Twenty years ago, Kaposi’s sarcoma (KS) was the oncologic counterpart to Winston Churchill’s Russia: a riddle, wrapped in a mystery, inside an enigma. First described by Moritz Kaposi in 1872, who reported it to be an aggressive skin tumor, KS became known over the next century as a slow-growing tumor of elderly men—in fact, most KS patients were expected to die with the tumor rather than from it. Nevertheless, the course and manifestations of the disease varied widely in different clinical contexts. The puzzle of KS came to the forefront as a harbinger of the AIDS epidemic. The articles in this issue of Viruses recount progress made in understanding Kaposi’s sarcoma herpesvirus (KSHV) since its initial description in 1994. Kaposi’s early microscopic studies revealed KS to be a complex tumor composed of disorganized endothelial cell proliferations forming blood-filled vascular clefts but also containing areas of organized micro-neovascularization and often with an inflammatory infiltrate. Pathologists and clinicians became suspicious of whether KS was a “true cancer”: most histopathologic examinations showed no clear demarcation of tumor margins, and the tumor often had a waxing and waning course. Although KS-like spindle cells could be grown in culture from KS lesions, these same cell proliferations could be also generated from blood, and were subsequently shown by Browning to be circulating endothelial precursor cells . Later iThe epidemiology of KS was complex as well, but proved critical in deciphering the biology of KSHV. Early on, KS was thought to be most common among elderly Ashkenazi Jewish men and later was also reported in isolated European ethnic groups, particularly Sicilians and residents of the Po Valley . By the + T cells, but how and why do AIDS patients get this cancer? Epidemiologists described yet again new patterns for “epidemic” or AIDS-KS: the disease occurred almost exclusively among the subset of gay and bisexual men with AIDS . Despite the variety of clinical manifestations for KS, all the different forms of KS had indistinguishable pathologic features suggesting it is a single cancer having a common underlying cause.In a landmark epidemiologic study, Valerie Beral, Harold Jaffe and colleagues sorted out the evolving epidemiology of KS, taking into account not only AIDS-KS patterns but also a century’s worth of observations on KS occurrence prior to the AIDS epidemic . Their cBy 1994, years of research had failed to reveal the identity of this agent. Over 20 different viruses, bacteria and environmental exposures had been proposed, but none of them fit the established patterns for KS. Many of these agents could be easily dismissed—the idea that recreational nitrite poppers act as mutagens for KS seemed potentially plausible for a subset of club-culture AIDS patients but certainly did not help explain KS among more sedentary gay men infected with HIV nor Africans or transplant patients. Other agents, such as cytomegalovirus (CMV), required years of replicative studies before the consensus conclusion was reached that CMV is not the cause for KS. HIV itself was promoted to cause KS by inducing inflammation. This inadequately explained KS occurring prior the AIDS epidemic or lack of KS among blood-recipients with AIDS and high HIV loads. The early 1990s was also a period of turmoil and frustration for scientists working on HIV/AIDS. Protest marches against AIDS and sit-in demonstrations at the U.S. Food and Drug Administration were commonplace. Enormous amounts had been learned about HIV, but death rates from AIDS continued to rise and the origins for KS remained obscure.In early 1993, we had just moved to New York City, and the Department of Pathology at Columbia University generously provided a 100 square foot laboratory and $20,000 start-up funding to Chang to perform clinical neuropathology research. Moore had been an epidemiologist at the Centers for Disease Control working on meningitis epidemics and refugee disasters in Africa. He took a job in the New York City Department of Health. We had been married for five years but had never worked together.At this time, several events contributed to our interest in searching for a KS pathogen. Genome scanning techniques then held promise for identifying large chromosome regions mutated in tumors, and Chang became interested in using these techniques. Moore had been involved in the control of a 1991 hemorrhagic fever epidemic in Nigeria. The cause turned out to be a variant yellow fever virus that was undetectable on standard diagnostic tests but could have been prevented with a cheap and effective vaccine had it been identified early in the outbreak.et al. generously provided a detailed RDA protocol but our first pilot project to RDA isolate lambda phage DNA spiked into a human tissue sample failed. The costs for this experiment in buying Taq polymerase alone nearly broke our meagre start-up budget. We did not have other options than to try again using an actual KS tumor without the typical optimization that normally would have been done.Molecular biology techniques developed for non-directed genomic searching—such as representational difference analysis (RDA)  describeIn early May of 1993, Anna Batistatou, then a resident in the Department of Pathology at Columbia, alerted us to her autopsy on a middle-aged man with AIDS-KS. This case turned out to be unusual for several reasons: firstly, the concern of contracting HIV infection from a slipped scalpel during an AIDS autopsy had driven this procedure to a trickle by 1993. Batistatous’s AIDS autopsy case would be one of the last ones performed at Columbia for several years and, although we did not know it at the time, our major local source for KS tissues was soon to be gone. Secondly, we later found that this patient’s KS lesions had the highest KSHV load of any KS tumors that we subsequently collected. A KS lesion and a control skin sample were taken for RDA analysis, which was performed by Chang and Melissa Pessin, a rotating Pathology resident. The iterative protocol began on August 12 and four RDA bands were isolated on September 7 and cloned by September 15, 1993.Given the possibility for PCR contamination, we chose to analyze the bands by the more time consuming, but dependable, Southern blotting technique. Detection would not be a problem since the KS agent should be present in every tumor cell. Further, a less sensitive technique would have the advantage of reducing false-positive results from a coincidental but non-causal infection. The films were developed on September 27, providing the first hint that something new might be present . The patBut there were mitigating concerns. We had earlier asked another Pathology resident, Anne Matsushima, for non-KS tissue samples from AIDS patients to use as negative controls. This would help ensure that we hadn’t isolated some opportunistic but unrelated pathogen DNA abundant in AIDS patients. She provided three AIDS lymphoma samples; one , Lane 2 With this paltry information and two unknown DNA sequences in hand, we began a collaboration with Ethel Cesarman and Dan Knowles. Out of 27 KS samples from the Hematopathology tissue bank, 25 were positive for the RDA fragments on blinded testing together with other control non-KS tissues from AIDS and non-AIDS patients. After breaking the code, we double-checked the two negative samples and found one was comprised of degraded DNA and the other was not actually KS but had been mislabeled. Slowly, the pattern began to emerge that actually did fit the Beral-Jaffe agent. The possibility that we were misinterpreting due to some trivial but subtle mistake was a constant concern. So there was palpable relief when we learned from Robin Weiss and Thomas Schulz in the UK, who agreed to test their own KS samples, that the same results were independently reproducible . As it tAnother four more months passed before the RDA fragments could be shown to be from a new human herpesvirus, similar to but distinct from any known herpesvirus. The next 15 months were filled with nearly nonstop work screening lambda phage libraries, sequencing the virus, isolating it in PEL cell culture, developing serologic tests and testing more tumor specimens. All successful tumor viruses go through a peer-review version of the Kübler-Ross stages: first there is shock and denial, then anger, next bargaining and depression, and, finally, acceptance . The ini


Photoacoustic imaging (PAI) and thermoacoustic imaging (TAI) are two emerging biomedical imaging techniques that both utilize ultrasonic signals as an information carrier. Unique advantages of PAI and TAI are their abilities to provide high resolution functional information such as hemoglobin and blood oxygenation and tissue dielectric properties relevant to physiology and pathology. These two methods, however, may have a limited detection depth and lack of endogenous contrast. An exogenous contrast agent is often needed to effectively resolve these problems. Such agents are able to greatly enhance the imaging contrast and potentially break through the imaging depth limit. Furthermore, a receptor-targeted contrast agent could trace the molecular and cellular biological processes in tissues. Thus, photoacoustic and thermoacoustic molecular imaging can be outstanding tools for early diagnosis, precise lesion localization, and molecular typing of various diseases. The agents also could be used for therapy in conjugation with drugs or in photothermal therapy, where it functions as an enhancer for the integration of diagnosis and therapy. In this article, we present a detailed review about various exogenous contrast agents for photoacoustic and thermoacoustic molecular imaging. In addition, challenges and future directions of photoacoustic and thermoacoustic molecular imaging in the field of translational medicine are also discussed. Photoacoustic imaging (PAI) is a burgeoning biomedical structural and functional imaging technique which utilizes ultrasonic signals as an information carrier that captures information about optical absorption property relevant physiology and pathology inside tissues. The basic process of the PAI excitation source is summarized below: 1) the tissue absorbs light energy after being irradiated by visible light or near-infrared light; 2) the absorber produces adiabatic expansion that induces photoacoustic signals . Be. Beet alet al., have reported the ability of silica-coated gold nanorods to provide a stable photoacoustic signal, which implies good imaging capabilities. These nanorods thus make silica-coated gold nanorods a promising imaging and therapeutic nanoagent for photoacoustic imaging and image-guided photothermal therapy . Fi. Fiet aloncology .3O4 magnetic nanoparticles comprise another type of agents. They are almost spherical with diameters ranging from 30–50 nm. Qin, et al., investigated the feasibility of using this nanoparticle as a contrast agent in thermoacoustic tomography (TAT) for hepatocellular carcinoma ex vivo detection [2[Gd(DTPA)] is one such paramagnetic ionic compound with seven unpaired electrons in the 4f orbital of the Gd3+ ion. The charged ions and unpaired electrons can interact with a microwave field and transform the absorbed microwave energy into heat. Qin, et al., verified the enhanced effect of NMG2[Gd(DTPA)] for thermoacoustic CT in vitro and in vivo experiments. They demonstrated that this contrast agent can increase the ionic conductivity and microwave absorption coefficient of tumors, thus making the boundary between tumor and normal tissue more obvious in TAI , which is made of superparamagnetic nanoparticles conjugated to folic acid (FA). These nanoparticles can then bind specifically to the surface of the folate receptor, a tumor marker. Nie, et al., have used a 6 GHz TAT system to successfully investigate that intravenous administration of the targeted nanoparticles to