Incorporating the characteristics of the single-layer graphene nanoribbon and the polyaniline molecule, we theoretically design a two-dimensional molecular device and investigate its transport properties by applying nonequilibrium Green's functions in combination with density-functional theory. The calculated results reveal that the arrangements of frontier molecular orbitals and the energy gap between the HOMO and the LUMO of an isolated polyaniline molecule are different between its two isolable states: full reduced leucoemeraldine base and full oxidized pernigraniline base. When a polyaniline molecule connects to two graphene nanoribbons as a two-dimensional molecular device, the conductance of its full oxidized pernigraniline base is much higher than the conductance of its full reduced leucoemeraldine base. The switch ratios of two bases' currents almost maintain a constant value before 0.8 V. In other word, the conductance switch behavior in our device is stable in a big bias region which makes it have a broader application in future logic and memory devices. This new material was first obtained by micro-mechanical cleavage in 20045 cm2/Vs)3 W/mK)69111315Graphene, one of the allotropes of elemental carbon, is a two-dimensional nanomaterial that has between one and ten layers of spBecause of its superior electronic, thermal, and mechanical properties, the graphene has been considered as a promising next-generation conducting material to replace traditional electrode materials in electrical and optical devices in order to reduce the size of the device. So far, the researches on single-layer graphene nanoribbons (GNRs) as a two-dimensional electrode have received increasing attention in experiment1718202122The polyaniline molecule has received considerable attention in recent years due to the controllable electrical conductivity25Firstly, we show the molecular orbitals of an isolated polyaniline at the leucoemeraldine base and the pernigraniline base in To identify the nature of molecular states, we give the density of states (DOS) of a polyaniline molecule and the projected density of states (PDOS) for three nitrogen atoms in Next, we connect a polyaniline molecule to two single-layer zigzag graphene nanoribbons as a device and study its electronic transport properties. The molecular devices are illustrated in 272829In order to explore the difference on conductance of SL and SP, their transmission spectra at the equilibrium state are shown in To explore the physical mechanism of the conductance switch behavior between the device SL and the devices SP, we plot their spatial local density of states (LDOS) on Fermi energy and the electrostatic potential distribution at zero bias in 31323435Moreover, the stable current switch behavior in large bias region is another unique character of our device. To explain it, we plot the transmission as a function of the energy and bias voltage in By applying density-functional theory and non-equilibrium Green's function method, we have investigated the quantum transport properties of a polyaniline molecule and two single-layer graphene nanoribbons as a two-dimensional molecular device. The arrangement of frontier molecular orbitals and the energy gap between the HOMO and the LUMO of a polyaniline molecule are different when the molecule alters among two isolable states by oxidation/reduction. A new broad and high DOS peak was induced by the three nitrogen atoms on full oxidized pernigraniline base. This suggests that the oxidation/reduction could be used to modulate the electronic structures of the polyaniline molecule. Once a polyaniline molecule connecting to two single-layer graphene nanoribbons as a two-dimensional molecular device, the calculated results show the conductance of SP device is much higher than the conductance of SL device. We demonstrate that the alignment between the Fermi energy of the electrode and the frontier molecular orbitals of two bases of the polyaniline molecule is a main reason of the conductance switch behavior in our device. The removing of hydrogen atoms on SP device enhances its spatial local density of states on the molecule and improves the electrostatic potential around the nitrogen atoms, resulting in the transfer of electrons in the junction. For SP device, the three transmission peaks in the vicinity of the Fermi level in turn come into bias window leading to the linear rise of the current. So, the current switch behavior in our device is stable in a big bias region which makes it having a broader potential in future logic and memory devices.ab initio code package based on nonequilibrium Green's functions and density functional theory37L/Rμ is the electrochemical potentials of the left and right electrodes and L/Rf are the Fermi distribution function for left and right electrodes40bV, the chemical potential of left electrode Lμ(bV) = −beV/2 and the chemical potential of right electrode Rμ(bV) = +beV/2. The energy region of the transmission spectrum that contributes to the current integral in the Landauer-like formula is referred to the bias window . The transport coefficient T can be calculated using the well-known formula, T = Tr[ΓL(E)RG(E)ΓR(E)AG(E)], where RG(E) and AG(E) are the retarded and advanced Green's functions, respectively, and The method in this paper is an K.Q.C. performed the device design and theoretical analysis, Z.Q.F. calculated geometrical properties, electronic structures, transmission spectra, and the I-V characteristics. All the authors discussed the results and wrote the manuscript.


The Editorial on the Research TopicSystems Biological Aspects of Pituitary TumorsPituitary adenomas are a category of neoplasms with a high degree of heterogeneity that occur in the central regulatory organ pituitary, which plays important roles in the hypothalamus–pituitary-targeted organ axis systems that impact on important physiological functions of human body –3. RapidHalupczok-Żyła et al.). Vitamin D deficiency and alteration in calcium–phosphate balance are associated with a wide spectrum of diseases, such as cancer, diabetes, cardiovascular disease, and respiratory disease. Data demonstrate that acromegaly patients are at the higher risk of vitamin D deficiency and have a tendency to a lower level of calcium and higher level of inorganic phosphate. Data suggest the importance of inorganic calcium–phosphate homeostasis in the pathogenesis of acromegaly patients (growth hormone pituitary adenomas) from the systemic view point. (ii) The second topic addressed the alteration in the chemokine network in pituitary adenomas from a systemic view . Chemokines are a category of inflammatory mediators that exert their roles through typical and atypical chemokine receptor signaling pathways. An alteration of chemokines and receptors is associated with cancer and inflammatory diseases. The chemokine network is proposed as the target of biomarker and new therapeutic approach for pituitary adenomas. (iii) The third topic addressed the proteomic variations in pituitary adenomas (Zhan and Wang). Proteomics is the key component of functional genomics and systems biology. Much progress has been achieved in pituitary adenoma proteomics to expand and enrich the systems biology analysis of pituitary adenomas. This topic emphasized the significance of variations in proteome and protein molecular networks for personalized and precise studies of pituitary adenomas. (iv) The fourth topic focused on molecular network variations in different subtypes of non-functional pituitary adenomas (Zhan and Long). That topic emphasized the integrative molecular networks derived from multiple omics data at the genome, transcriptome, proteome, peptidome, and metabolome levels, and on their variations in different subtypes of non-functional pituitary adenomas. These studies will benefit discovery of effective and reliable biomarkers and therapeutic targets for personalized and precise studies of highly heterogeneous non-functional pituitary adenomas.This present issue focuses on systems biological aspects of pituitary adenomas, which contains four topics. (i) The first topic addressed vitamin D status and calcium–phosphate homeostasis in acromegaly patients , and especially for different subtypes of pituitary adenomas , 9. SystFuture issues will collect different levels of omics studies, especially the integrative omics studies together with genetics and clinical information with developed advanced computational biology approaches.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular-scale electronics is mainly concerned by understanding charge transport through individual molecules. A key issue here is the charge transport capability through a single—typically linear—molecule, characterized by the current decay with increasing length. To improve the conductance of individual polymers, molecular design often either involves the use of rigid ribbon/ladder-type structures, thereby sacrificing for flexibility of the molecular wire, or a zero band gap, typically associated with chemical instability. Here we show that a conjugated polymer composed of alternating donor and acceptor repeat units, synthesized directly by an on-surface polymerization, exhibits a very high conductance while maintaining both its flexible structure and a finite band gap. Importantly, electronic delocalization along the wire does not seem to be necessary as proven by spatial mapping of the electronic states along individual molecular wires. Our approach should facilitate the realization of flexible ‘soft' molecular-scale circuitry, for example, on bendable substrates. Molecular wires with high conductivity and conformable flexibility are desirable in nanoelectronics. Here, Nacci et al. grow flexible molecular wires, composed of alternating donor and acceptor units, on a metal surface, and show that high conductance can be achieved without electronic delocalization. Back in 1974, Aviram and Ratner7910Molecular wires are key elements in nanoelectronics, in particular in single-molecule circuitry where the wires are responsible for electronic communication between various functional elements1The measurement of single-molecule conductance is experimentally difficult because one needs to bridge various orders of magnitude from the macroscopic electrodes to the molecular wire at the nanometre scale. Several methods have been developed in the last years, which can be essentially divided into two categories: on one hand statistical methods67131415121718Eg (at fixed chain length) is typically desired. Two main strategies have been followed over the yearsπ-conjugation has been enforced by ladder-type structures, such as oligoacenesπ-conjugated bridges leads to a significant lowering of Eg, as has been known for dyes for a long time and proven to be a viable strategy for developing organic optoelectronic materials2512Eg yet maintaining a somewhat flexible structure. Keeping the Eg of the molecular wire finite is beneficial to maintain its chemical stability, which is typically lowered by oxidation because of decreasing ionization potentials, that is, high-lying HOMO levels. Note that at zero band gap a high conductance has been determined in statistical measurementsIn order to design an ideal molecular wire with an optimal charge transport, a small (but not zero) highest occupied molecular orbital (HOMO)– lowest unoccupied molecular