All three psychostimulant drugs, through their effects on dopamine efflux, may have addictive liability although the effect of 3,4-CTMP on dopamine suggests that it might be most addictive and ethylphenidate least addictive.3,4-dichloromethylphenidate  and ethylphenidate are new psychoactive substances and analogs of the attention deficit medication methylphenidate. Both drugs have been reported on online user fora to induce effects similar to cocaine. In the UK, 3,4-CTMP appeared on the drug market in 2013 and ethylphenidate has been sold since 2010. We aimed to explore the neurochemical effects of these drugs on brain dopamine and noradrenaline efflux. 3,4-CTMP and ethylphenidate, purchased from online vendors, were analyzed using gas chromatography and mass spectroscopy to confirm their identity. Drugs were then tested in adolescent male rat brain slices of the nucleus accumbens and stria terminalis for effects on dopamine and noradrenaline efflux respectively. Fast cyclic voltammetry was used to measure transmitter release. Methylphenidate (10 μM) increased evoked dopamine and noradrenaline efflux by 4- and 2-fold, respectively. 3,4-CTMP (0.1 and 1 μM) increased evoked dopamine and noradrenaline efflux by ~6-fold and 2-fold, respectively. Ethylphenidate (1 μM) doubled evoked dopamine and noradrenaline efflux in both cases. 3,4-CTMP's effect on dopamine efflux was greater than that of methylphenidate, but ethylphenidate appears to be a weaker dopamine transporter inhibitor. Experiments using the dopamine D Concern has arisen over the past few years about “legal highs” or new psychoactive substances (NPS). Some of these NPS produce similar effects to cocaine, amphetamine, or MDMA (Ecstasy) and have changed the landscape of the UK drug scene by offering drug users the opportunity to use drugs without the risk of prosecution. The most prominent NPS is mephedrone, which was banned in the UK in 2010. The number of legal highs being sold on the Internet is increasing year-on-year. Deluca et al.  a main effect of drug concentration  and a drug X concentration interaction . Within the methylphenidate treated slices both 1 and 10 μM caused a significant increase in dopamine efflux (p < 0.05). Within the 3,4-CTMP treated slices, 100 nM caused an increase in evoked dopamine levels vs. controls (p < 0.05) while both 1 and 10 μM 3,4-CTMP increased evoked dopamine levels vs. both control and 100 nM (p < 0.05). Ethylphenidate did not significantly increase evoked dopamine levels at any concentration .Using a two-way ANOVA we compared the effects of methylphenidate, 3.4-CTMP and ethylphenidate on electrically evoked dopamine efflux in the accumbens Figures . There wp < 0.05 vs. both), 1 μM (p < 0.001 vs. both), and 10 μM (p < 0.001 vs. ethylphenidate and p < 0.05 vs. methylphenidate) while methylphenidate increased dopamine levels to a greater extent than ethylphenidate at 10 μM (p < 0.001).3,4-CTMP increased dopamine levels to a greater extent than both methylphenidate and ethylphenidate at 100 nM  = 0.0781, p = 0.925] and no drug X concentration interaction  but there was a main effect of drug concentration  all increased evoked noradrenaline efflux vs. controls.Using a two-way ANOVA we compared the effects of methylphenidate, 3.4-CTMP and ethylphenidate on electrically evoked noradrenaline efflux in the BNST Figures . There wF = 10.598, p < 0.001; Figure p < 0.001) and was nearly greater after haloperidol vs. yohimbine (p = 0.056). There was no significant effect of yohimbine vs. control (p = 0.659).Using a one-way ANOVA we compared the effects of haloperidol and yohimbine in the accumbens and BNST. In the accumbens there was a significant effect of drug [F = 12.477, p < 0.005; Figure p < 0.05) and control (p < 0.01). There was no significant difference in the effect of haloperidol vs. control (p = 0.999).In the BNST there was also a significant effect of drug [F = 20.073, p < 0.001; Figure p < 0.001) and desipramine (p < 0.005). There was no significant effect of desipramine vs. control (p = 876).Again using a one-way ANOVA we tested the effects of GBR12909 and desipramine on amine efflux in the accumbens and BNST. In the accumbens there was a significant effect of drug [F = 6.26, p < 0.005; Figure p < 0.05) and GBR12909 (p < 0.05). There was no effect on amine efflux of GBR12909 vs. control (p = 0.813).In the BNST there was also a significant effect of drug [The majority of drugs of abuse increase dopamine efflux in the nucleus accumbens  by eitheThere is a paucity of published research surrounding 3,4-CTMP, which was developed and tested in the 1990's as a potential treatment for cocaine abuse . It was Previous work from this lab has examined the effects of cocaine on accumbens dopamine efflux , 33. TheWe also found 3,4-CTMP to increase electrically evoked noradrenaline efflux, with 10 μM resulting in an ~2-fold increase in the BNST. We have been unable to find any published data relating to the effect of 3,4-CTMP on noradrenaline levels, but methylphenidate does block the noradrenaline transporter . Our daThere is a lack of published research on the effects of ethylphenidate on monoamine release. Ethylphenidate was first identified in two overdose victims who had co-administered large quantities of methylphenidate and ethanol , which r,Internet drug fora have provided some useful information on the effects of ethylphenidate, with some reports suggesting that it produces a less jittery, more euphoric high than methylphenidateWe found ethylphenidate to increases electrically evoked dopamine efflux by ~2-fold. The likely mechanism of action is by blocking the DAT; the same mechanism of action as methylphenidate . In prevThis study explored the neurochemical profile of the NPS methylphenidate analogs 3,4-CTMP and ethylphenidate as well as the parent compound methylphenidate. 3,4-CTMP was more potent than methylphenidate, increasing dopamine release ~6-fold, presumably caused by blockade of the DAT. Ethylphenidate has a weaker effect, modestly increasing stimulated dopamine release by ~2-fold. All 3 drugs increased noradrenaline efflux ~2-fold. At the concentrations tested, no drug increased basal levels of dopamine or noradrenaline, that is, they did not cause reverse transport as one might expect with an amphetamine-like drug. The effects of each drug on dopamine indicate that they have the potential to be addictive, especially 3,4-CTMP.in vitro work was carried out and animals were killed by schedule 1 methods, no UK Home Office License was needed. Thus this work was done in accordance with the UK animal scientific procedures act.This work was approved by the ethics committee at St George's University of London. Because only CD conceived of the work, undertook some experiments, did the statistics and wrote most of the manuscript. CR and VB undertook most of the experiments and helped analyse data. JR helped write themanuscript and conceived some of the work and analysed the NPS structures using mass spec.JR was employed by company TICTAC Communications Ltd. The other 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.