mice bearing tumors showed a five-fold greater thermoacoustic signal and a much longer elimination time than that of nontargeted nanoparticles.This portrays Fe3O4/polyaniline (PANI)’s potential for its use in targeted and guided cancer thermal therapy  agents should be of excellent water solubility, or they will be treated as foreign objects and rejected by the surrounding tissue; (4) they should be of proper biological compatibility and low toxicity in order to not cause adverse reactions to the tissues—the most major being damage to the endothelium, vascular system, blood-brain barrier (BBB), kidneys, and so on; (5) contrast agent should have a tendency to bind molecular probes , drugs or another contrast agent. They should have good surface modification and be able to effectively target specific receptors in target cells or tissues; (6) have a high stability, long half-life, and full dissolution in vivo in order to make them meet clinical requirements; (7) they should possess very good dispersibility in order to distribute evenly in the imaging region; (8) photoacoustic contrast agents should have a high light-to-ultrasound conversion efficiency, while thermoacoustic contrast agents need a high microwave-to-ultrasound conversion efficiency. Contrast agents need to be prepared and used discreetly in accordance with the provisions of the Food and Drug Administration (FDA).Effective photoacoustic or thermoacoustic contrast agents should significantly increase contrasts, effectively improve imaging depths or accurately provide molecular specific information. It is therefore prudent to follow these principles when choosing contrast agents: (1) photoacoustic or thermoacoustic contrast agents should be easily prepared and produced for a low cost due to future clinical applications; (2) the size of contrast agents has a significant impact on their distribution and drug delivery Despite many years of research, photoacoustic and thermoacoustic molecular imaging still has a lot of issues that need to be addressed promptly. The following future directions should be discussed: (1) There are limited categories of thermoacoustic contrast agents. Special superparamagnetism and bulk effect, the compounding of superparamagnetic nanoparticles, and their application in biomedicine will hopefully draw the attention of research fellows. Such methods show potential for the advance in multimodality technology of PAI, TAI and MRI; (2) The quantitative capability of photoacoustic and thermoacoustic molecular imaging is significant. It may help us differentiate benign from malignant lesions as well as obtain absolute tissue functional information. They are perhaps determinants of molecular typing. Besides, PAI and TAI can measure the amount of a drug at its site which will provide the theoretic foundation for ensuring the accuracy of injection doses for animals and patients; (3) The future research of contrast agents as a drug delivery system should emphasize its effectiveness and safety. The technical specifications of the system’s effectiveness and safety are not established yet. How to reduce toxicity and side effects as well as improve the drug targeting, solubility, stability, slow releasing action, and how to change tissue-targeted distribution and metabolism are key issues for this research; (4) Mono-modality imaging cannot obtain all the information required. Compared to low resolution ultrasound imaging, TAI can distinguish adsorbers which have the same acoustic impedance but different dielectric properties. PAI is also able to capture information about optical absorption with high resolution. Compared to PET and SPECT, these two imaging technologies have no risk of causing injury by radiation. Compared to MRI, PAI and TAI have quicker imaging speed. In comparison to OCT, they have longer imaging depths. Overall, PAI and TAI only demands imple equipment, have low cost, and are easy to operate, thus they have broad and applicable prospects. The combination of TAI and PAI is capable of revealing information such as water and ion concentration, blood volume, and oxygenation of hemoglobin. The preponderance complementarity of PAI, TAI, and other imaging modes will significantly improve medical high-tech, which will lead to more comprehensive sources of information about structural and functional physiology and pathology; (5) Unfortunately, the application of photoacoustic and thermoacoustic contrast agents in real time imaging and their ability to confirm lesions before treatment cannot currently meet the needs of modern medicine. Instead, targeted drug delivery systems, advanced physical therapy, image guided surgery, heat therapy , photothermal therapy, and magnet fluid hyperthermia are therefore the developing directionfor the future. Photoacoustic and thermoacoustic molecular imaging aim to integrate detection, monitoring, and reasonable treatment into one entity; (6) Contrast agent testings are too complicated, due to their specific animal testing experiments, longer experiment durations followed by clinical trials and high research and development costs. It has been concluded that the related research is unsystematic, has no unified standard, and is rarely used in the clinic due to various differences in species, individuals, and drug delivery systems. Therefore, the effectiveness and safety of photoacoustic and thermoacoustic molecular imaging play a key role in establishing better and more direct relations between basic research and clinical applications.Photoacoustic and thermoacoustic molecular imaging, working in tandem with powerful contrast agents, could provide groundbreaking opportunities for early diagnoses, precise lesion localization, molecular typing, drug delivery monitoring, image guided surgery, drug targeting therapy, and photothermic therapy of various diseases (such as cancer and cardio-cerebrovascular disorders). Photoacoustic and thermoacoustic molecular imaging, complemented by the development of such agents, will potentially bridge the gap between bench and bedside and further develop translational medicine as we know it.


IDH) mutations, tumor protein 53 mutations and the co-deletion of chromosome arms 1p and 19q, but were not closely aligned with histologic classes. These findings emphasize the potential for improved definition of clinically relevant disease subsets using integrated molecular approaches and highlight the importance of biomarkers for brain tumor classification.Lower-grade gliomas  are diffusely infiltrative neoplasms that arise most often in the cerebral hemispheres of adults and have traditionally been classified based on their presumed histogenesis as astrocytomas, oligodendrogliomas, or oligoastrocytomas. Although the histopathologic classification of lower-grade glioma has been the accepted standard for nearly a century, it suffers from high intra- and inter-observer variability and does not adequately predict clinical outcomes. Based on integrated analysis of multiplatform genomic data from The Cancer Genome Atlas, lower-grade gliomas have been found to segregate into three cohesive, clinically relevant molecular classes. Molecular classes were closely aligned with the status of isocitrate dehydrogenase ( Diffuse low-grade and intermediate-grade gliomas  are infiltrative neoplasms that arise most often in the cerebral hemispheres of adults , 2. HistIDH) mutations have been shown to be associated with a favorable prognosis in diffuse gliomas. The finding of co-deletion of chromosome arms 1p and 19q (1p/19q co-deletion) has been strongly linked with the oligodendroglioma histology and predicts better responses to radiochemotherapy. In order to determine if robust, clinically meaningful molecular classes of lower-grade gliomas could be identified and recognized by specific biomarkers, The Cancer Genome Atlas (TCGA) recently performed a comprehensive and integrated molecular analysis . Both the genomic properties and clinical behavior suggested that IDH wild-type lower-grade gliomas are immediate precursors to IDH wide-type glioblastoma.The third molecular subtype of lower-grade gliomas had wild-type mas Fig. . GeneticIDH, 1p/19q, TP53, ATRX, and TERT. Importantly, these molecularly defined subsets are cohesive and reproducible, whereas histologic classes, by themselves, are not. For example, tumors diagnosed as “oligoastrocytoma” were found in all three molecular classes and lacked a defining molecular signature. Previously, diagnostic criteria for oligoastrocytomas have been inconsistent, and many lower-grade gliomas with “ambiguous morphology” have been treated as oligoastrocytomas [IDH mutation have either 1p/19q co-deletion or a TP53 mutation, reflecting two distinct molecular mechanisms of oncogenesis; furthermore, no evidence exists for a biological or genetic signature specific to oligoastrocytoma. Based on these findings and others, molecular markers can be used to determine lineage rather than histologic appearance, and the proportion of “oligoastrocytoma” diagnosis among lower-grade gliomas will likely decrease.This unsupervised analysis of genome-wide molecular data identified three robust disease categories that can be recognized using well-established biomarkers, including ocytomas . ResultsIDH, 1p/19q, ATRX, TP53, and TERT) were capable of identifying new disease classes with high fidelity. In histologic classification, ambiguous morphology is common, resulting in low reproducibility and inter-observer concordance, leading to confusion in clinical management. Molecular classification represents an improvement in diagnostic practice, enabling practitioners to identify clinically distinct neoplasms and confidently direct appropriate standard therapies or clinical trials.Ultimately, the studies by TCGA indicate that advances in molecular profiling will allow neoplastic diseases to be defined using a new approach. Unsupervised clustering of whole-genome molecular data has ushered in a new age in which biological classes of disease can be precisely identified. In the case of lower-grade gliomas, well-established genetic markers (such as


A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein–protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for determining molecular tension appended by ligand-activated PPIs.The summary of the method is:•Molecular tension appended by protein–protein interactions (PPI) is visualized with a luciferase.•Estrogen activities are quantitatively illuminated with the molecular tension probes.