orbital (LUMO) gap Eg exhibit a high conductance around the Fermi level in single-molecule transport measurements.In analogy to inorganic semiconductors and insulators, where the incorporation of foreign atoms (dopants) into their crystalsThe target polymer structure was inspired by recent lines of work on alternating donor–acceptor polymers in the context of organic solar cells25n oligomers with covalent bonds between the DAD units6, which is the most abundant closed structure, has a curvature radius of ∼13.9±0.2 Å while (DAD)5 exhibits 11.5±0.2 Å  individual DAD monomer. By placing the STM tip at different positions over the molecule , the dI/dV spectrum at the acceptor location  group) reveals the presence of two clear features, one at low energy  and one at higher energy , respectively. At the thiophene  locations, the dI/dV spectrum increases monotonically (at positive bias) without showing any particular feature . At negative bias, electronic resonances cannot be clearly identified in STS because the HOMO states of the DAD monomer are not very well coupled to the Au(111) surface as we found in our calculations . In particular, the development of the electronic states as a function of the (DAD)ulations . This co2; 1) at the donor location, in agreement with our calculated spectra . Interestingly, it is spatially located at the site of the bithiophene within the molecular chain but no peak is visible at this energy position at the thiophene site for the DAD monomer  with increasing chain length is well established in the field of π-conjugated oligomersn chains, the dI/dV spectra show the persistence of these three states A1, A2 and D1 without any significant energy shift , the conductance maps of a chain reveal a dumbbell-like pattern  is not changing too much with V  passing through a single molecular wire junction is given byAfter characterizing the electronic structure of DAD chains when adsorbed flat on the Au(111) surface, electron transport was studied by pulling single oligomers with the STM tip off the surfaceh V ref. , the cure2/h To(E) being the electron energy-dependent contact conductance. L is the length of the molecular wire and β(E) the energy-dependent tunnel inverse decay length through the molecular wire that characterizes the conducting performance of the molecular wire. In a monoelectronic approach, β(E) depends on the effective mass m*(E) of the tunnelling electrons, on the HOMO–LUMO gap Eg, and on the relative position of the HOMO and LUMO electronic states relative to the Fermi level EF (refs β(E) remains non-zero), which has been confirmed experimentallyEg and the inverse decay length β(E) both equal zero if the electronic structure of the molecular wire is matching that of the electrodes.with 2 EF refs , 19, 43.I(z) was measured as a continuous function of the tip height, which reflects approximately the chain length in this electrode–molecule–electrode set-up  pulling curve (β (see I(z) curves n HOMO–LUMO gap (that is discussed below). This experimental finding is in very good agreement with the theoretical value βth=0.23 Å−1 in a pulling configuration  for a planar DAD polymer adsorbed on the surface. These are quite small β values in the band gap as compared with other molecular wires: 0.7–0.9 Å−1 for alkane9−1 for oligophenylene−1 for graphene nanoribbons−1 for thiophene chains−1 for polyfluoreneβ value is constant over the investigated oligomer lengths (−1 as compared with 0.3 Å−1 for polyfluorene). It should be mentioned that even smaller β values can be achieved at small electron energies if electronic states are located close to the Fermi level, as for instance in organometallic compoundsIn order to provide individual polymers for such experiments, lateral manipulation with the STM tip has been used to separate chains from each other. In the pulling experiments, the STM tip was positioned above the end of a given molecular wire, the STM feedback loop was disabled and the tip apex was then approached vertically towards the chain end until a contact was established. Afterwards, the tip was retracted and the current ng curve  exhibitse (β see . By perftion see  while we lengths  and thatI(z) pulling curves also show more complex oscillation structures with a periodicity of ∼11 Å  surface. On further pulling, two thiophene groups are then detached from the surface and they also rotate n chain is therefore caused by the pulling procedure and the resulting chain distortion  β value . This re β value . At firs β value .m* for a given chemical structure from independent complex band structure calculations  reveals a much larger band gap  and a reI/dV conductance maps were recorded in constant-current mode with the bias voltage referring to the sample with respect to the STM tip. The Au(111) sample was cleaned by repeated Ne ion sputtering (E=1.5 keV) and subsequent annealing up to 720 K. Molecules were evaporated from a Knudsen cell (at 445 K) on the clean Au(111) sample (at room temperature). Oligomers were produced from molecular building blocks via on-surface polymerizationMeasurements were performed under ultrahigh vacuum conditions with a low-temperature STM (Createc) operated at a temperature of 10 K. STS was performed with a lock-in amplifier (24 mV peak-to-peak modulation amplitude at 630 Hz frequency). STM images and d−1 was reached on the force for each atom. Positions of the atoms in the tip and on the surface were maintained fixed during this optimization. The interaction of the molecular wire with the Au(111) surface was described by means of van der Waals interactionsThe calculations of constant-current STM images and of electron transport properties through a molecular wire were carried out with the elastic scattering quantum chemistry techniqueThe monomer bis(5-bromo-2-thienyl)-benzobis, referred to as the Br-DAD-Br monomer, was synthesized in a linear sequence of six steps  starting52How to cite this article: Nacci, C. et al. Conductance of a single flexible molecular wire composed of alternating donor and acceptor units. Nat. Commun. 6:7397 doi: 10.1038/ncomms8397 (2015).Supplementary Figures 1-14, Supplementary Note 1 and Supplementary ReferencesAtomic coordinates  used for STM image calculations of DAD .Atomic coordinates  used for STM image calculations of the dimer (DAD)2 .Atomic coordinates  used for STM image calculations of an oligomer (DAD)10 and the electronic transport calculations through (DAD)10 .Slater atomic orbitals parameters used in ESQC calculations.


Current advances in molecular biology, together with the development of Biobanks as stable sources of biologic material, are enhancing the possibility of detecting genetic factors involved in the molecular pathogenic mechanisms of migraine, a complex neurological disorder classified as the seventh most disabling disease worldwide.To date, the migraine section of the Interinstitutional Multidisciplinary Biobank (BioBIM) of IRCCS San Raffaele Pisana has recruited 863 migraine patients and 400 healthy individuals as controls. Each biological sample has been associated with extremely detailed socio-demographic and clinical features of the donor. Thanks Our Biobank dedicated to migraine has proven to be a valuable resource to conduct molecular studies on this disease, allowing the identification of a new potential biomarker for detection of asymptomatic individuals at increased risk for migraine development, in addition to providing the basis for the design of more tailored and effective therapies.None.


We further performed a statistical analysis of the current–voltage characteristics of the molecular junctions in the two states. Within a single channel resonant tunnelling model, we obtained electronic couplings in the molecular junctions by fitting the current–voltage characteristics to the single channel model. The origin of the high conductance was attributed to experimentally obtained large electronic couplings of the direct π-bonded molecular junctions (ca. 0.15 eV). Based on analysis of the stretch length of the molecular junctions and the large electronic couplings obtained from the I–V analysis, we proposed two structural models, in which (i) mesitylene binds to the Au electrode perpendicular to the charge transport direction and (ii) mesitylene has tilted from the perpendicular orientation.We report on an experimental analysis of the charge transport properties of single mesitylene  molecular junctions. The electronic conductance and the current–voltage characteristics of mesitylene molecules wired into Au electrodes were measured by a scanning tunnelling microscopy-based break-junction method at room temperature in a liquid environment. We found the molecular junctions exhibited two distinct conductance states with high conductance values of ca. 10 G0 . The low electronic conductances prevent practical application of the molecular junctions for the molecular electronics. To bind a single molecule to metal electrodes, anchoring groups such as –SH [2 or (R)3–N [60 [Along with increasing interests in molecular electronics on the single molecular scale , much efh as –SH  and –NH2r (R)3–N –16 have r (R)3–N –20 inclur (R)3–N ,21–22 der (R)3–N ,19–20, C)3–N [60 –24, ethy)3–N [60 , and pyr)3–N [60 . The conI–V) characteristics of molecular junctions. Transition voltage spectroscopy [I–V characteristics. Energy level alignment of conduction orbitals with respect to Fermi level of metal electrodes has been examined for the molecular junctions of alkanedithiol, 1,4-benzenedithiol (BDT) and other molecules [I–V curve analysis [I–V characteristics to the Breit-Wigner model, the metal–molecule coupling Γ, and energy difference between Fermi level and conduction orbital ε0 are obtained. This I–V analysis has been applied for molecular junctions with a variety of anchoring groups. For example Γ = 60 meV and ε0 = 0.6 eV have been reported for BDT [0 = 0.5 eV for thiolated oligo(phenylene ethynylene) [The electronic structure of molecular junctions including metal–molecule couplings can be characterized by current-voltage  .I–V analysis for the direct π-bonded molecular junction system without anchoring groups, i.e., mesitylene molecular junctions -based break junction (BJ) technique [G0, which is at least 10 times larger than di-substituted benzene such as BDT [I–V characteristics of the mesitylene junctions, we demonstrate that the direct π-bonded system features larger metal–molecule coupling.In this study, we report on the unctions  to charaechnique . The conh as BDT  and 1,4-h as BDT . The mesh as BDT . Here, wI–V characteristics. G0, and the corresponding conductance histograms show a peak at 1G0  Au electrodes are repeatedly made just after the rupture of the Au contact to trap a single mesitylene. At the conductance range below 1G0, conductance steps preferentially appeared around 0.1G0 and 0.03G0  found in the present study can be hidden in background tunnelling currents.Mesitylene molecular junctions sandwiched by Au electrodes were prepared by the BJ method . We usedk at 1G0 ,d. Thesed 0.03G0 ,c, whichrding to , this coductance  and the ductance , we haveductance –42. In tductance , conductG0 to 0.7G0 for the high-conductance state and from 0.02G0 to 0.7G0 for the low-conductance state. Gaussian fits of the length distribution reveal average step lengths of 0.044 and 0.079 nm for high- and low-conductance states, respectively. The