –76. The  species , 47, 78.on) Fig. . Particuoduction .M. micrantha is its extremely fast growth and strong reproduction [M. cordata, M. micrantha grows very fast, e.g., the stem tip can elongate up to 20 cm a day under suitable conditions in summer [M. micrantha is comprised of genes involved mainly in cell growth, shoot/root development, seed germination, energy homeostasis , and other hormone-related processes that regulate plant growth and development . M. cordifolia seeds from Hillsborough Co. Florida, USA, were sown in a greenhouse, and one resulting seedling was used for RNA extraction experiments. The taxonomic identification of the plant material was undertaken by Dr. Ying Liu in Sun Yat-sen University. Voucher specimens  were deposited at the herbarium of Sun Yat-sen University (SYS). The seedlings were grown under the same condition for two months before their leaves were used for RNA extraction. One individual was sampled for each species, and total RNA was extracted separately from each individual using an improved CTAB method [Two seedlings, each representing B method  immediatFor each library, raw sequencing reads were first examined using FastQC  for qual− 6). Those with top-hits against sequences from non-plant organisms were excluded from downstream comparative, phylogenetic, and evolutionary analyses; while the remaining sequences were regarded as unigenes derived from the plant (‘unigenes’ hereinafter).After removing contigs with lengths < 200 bp, the longest sequence of each locus was selected to represent each gene, and the resulted sequence set was regarded as the non-redundant set of transcripts. To further improve reliability, the putative origin  of each non-redundant sequence was inferred by homology search against the NCBI non-redundant protein (NR) database  using BWA , and the− 6. Gene Ontology (GO) terms were then assigned based on the top hits following the BLAST2GO [To annotate the assembled unigenes, each unigene sequence was first aligned against the NR database using BLASTX with an E-value cutoff of 1eBLAST2GO  pipelineBLAST2GO . Fisher’M. micrantha, M. cordata, and M. cordifolia, the reads were mapped to their respective unigene sets using BOWTIE2 [M. micrantha-M. cordata and M. micrantha-M. cordifolia species pairs using the EdgeR package [P-values were corrected for multiple testing using the FDR method.For  BOWTIE2 . Only re BOWTIE2 . After e package , which tMikania species, putative orthologs of each species pair were first retrieved based on the bidirectional best hits of their proteome sequences using BLASTP. For each ortholog pair, protein sequences were aligned using ClustalW2 [For each species, the open reading frame and protein sequence of each unigene were obtained based on their BLASTX results against the NR database. To evaluate the sequence divergence among the three lustalW2  with deflustalW2 . The synlustalW2 .Mikania have diverged, transcriptome data of six other species from the Heliantheae alliance  and one species from Cichorioideae  were downloaded from the NCBI database and used in the phylogeny reconstruction and divergence time estimation, referring to the work by Huang et al. (2016) [M. micrantha, M. cordata, M. cordifolia, C. odorata, S. rebaudiana, A. artemisiifolia, H. annuus, A. montana, H. autumnale, and T. dubius) were combined to perform an all-against-all comparison using BLASTP with E-value cutoff of 1e− 10. All similar sequences were subsequently processed and clustered into gene families using OrthoMCL [To reconstruct a phylogeny and estimate the times that the three . (2016) . ProteinOrthoMCL . PutativH. autumnale) from other Heliantheae alliance species was set to 30.7–31.78 MYA, and the divergence of Heliantheae was set to 28.92–29.89 MYA as suggested by the estimation of Huang et al. (2016) [For each of the 456 ortholog groups, multiple alignments of protein sequences were performed using ClustalW2, and the corresponding coding-sequence alignments were obtained accordingly using PAL2NAL. For each species, all coding sequences from the alignments were concatenated to one supergene, based on which, the phylogenetic relationship of these ten species were reconstructed using the maximum likelihood method in PhyML  and a be. (2016) .Mikania species and also identify genes under positive selection, orthologous genes were identified using the bidirectional-best-hits method for the six species . The pairwise likelihood method in PAML was employed to calculate evolutionary parameters for each ortholog of the three Mikania species, including the total numbers of nonsynonymous (A) and synonymous (S) substitutions, Ka, Ks, and their ratio . After assigning GO annotations to these orthologs, the average Ka, Ks, and Ka/Ks values for each GO categories were obtained. To evaluate the statistical significance that the evolutionary rates of a