Oversupply of bulk saturated fatty acids (SFA) induces metabolic disorders and myocardial dysfunction. We investigated whether, without causing metabolic disorders, the uptake of individual dietary SFA species alters lipid profiles and induces myocardial dysfunction. C57BL/6 mice were fed various customized long-chain SFA diets , including a beef tallow (HBD), cocoa butter (HCD), milk fat (HMD) and palm oil diet (HPD), for 6 months. An isocaloric fat diet, containing medium-chain triglycerides, served as a control (CHD). Long-term intake of dietary long-chain SFA differentially affected the fatty acid composition in cardiac phospholipids. All long-chain SFA diets increased the levels of arachidonic acid and total SFA in cardiac phospholipids. The preferential incorporation of individual SFA into the cardiac phospholipid fraction was dependent on the dietary SFA species. Cardiac ceramide content was elevated in all mice fed long-chain SFA diets, while cardiac hypertrophy was only presented in mice fed HMD or HPD. We have demonstrated that the intake of long-chain SFA species differentially alters cardiac lipid profiles and induces cardiac dysfunction, without causing remarkable metabolic disorders. Obesity and type-2 diabetes are prevalent in Western countries and countries elsewhere that have adopted a Western lifestyle. In both conditions, lipotoxic cardiomyopathy commonly ensues, due to excessive fatty acid uptake by cardiomyocytes. The outcomes of patients with lipotoxic cardiomyopathy include severe heart failure, leading to death or heart transplantation, representing a major health problem. Cumulative evidence indicates that high-fat diets induce an elevation of blood triglycerides and free fatty acids, and persistent accumulation of lipids in the heart, leading to myocardial dysfunction, also described as lipotoxic cardiomyopathy ,2. CardiThe predominant SFA in Western diets are myristic acid (14:0), palmitic acid (16:0) and stearic acid (18:0), which display different metabolic properties . It has In this study, we hypothesized that individual dietary SFA species may directly induce differential changes in lipid profiles and myocardial dysfunction. Accordingly, we investigated whether the oversupply of individual SFA species, originating from commonly-used Western diet fats, including beef tallow, cocoa butter, milk fat and palm oil, could induce cardiac dysfunction and alter lipid profiles in mice, without causing metabolic disorders.This study conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23). All experimental procedures were approved by the Animal Use Subcommittee at the University of Western Ontario, Canada . Breeding pairs of C57BL/6 mice were purchased from The Jackson Laboratory . All animals were kept under controlled conditions at room temperature and constant relative humidity, with free access to food and water. The customized high-fat diets with major forms of long-chain SFA, including a high beef tallow diet (HBD), high cocoa butter diet (HCD), high milk fat diet (HMD) and high palm oil diet (HPD), representing diets rich in stearic acid (HBD and HCD), myristic acid (HMD) and palmitic acid (HPD), respectively, were all purchased from Harlan Laboratories. An isocaloric high-fat diet (CHD) was designed to contain medium-chain triglycerides as a control. The contents (% by weight) of protein, carbohydrate and fat in these high-fat diets were 20.9%, 44.8% and 20.2%, respectively. The calculated caloric intakes from these nutrients  were 18.8%, 40.3% and 40.9%, respectively. The detailed fatty acid compositions of the high-fat diets are summarized in w/w) and mice fed the control high-fat diet . Thus, we did not include a normal diet group in the current study, and all the results from dietary long-chain SFA groups were compared with those from the isocaloric control CHD group.In our preliminary experiment, of 6-month duration, there was no difference in cardiac function indices and metabolic parameters  between mice fed a normal rodent diet  were fed experimental diets for 3 or 6 months. Animal body weight was measured weekly among the 6-month diet groups. Mice were fasted overnight before terminal sample collection. Blood and heart tissues were collected immediately after animals were euthanized by cervical dislocation. Heart weight was recorded prior to the tissue being frozen at −80 °C. The serum was isolated and kept at −80 °C.Male C57BL/6 mice , while a significant decrease of 14:0 content was observed in HCD and HPD groups, compared with the CHD group. HPD induced a marked increase of 16:0 in serum lipids, whereas HBD-, HCD- and HPD-fed mice showed elevated 18:0 levels compared with CHD-fed mice. These alterations of individual lipid species in serum reflect the intake of constituent SFA in diets. However, the total SFA content was much higher, only in the HPD group, in comparison to the CHD group (at both three months and six months).In the present study, fatty acid profiles of total lipids in serum from mice fed different dietary SFA were analyzed by gas chromatography . An incrp < 0.05 or p < 0.01). Interestingly, the levels of docosahexaenoic acid  were greatly lower in mice fed CHD for six months, compared with those fed CHD for three months (0.25 ± 0.06 versus 0.44 ± 0.06), while slight changes in DHA level in serum lipids were documented in other high dietary fat diets, when a comparison was made between medium (3 months) and long (6 months) feeding periods. This might suggest that long-term intake of medium-chain SFA led to elevated transformation of DHA to other metabolites.As for monounsaturated fatty acids (MUFA), the level of palmitoleic acid (16:1) was 20–50% lower in both HCD and HPD groups, while the levels of oleic acid (18:1) were about 30% higher in both HBD and HMD groups, compared with the CHD group . The mosTo examine whether dietary SFA induced alterations in fatty acid composition in cardiac tissues, we extracted total fats from hearts of mice fed different high-fat diets and fractionated them into neutral lipids and phospholipids. The quantitative results for fatty acid profiles from neutral lipids and phospholipids are summarized in Phospholipids are one of the most abundant membrane lipid classes and play multiple pivotal roles in cell membrane function under physiological