•Renilla luciferase enhances the optical intensities after bending by PPI.Full-length  To date, several potential techniques have been established for determining protein–protein interactions (PPIs), including (i) Bioluminescence resonance energy transfer (BRET) based on energy transfer between bioluminescent donor and fluorescent acceptor proteins We previously developed a unique bioluminescent probe called “strain probe” for illuminating PPIs molecular tension probes emitting bioluminescence in response to molecular tension appended by PPIs in detail.In this method, we introduce how to fabricate •pcDNA 3.1(+) (Invitrogen)•a mammalian expression vector•HindIII, BamHI, KpnI, XhoI)Restriction enzymes (•African green monkey kidney fibroblast-derived COS-7 cells•A 96-well clear bottom microplate (Nunc)•Dulbecco’s modified Eagle’s medium (DMEM)•Fetal bovine serum •Penicillin/streptomycin •TransIT-LT1 (Mirus), a lipofection reagent•2; native estrogen)17β-Estradiol •Phosphate-buffered saline (PBS)•A lysis buffer •An assay buffer •Native coelenterazine (nCTZ)•A Bradford reagent for determining total protein amounts•Hank’s buffered salt solution (HBSS) buffer (Gibco)molecular tension probes consists of four different ingredients, i.e., a full-length luciferase, a pair of proteins of interest , and a flexible linker, where the luciferase is sandwiched between the two proteins of interest via a minimal length of flexible linkers (The basic design of  linkers . The lucRenilla reniformis luciferase (RLuc) easily receives tension from PPIs, whereas the flexible region in beetle luciferases relaxes the intra-molecular tension.Any luciferases basically have talent to vary their enzymatic activity more or less according to the molecular tension appended by an intra-molecular PPI. A globular marine luciferase may be advantageous over beetle luciferases, which consist of N- and C-terminal domains connected by a flexible hinge region molecular tension probe that is made of RLuc8 as a model luciferase sandwiched between the ligand-binding domain of the human estrogen receptor (ER LBD) as an intracellular receptor member of the nuclear receptor superfamily and Src homology domain 2 of ν-Src (SH2), based on our previous papers In this protocol, we exemplify a molecular tension probes is fabricated by conventional genetic engineering techniques including polymerase chain reaction (PCR) with an adequate primer set and its subcloning into a mammalian expression vector as follow  by PCR using a corresponding primer set flanked with unique restriction sites, BamHI and KpnI, for introducing unique restriction sites at the 5′- and 3′-terminals, respectively.Generate the cDNA segments encoding full-length 2.v-Src by PCR using corresponding primer sets flanked with the unique restriction sites, HindIII/BamHI, and KpnI/XhoI, respectively.Fabricate the cDNA segments encoding the ER LBD and the SH2 domain of 3.HindIII/BamHI; BamHI/KpnI; KpnI/XhoI; HindIII/KpnI, respectively (2Digest the above cDNA segments and the multiple cloning site (MCS) of a mammalian expression vector pcDNA 3.1(+) (Invitrogen) with the corresponding restriction enzymes, ectively B.24.Gel-purify the digested cDNA segments and the mammalian expression vector.5.pcDNA 3.1(+) (Invitrogen) to fabricate a cDNA construct encoding the molecular tension probe (single coding sequence) (Ligate the cDNA segments into the expression vector equence) B.6.pErs).3Confirm the sequences of the cDNA constructs in pcDNA3.1(+) vector with a genetic sequencer  .Seed the cells in 12-well culture plates, transiently transfected with an aliquot (1 μg) of 3.2, 37 °C) before bioluminescence measurement.5Further, incubate the cells for 16 h in the cell incubator (5% CO4.Stimulate the cells on the 12-well plates for 20 min with the vehicle (0.1% DMSO), 1 μM of 17β-estradiol  or 1 μM of 4-hydroxytamoxifen . Wash the cells once with phosphate-buffered saline .5.Lyse the cells with a lysis buffer  for 15 min in room temperature.6.Transfer an aliquot (10 μL) of the lysates into a 200 μL microtube and determine the bioluminescence spectra with a spectrophotometermolecular tension probe, ERS, as follows .Grow COS-7 cells in a 96-well clear-bottom microplate until a 70% fluency in the cell incubator (5% CO2.pErs (0.2 μg per well) using the lipofection reagent  and incubate them in the cell incubator for 16 h.Transiently transfect the COS-7 cells in the wells of the microplate with an aliquot of 3.−6 M of E2, or 10−6 M of OHT for 20 min.7Stimulate the cells with the vehicle, 104.Wash the cells once in the plate with PBS, and lyse them with a lysis buffer  for 15 min.5.Transfer the cell lysates to a 1.6 mL microtube and determine the subsequent bioluminescence intensities with a luminometer  immediately after adding an assay solution  dissolving nCTZ.6.Parallelly, measure the total protein amounts in the aliquot of the cell lysates with a Bradford reagent for the following normalization procedure.7.Normalize the RLuc luminescence intensities from Step 5 in a ratio of relative luminescence unit (RLU), that is, RLU (+)/RLU (−), where RLU (+) and RLU (−) are the luminescence intensities with 1 μg of cell lysate (Step 6) after the cells were incubated with and without a ligand, respectively, where the RLU is an amplified value of photon counts generated from the luminometer (arbitrary unit) B.The procedure from Steps 5 to 7 may be substituted by the following alternative C.5a. Transfer an aliquot of the cell lysates (10 μL) to each well of a fresh 96-well optical bottom plate.86a. Simultaneously inject an aliquot (50 μL) of the assay buffer dissolving nCTZ into the cell lysates on the plate using a multichannel pipette7a. Determine the optical intensities in the plate with the equipped controlling software (Image reader ver2.0) and analyze the optical images with the analysis software (Multi Gauge ver3.1).2) to the integrated time (second) and applied protein amounts (μg): i.e., RLU/μg/sec/mm2.8a. Normalize the optical intensities  until a 70% fluency.10Grow COS-7 cells in a 6-channel microslide  and keep them in a cell incubator  for 16 h.Transiently transfect the cells in the channels of the microslide with 3.−6 M OHT for 20 min before image acquisition.Stimulate the cells in the channels with vehicle (0.1% DMSO) or 104.Wash the cells in the channels once with a Hank’s buffered salt solution (HBSS) buffer (Gibco) and then fill the channels simultaneously with 80 μL of an HBSS buffer dissolving nCTZ using a multichannel pipet.5.Immediately transfer the microslide into an image analyzer  and determine the optical images with the equipped software (Image reader ver2.0 and Multi Gauge ver3.1).


Odocoileus virginianus) were found infected in a small geographic region of northwestern Minnesota. Genotyping of isolates determined both cattle and deer shared the same strain of bTB, and it was similar to types found in cattle in the southwestern United States and Mexico. Whole genomic sequencing confirmed the introduction of this infection into Minnesota was recent, with little genetic divergence. Aggressive surveillance and management efforts in both cattle and deer continued from 2010–2012; no additional infections were discovered. Over 10,000 deer were tested and 705 whole herd cattle tests performed in the investigation of this outbreak.Bovine tuberculosis (bTB) was discovered in a Minnesota cow through routine slaughter surveillance in 2005 and the resulting epidemiological investigation led to the discovery of infection in both cattle and white-tailed deer in the state. From 2005 through 2009, a total of 12 beef cattle herds and 27 free-ranging white-tailed deer ( Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) and an important zoonotic disease. The United States initiated a State-Federal Bovine Tuberculosis (bTB) Eradication Program in 1917 and has one of the world’s lowest bTB prevalences in cattle. Minnesota participated in the national eradication program since its inception and gained TB Free status in 1976, five years after the last TB infected cattle herd in the state was eliminated. With over thirty years without any known bTB infection in cattle, the 2005 discovery of a bTB infected animal from Minnesota identified through slaughter surveillance was unexpected.This paper documents the identification, testing, and epidemiology of an outbreak of bTB in beef cattle and white-tailed deer in Minnesota and the relationship between herds, and herds and deer based on cattle movement, infected deer locations, herd testing, and whole genome sequencing (WGS) of isolates from infected animals. There has been little documentation of molecular epidemiological investigations in bTB outbreaks that involve cattle and wildlife in the literature; this article provides a level of detail and data analyses not reported previously. The whole genome sequencing (WGS) technology supports the introduction and transmission theories of the epidemiological investigation in Minnesota. This information will serve to assist regulators, cattle producers, and epidemiologists in future bTB outbreaks.This introduction of bTB into Minnesota was identified through routine slaughter surveillance for bTB conducted by US Department of Agriculture (USDA) personnel at all US federally inspected slaughter plants. USDA veterinarians are trained to identify lesions in bovine carcasses that may be compatible with bTB infection and submit samples of lesions and associated lymph nodes from the animal for diagnostic testing to the National Veterinary Services Laboratories (NVSL) in Ames, Iowa. Carcasses with suspect lesions are held until diagnostic tests are completed. Positive tests result in condemnation of the carcass for food.All positive slaughter samples are traced back to a herd of origin through verification of the animal’s identification, sales records, and DNA testing. Subsequent investigation methods of a positive slaughter animal are outlined in the state—federal cooperative bovine eradication program document, Bovine TB Uniform Methods and Rules (UMR) . This inThe epidemiological investigation in Minnesota included quarantine and bTB testing of all cattle or bison herds with fence line contact with an infected herd and all herds that may have bought or sold animals from the herd over a designated span of time. The number of years required for tracing cattle movement in any herd varied based on the age of animals found infected in that herd, but did not exceed seven years for any herd due to the lack of records available. Both private and sale barn sales were traced to the extent feasible. Animals sold for slaughter or feeding did not have animal identification recorded on sales documents so specific animals could not be traced. Often these animals were commingled into larger groups so the group was traced even as it was subdivided and resold to multiple owners. If trace animals were found in a herd in Minnesota, the animals were indemnified, euthanized and tested for bTB. If no trace animals remained in the herd, the herd was tested for bTB using live animal testing and the exposed animals were traced through the subsequent owner’s records of movement. Producers that sold animals to bTB infected herd owners had their herds quarantined and tested for bTB. When traces led to other states, the State Veterinarian and the USDA Area Veterinarian in Charge for each state were notified.When bTB infection is found in wildlife, additional surveillance and testing requirements for livestock are required as outlined in the Bovine TB UMR . These r2 in the area where the positive beef cattle herds were identified. They were known to feed in areas also used by cattle for feeding [Another component of the epidemiologic investigation was to determine if wildlife species were involved in the outbreak. White-tailed deer are susceptible to bovine bTB infection and were present at 2–3 deer/kmAll cattle twelve months of age and older were tested using the caudal fold tuberculin (CFT) test as described for a ‘whole herd test’ in the Definitions section of the Bovine TB UMR . The intMycobacterium tuberculosis complex IS6110 DNA (M. tb complex) [Mycobacterium bovis (M. bovis). All cattle necropsied for bTB testing were euthanized and disposed of through rendering, alkali digestion or burial.Necropsies were performed by state and federal veterinarians on cattle for evidence of bTB. Necropsy procedures included palpation and incision of retropharyngeal, mandibular and parotid lymph nodes from the head; tracheobronchial and mediastinal lymph nodes from the thorax; palpation of lungs and parietal pleura; incision of gastrohepatic, cranial and caudal mesenteric lymph nodes in the abdomen, palpation of spleen, liver and uterus; and incision of deep popliteal, subiliac, mammary, medial iliac, superficial cervical, caudal deep cervical, and prescapular lymph nodes in the carcass. Liver, lung, or other organ tissues were only submitted for further diagnostics if gross lesions were observed in the tissue. Tissue samples were submitted to the National Veterinary Services Laboratory  in formalin (fixed) and borate (fresh). Pathologists examined formalin fixed tissue for lesions consistent with bTB. If acid fast stained bacteria were observed within a granuloma lesion on histopathology, the fixed tissue was tested by conventional