group of genes differ between two species pairs , a 2 × 2 contingency table was built, with the four entries being the total A and S values in either of the two species pairs. Fisher’s exact test was then applied to the table to test statistical significance that evolutionary rates differed between the two species pairs [To examine the type of genes that showed accelerated evolution among the es pairs . To evales pairs .Mikania species using the free-ratio model implemented in the codeml module of PAML, and median values were selected to represent the lineage-specific values since the median is more robust and less influenced by outliers than the mean. To detect candidate genes that have undergone positive selection in M. micrantha, the optimized branch-site model implemented in the codeml module of PAML was used, with M. micrantha as foreground branch and all other branches in the tree as background branches. A likelihood ratio test (LRT) was performed to assess the difference between the results of null and alternative models, and the LRT P-values were further tested using the FDR method with a conservative criterion of 0.05 to correct for multiple comparisons. GO categories with significantly higher representation of PSGs than background levels were detected using the Fisher’s exact test in GOBU. The abundance of each PSG was measured with the normalizing statistic FPKM (fragments mapped per kilobase of exon per million reads mapped) calculated using RSEM [For lineage-specific analysis, values of Ka, Ks, and Ka/Ks were estimated for each ortholog of the three ing RSEM .Additional file 1:M. micrantha, M. cordata, and M. cordifolia assemblies. (PDF 18 kb)Top-hit species categories of the non-redundant sequences of Additional file 2:M. micrantha, M. cordata, and M. cordifolia. (PDF 202 kb)Length (a) and GC (b) distributions of the assembled unigenes of Additional file 3:M. micrantha, M. cordata, and M. cordifolia unigenes. (a) Mapping depth distribution of assembled unigene sequences. Histogram shows the percentages of bases with certain ranges of coverage depth. (b) Identity distribution between assembled unigenes and their corresponding sequences from public databases. (PDF 132 kb)Assessment of per-base sequence accuracy of Additional file 4:Downloaded nucleotide sequences from public databases and their alignment statistics. (PDF 117 kb)Additional file 5:Sanger sequencing-derived sequences and their alignment statistics. (PDF 179 kb)Additional file 6:M. micrantha, M. cordata, and M. cordifolia ortholog pairs. (PDF 35 kb)Sequence comparison of Additional file 7:M. micrantha, M. cordata, and M. cordifolia unigenes. (a) Histogram plot of the data distribution based on search against multiple public databases. (b/c/d) Species/E-value/similarity distributions of top hits for the unigenes based on BLAST search against NCBI non-redundant protein (NR) database. (PDF 414 kb)Similarity search and annotation of Additional file 8:M. micrantha compared with both M. cordata and M. cordifolia. Significance was tested using Fisher’s exact test and control of false discovery rate (FDR) with P-value cutoff of 0.05. (XLSX 37 kb)GO terms significantly enriched in Additional file 9:. (a) M. micrantha-M. cordata. (b) M. micrantha-M. cordifolia. (c) M. cordata-M. cordifolia. Significance was tested using Fisher’s exact test and control of false discovery rate (FDR) with P-value cutoff of 0.05. (XLSX 31 kb)KEGG pathways with genes significantly differed between two speciesAdditional file 10:M. micrantha, M. cordata, and M. cordifolia unigenes. A total of 11,127, 11,497, and 29,854 unigenes, respectively, were grouped into 26 clusters of ortholog group terms. (PDF 74 kb)KOG classification for Additional file 11:Number of differentially expressed genes (DEGs) under different dispersion values. (PDF 49 kb)Additional file 12:M. micrantha compared with both M. cordata and M. cordifolia. (XLSX 246 kb)List of genes that were up- and down-regulated in Additional file 13:M. micrantha. (XLSX 46 kb)GO categories over-represented in the list of up-regulated genes in Additional file 14:a) M. micrantha-M. cordata. (b) M. micrantha-M. cordifolia. (c) M. cordata-M. cordifolia. (XLSX 19 kb)Top 10% GO categories displaying the highest Ka/Ks ratios between two species.  M. micrantha-M. cordata vs. M. cordata-M. cordifolia. (b) M. micrantha-M. cordifolia vs. M. cordata-M. cordifolia. (XLSX 36 kb)Identification of GO categories with accelerated evolution between invasive and non-invasive Additional file 16:M. micrantha, M. cordata, and M. cordifolia. (PDF 41 kb)Distribution of branch-specific Ka/Ks for Additional file 17:a) M. micrantha, (b) M. cordata, and (c) M. cordifolia. (XLSX 39 kb)Positively selected genes identified in  with P-value cutoff of 0.05. (XLSX 22 kb)Functional categories overrepresented among genes under positive selection in