and pathological conditions. Early reports have illustrated that the fatty acid composition of phospholipids changes in response to dietary fat intake ,16,17. OFor MUFA, all the dietary fat diets resulted in a significant reduction in the level of eicosenoic acid (20:1) in cardiac phospholipid fractions from mice fed high SFA diets for three and six months, compared with those of CHD. Moreover, the level of 16:1 was also decreased in mice fed HPD for three and six months. As for PUFA, consumption of HMD for three and six months resulted in a significant increase in docosapentaenoic acid (22:5), whereas consumption of HPD for three months enhanced the level of 22:5 compared with CHD . There wIn this study, levels of several individual ceramide species  in heart tissues were slightly increased in response to feeding HBD, HCD, HMD and HPD for three months, compared with those in the CHD group . The totLevels of malondialdehyde in heart tissues were significantly increased in response to feeding HBD, HMD and HPD for three months and six months . HoweverThe major findings of this study are that uptakes of all dietary long-chain SFA induced cardiac dysfunction, without causing significant changes in blood pressure, fasting blood glucose, glucose tolerance and insulin resistance in mice, suggesting that over-supply of individual SFA may compromise myocardial function by mechanisms independent of the majority of metabolic disorders. The changes in blood fatty acid profile, altered fatty acid composition in cardiac phospholipids and increased cardiac ceramide and malondialdehyde contents, induced by varied SFA consumption, highlight the potential mechanisms by which dietary SFA intake compromised cardiac function. Thus, this study lays the foundation for future studies about the influence of dietary SFA intake on the heart. To the best of our knowledge, this is the first study that has comprehensively investigated the effects of various SFA diets intake on the heart in vivo.It has been recognized that the type of dietary fats consumed in a diet may be a key determinant for heart health ,19. SFA,w/w), or calculated as about 40% energy from fat, because they altered blood lipid profiles but did not change most vital metabolic profiles, including body weight, blood pressure level, fasting blood glucose level, insulin and glucose tolerance. Thus, these SFA diets allow us to investigate the link between individual SFA oversupply and cardiac changes, without the complications associated with metabolic disorders. It is should be noted that there were two exceptions to the lack of changes in body weight noted above. Specifically, after three months of HPD feeding as well as six months HMD feeding, significant increases in the body weight of mice were noted. The difference in body weight between HPD and CHD groups diminished after six months feeding, hence, so far, it is still unclear whether HPD affected the body weight of mice. Nevertheless, the increased body weight induced by HMD feeding was consistent with a previous study in which mice fed a Western diet, containing 21% milk fat, gained 25–35% more body weight than control mice [In this study, we examined several SFAs commonly found in the diets of our population, including palm oil (HPD), milk fat (HMD), beef tallow (HBD) and cocoa butter (HCD), which represent diets rich in myristic acid (HMD), palmitic acid (HPD), and stearic acid (HBD and HCD), respectively. Because we did not see any differences between CHD and normal diet, in terms of body weight, blood glucose, blood pressure, cardiomyocyte size and myocardial function, in our preliminary experiments, we used CHD as a control diet for this study. As a control diet, CHD only contained short- to medium-chain SFA (6:0 to 10:0), whereas the major forms of SFA in HBD, HCD, HMD and HPD were long-chain SFA (14:0 to 18:0). Notably, we chose the total fat content of high SFA diets with 20% , whereas the major forms of SFA in other high dietary SFA groups were long-chain SFA (14:0 to 18:0), the intake of long-chain SFA induced cardiac dysfunction independent of the SFA species.High-fat diets have been implicated in cardiac hypertrophy in several animal-based studies ,22,23. IConsidering that the type and level of blood fatty acids are closely associated with the cardiac fatty acid composition, the investigation of fatty acid profile changes in heart tissues provided useful data for further analyzing the effect of the SFA diet on cardiac function. Previous studies have demonstrated that modulation of membrane fatty acid composition in the heart by dietary fats may affect membrane fluidity or permeability, phase behavior and membrane fusion, leading to the perturbations in membrane-bound enzymes and receptors, which ultimately impacts cardiac function ,24,25. IAnother interesting finding is that after a 6-month feeding, all SFA diets resulted in a significantly increased content of AA in the cardiac phospholipid fraction. AA belongs to the n-6 PUFA family and is a key substrate for the synthesis of eicosanoids, including prostaglandins, thromboxanes and leukotrienes, which exhibit pro-inflammatory activity in various types of cells . NumerouCeramides, one of the component lipids of cell membranes, are thought to be cardiac lipotoxic, and accumulate in the hearts of animals fed high-fat diets. Accumulation of ceramides has been implicated in apoptotic cell death in cardiomyocytes ,12 and tAs a product of the degradation of polyunsaturated lipids, malondialdehyde is a typical indicator of oxidative stress. Significant increases in malondialdehyde levels in the HBD, HMD and HPD groups suggests the occurrence of oxidative stress, which may mediate cardiac dysfunction. However, HCD could not induce oxidative stress in mouse hearts. This suggests that not all forms of long-term dietary long-chain SFA intake share the same mechanisms of myocardial dysfunction.Long-term dietary SFA intake can induce cardiac dysfunction without significantly altering metabolic profiles in mice. The altered fatty acid composition of the cardiac phospholipids is dependent on different dietary SFA types. Accumulated total SFA and AA in cardiac phospholipids and increased ceramide levels in the heart may be responsible for SFA intake-induced cardiac dysfunction in mice.