PCR for complex) . Fresh tDeer surveillance was primarily conducted between 48° 11_N and 49° 0_ N latitude and 94°56_W and 95°58_W longitude in northwest Minnesota. All deer collected in the surveillance effort were killed by gunshot or archery and were taken by a) hunters licensed with the State of Minnesota under the authority of the Commissioner of Natural Resources or b) USDA APHIS Wildlife Services (WS) staff contracted by the same Commissioner. USDA APHIS WS staff collected deer on public lands and on private lands with signed authorization from landowners. All deer collected were examined by Minnesota Department of Natural Resources (DNR) personnel trained to detect lesions suspicious of bTB. These lesions included any abnormally colored (e.g. brownish-tan) foci in lymph nodes , lungs, and body cavities (particularly thoracic cavity). Sampling procedures included a visual inspection of the chest cavity of the deer. All animals with suspicious lesions were confiscated  and submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) for further diagnostic testing. In animals without suspicious lesions, three pairs of head lymph nodes  were removed and submitted to MVDL for further testing and the carcass released to the hunter.M. tb complex and M. avium by PCR as described for cattle [Further testing at the MVDL included visual inspection of the lymph nodes by supervised technicians and lab personal for the presence of gross lesions. If gross lesions were detected in the lymph nodes, histopathology of the lesion using routine hematoxylin and eosin stains and routine acid fast stain was performed. Fresh frozen and paraffin embedded fixed tissue from the individual animal were submitted to the NVSL for culture and testing for r cattle . If no gDeer with visible lesions submitted to the MVDL were disposed of via alkali digestion. In cases of hunter-harvested deer that cultured positive for bTB but no visible lesions were detected at the time of sampling, individuals were contacted regarding test results and could voluntarily surrender venison for alkali digestion; the remainder of those carcasses were placed in a landfill.M.tb complex with commercial DNA probes . Species determination and genotyping was initially conducted using spoligotyping and variable number tandem repeats (VNTR). [Tissues were trimmed, placed in 0.06% sodium hypochlorite for 15 minutes, homogenated with phenol red broth and then decontaminated for 7–10 minutes using sodium hydroxide as described previously . Tissue https://github.com/USDA-VS). Briefly, reads were aligned to the reference genome AF2122/97, NCBI accession number NC_0002945, using BWA and Samtools [To obtain the whole genome sequences, isolates associated with this outbreak were sequenced on a MiSeq instrument  using 2x250 paired-end chemistry and the Nextera XT library preparation kit . FASTQ files from the instrument were put through the NVSL in-house pipeline  and constant population size or Bayesian skyline [M. bovis isolated from outbreaks throughout the United States (unpublished data), an informative lognormal prior was used for the molecular clock rate . All other priors were the default priors (https://code.google.com/p/beast-mcmc/wiki/ParameterPriors).Isolates were further characterized using molecular clock phylogenetic analysis to estimate times to introduction and transmission of bTB within the outbreak . The tim v.2.1.3 . Tip dat skyline  demograp skyline ; S2 Textth generation of the combined posterior parameter distributions of the 5 chains after a 10% burn-in . The Tracer v1.6 program [All permutations of the molecular clock models and demographic models were fit for the Minnesota isolates in addition to 6 sequenced isolates recovered from a herd in Texas, which were the most closely related isolates (based on SNP identity) in the NVSL archive to estimate phylogenetic relationship and time of divergence. The same analysis was performed for only the Minnesota isolates to infer the phylogenetic relationships, divergence timing, and demographic reconstruction of the well-sampled Minnesota outbreak. Model comparison methods are presented in  program  was usedA maximum clade credibility tree was created with median and 95% HPD heights (in years) for nodes with greater than 0.40 posterior probability using Fig Tree v1.4.2 . PairwisM. bovis was confirmed at the NVSL, federal and state animal health officials determined whether the herd would be declared ‘Affected’ [If evidence of ffected’ . IndemniIn addition to local surveillance, statewide surveillance for bTB in both cattle and deer was conducted to meet USDA requirements for the state’s cattle TB status. All cattle 12 months of age and older in a herd (a whole herd test) were tested using the CFT test. Herds were tested from a random selection of cattle herds throughout Minnesota outside of the TB affected area between June 2006 and December 2007. Samples collected from hunter harvested deer collected outside of the TB affected area during the 2007 fall hunting season were tested for bTB.A five year old beef cow was identified with lesions suspect bTB lesions at a slaughter plant in Wisconsin in February 2005. The subsequent epidemiological investigation identified twelve bTB infected beef cattle herds between July 2005 and January 2009, all within 43 miles of each other in Beltrami, Marshall, and Roseau Counties in northwestern Minnesota , S1 Tab. A totalSurveillance in free-ranging white-tailed deer was initiated in late 2005 and occurred over eight years through the 2012 hunting season with 10,667 animals tested for bTB. The majority of the sampling was conducted through hunting ; however agency sponsored sharpshooting conducted in the winters of 2007–2010 in key areas where previously infected deer were found accounted for an additional 2,613 deer tested. Landowner shooting permits collected 215 deer. In total, 27 TB positive deer were identified, all within a 10 mile radius of the index cattle farm .M. bovis. All infected cattle had visible gross lesions, primarily found in head and thoracic lymph nodes. Of the 37 infected cattle, eight had gross lesions of bTB found only in head lymph nodes and 19 animals had gross lesions found only in thoracic lymph nodes. In two animals, gross lesions were found only in the abdominal lymph nodes. Five animals had lymph node lesions found in more than one area of the body—head, thorax, or abdomen, and one had lesions in a prescapular lymph node. Nodular or granuloma lesions were found infrequently in organ tissues; one animal has lesions in the lung and spleen and two different animals had lesions found in the liver  had not previously been identified in USA origin cattle or from cattle originating from Mexico that were slaughtered in the USA. However, the parent spoligotype SB0140, differing by only one spacer region, was commonly found in Mexican origin imports.M. bovis lesions). Ten cattle from six herds, Herds A, B, D, E, F, and J, and eleven deer had the same WGS SNP profile, which was the MRCA for all the isolates in the outbreak.  from the most recent isolate . The median date of MRCA of isolates from cattle was 2002  and the median date of MRCA from white-tailed deer was 2004  of transmission was capable of dispersing M. bovis at a scale similar to the extent of the outbreak.The investigation and subsequent genomic sequencing and analysis of isolates from infected animals in this outbreak all point to a recent introduction of bTB into Minnesota and subsequent spread between cattle and deer in the area of the outbreak. Genomic sequencing clusters the isolates closely together based on the low number of mutations from the first strain introduced into Minnesota and given the few mutations that occurred, transmission had not been going on over a prolonged period of time. Additional evidence for this is in the age of infected deer identified, as there was an absence of younger animals found with the disease as the surveillance progressed. This further supports the disease was not established in deer; thus preventing the development of a reservoir . The cluS1 FigDark vertical dashed line is the median estimate of time of most recent common ancestor (MRCA) and light vertical dashed line is the lower 95% HPD estimate of time of MRCA. The plot begins at the 95% upper HPD estimate of time of MRCA.(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableA. SNP Chart of Whole Genome Sequences of Isolates from Infected Animals in the Outbreak. B. SNP Statistics of Whole Genome Sequences of Isolates from Infected Animals in the Outbreak.(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


In order to get the dynamic molecule model from the static one, the molecular dynamics (MD) simulation needs to be performed. Some software sets such as GROMACS are used for that purpose. Unfortunately they lack GUI. The Dynamics PyMOL plugin allows researcher to perform MD simulations directly from the PyMOL software by GUI-based interface of GROMACS tools. This paper describes many improvements introduced into the Dynamics PyMOL plugin 2.0 including: an integration with ProDy library, possibility to use the implicit solvents, an ability to interpret the MD simulations, and implementation of some more GROMACS functionality. Structures of molecules obtained from X-ray crystallography , nuclearPerforming molecular dynamics simulation is a relatively complicated task, therefore every option to reduce level of complexity of calculations is welcome. One of such options is the reduction of the number of interacting centers through the use of an implicit solvent model. Although the explicit solvent is still the standard solution model in molecular dynamics simulations and GROMACS offers TIP3P, TIP4P, SPC, and their variants as a water model , the calThe results of the molecular dynamics simulation can be visualized as an animation. Those animations give researchers important information about movement of the molecule, but usually they are not enough. In order to obtain more data from the system it is critical to perform proper analysis of the MD results. The correct interpretation of MD data can produce static images presenting the results of dynamic behavior of the molecule shown in the form of, for example, vectors pointing into direction of the highest displacement of the molecular fragment. It also provides quantitative interpretation of the results, which can be used for comparison with other simulations or for further analysis. Two methods are particularly useful in aiding the interpretation of MD results, these are the normal mode analysis (NMA) and the principal component analysis (PCA).