stretch length of the low-conductance state is larger than that of the high-conductance state. It should be noted here that the length does not directly correspond with the actual gap size. The breakage of the Au atomic contact leads to the immediate formation of a nanogap of finite size as a result of the elastic response of the electrode banks. In case of Au atomic contact, the width of the gap thus created is typically 0.4 nm [G0, the larger stretch length for the low state of 0.03G0 can be interpreted as a result of tilted orientation of the mesitylene junction upon stretching. We will discuss this matter in a later section.To estimate the effective separation of electrodes for the high-conductance and the low-conductance state, we analysed the electrodes separation length in the conductance traces. y 0.4 nm . Considey 0.4 nm  for the I–V characteristic to obtain the electronic couplings of single molecular junction. In previous research [I–V characteristics of the mesitylene junctions by sweeping a wide bias range of ±1 V at fixed electrode separations. I–V curves of mesitylene molecular junctions. In addition to the almost linear increase of the current at low bias voltages, a non-linear increase of the current is apparent at high bias voltages. In the high-bias range, the Fermi level of the Au electrode moves close to the energy levels of the mesitylene conduction orbitals. Thus, the charge transport changes from non-resonant to resonant transport. Two clear distributions are present in the 2D histogram  and above 2400 nA (above 0.1G0) at +0.3 V are selected for low and high states, respectively. The conductance windows are chosen to separate the I–V curves into two states with the conductance values of 0.1G0 and 0.03G0 observed in We examined research , the curistogram . This reistogram . To obtaThe transmission of single molecular junction in a single channel resonant tunnelling model is represented by 0, ΓL, and ΓR are the energy alignment of the conduction orbital, and the strength of the coupling between molecule and left and right electrodes, respectively. The current in the molecular junction is represented bywhere εn is the number of bridging molecules. The formula for the current in molecular junctions . For both of the high and low conductance states the couplings were found to be symmetric (α = ΓL/Γ ≈ 0.5). If the high conductance state is due to formation of multi-molecular junctions, we should also observe a fundamental conductance state that comes from the formation of a single molecular junction. Then we checked whether the I–V curve of the high state can be fitted by multiple formation of the observed low conductance state. The conductance of the high state is 3–4 times higher than that of the low state. The fitting results are Γ_H,n=3 = 0.098 eV, ε0_H-n3 = 0.32 eV for n = 3, Γ_H-n4 = 0.076 eV, ε0_H-n4 = 0.37 eV for n = 4. The ε0_H-n3 and ε0_H-n4 are roughly half of ε0_L. This mismatch indicates that the high state cannot be explained by multiple formation of molecular junctions. In other words, the high state is not due to the formation of multiple molecular junctions because there is no fundamental conductance state in lower conductance regions for the high state. The I–V analysis of the statistically averaged data revealed the high and low states have different electronic structures. In contrast to BDT junctions with the anchoring group [Here, Γ = ΓFinally, we comment on structural models for the experimentally observed two states of the mesitylene-molecular junctions. In conductance traces, plateaus corresponding to the low conductance state appear after the plateau corresponding to the high conductance state . The strI–V measurements. In the conductance measurement and I–V measurement, two conductance states (a high-conductance and a low-conductance state) were identified. The energy level alignment and the electronic couplings of mesitylene to Au electrodes were determined by fitting of statistically averaged I–V curves. The origin of the high conductance for the direct π-bonded molecular junctions was ascribed to the experimentally obtained large electronic couplings of ca. 0.15 eV for the two states. Based on the stretch length of the conductance trace and the large electronic coupling obtained from the I–V analysis, we proposed two structural models.We fabricated single molecular junctions of mesitylene with the STM-BJ method and performed conductance and I–V experiments, the STM tip was approached to the substrate until a metal junction with conductance of 6.5G0 was formed in air at room temperature. Subsequently, the tip was withdrawn for 10 nm at a tip speed of 38 nm/s to break the Au junction and to prepare the molecular junction. The tip was held and the bias voltage was simultaneously swept at 100 kHz frequency to record one I–V curve. The bias voltage range was ±1 V. Then, the junction was broken by pulling away of the tip from the substrate. We repeated this process and obtained more than 1000 I–V curves. The I–V characteristics of molecular junction were obtained by removing I–V curves of Au junctions or vacuum gaps. The I–V curves for the vacuum tunnelling are defined as I–V curves with less than 100 nA current at a bias of 1 V (ca. 0.0013G0) while I–V curves with less than 10000 nA current at a bias of 0.2 V (ca. 0.65G0) were assigned to charge transport through metal contacts. More than 95% of the I–V curves recorded in blank experiments fall into either the vacuum tunnelling or the charge transport through metal contacts.Conductance measurements were performed using an electrochemical scanning tunnelling microscope . Au(111) substrate was prepared by thermal deposition of Au on cleaved mica surface at 625–675 K. The Au substrate was cleaned by flame annealing before use. Then the Au substrate was fixed in a Teflon liquid cell with the Viton O-ring. Pure mesitylene solvent was poured into the cell. STM-BJ experiments were performed as reported previously. The STM tip was prepared by mechanically cutting Au wire  . In the


Molecular imaging differs from traditional imaging in that imaging agents or probes enable the visualization of the cellular or molecular function and followup of the molecular process in living subjects. It has been shown to be an effective noninvasive technology on improving diagnosis, prognosis, planning, and monitoring of personalized medication. Molecular imaging is a diverse technology which includes various modalities, for instance, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), computed tomography (CT), ultrasound (US), and optical imaging . In the era of personalized theranostics (therapeutics and diagnostics), molecular imaging with the specific probe(s) would allow the clinical physicians to select the right patients for the appropriate treatment.EGFR:1907, targeting the extracellular domain of epidermal growth factor receptor (EGFR), which was newly developed and used for detection of hepatocellular carcinoma (HCC). They observed that EGFR-expressing HCC lesions could be specifically detected by Ac-Cys-ZEGFR:1907. PET imaging might have better diagnostic value than optical imaging based on the same Affibody. The early and sensitive detection of HCC based on molecular cancer markers, such as EGFR, is a critical step in improving the currently dismal prognosis of HCC patients. Dr. H. Ma et al. presented a comparison study on 99mTc-N-DBODC5 and 99mTc-MIBI in patients with coronary artery disease. Their study results demonstrated that 99mTc-N-DBODC5 and 99mTc-MIBI MPI provide comparable diagnostic information for patients undergoing exercise rest for detection of CAD. In addition, 99mTc-N-DBODC5 does not exhibit the disadvantages of 99mTc-MIBI in their study.Topics covered in this special issue include advances in biomarkers in preclinical drug discovery, molecular imaging modalities in disease management, image-guided therapy approach of diseases, and imaging technology in drug development. For example, Dr. Z. Cheng's group at Stanford University reported an Affibody molecule, Ac-Cys-ZStem cell therapy is a promising therapeutic approach for some human major diseases. Molecular imaging plays an important role in terms of noninvasive, in vivo trafficking and evaluation of short-term or long-term therapeutic response. In this issue, molecular imaging in stem cell therapy for spinal cord injury and stroke were reviewed by F. Song et al. and F. Chao et al., who provided up-to-date information and future perspectives on these hot topics. Recently, molecular imaging, especially PET, has been introduced to evaluate the functional or metabolic changes in cerebral ischemia after administration of traditional Chinese medicine. Most of these works were done by the group from the Second Hospital of Zhejiang University School of Medicine. Z. Wang et al. from the same group reviewed molecular imaging in applications of traditional Chinese medicine in neurological disease.New technologies, such as microfluidics for synthesis of PET tracers and acoustic droplet vaporization, were included in this special issue. Over the last decade, microfluidics and lab-on-a-chip (LOC) technology have boomed as powerful tools in the field of organic chemistry, which potentially provides significant help to the PET chemistry. Microfluidic radiolabeling technology and its application for synthesis of peptide-based PET tracers are summarized and discussed in the review paper by Dr. Y. Liu et al. Acoustic droplet vaporization (ADV) is a physical process in which the pressure waves of ultrasound induce a phase transition that causes superheated liquid nanodroplets to form gas bubbles. The bubbles provide ultrasonic imaging contrast and other functions. C.-Y. Lin and W. G. Pitt reviewed the literature regarding the use of ADV in clinical applications of imaging, embolic therapy, and therapeutic delivery.131I radioablation on the important lymphocytes subtypes of regulatory T and B cells in patients with papillary thyroid carcinoma and the prognostic value of interim and final FDG-PET in major histotypes of B cell NHL patients treated with rituximab-containing chemotherapy have been discussed in this special issue.An interesting study on abacus training in Chinese kids using functional magnetic resonance imaging (fMRI) found that abacus training modulates the neural correlations of exact and approximate calculations. Additionally, findings on the influence of In summary, molecular imaging could be applied to target characterization, underlying disease progression, and evaluation of therapeutic response after administration of western medication, stem cell, and traditional medicine. This special issue provides a platform of efficacy of personalized medication from molecular imaging technology which may have high impact on patient care and drug development.Hong ZhangMei TianIgnasi CarrioAli Cahid CivelekYasuhisa Fujibayashi