Besides an AIDS-defining illness, Kaposi sarcoma (KS) is also seen in individuals on long-term immunosuppressant therapy. We report KS in a 70-year-old immunocompetent man, which initially mimicked acute flare of ulcerative colitis (UC). He was hospitalized multiple times for complaints of watery diarrhea and tenesmus. Despite treatment with mesalamine, short courses of methylprednisolone, and one dose of infliximab, his symptoms improved only partially. He underwent colonoscopy, which revealed mild active colitis and a mass in the ascending colon. After treatment of acute flare with methylprednisone and mesalamine, he underwent total colectomy with end ileostomy. The histopathology confirmed stage I adenocarcinoma of colon. He continued to experience watery diarrhea, which was attributed to intractable UC, and he underwent protectomy several weeks later. The histopathology of rectum revealed KS. After surgery, watery diarrhea resolved completely. Review of literature suggests KS has been rarely reported in immunocompetent individuals with inflammatory bowel disease. The occurrence of primary intestinal KS without cutaneous involvement is uncommon.2 Moreover, development of KS in patients with inflammatory bowel disease is rarely reported.3 We report a case of an immunocompetent patient with ulcerative colitis (UC) with chronic active proctitis who presented with intractable diarrhea for prolonged period and received multiple treatments for acute flare of UC. Due to refractory disease he underwent protectomy and was diagnosed with KS on histopathological examination of rectal tissue.Kaposi sarcoma (KS) is an acquired immune deficiency syndrome (AIDS) defining illness and is associated with human herpes 8 virus (HHV-8) infection.A 70-year-old man with history of hypertension and UC for 4 years presented to the emergency room with worsening of watery diarrhea, tenesmus, intermittent episodes of generalized abdominal pain, and nausea without any vomiting. He denied history of any fever, chills, anorexia, or weight loss. On initial presentation, his vital signs were stable. General physical examination revealed soft abdomen but mild tenderness was present over all quadrants with slightly increased bowel sounds. Cardiovascular, pulmonary, skin, and extremities examination was normal.Clostridium difficile antigen tests were also negative.Laboratory investigations showed mild leucocytosis (white blood cells 11 400/UL), anemia (hemoglobin/hematocrit 9.9/31.8 g/dL), mild hypoalbuminemia , raised erythrocyte sedimentation rate (141 mm/h), and C-reactive protein (12.74 mg/dL). Kidney, liver, and human immunodeficiency virus (HIV) panels were normal. Stool analysis revealed presence of pus cells but ova and parasites were absent. Stool culture and Prior to this admission, the patient was hospitalized multiple times over the last several months for similar symptoms. He received multiple treatments for acute exacerbation of UC including mesalamine and short courses of methylprednisolone. He witnessed only partial improvement in the symptoms without complete resolution. For most of these episodes, he received intravenous methylprednisone during his hospital stay, and oral prednisone in tapering doses after discharge. Prior to this admission, he also underwent colonoscopies for these symptoms on 2 occasions, with the biopsy results showing chronic active inflammatory changes. The last colonoscopy was performed 8 months prior to this admission. His symptoms recurred soon after corticosteroids were stopped. Currently, he experienced worsening of symptoms despite on maintenance mesalamine therapy. He received one dose of infliximab but was discontinued due to reaction and intolerance. However, he never received any immunomodulatory drugs such as azathioprine, 6-mercaptopurine, or cyclosporine.On this admission, colonoscopy revealed a mass-like lesion in ascending colon with active colitis with marked colitis endoscopically and active chronic colitis microscopically. The biopsy of the mass showed low-grade dysplasia and focal high-grade dysplasia. As he was at a high risk of adenocarcinoma in UC and biopsy proven high-grade dysplasia, he underwent total abdominal colectomy with end ileostomy. He tolerated the surgery well and was discharged to the rehabilitation center on mesalamine and oral corticosteroids in tapering doses. The histopathology report of surgical specimen from total abdominal colectomy showed 0.3 cm adenocarcinoma arising in association with tubulovillous adenoma invading the submucosa. This mass was a tubulovillous adenoma (dysplasia seen on biopsy) with focal adenocarcinoma on resection. Proximal, distal, and mesenteric margins were negative for tumor. Pathologically, it was reported as stage pT1 tumor. Besides, histological changes consistent with chronic active UC were also present throughout the specimen .However, the patient still remained symptomatic and continued to have watery diarrhea with tenesmus. A 3-stage panproctocolectomy with creation of ileoanal J-pouch was recommended. As a first stage, patient was scheduled semi-urgently for robotic-assisted subtotal colectomy with end ileostomy. For refractory UC he underwent protectomy with ileal pouch-anal anastomosis. The biopsy of the rectal specimen from total protectomy showed up to 4 mucosal/submucosal nodules (10 mm in greatest diameter) and with 1 out of 34 lymph nodes involved by KS. On immunohistochemistry, lesional cells were positive for CD34 . Kappa aThe patient tolerated the procedure well. His symptoms resolved completely after surgery and is in remission currently.4 KS is seen in following 4 forms: (1) classical, (2) AIDS-associated, (3) iatrogenic, and (4) endemic.5 All forms are associated with HHV-8 infection.69 The most common form seen in the United States is in association with AIDS followed by iatrogenic.10 However, the prevalence of KS has decreased in the highly active antiretroviral treatment era.KS is rare and it primarily affects the skin.11013 The clinical manifestations of KS in UC is quite variable. Most of the patients in previous case reports presented with bloody or watery diarrhea, abdominal pain, weight loss, and cachexia.1214 Complications like severe bleeding or toxic megacolon have also been reported. Most of these patients had refractory UC requiring long-term immunosuppression.1214 Majority of these patients also develop cutaneous lesions before or after the diagnosis of KS.12UC is a chronic inflammatory bowel disease that causes inflammation and ulcers in the digestive tract. Few case reports have