α atoms are considered. The method is simple, efficient, and provides reliable results, therefore, an ENM method has emerged as the preferred approach to perform NMA on proteins containing several thousands of residues [The normal mode analysis method has been successfully used  to deterresidues . One of residues .Another approach to analyze the protein dynamics is to use the principal component analysis method . The resYersinia enterocolitica proteins [One of the new and promising software for protein structural dynamics analysis is ProDy . It alloproteins .The Dynamics PyMOL plugin is written in Python  programmIn order to perform the molecular dynamics calculation, the GROMACS package is needed. The designed plugin is now compatible with the latest GROMACS 5.0 , but canThe plugin is designed for Unix-like systems and it requires Linux, Unix or Mac OS X in order to run.The plugin can be installed by PyMOL built-in plugin installation tool  and after this procedure is added to the Plugin submenu in the menu bar. On Ubuntu Linux the latest stable version of the plugin is also available by the PPA repository  and can The Fig. The Dynamics PyMOL plugin can be started by clicking on top PyMOL menu bar Plugin- > dynamics. The software is meant to be as easy to use as possible. It takes as an input a PDB formatted molecule model and it creates PDB multimodel molecule file as an output, which could be then easily stored and viewed.The most important improvement, which was introduced to Dynamics PyMOL plugin is integration with ProDy library. It is fully optional and lack of this library neither will prevent the plugin from working, nor cripple any of its other functionality. ProDy allows the researcher to calculate the normal modes, which can be used afterward to display results of molecular dynamics simulation as vectors, the resulting images are sometimes called “the porcupine plots”. It also allows the researcher to calculate the Kirchoff matrix, which then can be used to display contact Map and cross-correlations. Figure ProDy is the python library meant to be used from command line. It saves results in the nmd format . Normal Integration with ProDy enables also a few additional options in the molecular dynamics interpretation window, including possibility to display the Contact Map and Cross-correlations. Figure Implicit solvation is a feature introduced to GROMACS to lower compute power needed for performing the molecular dynamics simulations . In contAnother big improvement to the Dynamics PyMOL plugin is the introduction of a molecular dynamics interpretation window displayed after all calculations are finished. Figure There are many other smaller improvements introduced to the software to date, which extends its capabilities and hopefully makes it more user friendly. First of all, until now, only the GROMACS pdb2gmx  was usedThe time of molecular dynamic simulation is one of the most important factors in visualization of its results. In accordance to GROMACS specification it can be set by changing the value of time step for integration (dt) and the number of steps (nsteps) in molecular dynamics options . The addThere are also some improvements in the water box default options. The water box shape (editconf -bt) is now, by default, a dodecahedron instead of cubic. The volume of a dodecahedron, with the same periodic distance, is 0.71 times that of the cube , which sSetting the correct molecular dynamics conditions may be of a “trial and error” nature. If the GROMACS software encounters an error during the process, the simulation will stop and the error message will be displayed in the new window. The new feature, which is now introduced, is a LOG button present in the calculation and interpretation window. It will display the full GROMACS output and therefore it allows the researcher to examine all warnings and errors and assist in solving the issues with improperly set dynamics conditions. This functionality is especially useful if no fatal error was encountered, but still simulation did not proceed as expected and the researcher wishes to analyze the cause.We polished the plugin to be more user friendly, the pop-up balloons with some explanations will now appear after hovering a mouse cursor over an option or stopping cursor movement for some time.With the support of Mac OS X users , the pluThe scroll bars and progress bars where ported from Tix to ttk, which removes Tix from plugin dependencies and therefore make the plugin easier to install .https://github.com/tomaszmakarewicz/Dynamics. The Ubuntu Linux users can install it from official PPA repository: https://launchpad.net/~tomaszm/+archive/dynamics.The Dynamics PyMOL plugin was enhanced with tons of features including: displaying the results of MD with vectors, options to use implicit solvents, window supporting an interpretation of MD, ability to use x2top as an optional GROMACS tool, easy set up of the desired length of simulation, water box optimization, better GROMACS error and warning handling, support for Mac OS X, removing Tix dependence. With all these changes the software is marked as version 2.0. The plugin is now much more useful and provides easier and better solutions for many molecular dynamics simulation use scenarios. The software remains to be open source like GROMACS and PyMOL and provides a full open source set of utilities for viewing, setting, and performing molecular dynamics simulation using only the easy GUI tools. This allows the researcher to obtain all of those programs free of charge and modify them as he or she wishes. The source code of the plugin is available under the terms of GPLv3 license from The dynamics PyMOL plugin version 2.0 is an important step toward easy and yet powerful molecular dynamics simulation using GUI. It introduces many features, which allows for more comprehensive use of GROMACS software and also introduces additional capabilities offered by ProDy. Nevertheless, researchers need to remember that performing molecular dynamics simulation is a complex work and in many cases the default plugin settings will not produce the proper results. One could encounter errors and familiarization with GROMACS manual may be required in order to set dynamics conditions properly. This plugin should work correctly on the wide range of Unix and Unix-like operating systems as long as dependency software is installed, but Microsoft Windows operating system is not yet supported.The software is open source and free of charge, which gives researchers easy access to it. On the developer side the feedback from users allows correction of bugs and introduction of new requested features. The plugin is constantly improving and in the future it should incorporate new GROMACS capabilities and still make them as easy to use as possible.


It was an error in the structure of dopamine o-quinone published in the commentary on Evaluation of models of Parkinson's disease since the benzene ring contained an extra double bound between the carbonyls. The actual structure of dopamine o-quinone is the correct structure.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.Supported by University of Chile (ENL014/14).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.


The problem of manufacture and reproduction of materials will be quite different … inspired by biological phenomena in which chemical forces are used in a repetitious fashion to produce all kinds of weird effects (one of which is the author) … Richard P. Feynman (1959)It has long been appreciated that molecular motors and machines are central to almost every biological process. The harvesting of energy from the sun, the storing of energy, transporting cargoes around the cell, the movement of cells, generation of force (at both the molecular and macroscopic levels), replication, transcription, translation, synthesis, driving chemical systems away from equilibrium, etc.—virtually every biological task involves molecular machines. Given thThere’s plenty of room at the bottom Sto Sto49] SUltimately, we can do chemical synthesis. A chemist comes to us and says, ‘Look, I want a molecule that has the atoms arranged this and so; make me that molecule.’ Richard P. Feynman (1959)12, Figure From polyketide synthase to DNA polymerases and the ribosome, one of the key uses of molecular machines in biology is for the construction of other molecules. In 2013 an artificial small-molecule machine was invented that assembled a tripeptide of specific sequence by travelling along a track loaded with amino acid building blocks The future for the field of artificial molecular machines appears very bright. There is already a working nanotechnology based on molecular machines that perform numerous useful tasks: it is called Biology. The natural world shows us just how exquisite and diverse the functions are that can be carried out with molecular machines. Advances in artificial systems over the past 25 years mean that chemists now have the know-how and synthetic tools available to enable them to make suitable machine architectures . They can switch the position of components , they understand how to use ratchet mechanisms to create motor mechanisms, and are learning how to introduce them into more complex molecular machine systems.However, there are still basic challenges to overcome. In contrast to motor proteins, powered by ATP hydrolysis or proton gradients, there are as yet no chemically driven synthetic small-molecule motors that can operate autonomously , the closest counter-examples being the over-crowded alkene motors designed by Feringa and co-workers that rotate continuously under irradiation with light. Furthermore, although there have been some notable successes in using artificial molecular machines to bring about property changes, few have been shown to perform useful tasks that cannot be accomplished by conventional chemical means. This contrasts with the essential roles played by biological machines in numerous cellular processes. When this last step happens—and the rapid advances of the last few years suggest that that time is not too far away—then artificial molecular machines will start to become the extraordinary nanotechnology that Feynman predicted. Making that happen, as he suggested, will dou


In this review the recent progress in molecular platforms that form rigid and well-defined contact to a metal surface are discussed. Most of the presented examples have at least three anchoring units in order to control the spatial arrangement of the protruding molecular subunit. Another interesting feature is the lateral orientation of these foot structures which, depending on the particular application, is equally important as the spatial arrangement of the molecules. The numerous approaches towards assembling and organizing functional molecules into specific architectures on metal substrates are reviewed here. Particular attention is paid to variations of both, the core structures and the anchoring groups. Furthermore, the analytical methods enabling the investigation of individual molecules as well as monomolecular layers of ordered platform structures are summarized. The presented multipodal platforms bearing several anchoring groups form considerably more stable molecule–metal contacts than corresponding monopodal analogues and exhibit an enlarged separation of the functional molecules due to the increased footprint, as well as restrict tilting of the functional termini with respect to the metal surface. These platforms are thus ideally suited to tune important properties of the molecule–metal interface. On a single-molecule level, several of these platforms enable the control over the arrangement of the protruding rod-type molecular structures  with respect to the surface of the substrate. Molecular electronics, as motivated in the 1970s from a rather theoretical point of view by Kuhn and Möbius , tries tSince the first electrical measurements on benzene-1,4-dithiol molecules in 1997 –11. CurrThe ultimate goal of molecular electronics is to use assemblies of molecules or even single molecules as functional building blocks and to integrate them into electric circuits between the macroscopic electrodes, where a sufficiently strong binding between two terminal anchoring groups of the bridging molecule and the metal electrode is achieved. Only if this requirement is met, the control over the electronic properties of single-molecule devices becomes possible, which is of paramount importance for molecular devices. Furthermore, not only the effect of anchoring groups, but also accurate measurements of the molecular conductance over the functional core and molecular wire is crucial to fulfill requirements for molecular electronic devices.At present, the field of molecular electronic is far from maturity. The influence of the junction geometry on the electrical characteristics of single molecules remains a fascinating and important area of research. Early experiments have shown that the electrical characteristics of junctions of symmetric molecules are not necessarily symmetric under bias voltage . FurtherThe organization of the molecules within the junction is usually based on some sort of