CFI) in 2266 individuals with AMD and 1400 without, identifying 231 individuals with rare genetic variants. We evaluated the functional impact by measuring circulating serum factor I (FI) protein levels in individuals with and without rare CFI variants. The burden of very rare (frequency <1/1000) variants in CFI was strongly associated with disease (P = 1.1 × 10−8). In addition, we examined eight coding variants with counts ≥5 and saw evidence for association with AMD in three variants. Individuals with advanced AMD carrying a rare CFI variant had lower mean FI compared with non-AMD subjects carrying a variant (P < 0.001). Further new evidence that FI levels drive AMD risk comes from analyses showing individuals with a CFI rare variant and low FI were more likely to have advanced AMD (P = 5.6 × 10−5). Controlling for covariates, low FI increased the risk of advanced AMD among those with a variant compared with individuals without advanced AMD with a rare CFI variant , and also compared with control individuals without a rare CFI variant . Thus, low FI levels are strongly associated with rare CFI variants and AMD. Enhancing FI activity may be therapeutic and measuring FI provides a screening tool for identifying patients who are most likely to benefit from complement inhibitory therapy.To assess a potential diagnostic and therapeutic biomarker for age-related macular degeneration (AMD), we sequenced the complement factor I gene ( CFH) (Age-related macular degeneration (AMD) is a common disease with multifactorial etiology ,2. In adCFH) ,7, furthCFH) .CFI) with advanced AMD (AAMD)  –11. Thes of AAMD . The fac of AAMD ,14.CFI genetic variants described in AMD patients are missense mutations of uncertain biologic relevance. Considering the loss-of-function CFI variant association with AMD, the potential of agents that prevent C3 activation , including ones classified as benign, possibly damaging, probably damaging, or loss-of-function by PolyPhen2. Variants classified as loss-of-function or probably damaging among variants with counts <5 in AAMD and non-AAMD (45 in AAMD versus 3 in non-AAMD) were significantly related to AMD (P = 6.1 × 10−7) . Overall, there were far more rare, non-synonymous CFI variants in all categories combined in the AAMD group than the non-AAMD group  . Underscoring the matching of the case and control groups used in the sequencing analysis for ancestry and data quality, synonymous variants in CFI showed no difference .In our study of 3666 AAMD and non-AAMD individuals Fig. , 71 diffCFI variants with counts in AAMD or non-AAMD groups ≥5. No individual CFI genetic variant was highly associated with AAMD, although three were nominally associated: p.P553S (benign) with odds ratio (OR) of 2.69, P = 0.027; p.R406H (possibly damaging), OR 0.10, P = 0.015; and p.A240G (probably damaging), OR 7.43, P = 0.023 . This number of AAMD-associated variants (three of eight) is higher than would be expected by chance (P = 5.8 × 10−3).In addition, we examined the eight CFI, of which 79 (69%) had AAMD. These rare genetic variants occurred throughout the gene with amino acid changes in both heavy and light chains of FI and with no obvious mutational hot spots .Of the 209 individuals who had serum analyses, 114 subjects had a rare genetic variant in CFI variants analyzed for association with AAMD. Among the five individuals with the p.A240G risk variant and classified as probably damaging , four of the five had low serum FI levels. All individuals carrying the p.P553S risk variant and classified as benign  had normal FI levels. Among the three individuals with the protective p.R406H variant classified as possibly damaging , the serum FI levels were normal. The previously reported p.G119R risk variant . Among individuals without a CFI variant, there was no significant difference between AAMD (40.1 µg/ml) and the comparison group (39.2 µg/ml). Among all individuals with AAMD, the mean FI level was significantly lower among those carrying a rare genetic variant (P = 0.03). A larger percentage of individuals with AAMD and a rare CFI variant had low serum FI, compared with the comparison groups: AAMD without a variant, non-AAMD without a variant, and non-AAMD with a variant (P < 0.001). Therefore, rare CFI variants are lowering serum FI levels and contributing to AMD risk, whereas serum FI is not contributing to AMD risk among individuals without a variant.As shown in Figure CFI variant status and low serum FI levels. Without adjustment for covariates, having low serum FI strongly increased risk of AAMD among those with a variant  compared with individuals without AAMD and without a variant. After adjustment for age, gender, body mass index (BMI) and smoking, the OR was 19.0, P = 1.1 × 10−5. When comparing AAMD with a variant to AAMD without a variant, the OR's for low serum FI were 7.8 in the unadjusted model (P = 7.5 × 10−5) and 7.2 (P = 5.1 × 10−4) for the adjusted model. For analyses only among individuals with a rare variant, low serum FI was associated with AAMD with OR 11.2 in the unadjusted model (P = 1.8 × 10−4) and 13.6 (P = 1.6 × 10−4) in the adjusted model. Thus, low serum FI is a risk factor for AAMD in association with a rare CFI variant.Figure P = 5.6 × 10−5). Males tended to have lower FI levels (P = 0.02) and there was no association with age (P = 0.68). There was also no association with smoking or BMI (data not shown).Table CFI variant, 33 had low FI levels. On the other hand, among the 35 subjects without AAMD and a CFI variant, only 2 had low FI levels. Twenty-one different genetic variants were seen in association with low FI levels.As shown in Figure in silico prediction of functional consequence of the genetic variant. FI levels were significantly lower in individuals with probably damaging or loss-of-function CFI variants compared with those with benign or loss-of-function variants  as well as compared with those with no CFI variant .Figure CFI, FI levels were assessed in relation to the common variant associated with AMD, rs10033900, near the CFI gene , indicating no significant difference in FI levels related to the common CFI variant.In the 95 individuals with no rare genetic variants in CFI gene . The meaCFI variant associated with low FI levels (type 1 CFI mutation) and 13 individuals with grade 1 only and no CFI variants. Only three individuals with a type 1 CFI mutation had low C3 levels . There was a positive correlation between the levels of FI and C3 in these 32 individuals with a type 1 CFI variant  .C3 levels were measured in 32 individuals with AAMD and a CFI variants commonly have low serum FI antigenic levels. This result, observed in association with 21 variants, is highly suggestive of a variant causing a structural effect on the mutant protein, impairing secretion. Further, this provides compelling data corroborating previous genetic evidence for the role of impaired FI-mediated regulation of the complement system's alternative pathway in the pathogenesis of AMD  compared with those without AAMD (13.6-fold higher). Type 1 CFI mutations accounted for 42% of the AAMD group but only 6% of the non- AAMD group.In this study, we demonstrated that individuals with AAMD and rare s of AMD –11. AmonCFI, CFH and CD46 genes) which will respond to the complement inhibitor, Eculizumab and the non-complement-mediated forms (e.g. DGKE gene) that do not  of FI is associated with atypical Hemolytic Uremic Syndrome (aHUS: MIM 612923) . FI is ot do not .Twenty-one distinct genetic variants were identified in association with low FI levels in the AMD population. Of these variants, 10 have been seen in aHUS patients associated with low levels  p.P50A –22, p.P6P = 0.15) but had low FI in all individuals tested  remains unclear. Of the type 2 variants, p.P553S, classified as benign by PolyPhen2, increased risk for AMD (OR 2.7), while p.R406H classified as possibly damaging, was protective (OR 0.10). Functional analyses of the type 2 variants p.G261D and p.R406H have not shown a loss of activity   ,40. We dCFI knockout mouse, C3 circulates as C3b with no evidence of further degradation products   . This mib, C3dg) ,42. Our Our hypothesis to explain the AMD/FI association is straightforward: increased complement alternative pathway activity in the retina results from or is secondary to low FI levels; i.e. for a given degree of retinal injury or damage, there is an excessive (undesirable) degree of complement activation which is particularly damaging to the retina. The hyperinflammatory response may affect the retinal pigment epithelium, drusen, or the local vasculature, leading to an acceleration of the disease process. This conforms to established complement risk factors for AMD which can cause an accelerated C3b feedback cycle. Unsurprisingly, it has long been known that elevating serum FI concentration can downregulate this C3b amplification loop .CFI-associated aHUS . Using the same primers previously shown to produce high quality amplicons covering the entire exon sequence and adjacent splice sites .Figure CFI . We expaCFI . In thatce sites , we sequCFI rare variants on serum FI levels, we first identified individuals with a variant and with AAMD for whom serum samples were available (n = 79). We identified three comparison groups from the sequenced individuals with similar mean ages at the time of blood draw compared to advanced AMD cases carrying a variant: (i) subjects with AAMD but no variant (n = 48); (ii) subjects without AAMD and without a variant (n = 47); and (iii) subjects without AAMD but with a variant (n = 35)  Fig. . The totCFI rare variant status was assessed using a one-way ANOVA. Pearson's Chi-square test was used for gender, BMI, smoking status and low serum FI level.We obtained information on covariates including age at blood sample collection, gender, BMI and smoking from our AMD study databases as previously described ,17,44–50CFI rare variant status and low serum FI, we classified individuals according to their age (<80 yrs or 80+ yrs), gender, BMI and smoking status. The OR for non-adjusted and covariate adjusted associations were computed using exact binomial probabilities by PROC FREQ procedure with EXACT option of SAS 9.3.To calculate the OR of the association between AAMD/CFI genetic variant, we used binary logistic regression analysis to analyze the association between a subject's AAMD status and serum FI, controlling for age at blood sample collection, gender, smoking and BMI status. Subjects with AAMD were compared to individuals with a rare CFI variant but without AAMD. For the rare CFI variants (defined as a frequency <1% in either the AAMD or non-AAMD groups), we tested variants with a frequency ≥5 among AAMD and non- AAMD groups separately. For variants with frequency <5, we grouped variants into categories based on PolyPhen2 , K08AR055688 (S.R.), U01HG0070033 (S.R.), F30HL103072 (M.T.), R01-AI041592 (J.P.A.), U54 HL112303 (J.P.A.); Edward N. & Della L. Thome Memorial Foundation (J.P.A.); the Doris Duke Clinical Scientist Development Award; the Protein Core Facility of the Rheumatic Diseases Core supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health, under award number P30 AR48335 (J.P.A.), Massachusetts Lions Eye Research Fund, Inc. (J.M.S.); the Foundation Fighting Blindness (J.M.S.); Research to Prevent Blindness Challenge grant to the New England Eye Center, Department of Ophthalmology, Tufts University School of Medicine; American Macular Degeneration Foundation (J.M.S.); and the Macular Degeneration Research Fund of the Ophthalmic Epidemiology and Genetics Service, New England Eye Center, Tufts Medical Center, Tufts University School of Medicine. D.K. is a Wellcome Trust Intermediate Clinical Fellow and is funded by Fight for Sight. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.This work was supported in part by Conflict of Interest statement. None declared.


Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target. Drosophila neuroblasts  × 100.Asynchronous proliferating HeLa and HeLa-HSET-GFP cells were grown on coverslips to a confluency of ~70% and then incorporated with 10μM BrdU for 1h followed by fixation with 70% ethanol at room temperature and immersion in 0.07 N NaOH for 2 minutes . Coverslips were then incubated in 2% bovine serum albumin/PBS.0.05% Triton X-100 at 37 °C for 1h followed by immunostaining using a 1:1000 dilution of Anti-BrdU-FITC antibody . BrdU positive cells, indicative of cell proliferation, were captured on a Zeiss Axioplan-2 fluorescence microscope (20X objective).MDA-MB-231 cells were transiently transfected with control vector, HSET-pEGFP plasmid or SMARTpool siRNA as described above and lysates were collected. Cell lysates were clarified by centrifugation at 10,000 rpm, and the supernatants (500 μg of protein) were subjected to immunoprecipitation with 4 μl of anti-HSET or anti-survivin antibodies. After overnight incubation at 4°C, protein A-agarose beads were added and left at 4°C overnight. Immunocomplexes were then subjected to Western blot analysis as described previously. Western blot analysis with anti-ubiquitin antibody  was performed by first incubating the PVDF membrane with 0.5% glutaraldehyde/PBS pH 7.0 for 20 min and then probing for the antibody.


Clinical genomics is now a reality and lies at the heart of individualized medicine efforts. The success of these approaches is evidenced by the increasing volume of publications that report causal links between genomic variants and disease. In spite of early success, clinical genomics currently faces significant challenges in establishing the relevance of the majority of variants identified by next generation sequencing tests. Indeed, the majority of mutations identified are harbored by proteins whose functions remain elusive. Herein we describe the current scenario in genomic testing and in particular the burden of variants of uncertain significance (VUSs). We highlight a role for molecular modeling and molecular dynamic simulations as tools that can significantly increase the yield of information to aid in the evaluation of pathogenicity. Though the application of these methodologies to the interpretation of variants identified by genomic testing is not yet widespread, we predict that an increase in their use will significantly benefit the mission of clinical genomics for individualized medicine. Now, 16 years after completion of the draft sequence, genomic assays are an increasingly prevalent component of clinical testing. Oncology, hematology and the diagnosis of rare, Mendelian genetic disorders (diagnostic odyssey) have particularly benefited from the increased genetic testing resolution afforded by modern sequencing technologies. The commercialization of clinical assays to profile patients’ somatic or germline genomes is creating the potential for higher resolution diagnoses and individualized treatment options. Stories of the resounding success of such efforts have been widely and justifiably publicized, and have now captured imaginations well beyond the laboratory or clinic; even so far as the White House and US Congress. The President’s Precision Medicine InitiativeWhile sequencing technologies enable the interrogation of entire genomic, transcriptomic and epigenomic repertoires, current clinical implementations remain heavily biased toward the protein coding component of the DNA, either in the form of whole-exome sequencing or gene panel testing. This is a reflection of both the relative maturity of the field of molecular genetics and the clinical familiarity with both it and the concept of molecularly targeted therapy, which currently represents the major paradigm of tailored cancer treatment. Limiting clinical interrogation of a patient genome to a coding subset may seem counterintuitive in the era of whole-genome sequencing, but the reality is that our understanding of non-coding genomic variation and its role in disease remains in its infancy, particularly with regard to clinical actionability. The explosion of genomics research has increased our understanding of the human genome and the wide-ranging functions it encodes, however most regions of the genome remain uncharacterized and poorly understood. Even with genomic testing limited to protein coding regions, the majority of sequence variants detected go unreported or are classified as variants of uncertain significance (VUSs) due to their uncertain impact on normal physiological function.3.For the researchers and clinicians on the ground of this nascent field, enthusiasm is high but it is tempered with frustration due to the as-yet high rate of cases for which genomic testing remains insufficiently informative (around 75% for diagnostic odyssey)A recurring scene is the convening of large, multidisciplinary teams of clinical and research staff to pore over lengthy lists of genomic sequence variants, exchange professional opinion and debate next steps. However, in the end, these involved efforts often fail to identify variants with highly confident causal or mechanistic relationships with the disease phenotype. In oncology, the knowledge gained for treatment selection is often compelling, but with little prior evidence, most are difficult to actualize. For example, identification of a novel missense mutation in the functional domain of a known, druggable oncogene might logically appear to be a therapeutic target, but no information may exist linking the mutation to drug efficacy. It is this disconnect between novel genotypes identified and their link to the patient’s phenotypes which thwarts our ability to further improve clinical decision making. Thus, there is a significant need to overcome this critical challenge to expand the success of genomic testing in individualized medicine.4. These algorithms utilize varied information including evolutionary conservation, genomic position or basic protein-level structural information to assign probabilistic scores or categorical predictions of pathogenicity. Their accuracy varies widely, with alternative tools often producing conflicting predictions for the same variant. Furthermore, the predictions often lack contextualization in terms of biological effect. Algorithmic categorization of a variant as pathogenic offers little insight into the nature of its phenotypic effect and less indication still of whether it is relevant clinically or how to act upon it. Many researchers continue to work to improve these methods, but the burden of VUS interpretation persists and the need for a means to address this burden and facilitate clinical interpretation is widely felt within the field.The most mature applications of clinical exome and panel testing are the detection of focal substitutions , insertions and deletions (INDELS), or copy number changes. While copy number gains or losses can intuitively be interpreted as potential gain-of-function or loss-of function events respectively, point mutations and INDELS are less easily characterized in terms of their effects on protein function. Wet-lab assays designed to ascertain functional relevance of specific mutations are a highly desired solution, but they remain cumbersome, time-consuming and cost prohibitive in the great majority of cases. They are therefore misaligned with the high throughput nature of modern genomics. Meanwhile, computational methods of predicting the pathogenicity of genomic sequence alterations exist and are both amenable to high-throughput predictions and widely applied in the field, to the extent that they form a component of the American College of Medical Genetics and Genomics (ACMG) guidelines for the clinical interpretation of sequence variantsThe aforementioned computational tools largely leverage genome-centric information. While there is much proven value to these data, the biological reality comprises many additional layers of complexity. Mutations affect atomic-level biophysical changes that may alter the structure and biochemical function of the genome's protein products. While we detect variants in DNA, we typically interpret their effect by inferring or confirming their deleterious effect on encoded proteins . Even thin silico that would be difficult, costly, or intractable in the lab. Similar to laboratory experiments, computational calculations and simulations are most interpretable when they are well designed, test a specific hypothesis, contain positive and negative controls, connect the data generated to the pre-existent knowledge in the field, and allow drawing further functional inferences. When all these conditions are met, the three dimensional and dynamic representation of the modeled mutations may add a significant value to the interpretation of a genomic test’s finding.Computational biophysics and biochemistry aim to understand molecular function in a dynamic manner at an atomic resolution differing from their wet-lab counterparts methodologically but sharing the same goals. The most obvious advantages of computational approaches include the potential to test hypotheses6.Ab initio modeling is also possible, albeit with lower confidence. The model itself contains information about the linear amino acid sequence of the protein, along with the relative spatial coordinates of its atoms. Precise mathematical and biophysical parameters in the form of a force field are applied to the model to calculate energetic characteristics of the system. The methods are often mature and under reasonable conditions can be expected to produce a model with error comparable to that of a typical structure solved by nuclear magnetic resonance spectroscopy.Motivated by the conditions described, we have begun to apply molecular protein modeling and dynamic simulation techniques in the interpretation of genomic variants identified by next generation sequencing. These methodologies abandon the practice of regarding mutations as occurring in linear strings of nucleotides or amino acids and instead offer a three dimensional, dynamic view, at an atomic resolution. Furthermore they are inherently compatible with the current state of clinical genomics testing and its predominant focus on point mutations within protein coding regions. The methods involve the computational generation, optimization and verification of a protein structural model, often based on homology to an experimentally determined protein structureinter-molecular interactions - for example between an enzyme and its substrate - can also be modeled. To illustrate their function, many enzymes are analogized to traditional tools. One example likens proteases and scissors; if a variant inhibits the closing motion of the scissors about their fulcrum, the blades cannot function; if a variant blocks entry of a material between the blades, then that material can no longer be cut. The reality, of course, is more complex. Proteins are flexible polymers that only achieve mechanistic accuracy by folding into complex and specific three dimensional conformations that restrain or focus thermal fluctuations towards collective motions that are typically part of the mechanism itself. Regions of the properly folded structure’s surface that interact with other molecules, either for chemical modification (e.g. phosphorylation) or structural contacts (e.g. α/β tubulin assembly), can also be critical for function and modified by variants. In addition to the native shape of a protein, the ability of the linear amino acid polymer to achieve that shape is critical. Protein folding often occurs through progressive assembly of local structural elements or intermediates. If an intermediate is either stabilized or destabilized by a variant, the ability of the protein to achieve the native fold could be altered. Molecular modeling allows us to visualize the manner in which proteins are folded to create a functional structure and to accurately simulate how this is disrupted by mutational events. Because we can visualize atomic bonds, mutations which disrupt7. A model is by nature an approximation of reality. Nonetheless, current techniques have achieved suitable accuracies such that they are frequently used11 in applications including drug design, virtual screening, protein engineering and site-directed mutagenesis. With this in mind, their relatively slow uptake in the clinical setting is somewhat surprising. This fact may simply reflect the relative nascence of clinical genomics and the tendency of specialists to seek out fields in which their specialty is already known and accepted.Of course, molecular modeling methods have limitations. The generation of a reliable model is often dependent on the pre-existence of experimentally determined homologous structures and even where these exist, they may provide only partial information13 and enable a researcher or clinician to experiment with core modeling methods in the absence of extensive technical expertise. Such independent or collaborative exploration will open the door to deeper understanding of biological mutations and has the potential to inform clinical thinking. Wider use of these methods will enable the field to progress toward standardization of methodologies and approaches, and aid in the formulation of clinical best-practices that will serve to strengthen initiatives like the ACMG standards.We encourage those working within or in proximity to the clinical genomics setting to engage in or promote increased exploration of these methods in their work. Our initial experiences of applying such techniques at the clinical-research boundary of genomics-driven oncology, hematology and diagnostic odyssey