been published highlighting the association of KS and UC.12 or due to long-term immunomodulatory treatment with azathioprine or 6-mercaptopurine.13 This patient received corticosteroids intermittently but most likely KS developed prior to these exacerbations.This case is unique due to several reasons. First, KS was diagnosed in this patient without any underlying immunodeficiency. In the previous reports of intestinal KS with UC most of the patients were immunodeficient, either due to HIV12 Most of the patients in prior case reports developed cutaneous forms of KS during the course of illness. This patient did not have any skin lesions suspicious of KS.Second, the diagnosis of primary gastrointestinal KS in the absence of any skin lesions is difficult to make.Third, this patient underwent colonoscopy multiple times but did not have any lesions with appearance suspicious of KS. Most of the patients reported in the past had colonic nodular lesions that raised suspicion of KS and was later confirmed on biopsy. Moreover, the coexistence of adenocarcinoma, KS, and UC has not been described previously.15 In vitro studies have highlighted the possible carcinogenic effect of immunomodulatory drugs.16 However, clinical studies have failed to show any causal effect of immunomodulatory drugs on development of KS.16 UC is a well-known precursor of colonic adenocarcinoma; however, its role in the genesis of KS is unlikely and has not been proven.17The concurrent diagnosis of KS in UC patients without underlying immunodeficiency is the matter of active research.Kaposi sarcoma can be seen in ulcerative colitis patients without any underlying immunodeficiency or in the absence of long-term immunosuppressive treatment. The clinical manifestations of Kaposi sarcoma in ulcerative colitis is variable and may be confused with acute exacerbation of ulcerative colitis often delaying the diagnosis. In the patients with ulcerative colitis, Kaposi sarcoma can affect only gastrointestinal tract in the absence of skin lesions.


After the publication of this work , an erro


One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5–15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria. Clostridium difficile, Bacillus anthracis, and Bacillus subtilis, contain very high concentrations of the small molecule dipicolinic acid (DPA). This molecule displaces water in the spore core where it plays an integral role in spore resistance and dormancy. DPA and its contribution to spore dehydration were discovered in 1953 but the molecular basis for its accumulation in the spore has remained unclear. The developing endospore resides within a mother cell that assembles protective layers around the spore and nurtures it by providing mother-cell-produced molecules. DPA is produced in the mother cell at a late stage in development and then must be translocated across two membranes into the spore core. Here, we report the discovery of the missing DPA transporter, homologs of which are present in virtually all endospore-forming bacteria. Our data provide evidence for a simple two-step transport pathway in which the mother cell nurtures the developing spore by sequentially moving DPA across the two membranes that surround it.All pathogenic and non-pathogenic bacteria that differentiate into dormant endospores including  The mother cell plays a central role in this process packaging the prospective spore (the forespore) in a series of protective layers and nurturing it by providing mother-cell-produced molecules . Theriewed in –6. In thiewed in  but the B. subtilis initiates the developmental process of sporulation . Theiewed in , 12 thatiewed in –16. At tiewed in . Upon sp2+ is thought to protect the spore DNA and also displace water resulting in core dehydration and increased heat resistance of macromolecules [This eight-hour morphological process is driven by sporulation-specific sigma factors that control developmental gene expression in a stage- and compartment-specific fashion , 10, 17.olecules , 22.Bacillales and Clostridiales orders contain high concentrations of DPA [It has been appreciated for over fifty years that dormant spores derived from the s of DPA , 23, yetspoVFAB and ∆ylbJ (spoVFAB operon encodes the DPA synthase that converts dihydroxydipicolinic acid into DPA [spoVFA and ∆spoVFB mutants improved by more than 100-fold to 0.2% (spoVFA ∆gerA (or ∆spoVFB ∆gerA) spores appearing phase grey-white Fig 1A). into DPA . Cells l to 0.2% . Furtherey-white .ylbJ gene encodes a membrane protein of unknown function that is expressed under the control of the mother cell transcription factor SigE [ylbJ described below we have renamed the gene spoVV. The ∆spoVV mutant is one of the last uncharacterized sporulation mutants that has a strong defect in spore formation. Cells lacking SpoVV are reduced in sporulation efficiency by >1,000,000-fold as assayed by resistance to wet heat [spoVV mutant improved dramatically  to 0.33% [spoVFA, a strong cytological suppression was also observed in the ∆spoVV ∆gerA double mutant ylbJ genespoVV and ∆spoVFA mutants. For these assays, concentrated spore suspensions with equivalent optical densities are boiled to release DPA, which is then quantified using a colorimetric assay [spoVFA and ∆spoVV mutants we included a ∆gerA mutation. However, even in this background sporulated cells had many phase dark and hollow spores [To investigate this possibility, we sought to quantitatively compare the DPA levels in spores derived from wild-type and the ∆ic assay , 28. To w spores  Fig 1A)spoVV andw spores  yielded spoVFA mutant was reduced ~20-fold in DPA content  that were also suppressed by ∆gerA [Analysis of DPA content in these homogenous spore populations revealed that the ∆ content , as repo content  althoughby ∆gerA  producedSpoVV is a polytopic membrane protein with remote homology  to conceThis localization pattern has been reported for a large number of membrane proteins that are produced in the mother cell under the control of SigE , 32–36. spoVV-gfp to a promoter (PyeeK) recognized by SigK [To investigate whether SpoVV is involved in membrane transport of DPA, we sought to localize the protein to the peripheral membranes of the mother cell. If SpoVV functions in DPA transport, DPA should accumulate in the sporulation medium under these conditions. To specifically localize SpoVV in the peripheral membranes, we took advantage of an observation made over a decade ago that membrane proteins produced in the mother cell after engulfment is complete are inserted exclusively into the peripheral membrane and