self-assembly using chemisorption or physisorption methods to form monomolecular layers between both electrodes. In many cases, either self-assembled monolayers (SAMs)  or Langm2) methyl tripodal platforms 3 and 34  to reali33 as well as monopodal platforms bearing both a redox-active oligothiophene tail group for CVs, and a (triisopropylsilyl)ethynyl tail group for XPS measurements were evaluated. CVs of the redox-active tripodally modified gold electrodes displayed a reversible one-electron redox wave at a surface coverage of 7.1 × 10−11 mol·cm−2. Furthermore, they examined the electrochemical stability of self-assembled monolayers on gold electrodes and found that 30% of the tripodal molecules still remain on the surface after 10 scans within the range of 0–0.55 V. XPS measurements revealed that the π orbitals of the pyridines contributed to the physisorption of the tripodal platform on gold. Measurements of single-molecule conductance were successfully carried out using modified STM techniques for single-molecule junctions that consisted of the tripodal anchors and a diphenyl acetylene linker 34. The single-molecule conductance of a metal–molecule–metal junction based on the pyridine-terminated tripodal structure 34 exhibited conductance values of (5 ± 1 × 10−4G0), about two orders of magnitude higher than that of the previously reported monopodal pyridine analogue 35  and three benchmark tripods were further studied by XPS and FTIR measurements on gold substrates [37 , fitted to the single or aggregated molecule, placed perpendicularly to the Au(111) surface. This STM-junction operating in the tunneling regime was irradiated with a short excitation pulse to measure the molecular electroluminescence when excited by local electron tunneling. Electroluminescence from the excited molecules is a strongly unipolar process and depends on the polarity of the applied bias as revealed by STML spectra, which displayed electroluminescence exclusively at positive bias polarity (ca. 1.9 V). They attributed this unipolar behavior to both, the energy alignment determined by the position of the frontier molecular orbitals to the Fermi level of gold at the molecular interface and to the molecular tip–molecule–gold junction asymmetry. Based on these results, it was suggested that at a positive tunneling bias, a photo-excited hot electron from the STM tip resonantly tunnels into an excited state of the porphyrin molecule 39  tips. The silicon core in these tripodal platforms provides a suitable template for the required construction of the three legs and the probe arm, each length being variable using different rigid oligo(phenylene ethylene)s. However, self-assembly of these 41 –Fe(II) oligomeric wires 45 on Au(111) (Recently Nishihara and co-workers employed the tetraphenylsilane scaffold bearing in the  Au(111)  and meas3-hybridized Si core, which is known to provide some σ–π conjugation with the aromatic substituents. We note that all tripodal adsorbates reported so far adopted non-π-conjugated aliphatic thiols as an anchoring group on the metal surfaces, such as sulfanylmethyladamantanes and sulfanylmethylphenyl. The formation of the SAMs of a tripodal terpyridine anchor ligand on gold was optimized to ensure that all sulfur atoms are chemisorbed, which was determined by XPS and IR measurements. The bottom-up fabrication of bis(terpyridine)–Fe(II) oligomer wires from the SAM of a tripodal terpyridine anchor ligand on gold proceeded quantitatively as determined by CV, and the perpendicular arrangement of molecular wires on a surface was corroborated by AFM and cross-sectional scanning electron microscopy (SEM). The obtained thickness of the film was in good agreement with the height estimated by molecular modelling. Finally, intrawire electron transport behavior was investigated and found that the tripodal scaffold realized fast electron transfer through the oligomeric wires, showing large k0et values.The fast electron transfer in these structures should be also supported with the sp46  and also corroborate the validity of the molecular design by a protruding rigid-rod molecular subunit, which was revealed by UHV-STM experiments (A first model platform eriments . All tog49 deposited on Au(111) was investigated in a UHV-STM and the experiments showed the tripodal shape of isolated molecules on the surface. However, the molecules most frequently lie on the surface and are rather attached via two legs while the third leg protrudes outwards. This arrangement probably arises from a strong interaction of the side and head part of the molecule with the Au(111) and also due to the presence of methylsulfanyl anchoring groups, which are exhibiting a lower affinity to the metal surfaces. In a further study, the conductance behavior of the feet of these tripodal structures with respect to their position and coupling to the surface electrode with the submolecular resolution of a STM was investigated and the results were supported by calculations of the electronic structure simulating the conformation of the molecule on the surface by DFT with dispersion corrections [We noted that also several organometallic complexes have been recently employed to serve as molecular platforms for the metal surfaces. Our group recently synthetized and employed a series of tripodal M(III) complexes  functionrections .50  complex formed islands of dimers which exhibit a medium range order. Another organometallic tripodal platform was synthetized by Launay, Rapenne and co-workers and mounted on Au(111) surface [51  complexes 50  comprisi surface ,133–134.face [51  consisti52 (fluorinated phenanthrene derivative) or a non-polar 53 (pyrene derivative) rotator and served as a dipolar and nonpolar altitudinal molecular rotors  of self-assembled monolayers provided information concerning the average orientation of the rotator with respect to the gold. Air stability measurements of such monolayers on gold showed that the sulfur-containing tentacles start to be detectably oxidized within hours, as determined by XPS. These oxidized molecules can then be washed away in a polar solvent. Detailed molecular dynamic simulations of the altitudinal rotor on a gold surface using the universal force field (UFF) potential, showed synchronous and half-synchronous unidirectional nature of the rotational hysteresis around the horizontal axle in one MMP/PPM pair of conformational enantiomers. Furthermore, the effect of the metal on the motion of adsorbed surface-mounted molecular rotors has been approximated by using the image charges and by implementing a Langevin molecular dynamics with electronic friction. Each of the rotors can exist as three pairs of enantiomers , where these symbols correspond to the helical P/M symmetry of both tetraarylcyclobutadienes and the rotator in 52. In 52 representing maximal  and minimal  rotator–tentacle interaction on the surface. While in conformation B the tentacles do not interfere with rotation and the rotation is energetically possible for all pair of enantiomers, the rotational energy barriers for all three stereoisomers in conformation A exceed 30 kcal/mol and hamper rotation of the rotator on the surface [They fabricated monolayers and submonolayers on gold and this surface attachment of altitudinal molecular rotors provided with ten –HgSCHtroscopy . These rtroscopy . The STM surface . The sur surface –139.54 (TATA) or trioxatriangulenium 55 (TOTA) cation (Another platforms based on flat triazatriangulenium ) cation  were synC3-symmetrical trioxa- or triazatriangulenium platforms are oriented perpendicular to the metal surface. In the case of TATA platform the size and the lateral arrangement of these platforms in the densely packed hexagonal array on gold is determined by the length of the alkyl side chains at three outer nitrogen atoms. Although, the interaction of functionalized TATA platforms with gold surfaces is mainly based on weak dispersion forces, the binding energy of these platforms is surprisingly higher than that of thiols to gold. It was also found that, despite the presence of a sp3-hybridized carbon atom in the conduction path of the TATA unit, the TATA platform exhibits a contact resistance only slightly larger than that of the thiols [3-hybridized carbon atom. Several different derivatives of TATA platforms on Au(111) surfaces uprightly functionalized with ethynyl, phenyl, azobenzene, zinc-porphyrins were synthetized and adlayers have been studied by using STM, XPS, CP-AFM, gap-mode surface-enhanced Raman spectroscopy (SERS), infrared-reflection absorption spectroscopy (IRRAS), cyclic voltammetry (CV), and quantum chemical calculations [2) hampers the lateral interactions between the perpendicular functional moieties in the monolayer. In several studies, Herges and Magnussen addressed the features and advantages of these TATA platforms, in particularly to control the spatial arrangement of functional molecules on metal surfaces and to create a free volume for sterically demanding operations in densely packed self-assembled monolayers, e.g., in surface-mounted molecular switches based on azobenzenes [This “platform approach” allows for a comprehensive spatial and lateral control of the molecular arrangement and orientation on metal surfaces. The functional moieties attached to the central carbon atom of e thiols . The TATulations –146. Usibenzenes ,146, azibenzenes . The “plIn this review several approaches to control the spatial arrangement of molecular structures at planar solid substrates have been presented. In several cases the control over the lateral order of these large footprint structures was equally interesting. The main focus was set on tripodal organic architectures and, to the best of our knowledge, organic model compounds exposing three anchor groups for noble metal substrates are discussed comprehensively. In contrast to that, alternative concepts profiting, e.g., from an even larger number of anchor groups or from the van der Waals interaction of extended aromatic systems are only represented with particular appealing examples.While all platforms exposing multiple anchor groups exhibited an increased stability of the molecular monolayer compared with analogues comprising only a single anchor group, the extent of anchor groups really forming covalent bonds with the substrate and thus also the perfection of structural control at the interface varies considerably between the various design concepts. Also other important structural features such as the spatial control over the protruding molecular subunit, the nature of its coupling to the substrate, or the lateral arrangement of neighboring molecules vary considerably between the different presented examples. The same is also true for chemical aspects such as the modularity of the approach defined by the ease to alter the protruding subunit for a particular footing structure.The large variety of the different molecular platforms is not surprising because they were not all optimized for the same purpose, and the requirements for an ideal footing structure differ considerably between different applications and are in some cases even in contradiction with each other. While for example a strong electronic coupling between the substrate and the protruding subunit is preferable for electronic devices, it leads to immediate quenching of a molecular excited state in labeling applications and thus, insulating features are more appealing for the latter.In spite of the large number of already synthesized and investigated multivalent molecular platforms, there remains a rich structural variety to explore. The structure–property correlations of the model compounds reported so far reveal molecular design rules supporting the further development of molecular footing structures. The ideal platform for a particular application still has to be custom-built and we are looking forward to many interesting structures still to be found.