have been encouraging and have begun to inform decision-making. We are observing clear initial benefits in regard to variant prioritization, interpretation and validation, with several initial publications in preparation to highlight the value obtained. Of course, not everyone will possess the necessary scientific knowledge or technical skills to deploy these methods directly, but we propose that inter or intra-institutional collaborations may enable those lacking the requisite expertise to identify and access appropriate resources. Alternatively, several freely available online solutions existAs we alluded to earlier in this article, wider ‘omics’ research is a broad and varied landscape of many alternative techniques that are continually revealing new findings with potential diagnostic and therapeutic relevance. Applications such as transcriptomics and epigenomics are beginning to develop clinical presence, but currently they lag behind DNA-based methods. Within the realm of DNA sequencing, the potential clinical relevance of non-coding variation is recognized and bioinformatics developments as well as major experimental initiatives like The Encyclopedia of DNA Elements (ENCODE) bring us closer to routine exploitation of such findings in the clinic. Multi-omic and systems-level research are also widening the scope in which we consider the molecular underpinnings of disease and these promise to have productive futures in clinical settings. Furthermore, novel treatments including oligonucleotide-based and immunogenic therapies are emerging, and move beyond the current prevailing targeted therapeutic paradigms. The case we have made for increased use of protein modeling focuses intentionally on the current state of clinical genomics testing and related diagnosis and treatment decision-making, rather than within these burgeoning research areas. Our opinion is that protein modeling satisfies a current and real need within clinical genomics and that its application should be immediately explored and encouraged. This is not to say it’s clinical niche will be short-lived as many emerging techniques may well benefit from similar modeling methods being applied in either central or supportive roles, for example in nucleic acid structural modeling or in predicting neo-antigen affinities for MHC-class molecules.In summary, genomic testing is assuming an increasingly prominent role in the clinical identification, prioritization, and interpretation of disease-associated genomic variants. While a few of these variants are known to be pathogenic, knowledge of the deleterious effects of the majority remain elusive. Laboratory methods remain gold-standards for functional characterization, but are generally incompatible with large-scale characterization of variant effects. Predictive algorithms are in some instances successfully applied to differentiate pathogenic variants from variants of uncertain significance, however these methods largely ignore measures of protein structure, energies, molecular bonds, intermolecular interactions, post-translational modification effects, protein aggregation, and stability. Conversely, this information lies at the heart of molecular modeling and dynamic simulation, which collectively equip us more fully to grapple with interpreting the effects of VUSs. We strongly believe that these methodologies can be major components in forging the analytical pipeline for interpreting genomic testing in individualized medicine and advocate their increased deployment within variant characterization efforts. A clearer and more pointed presentation of key points is provided. A broader discussion may begin on issues that remain open, such as large-scale crystallization efforts/ 'universal' protein modeling or on the role of mutations in non-coding DNA. This may well form the basis for additional articles on this topicI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This revision significantly improved from the original article. Neoantigen prediction is a good example, where atomic level modelling can significantly contribute to clinical decisions.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. highlight to importance of analytical methods such as “molecular modeling and molecular dynamic simulations" as tools that can significantly increase the interpretation of the functional impact of mutations/polymorphisms currently classified as variants of unknown significance (VUSs).In this article Oliver the current limitation of these methodsthe complexity of these nucleotide changes in e.g. coding and non-coding genomic regionsintegration with other high throughput technologies and thus increase challenges for multimodal effects in such simulationsHIV is given as an example, however other clinical examples, their associated specificity and limitations should be provided to make this article relevant for a wider audience. While the topic of molecular dynamics simulation for VUSs is worth discussing, the article could expand onI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors of this article "highlight a role for molecular modeling and molecular dynamic simulations as tools that can significantly increase the yield of information to aid in the evaluation of pathogenicity." Molecular dynamics simulations are indeed excellent tools to study protein and nucleic acid structures, protein-ligand, protein-protein and protein-nucleic acid interactions. These structural changes and interactions are fundamental in molecular biology and thus molecular simulations are an excellent tool to increase our understanding of these biological processes. In particular the proposed method may help the understanding of coding variants associated with amino acid substitution. In addition, RNA structure can be modeled, which can be informative about some micro-RNA variants. However, these methods are not without limitations and this paper does not state these limitations explicitly. Pathogenic effects come in all strength and complexities. Most often pathogenicity is not associated with a single variant, but with a combination of several variants and the effect of a particular variant  depends on the genotypes of other genes. Most likely, variants will stay of unknown significance unless we develop a system level understanding of biology. We may reach a point that we can simulate viral functions at molecular level, but it will not be applied to human variants any time soon. Molecular modelling has some limitations and requires expert knowledge to apply it properly. While I agree that molecular modelling is an excellent tool to understand molecular biology, we need to develop a system level of understanding of the connection between molecular biology and genetics before we can apply molecular simulations. Currently, molecular modeling can make practical contribution to understand variants associated with strong effects or the molecular basis of some Mendelian disorders.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In this article the Authors seek ways on how to better interpret the functional impact of mutations/polymorphisms currently classified as variants of unknown significance (VUSs). To tackle such a problem, the authors propose “molecular modeling and molecular dynamic simulations as tools that can significantly increase the yield of information to aid in the evaluation of pathogenicity.” Expectations are that this “will significantly benefit the mission of clinical genomics for individualized medicine.”The idea is good and deserves to be published. Most of all, it deserves to be extensively discussed.Points to be discussedAs much as the idea is good, it would benefit from better highlighting its (current) limitations.Ab initio molecular modeling is a difficult enterprise. It can rarely be performed to some extent of accuracy by automatized engines/softwares and often reaches good structure prediction in around 60% of cases.Several large-scale, tentatively whole-coding genome, protein crystallization initiatives have been undertaken. Discussion of the state of the art of these initiatives may provide metrics on what could be expected from real-life application of the Authors’ proposal, and how this is expected to evolve in the near future.Most mutations/polymorphisms are detected in non-coding regions of DNA. This is hardly surprising given that non-coding DNA represents approximately 97% of the total content of a mammalian cell. The authors mention mutations in non coding regions, but this deserves being more extensively discussed in the publication. Suggestions Discussion of the points above and of the suggestions below, may possibly lead to extend the proposed approach to additional layers of prediction.Algorithms are there that do recognise promoter, enhancer and super-enhancer regions with increasing accuracy. Clearly, allocating a mutation/polymorphism in one such region would increase its investigational and possibly medical value.Additional regions of interest are CpG islands upstream or, less reliably, within coding regions.Additional regions of interest are anchoring sites of DNA loops to distinct nuclear/nuclear membrane regions.Additional regions of interest are those hosting the increasing family of non-coding RNA (NCRNA), including long NCRNA and miRNA, that have been shown to play distinct regulatory functions and may have roles in cancer development.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


We report atypical case of Kaposi Sarcoma (KS) in a 32-year-old human immunodeficiency virus- (HIV-) infected female, involving only the tongue. Viral loads and CD4 T cells were measured and were 65,000 cps/mL and 10 cells/mL, respectively. This patient was newly diagnosed and had no history of antiretroviral therapy (ART), radiotherapy, or immunosuppressive drugs prior to this admission. Clinically, there were no dermatological features of KS lesions which are purple, red, or brown and which may be flat, raised (plaques), or bumpy (nodules) except for fungating and protruding enlarged tongue which was necrotic. Histologically, it was proven to be the most common type of KS “epidemic” or AIDS-related KS. Kaposi Sarcoma (KS) is the soft tissue cancer that develops from the cells that line lymph or blood vessels . It is cEpidemic KS among HIV patients is most commonly reported in Africa since the 1980s seen in about 40% of all HIV/AIDS patients. It has been linked with other diseases like primary effusion lymphoma . Its pat2. She was chronically ill with big necrotic and fungating protruding large tongue filling the whole oral cavity. The tongue has mixed lesions, that is, necrotic tissues and fresh wounds with easy bleeding plaques on the dorsal aspects of the tongue. She had also fungal infection (dermatophytes) of the upper lip  and hepatitis C were all negative. Our working diagnoses were World Health Organization (WHO) Clinical stage 4 HIV disease, septicaemia, oropharyngeal candidiasis, upper airway obstruction, and lingual carcinoma with malnutrition. She commenced first-line ART  alongside antibiotics Ampicillin and Metronidazole, Flucanzole, and oxygen therapy. On the fourth day in the ward, the condition of the patient changed and became dyspnoeic with oxygen saturation of 40% on oxygen therapy. Intubation was tried but was unsuccessful due to enlarged tongue occluding the oropharynx; tracheostomy was performed but patient condition deteriorated and she passed away. Histology results came out after one week and showed extravasations of red blood cells, collagen splitting, and spindle cells tumours  female who was referred to our tertiary hospital in June 2015 with two weeks' history of recurrent fever, inability to swallow, weight loss, enlargement and protrusion of the tongue, and difficulty in speech. Her weight on time of admission was 43 kgs and height was 158 cm: Body Mass Index (BMI) was 17.22 Kg/mpper lip . This to tumours . Pneumocystis jirovecii pneumonia may mimic radiographic finding of pulmonary KS. Typical feature of pulmonary KS includes hilar lymphadenopathy, perihilar lymphadenopathy, lower zone infiltration, and effusions [This case of histologically proven KS of the tongue in this patient with confirmed HIV infection illustrates that KS can present with only one feature of tongue enlargement. This was atypical presentation because there was no single lesion which was seen on the skin or from radiographic findings and ultrasonography. This was misdiagnosed as lingual carcinoma of the tongue or neurofibromatosis or fungal infection of the tongue. Routine tissue biopsy of the lesions among HIV positive patients is essential for the correct diagnosis. This case report had atypical presentation because it is very rare to see KS affecting only one organ because it has multiple sites. Skin is the most common site for KS to occur; it may involve the skin itself or the skin with other sites , 12. In ffusions , 14. In Patients with KS need to be diagnosed and treated promptly to reduce mortality and morbidity. Atypical presentation of KS which involves only the tongue can happen and it is difficult to diagnose and treat. High index suspicion for KS in HIV infected individuals especially with CD4 count <200 cps/mL will be helpful in early diagnosis , 12. ThoWe report the case of a 32-year-old female who died within 5 days since admission due to airway obstruction secondary to KS of the tongue confirmed histologically. She was initially suspected to have lingual carcinoma with upper airway obstruction. This case report underscores the importance of histology in diagnosing different types of malignancies as well as other opportunistic infections among HIV patients.