are unable to re-localize around the outer forespore membrane because these membrane compartments are topologically distinct . We fuse by SigK  that becTo investigate whether DPA accumulates in the sporulation medium, we harvested cultured supernatant from sporulating cells at hour 7 and analyzed DPA levels. In cells lacking SpoVV, there was a small increase in DPA in the culture medium compared to wild-type . HoweverspoVFAB operon to a strong IPTG-inducible promoter (Phyperspank) and spoVV to a xylose-inducible promoter (PxylA). Both fusions were inserted in single copy in the B. subtilis genome. Cells harboring Phyperspank-spoVFAB with and without PxylA-spoVV were grown in minimal medium in the presence of xylose. DPA accumulation in the culture medium was then analyzed before and every hour after the addition of IPTG and expression of the DPA synthase. In support of the idea that SpoVV is the only sporulation-specific protein required for DPA transport, DPA accumulated to high levels in the medium during the induction time course (meso-diaminopimelic acid (mDAP) required for cell wall synthesis [To investigate whether any other sporulation proteins are required for SpoVV-dependent transport of DPA, we sought to reconstitute DPA secretion in vegetatively growing cells. A previous study found that expression of SpoVFAB under non-sporulating conditions was sufficient to produce DPA . Accordie course . Productynthesis . AccordiNW from Vibrio cholerae and Neisseria wadsworthii, respectively, for which high resolution crystal structures have been determined [spoVV-gfp fusion and tested whether they could complement the ∆spoVV null mutant. Three of the five SpoVV mutants  had strong sporulation defects while the other two  had more mild phenotypes (SpoVV is predicted to contain eight transmembrane segments and is annotated as a member of the Gate family of transporters (PFAM07670). Remote homology searches using HHPred  identifitermined –50. Thesenotypes . Analysienotypes . Importaenotypes . MoreovespoVA locus. While it is possible that SpoVV and the SpoVA proteins interact across the intermembrane space, we favor the idea that they function independently. During engulfment, the distance between the inner and outer membranes is already quite large (~22 nm) [Since its discovery in 1953, DPA has been found to be both a universal and specific component of bacterial endospores , 51, 52.(~22 nm)  and cort(~22 nm) .We contrast the simple intracellular transport pathway described here with a seemingly more complicated one that has been proposed to function at an earlier stage in sporulation. During engulfment a complex composed of SpoIIQ synthesized in the forespore and the proteins in the SpoIIIA operon and GerM produced in the mother cell is assembled across the inner and outer forespore membranes , 34, 56.spoVV, are repressed by SpoIIID shortly after they are activated [The DPA transport pathway described here exemplifies how intimately the sporulation gene expression programs are coordinated with morphogenesis . The Spoctivated . Thus, Sctivated . After mctivated . And finctivated . ProductB. subtilis strains were derived from the auxtrophic strain168 [Bacillus knock-out (BKE) collection [B. subtilis. All BKE mutants were back-crossed twice into B. subtilis 168 before assaying and prior to antibiotic cassette removal. Antibiotic cassette removal was performed using a temperature-sensitive plasmid that constitutively expresses Cre recombinase [B. subtilis strains were constructed using genomic DNA and a 1-step competence method. Tables of strains  at a final concentration of 50μM. Live-dead staining was performed with propidium iodide  at a final concentration of 5μM. Exposure times were typically 100, 200 and 400 ms for TMA-DPH, GFP, and PI, respectively. Image analysis and processing were performed using MetaMorph software .Vegetative and sporulating cells and dormant spores were collected by centrifugation at 6,500×g for 1 min and immobilized on 2% agarose pads. Phase-contrast and fluorescence microscopy were performed using an Olympus BX61 microscope equipped with an UplanF1 100× phase contrast objective lens and a monochrome CoolSnapHQ digital camera (Photometrics). Membranes were stained with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene 2O prior to assaying and/or imaging.Spores produced by resuspension at 37°C were harvested 30 h after the onset of sporulation. The spore pellet was suspended in 25 mL of TE containing 30 mg of lysozyme, incubated for 1 h at 37°C with shaking and then 3 mL of 20% SDS was added and incubated for an additional 20 min. The spore suspension was exhaustively washed with ddH600 of 5 was incubated at 100°C for 30 min and then placed on ice for 15 min. The spores were pelleted by centrifugation  and the supernatant was assayed for DPA content. For the experiments in which DPA was assayed in the culture medium, 2 mL of vegetatively growing or sporulating cells were pelleted by centrifugation  and the supernatant was assayed directly for DPA content. The supernatant (400 μL) was mixed with an equal volume of ddH2O and then incubated with 200 μL of solution 1 containing 50 mM sodium acetate (pH 4.6), 8.25 mM L-cysteine, 24.22 mM (NH4)2SO4 and 44.76 mM FeSO4. The reaction was incubated at room temperature for 2 min and the supernatant was then collected by centrifugation  and its absorbance measured at 440 nm. Commercial DPA (Sigma-Aldrich) was used to generate a standard curve. Because rich medium interferes with the colorimetric assay, for the experiments in which DPA levels were assayed in the culture supernatant, minimal medium was used for the vegetatively growing cells and sporulation was induced by resuspension.Spore DPA content was assayed as described previously . BrieflyVibrio cholerae concentrative nucleoside transporter. The structure of the transporter in complex with uridine  was useID 4PD6; . The tem600 at the time of harvest. Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto Immobilon-P membranes (MilliporeSigma) and blocked in 5% non-fat milk in phosphate-buffered saline (PBS)-0.5% Tween-20. The blocked membranes were probed with anti-GFP [Sporulating cells induced by resuspension were collected at hour 3 of sporulation by centrifugation and whole-cell lysates were prepared as described previously . Samplesanti-GFP  and antianti-GFP  antibodiS1 FiggerA for clarity. Scale bar indicates 2 μm.Representative phase-contrast images of the indicated strains sporulated for 30 h at 37°C in liquid DSM are shown. Sporulation efficiencies are indicated above each image. Strains lacking the B subunit of the GerA receptor (GerAB) are designated ∆(PDF)Click here for additional data file.S2 FiggerA for clarity. We note that the homogenous spore populations shown in Representative phase-contrast images of the indicated strains sporulated by resuspension for 30 h at 37°C. Sporulation efficiencies are indicated above each image. Strains lacking the B subunit of the GerA receptor (GerAB) are designated ∆(PDF)Click here for additional data file.S3 FigA. Growth curve of wild-type and the strains engineered to over-express the DPA synthase in the presence and absence of SpoVV. Indicated strains were grown in minimal medium supplemented with 33 mM xylose. When the cultures reached an OD600 of 0.3, IPTG was added  to induce expression of SpoVFA and SpoVFB. Samples were collected before and after IPTG addition at the indicated times to assay DPA levels in the medium. B. Representative phase-contrast and fluorescence microscopy images of the indicated strains collected 3 h after the addition of IPTG. Membranes were stained with TMA-DPH  and membrane permeability was assessed with propidium iodide (PI) . The number of PI-positive cells was quantified for each strain. Scale bar indicates 2 μm.(PDF)Click here for additional data file.S4 FigB. subtilis SpoVFA, SpoVFB, SpoVV, and the SpoVA proteins served as queries in a BLASTp search against the NCBI ‘nr’ database with an e-value cutoff of 1x10-4. This analysis was performed through the Harvard Medical School Research Computing Orchestra cluster. The phylogenetic tree was constructed in PhyloT (http://phylot.biobyte.de/) and the BLASTp search results were plotted against the tree. The tree was visualized and annotated using the Interactive Tree Of Life web-based tool .Phylogenetic tree showing the co-occurrence of the DPA synthase (SpoVFA and SpoVFB) and transporters (SpoVV and SpoVAA-AF) in a diverse set of 1,773 bacterial taxa. The amino acid sequences of (PDF)Click here for additional data file.S1 Methods(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Bovine tuberculosis (bTB) still remains a major zoonotic bacterial disease affecting livestock and humans worldwide. The disease remains a poorly managed tropical disease in most developing countries of the world; where in addition to productivity losses and significance in international trade, it posed a major public health threat to both humans and animals. A retrospective study was designed to investigate the occurrence of bTB lesions at Bauchi municipal abattoir.n = 56,070 males and n = 52,570 females were slaughtered at the municipal abattoir during the study period.The study utilized abattoir records spanning a period of 10 years (2004-2013). The records indicated that a total of 1,08,638 heads of cattle comprising n = 1230 (1.13%)  had tuberculous lesions. The annual occurrence during the study period varied significantly (p<0.001) from 0.53%  to 1.87%  in 2010 and 2012, respectively. Females had a significantly higher (p<0.001) prevalence of 2.10%  compared with the males 0.23% . The distribution of suspected gross bTB lesions in different organs showed 11.87% in the lungs, 5.93% in the liver, 1.14% in the heart, and 0.49% accounted for generalized bTB. However, none was observed on the lymph nodes and intestines.Of these heads, It can be concluded that bTB persists in Bauchi State with annual variations during the study period. This study highlights the importance of meat inspection as an important tool for detecting the presence of bTB lesions. Animal production in Sub-Saharan Africa is facing a serious challenge due to increased demand for meat, milk, egg, and other animal products as a result of demographic growth, economic development, and urbanization . The exiMycobacterium bovis, which is characterized by the formation of tubercles mainly in the lungs, intestine, liver, and lymph nodes [M. bovis is considered the most frequent route of infection among cattle, but infection by ingestion of contaminated materials has also been reported [M. bovis can also be transmitted through ingestion of the organism from contaminated farm environment. In recent times, M. bovis infection in cattle rarely manifests in clinical form; it is now commonly seen in apparently healthy cattle without manifestation of the clinical disease, as such, there is a need for sustained routine surveillance [bTB is a chronic bacterial zoonotic disease of animals and man caused by ph nodes -7. The dph nodes ,9. In adph nodes ,11. Exporeported . In humareported . bTB is reported . M. bovieillance . This iseillance .Mycobacterium tuberculosis and M. bovis. And since the real incidence of M. bovis on human health remains unknown, it is imperative to pursue the advancement of eradication of bTB worldwide with particular interests to be focused mainly in developing countries where the risk of infection with bTB is high [Zoonotic tuberculosis poses a significant public health challenge worldwide, especially in developing countries, Nigeria inclusive where there are deficiencies in prevention and control strategies . The sit is high .Nigeria shares an extensive border with Chad and Cameroon Republic in the Northeast and Northwestern part of the country, respectively. These borders are porous to the movement of cattle both for market and those in search of grazing land. Previous reports based on abattoir records in Cameroon and Chad Republic revealed the prevalence of bTB as 1.03% and 7.3%, respectively ,19. In NM. bovis using PCR [Mycobacterium. This diagnostic technique has not been fully explored in some parts of Nigeria due to its cumbersomeness, long incubation period, the requirement of biosafety level 3 containment, and most often unsafe, inadequate, and ill-equipped laboratories [The situation has become such that there are little or no data on the prevalence of bTB for control policy in Nigeria. Detection of bTB in cattle and other susceptible animal species is usually carried out using history of the disease in a herd, clinical and necropsy findings, abattoir meat inspection, and tuberculin skin testing. However, a definitive diagnosis of bTB is made based on bacteriological culture, colonial morphology, biochemical test and molecular detection of sing PCR . In Nigeratories . Thus, pratories . InterveThus, the current study was designed to investigate the occurrence of bTB gross lesions among slaughtered cattle in the state municipal abattoir utilizing records retrospectively for the period 2004-2013.This study was approved by the Research and Ethics Committee of the Faculty