An influential model suggests that dopamine signals the difference between predicted and experienced reward. In this way, dopamine can act as a learning signal that can shape behaviors to maximize rewards and avoid punishments. Dopamine is also thought to invigorate reward seeking behavior. Loss of dopamine signaling is the major abnormality in Parkinson’s disease. Dopamine agonists have been implicated in the occurrence of impulse control disorders in Parkinson’s disease patients, the most common being pathological gambling, compulsive sexual behavior, and compulsive buying. Recently, a number of functional imaging studies investigating impulse control disorders in Parkinson’s disease have been published. Here we review this literature, and attempt to place it within a decision-making framework in which potential gains and losses are evaluated to arrive at optimum choices. We also provide a hypothetical but still incomplete model on the effect of dopamine agonist treatment on these value and risk assessments. Two of the main brain structures thought to be involved in computing aspects of reward and loss are the ventral striatum (VStr) and the insula, both dopamine projection sites. Both structures are consistently implicated in functional brain imaging studies of pathological gambling in Parkinson’s disease. Pathological gambling can be conceptualized as a disorder of reward and punishment processing, whereby the gambler selects an immediate but risky opportunity to obtain money over the larger, more probable opportunity to save money . Other factors such as outcome ambiguity and variance (sometimes referred to as risk) also affect individual choices  imaging allows for changes in endogenous levels of dopamine to be inferred from changes in the binding of the [11C]raclopride PET studies reported a greater reduction of VStr binding potential (an index of dopamine release) during gambling   . It is conceivable that enhanced mesolimbic transmission is also a risk factor in the general population (Buckholtz et al., 2 receptor agonism alone is sufficient for the development of the addictive syndrome. While combined D1/D2 agonists such as L-Dopa may themselves be addictive (Lawrence et al., 2 agonists are not typically administered compulsively; rather, they have the ability to promote other addictions such as pathological gambling (O’Sullivan et al., 1 receptor stimulation is reinforcing while D2 receptor stimulation inhibits the inhibitory indirect pathway. We suggest that D2 agonism, in vulnerable individuals, has the effect of “releasing the brake” on reinforcement systems, thus facilitating the development of impulse control disorders. The time-locked nature of the D2 effect, and the fact that addictive behaviors typically resolve upon discontinuation of the dopamine agonist, is consistent with the theory that tonic dopamine has an invigorating effect on reward seeking behavior (Niv et al., The causal association between dopamine DWe note however that other mechanisms besides dopamine-mediated disruption of responses to reinforcing events and stimuli may play a role. For example, Averbeck et al.  have proThe 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.


Pyogenic granuloma is a benign vascular tumor that may affect the gastrointestinal tract. This report describes a rare case of sigmoid-colon pyogenic granuloma in a 4-month-old boy causing intussusception. Resection and anastomosis were curative. The mother had history of high dose of progesterone exposure during initial weeks of conception for vaginal bleeding. This may point towards etiology of the lesion. A 4-month-old boy was referred with sudden onset of colicky abdominal pain, vomiting, and rectal prolapse. Ultrasonic evaluation of the abdomen revealed intussusception. Contrast enema successfully reduced the intussusception and revealed a 2cm x1.9cm tumor-like lesion in the sigmoid colon that partially occluded the lumen . Routine blood tests were normal. Diagnostic single-port transumbilical laparoscopy was immediately performed which revealed a serosal tumefaction at the sigmoid colon level. Laparoscopy was converted to open surgery which confirmed the lesion. The colon was incised opposite to the mass revealing a polypoid, sessile, ulcerated tumor. Segmental resection-anastomosis of the sigmoid colon was performed. The post-operative course and the two years follow-up were uneventful.Detailed obstetric history revealed that during initial weeks of pregnancy there was frequent vaginal bleeding that was treated with micronized progesterone vaginal ovule (dosed 200 mg) for nineteen weeks and a weekly intramuscular injection of 340 mg of 17-hydroxyprogesterone caproate (17-OHPC) for seventeen weeks (maintenance therapy). The child was born by cesarean section at 34th week of gestation following the onset of maternal hypertension and interruption of fetal growth.Histopathological examination showed a submucosal lesion with ulcerated mucosal lining and consisted of a lobular hemangioma in a fibrous matrix . Antibodies against CD31 highlighted closely packed capillaries , while antibodies against estrogen receptors (SP1 Ventana) and progesterone receptors (1E2 Ventana) were negative.The etiology of pyogenic granuloma remains unclear. Supposed predisposing factors reported in literature are trauma, angiogenic stimulating factors, and hormones overgrowth as observed in pregnancy or in peripubertal age.[1] Hormonal imbalance during pregnancy associated with bacterial plaque and gingival inflammation was supposed to cause pyogenic granuloma on the oral mucosa. Generally, the lesion appears in the second or third month of pregnancy and disappears after the delivery.[2] Estrogen promotes granulation tissue formation and enhances macrophage production of vascular endothelial growth factor (VEGF), favoring the development of pyogenic granuloma in pregnancy.[3] Progesterone also promotes VEGF expression and enhances chronic tissue reaction in gingival tissue.[4] Both progesterone and estrogen inhibit granuloma cell apoptosis.[4]17-OHPC is a very useful therapy in reducing preterm birth rates in women and is detectable in maternal and fetal blood for at least forty-four days after the last injection as micronized progesterone that also helps to limit preterm delivery.[5] The voluminous size of the granuloma in our case probably be explained referring to an early origin of the lesion in pregnancy, after some unidentified mucosal damages, and the subsequent early exogenous pharmacological stimulation. Prolonged and intense progesterone exposure in the first twenty-three weeks of pregnancy may have promoted the considerable growth of the polyp. Furthermore, in the absence of extracellular hormone stimulation (as it happens after birth) progesterone receptors degrade , explaining receptor negativity at the histology.Finally, according to Whitaker the quantity of estrogen or progesterone receptors in pyogenic granuloma is less important in the pathogenesis of this lesion than the circulating hormone levels.[6] This case is possibly another example of how diverse progesterone effects are, but further studies are required to support this hypothesis and to find hormonal markers to detect these lesions after birth. To conclude, pyogenic granuloma is a rare clinical entity that can act as a lead point in intestinal intussusception. Its association with full-dose hormonal supplementation during pregnancy could explain rapid growth of these lesions and possible complications. Women receiving these hormones during initial days of pregnancy must be educated about development of these complications in the baby after birth.Source of Support: NilConflict of Interest: None declared


Here, we investigated whether this difference could be due to differences in the degree of dopamine receptor activation in the NAc. First, we observed that although rats given ad libitum access to chow in their home cages approached a food receptacle in response to reward-predictive cues, the number of such approaches declined as animals accumulated food rewards. Intriguingly, cued approach to food occurred in clusters, with several cued responses followed by successive non-responses. This pattern suggested that behavior was dictated by transitions between two states, responsive and non-responsive. Injection of D1 or D2 dopamine receptor agonists into the NAc dose-dependently increased cue responding by promoting transitions to the responsive state and by preventing transitions to the non-responsive state. In contrast, antagonists of either D1 or D2 receptors promoted long bouts of non-responding by inducing transitions to the non-responsive state and by preventing transitions to the responsive state. Moreover, locomotor behavior during the inter-trial interval was correlated with the responsive state, and was also increased by dopamine receptor agonists. These results suggest that activation of NAc dopamine receptors plays an important role in regulating the probability of approach to food under conditions of normative satiety.Dopamine receptor activation in the nucleus accumbens (NAc) promotes vigorous environmentally-cued food-seeking in hungry rats. Rats fed  For a hungry animal, the decision to respond to a food-predictive cue is a trivial one. Hungry, well-trained animals respond to nearly every cue signaling food availability. The likelihood and vigor of these responses, however, is lower in the normative state of satiety. What are the neural mechanisms that set the probability of approach to food under such conditions? Because responding to food-predictive cues in the absence of caloric need likely contributes to elevated calorie consumption  is critical for cued approach toward food-associated objects under conditions where a rat's starting position varies from trial to trial. Under these conditions, injection of either D1 or D2 dopamine receptor antagonists into the NAc core reduces the proportion of cues to which animals respond by increasing the latency to initiate approach Nicola, . These ead libitum in their home cages in order to induce a state of relative satiety, which greatly decreased the probability that animals would respond to a given cue presentation. This lower response probability allowed us to assess whether dopamine receptor agonists increase that probability, which is not possible in hungry animals because they respond to nearly every cue. We found that blocking dopamine receptors decreased responding whereas activation of the same receptors increased responding. These results suggest that response probability and food seeking in relatively sated animals is actively regulated by NAc dopamine.To test this hypothesis, we asked whether pharmacologically blocking and tonically activating NAc dopamine receptors in non-food-restricted animals could, respectively, attenuate and promote cue responding. In the experimental phase, rats had access to food and water Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Albert Einstein College of Medicine.Eight male Long-Evans that weighed 275–300 g were purchased from Harlan and singly housed on a 12 h light/ dark cycle. All experiments were conducted in the light phase. Animal care was identical to previously published accounts Nicola, . Upon arBehavioral training took place in operant chambers (30 × 25 cm) purchased from Med Associates. Experiments were conducted in sound-attenuating cabinets with blue house lights illuminated. A constant white noise (65 dB) was played within the chamber to limit distractions from outside noise. Operant chambers were equipped with a reward receptacle on one wall. A photobeam located across the front of the receptacle measured receptacle entry and exit times. A syringe pump, located outside the chamber, was used to deliver liquid sucrose reward into the reward receptacle. Behavioral time stamps were recorded with a resolution of 1 ms.ad libitum in their home cages from that point until the end of experiments. After task performance stabilized, the