Mycobacterium tuberculosis and bovine tuberculosis in a community, consisting of humans and African buffalos, is presented. The buffalo-only component of the model exhibits the phenomenon of backward bifurcation, which arises due to the reinfection of exposed and recovered buffalos, when the associated reproduction number is less than unity. This model has a unique endemic equilibrium, which is globally asymptotically stable for a special case, when the reproduction number exceeds unity. Uncertainty and sensitivity analyses, using data relevant to the dynamics of the two diseases in the Kruger National Park, show that the distribution of the associated reproduction number is less than unity . Crucial parameters that influence the dynamics of the two diseases are also identified. Both the buffalo-only and the buffalo-human model exhibit the same qualitative dynamics with respect to the local and global asymptotic stability of their respective disease-free equilibrium, as well as with respect to the backward bifurcation phenomenon. Numerical simulations of the buffalo-human model show that the cumulative number of Mycobacterium tuberculosis cases in humans  decreases with increasing number of bovine tuberculosis infections in humans .A new model for the transmission dynamics of Mycobacterium tuberculosis (MTB) and bovine tuberculosis (BTB) are chronic bacterial diseases, classified amongst the closely related species that form the M. tuberculosis complex (MTBC)   are the BTB transmission rate (βB), the recovery rate of buffalos (γB1), and the BTB-induced mortality in buffalos (δB).outliers ). Thus, IBB + IMB) as the response function. This figure shows a distribution of the number of symptomatic buffalos lying in the range [20–130]. Hence, this study shows that, using the parameter values and ranges relevant to BTB-MTB dynamics at the Kruger National Park, a BTB outbreak could cause no more than 130 confirmed cases (of BTB and MTB) in the park. The associated PRCC values  are depicted in B), the natural (μB), and the disease-induced (δB) death rate of buffalos. Hence, this study shows variability in the top-ranked PRCC values on the chosen response/output function.Similarly, Having fully studied the dynamics of the bovine-only model , the fulIt can be shown, using the approach in TB model .θMM = θBB = 0. The justification for this assumption is based on the fact that contact between humans and buffalos in the Kruger National Park are tightly controlled . The DFE of the BTB-MTB model  are set to zero. This is due to the reinfection of exposed and recovered humans . To illustrate this fact, it is shown that the DFE (Ef0) of the BTB-MTB model  is less than unity.It can be shown, as in TB model  is GAS iEf0, of the BTB-MTB model :θMM = 0,no transmission of BTB from buffalos to humans: θMM = 0.25,low rate of transmission of BTB from buffalos to humans: θMM = 0.50,moderate rate of transmission of BTB from buffalos to humans: θMM = 0.75.high rate of transmission of BTB from buffalos to humans: The effect of BTB  on the spread of MTB in the human population (within the park) is assessed by simulating the BTB-MTB model  using paθMM).The simulation results obtained, depicted in θHH) are considered, namely, none (θHH = 0), low (θHH = 0.25), moderate (θHH = 0.50), and high (θHH = 0.75). The results obtained, depicted in Similar plot is generated to assess the effect of MTB  on the spread of BTB in the buffalo population. Here, too, four transmission levels of the associated parameter , the buffalo-only model is shown to have unique endemic equilibrium whenever its reproduction number exceeds unity (R0 > 1). This equilibrium is shown to be globally asymptotically stable for the special case where the disease-induced mortality in buffalos is negligible (δB = 0).In the absence of the reinfection of exposed and recovered buffalos (R0) is chosen as the response/output function, reveals that the three main parameters that govern the disease dynamics are the BTB transmission rate, recovery rate of buffalos, and BTB-induced mortality rate. Similarly, three parameters  are identified as the main influential parameters for the case where the number of symptomatic buffalos (with BTB or MTB) is the chosen output function.Detailed uncertainty analyses of the buffalo-only model, using a reasonable set of parameter values and ranges  relevantθRB and θEB) are set to zero. This is due to the reinfection of exposed and recovered humans . It is shown that this model does not undergo backward bifurcation in the absence of reinfection of exposed and recovered host(s) . For this case, it is shown that the DFE of the BTB-MTB model ). In both models, the backward bifurcation phenomenon is shown to arises due to the reinfection of exposed and recovered host(s).Numerical simulations of the BTB-MTB model show that an increase in the cumulative number of BTB infection leads to a marked reduction in the cumulative number of new MTB cases in humans. Similarly, an increase in the cumulative number of MTB infection led to a significant decrease in the cumulative number of new BTB cases in buffalos.A new deterministic model for the transmission dynamics of BTB and MTB in a community of humans and buffalos is designed and rigorously analyzed. Some of the main findings of the study are as follows.


Dopamine neurotransmission has long been identified as a key component of prefrontal cortical function and flexible cognition; however, flexible behavior does not depend solely on dopamine. Together, multiple systems allow fast and flexible behavior in the face of a constantly changing environment. This Research Topic illuminates work by researchers who are leading efforts to understand how different neural systems yield flexible behavior.Yen et al.).The psychostimulant amphetamine is, paradoxically, a useful treatment for Attention Deficit Hyperactivity Disorder (ADHD) symptoms. Here, Yen and colleagues demonstrate in a mouse model of ADHD that the calming effect of amphetamine is due to decreased GSK3B, initiated through NMDA receptor signaling (Mizuki and Fujiwara).Stress is known to be under the partial regulation of oxytocin, and oxytocin levels in adults may be influenced by severity of childhood maltreatment. The inconsistent quality of results on this topic are tackled by Mizuki and Fujiwara, who were able to demonstrate a correlation of severity of childhood maltreatment with diminishing oxytocin levels in humans . This information is critical in developing individualized treatment plans for treating maladaptive behaviors.A key challenge in combatting drug addiction is the incentive motivational property of a stimulus. This is known to develop through Pavlovian Instrumental Transfer (PIT). Garofalo and di Pellegrino were able to show, for the first time in humans, that Sign-Tracker responses were biased by reward-paired cues while Goal-Trackers were not . These data suggest the usefulness in physical exercise to promote improved flexible cognition in adolescents.Physical exercise is known to promote better cognition, but the mechanism remains unknown. In their article Berse and colleagues identified a particular single nucleotide polymorphism in the Dopamine Transporter (DAT1/SLCA6A3) gene which contributed to improved cognition under physical exercise conditions in adolescent humans . These results fail to replicate an earlier and smaller experiment, and the authors propose that the difficulty in diagnosing ADHD may lie in a number of false-negative studies.Altered neurochemistry in the anterior cingulate cortex (ACC) is thought to be a root cause of the behavioral symptoms of ADHD. However, when Endres and colleagues conducted a large single voxel proton scale magnetic resonance spectroscopy study, they identified no neurometabolic differences in ACC or cerebellum between control and experimental groups (Bissonette and Roesch). Importantly, these populations of neurons also modified their activity based on feedback, significantly increasing activity for rewarded outcomes and significantly decreasing activity for non-rewarded choices. These results demonstrate how the mPFC signals the need for behavior to become flexible.Flexible behavior is known to depend on the medial prefrontal cortex (mPFC). Bissonette and Roesch demonstrate how neural correlates of rules and conflict are encoded and signaled by mPFC neurons as rats modify their behavior in the face of changing contingencies (Raver and Lin). This review serves focuses attention on how the BF salience system is a critical part of signaling attention towards relevant stimuli, promoting flexible behavior.Raver and Lin present a very cogent review of the role that the basal forebrain plays in signaling arousal, attention and decision-making. They discuss relevant literature focusing on how a population of BF non-cholinergic neurons encode motivational salience through ensemble burst firing . These findings are important for interpreting data that manipulates the mPFC and uses reward-based learning paradigms.Most animal research uses reward-based learning paradigms, requiring animals to consume their reward. In this research topic, Parent and colleagues demonstrate how cholinergic and ghrelinergic signaling—both working through KCNQ channels in the mPFC—are critical for regulating the duration of licking. These data suggest that natural variation among animals in their tendency towards either sign or goal-tracking may be correlated with drug addiction vulnerability.Sign-tracking in animals is associated with increased sensitivity to food cues. Nasser and colleagues demonstrate that sign-tracking, but not goal-tracking, rats are more sensitive to food associated cues and that the degree of sign-tracking correlated with a failure to suppress responses during devaluation . These results suggest a role for LC not during initial learning, but during situations which require flexible shifts in behavior.The locus coeruleus (LC), which provides noradrenergic projects to the cortex, is important for attention. Janitzky and colleagues demonstrate that optogenetically suppressing LC activity during a set shifting task impaired performance on reversal and extra-dimensional set-shifting but not on learning of intra-dimensional shifts or compound discriminations . This information is necessary to guide future research on how LHb activity may be important for flexible behavior.The lateral habenula (LHb) acts as a gatekeeper for information about negative outcomes being transmitted to the midbrain dopamine system. Baker and colleagues provide a thorough review of the LHb literature and present data to support a hypothesis whereby LHb neural activity is important for the maintenance of goal-directed actions to new response types during changing contingencies . These data provide further support that frontal cortical neural activity works best with a basal level of dopamine.Particular behavioral responses need to be organized over time, allowing appropriate actions to occur at the right time. Parker, Ruggiero and Narayanan demonstrate that blockade of D1-type dopamine receptors in the medial frontal cortex leads to altered interval timing performance by impacting ramping patterns and differentially field potential activity without impacting baseline firing rates of neurons (Hernandez and Cheer). This review summarizes the literature around how different aspects of the endocannabinoid system may modulate dopamine release.Many neural systems interact while animals engage in complex behaviors and make decisions. Hernandez and Cheer provide a thorough review of the literature surrounding the endocannabinoid system and how endocannabinoid and dopamine systems may interact while animals make decisions .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ibuprofen is a well-known agent used to treat patent ductus arteriosus in preterm neonates in the first days of life. In the current case report we illustrate the potential use of ibuprofen in two preterm neonates 60 and 88 days after birth, respectively. To our knowledge, this is the first report on the effects of ibuprofen on patent ductus arteriosus in preterm newborns after months of life. These cases suggest that the ductus arteriosus does not become refractory for ibuprofen after the first days of life. Late closure of the duct with ibuprofen might still improve the cardiorespiratory condition and prevent infants from surgical closure. Controlled trials are necessary to further study these findings.  In preterm neonates, the ductus arteriosus often remains open after birth. Pharmacologically, this patent ductus arteriosus (PDA) can be closed with Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), such as ibuprofen and indomethacin . There iCase 1 was a preterm boy born by caesarian section because of maternal preeclampsia after 30 weeks and 5 days, after maternal corticosteroids, with a birth weight of 1280 grams and Apgar scores of 9 and 9 after 1 and 5 minutes, respectively. He was treated twice with surfactant because of respiratory distress syndrome and subsequently treated with nasal CPAP. Staphylococcus aureus sepsis at that time. Unfortunately, the bacteremia persisted under antibiotic treatment and an intracardiac thrombus was found. The patient was treated with low molecular weight heparin and antibiotics for more than 6 weeks. During this period, several cardiac ultrasounds were made and all showed the PDA. The patient was cardiorespiratory stabile while treated with diuretics. CPAP was weaned successfully.The first cardiac ultrasound was made at the 9th day of life and showed a large hemodynamically significant PDA. The PDA was not treated, because of aAt the age of 58 days, weeks after recovery from the bacteremia, the patient's respiratory condition worsened and he showed increased oedema. He needed more respiratory support; Optiflow support was started with oxygen therapy up to 30%. Cardiac ultrasound showed a hemodynamically significant PDA see . IntraveCase 2 was a preterm born girl, first of twins, born after 25 weeks of gestation with a birthweight of 645 grams and Apgar scores of 1, 6, and 8 after 1, 5, and 10 minutes, respectively. She received surfactant once via an INSURE  method. She needed noninvasive ventilation from postnatal day 5 until day 43.A hsPDA was found  at postnatal day 6 and treated with intravenous ibuprofen during the next 6 consecutive days (dosages: 10-5-5-10-5-5 mg/kg). Afterwards, the ductus unfortunately turned out to be still open and diuretics were started to reduce pulmonary fluid overload. Because the cardiopulmonary condition stabilized surgical ductus ligation was not performed. The patient developed a severe BPD with 36% oxygen and 6 liters/minute Optiflow at postmenstrual age of 36 weeks.Because her cardiopulmonary condition worsened at the age of 88 days (PMA 37 + 4 weeks) a new cardiac ultrasound was performed. A hsPDA was still present. We decided to treat the patient again with ibuprofen during a total of 6 days . Cardiac ultrasound see  again reTo our knowledge, these are the first reported cases of ibuprofen therapy for PDA in premature infants treated after postnatal ages of 2-3 months. In most patients described in literature PDA treatment is started within the 72 hours of life  or withiThe first patient had a relatively old gestational age compared to other preterm neonates treated with ibuprofen reported in the literature. In patients born after 30 weeks of gestation, the ductus arteriosus often closes spontaneously. It is unlikely, however, that the detected effect of the ductus was a spontaneous phenomenon. Initially, in the first patient, the patent duct was not treated because of ongoing sepsis but was open on repetitive ultrasounds. Weeks after appropriate treatment of the sepsis, the duct was large and became hemodynamically important. The ibuprofen dosages used are relatively high, compared to those advised during the first days of life. Ibuprofen clearance increases during the first weeks of life and the increased dosages seem to be adequate . PharmacIn conclusion, our case reports suggest that ibuprofen might still be effective for PDA closure in preterm neonates even after 60 days of life. As a consequence, it should be taken into account as a potential treatment option in case of prolonged hsPDA.


Serial crystallography at a synchrotron has been used to obtain time-resolved atomic resolution density maps of enzyme catalysis in copper nitrite reductase. Similar XFEL studies, intended to out-run radiation damage, will also soon appear. The 1958 Nobel prize to Beadle and Tatum for proposing that each gene is responsible for a distinct enzyme is now seen as both foundational to molecular biology and genetics, albeit oversimplified. Some genes, for example, code for functional RNAs, while others code for non-enzymatic proteins such as collagen. Yet enzymes remain fundamental to life on earth, catalyzing at least 5000 biochemical reactions (so far identified). Enzymes can increase reaction rates by huge factors, from millions of years to milliseconds per event, so that, from meat tenderizer to washing powder, to muscle contraction, cargo transport in the cell, ion pumps, infection and digestion, no molecular machine is more fundamental to biological function than the enzyme.How do they work? Fischer’s 1894 ‘lock and key’ model, establishing the enzyme and substrate model, was improved by Koshland to include induced fit and molecular recognition, and it has long been understood that enzymes lower the Gibbs free (activation) energy by stabilizing an intermediate state, providing an alternative reaction pathway, or by destabilizing the substrate ground state, all of which can now be understood in terms of an energy landscape. Michaelis and Menten first proposed the kinetics for a two-step model for conversion of substrate to product. Allostery, feedback, inhibitors (including drugs and poisons) and activators are all now known to be important in enzyme regulation.et al.  of recombinant copper nitrite reductase in sodium nitrite for an hour at room temperature before transferring them to a cryoprotectant and plunging into liquid nitrogen, to trap the structure of the room temperature complex. At this stage the reaction does not proceed because no reducing agent is present. The required electrons are provided by free radicals generated by the very X-rays used to image the structure.The prospect of imaging such molecular machines during their catalytic cycles by a kind of atomic resolution X-ray molecular movie is brought a step closer in this issue in the paper by Horrell  al. 2016. Their a2O.The authors then used the Diamond synchrotron fitted with a new fast shutterless detector to obtain 45 low-dose Bragg diffraction datasets in 19 s each from the same regions of the crystal extending to almost 1 Å resolution. This interval spans the catalytic cycle of nitrite reduction, a vital process in agriculture and in the formation of the greenhouse gas Net al., 2002et al. find little reduction in resolution during the collection of their low-dose ‘molecular movie’, in which the time-resolved density maps do show conversion of substrate at a catalytic copper center from nitrite to nitric oxide, suggesting  that they are seeing chemistry relevant to catalysis, not predominantly radiation damage.Previous observations of radiation-induced reactions in protein crystallography include those devoted to horseradish peroxidase  may allow us to make movies of enzyme kinetics. The interpretation of these is not straighforward, since from a crystal of reacting molecules one reconstructs a spatially periodic average density map of the sum of all stable intermediate states as a function of time, representing the total kinetics, rather than the dynamics of molecular transformations at the atomic scale. The latter occur too rarely and rapidly to be imaged or even simulated on the correct timescale. It remains to be seen if the often-cited advantage of EXFELs to ‘outrun’ radiation damage, using brief pulses instead of sample cooling, will allow imaging at the physiological temperatures which provide the correct energy for the reaction, and so provide a better approach, also based on serial crystallography. Studies of enzyme dynamics have been proposed at EXFELs (Schmidt, 2013