sucrose concentration of the liquid reward was reduced from 10% to 3%; the volumes were not changed. Behavior was monitored daily until asymptotic task performance was achieved.Animals were food restricted during the initial training stages. The first stage of training required that the animals enter the food receptacle, which triggered delivery of 10% liquid sucrose. After a 10 s delay to allow for reward consumption, animals had to leave the receptacle and re-enter it in order to earn additional reward. In subsequent training stages, delays of 20 s and then 30 s were introduced between reward availability. Criterion performance was set at 100 rewards earned in 1 h. After criterion performance was established with a 30 s delay between reward availability, two auditory cues were introduced that predicted either a small or large reward (150 or 250 μl of 10% sucrose solution in water). The auditory cues consisted of a siren tone (which cycled in frequency from 4 to 8 kHz over 400 ms) and an intermittent tone ; cues were assigned to large and small reward randomly for each rat and the cue-reward magnitude relationship remained constant across training and experiments for a given rat. Reward delivery was contingent on the rat entering the reward receptacle during the cue presentation, at which point the cue was terminated. Cues were on for up to 5 s. The inter-trial interval was chosen pseudorandomly from a truncated exponential distribution with a mean of 30 s. Once animals responded to > 80% of the cues, animals were fed After behavioral performance stabilized, bilateral guide cannulae targeting the NAc core were chronically implanted as described previously Nicola, . BrieflyDrugs were purchased from Sigma and freshly dissolved in 0.9% sterile saline on the day they were used. Drug doses per side were: “D1 agonist low,” 0.1 μg SKF81297; “D1 agonist high,” 0.4 μg SKF81297; “D1 antagonist,” 1.1 μg Schering 23390; “D2 agonist low,” 1 μg quinpirole; “D2 agonist high,” 10 μg quinpirole; “D2 antagonist,” 2.2 μg raclopride.As previously described Nicola, , rats weOn test days, the rat's position was recorded using an overhead camera (30 frames/s) and automated tracking system (either Plexon Cineplex or Noldus Ethovision). This system tracked the x and y positions of red and green LEDs attached to the rat's head. As previously described Nicola, , to deteOne rat failed to re-attain pre-surgery performance levels after cannula implantation and thus was not subjected to microinjections. The cannulae from a second rat became clogged and consequently some microinjections were not performed. Thus, data were obtained from 7 microinjections for some experiments and 6 for others. Behavioral time stamps and raw video tracking position data were exported and analysis was performed with custom routines in the R statistical computing environment  significantly influenced response ratio. Two-tailed Welch's t-tests were used to compare response ratios for each drug to saline. P values for post-hoc t-tests were corrected using the Sidak multiple comparisons correction procedure. The significance threshold for all statistical tests was set at p < 0.05. The results from all statistical tests can be found in Table In Figures Post-hoc two-tailed Sidak-corrected Welch's t-tests were used to compare both pause number and total time spent in pauses in each drug and saline.In Figures Figures 4A,C,F,H, each trial t was coded as eliciting a response (R+) or failing to elicit a response (R−). We then calculated the empirical probability of the occurrence of R+ or R− at t+1. This procedure results in 4 probability measures, each of which is associated with a unique pattern of response and no response on the two consecutive trials, t and t+1: P(R+R+), P(R+R−), P(R−R−), P(R−R+). When these probabilities are arranged so that each couplet that begins with the same response type (R+ or R−) is on the same row of a 2 × 2 matrix, each row sums to one; i.e., the matrix is right stochastic. In Figures 4A,C,F,H, we plotted (separately for each drug) the mean probabilities for each couplet with the row values of these matrices on the same axis. For example, P(R+R+), P(R+R−) are on the vertical axis because each couplet begins with an R+. Because each row of each matrix sums to one, the matrix values are all positive, and the rat can freely transition from a responsive (R+) to non-responsive state (R−), and vice versa, the stochastic matrix can describe a Markov chain for which a stationary probability vector π can be calculated. These probability vectors are estimates of the probability of finding the rat in the responsive and non-responsive state at a steady state of the Markov chain .In Figures Animals were deeply anesthetized with Euthasol and decapitated with a guillotine. Brains were quickly removed from the skull and then fixed in formalin. Prior to slicing with a cryostat, brains were cryoprotected by immersion in 30% sucrose for several days. Sections (50 μm) were stained for Nissl substance to visualize cannula and injector tracks. Estimates of injection sites for each animal are shown in Figure We trained 8 rats to respond to distinct auditory cues that predicted either a small or large sucrose reward Figure . Even thBy the time performance stabilized, 4 days after switching to 3% sucrose rewards, a difference in responding to large and small reward cues was evident toward the beginning of the session  Figure , dark reP(R+R−) = N(R+R−) / N(R+); see Methods section Data analysis). Note that the probability of transitioning out of a state is therefore 1 minus the probability of remaining in a state  = 1 – P(R+R+)). Thus, in Figures The increase in cue responses caused by the D1 and D2 agonists, as well as the greater time spent in the responsive than the non-responsive state, could be explained either by an increased probability of transitioning from the non-responsive to the responsive state, or conversely, a decreased probability of transitioning from the responsive to the non-responsive state (or both). To determine which of these was the case we implemented a simple two state Markov model Figure  by calcuP(R+R+) > P(R+R−); Figure P(R−R−) > P(R−R+); Figure In the first hour of behavioral testing, saline-treated rats tended to cluster their cue responding: if they responded to one cue, the probability of a response to the next cue was greater than that of a non-response  or a trend to an increase  Figure . Thus, tCue-elicited approach is strongly dependent on the mesolimbic dopamine projection from the VTA to the NAc only in very specific circumstances: those in which responding involves “flexible approach” Nicola,  [also caad libitum access to chow. The dependence of taxic approach on mesolimbic dopamine is therefore not a function of nutrient deficit or the subject's state of hunger. Indeed, the present results support a role for mesolimbic dopamine in promoting cue-elicited approach to high-calorie food even in the absence of a homeostatic need for calories, supporting the hypothesis that this circuitry contributes to overeating and obesity  task in which the PS predicted 10% sucrose reward on only 15% of trials, the response probability was lower, and pharmacological blockade of dopamine reuptake increased this probability  to show that responding to NS presentation is not increased by the agonists. We did not include an NS in our design. We hypothesize that had we done so, we would have observed an increase in locomotion during the NS  but not an increase in receptacle entries. This hypothesis is based on several observations indicating that the increased entry probability after cue presentation was not a result of increased generalized locomotion. First, the increase in locomotion during the inter-trial interval caused by the agonists was decoupled from the increase in cue responding, occurring even during intervals that were followed by a non-response to the cue Figure . Second,Another difference between the current task and our previous studies using DS and PS tasks is that we presented two reward-predictive cues, which predicted large and small volumes of sucrose, rather than one reward-predictive cue and one non-reward-predictive stimulus (NS). We included dual reward-predictive cues in the task design in order to assess whether manipulations of NAc dopamine receptors differentially influence behavior triggered by cues predictive of different reward magnitudes. However, we could not conduct such an analysis because the animals did not robustly differentiate between the two cues. When the reward was 10% sucrose, there were no significant differences in response ratio between large- and small-reward predictive cues; and when the reward was 3% sucrose, a small (~20%) difference was observed only in the first hour of the session Figure . More read libitum chow-fed animals was a decline in cue response probability over the 2 h session, which was far more pronounced when the reward was 3% sucrose than when it was 10% sucrose. Rats given free access to sucrose show a similar decline in lick rate from the beginning of the session, which is attributable to satiation: post-ingestive nutrient detection mechanisms signal to the brain, resulting in reduced consumption  rate of responding and greatly reduced the magnitude of the normal decline in responding from the first to second hour compared with the control condition Figures , essentiAn alternative hypothesis is that the dopamine drugs did not interfere with reinforcement, but with a neural mechanism that directly activates cued approach behavior. This proposal is supported by our previous studies demonstrating that a large proportion  of NAc neurons are excited by cues in a DS task  conditions, response clusters were longer and more frequent toward the beginning of the session, becoming shorter and less frequent toward session end; and necessarily vice-versa for non-response clusters. This pattern suggest that there are two states, responsive and non-responsive Figure , which fThe effects of the dopamine agonists and antagonists on transition probabilities are consistent with the hypothesis that response state is a function of dopamine receptor occupation. Thus, when NAc dopamine levels reach and exceed a threshold, the animal is in the responsive state; below this threshold, the animal is in the non-responsive state. Testing this hypothesis would require measuring tonic dopamine levels as animals perform this or a similar task; the hypothesis predicts that dopamine levels should be higher during response clusters than non-response clusters. Although to our knowledge previous microdialysis studies have not examined whether fluctuations in dopamine level correlate with local taxic approach probability, a previous investigation found that NAc dopamine levels were higher when food pellets were dropped into receptacles at 45 s or 4 min intervals  than when food was freely available (a situation that minimizes the need for taxic approach) , but also that dopamine levels are set by the rate of reinforcement (Niv et al., ad libitum-fed animals observed here is consistent with many recent studies of regulation of dopamine neurons by messengers, such as cholecystokinin, orexin, ghrelin, leptin, insulin and glucagon-like peptide 1, that signal the body's nutrient status detected via various mechanisms. In general, signals that report nutrient deficit increase dopamine neuronal activity, whereas signals that report satiety or nutrient surfeit decrease it (Ladurelle et al., ad libitum-fed animals—is therefore ideal for further investigation of the regulation of dopamine dynamics by nutrient state, reinforcement rate, and other parameters, and of the mechanism by which these variables impact NAc dopamine-dependent behavior.The low dopamine-dependent response probability in JD designed and conducted the experiment, analyzed the data, and co-wrote the paper. SN advised JD on design and analysis and co-wrote the paper.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.