of Veterinary Medicine, University of Maiduguri, Nigeria. Approval for data collection was obtained from the Chief Veterinary Officer (CVO) and the Abattoir Manager of Bauchi State, Nigeria.2 representing about 5.3% of Nigeria’s total landmass  indigenous and exotic breeds of cattle [et al. [In this study, females had a significantly higher occurrence of bTB lesions compared with the male cattle . This aget al.  who repot et al.  reportedf cattle . This sh [et al. , where tet al. [et al. [Detection of bTB in Nigeria among slaughtered cattle at abattoir poses a great danger of contracting the disease by abattoir workers and butchers, who wear minimal protective clothing and use bare hands in processing meat of these slaughtered animals. This further confirmed that control measures for bTB are not in place or inadequately applied. This agrees with the works of Corner et al. , who rep [et al.  also sup [et al. -45. This [et al. ,40. Seve [et al. ,35,46. P [et al.  also rep [et al. ,48. ThisIt is important to note that other diseases such as Nocardiosis could present similar bacterial nodular lesions as bTB and therefore could be mistaken for bTB during routine meat inspection. Therefore, this signals for proper meat inspection procedures in Nigerian slaughterhouses and abattoirs, which do not have adequate facilities to confirm TB and similar chronic infections ,49. CornM. bovis in and around Bauchi state. We also recommend increased awareness campaign to herdsmen, farmers, and meat sellers on the zoonotic importance of M. bovis.The result of this study showed that even though the prevalence of bTB is low, the disease persists in Bauchi State and still poses a significant public health risk to humans. There is a need for routine surveillance, regular screening, and vaccination of cattle and other livestock for SMJ conceived and designed the study. NNA and AAB conducted and revised the study. TMH analyzed the data and MDG retrieved the data from abattoir records and entered into excel for processing. SMJ, NNA, and AAB drafted and revised the manuscript. All authors read and approved the final manuscript.


O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) has been widely studied. Pyridone methylsulfone hydroxamate (PMH) compounds can effectively inhibit the catalytic activity of LpxC. In this work, the three-dimensional quantitative structure-activity relationships of PMH inhibitors were explored and models with good predictive ability were established using comparative molecular field analysis and comparative molecular similarity index analysis methods. The effect of PMH inhibitors’ electrostatic potential on the inhibitory ability of Pseudomonas aeruginosa LpxC (PaLpxC) is revealed at the molecular level via molecular electrostatic potential analyses. Then, two molecular dynamics simulations for the PaLpxC and PaLpxC-inhibitor systems were also performed respectively to investigate the key residues of PaLpxC hydrolase binding to water molecules. The results indicate that orderly alternative water molecules can form stable hydrogen bonds with M62, E77, T190, and H264 in the catalytic center, and tetracoordinate to zinc ion along with H78, H237, and D241. It was found that the conformational transition space of PaLpxC changes after association with PMH inhibitors through free energy landscape and cluster analyses. Finally, a possible inhibitory mechanism of PMH inhibitors was proposed, based on our molecular simulation. This paper will provide a theoretical basis for the molecular design of LpxC-targeting antibiotics.As an important target for the development of novel antibiotics, UDP-3- Acinectobacter baumannii, carbapenem-resistant Klebsiella pneumoniae, and Pseudomonas aeruginosa. Most of these “superbugs” are Gram-negative (G−) bacteria possessing complex cell membranes, which strongly obstructs antibacterial agents’ ability to penetrate into the cell. This makes the treatment of MDR bacterial infection much more difficult.Bacterial drug-resistance has become a public health problem worldwide and a serious threat to human health ,2. Recen− bacteria, which can cause fever, microcirculatory disturbance, and even shock imidazol-3-one derivatives as LpxC inhibitors, and these compounds were able to effectively inhibit partial strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae  containing systems . In this force field, a pseudo-atom was introduced to guide the ligand coordinating with zinc, and the geometrical characteristics and bond strength of Zn(II) or Zn2+ were considered sufficiently, which greatly improved the accuracy of molecular docking for zinc-containing molecular systems [2+ coordinates of PaLpxC (PDB entry: 3UHM), and built a 40 Å × 90 Å × 60 Å rectangular box with a grid space of 0.375 Å. 128 conformations were collected in each docking, and the conformation of the lowest energy in the largest conformation cluster was defined as the near native conformation for the subsequent 3D-QSAR analyses.ChemBio3D Ultra 12.0  was used to construct the molecular structures of the PMH LpxC inhibitors except for Cmpd # 290, and energy optimization of these structures was performed under the MM2 molecular force field with the convergence condition of root mean square (RMS) less than 0.0001 kcal·molYL-X 1.3  and mole package  was used systems . In the 50 value of the experiment was converted to a negative logarithm pIC50 form. Before constructing the 3D-QSAR models, it was necessary to align the compound molecules. The quality of molecular alignment has a great influence on the reliability of the 3D-QSAR models. In molecular alignment, ensuring all molecules using the construction of 3D-QSAR models with the largest similar spatial orientation of molecular fields was helpful to improve the similarity of the steric and electrostatic fields computed. Considering that the 3D-QSAR models assume that all molecules are in the same active binding mode at the same site, the crystallographic structure of Cmpd # 290 was selected as the template molecule, and molecular alignment was based on the pyridone methylsulfone hydroxamate commom substructure. 3D-QSAR analyses were completed in SYBYL-X 1.3 [3 hybridized C+ probe walks in the space around the molecules, the interaction between the probe and molecules was calculated, and the energy values of the interaction with different space coordinates were recorded to obtain the steric and electrostatic field data; CoMSIA was similar to CoMFA, but in addition to the analyses of the steric field (S) and electrostatic field (E), the hydrophobic field (H), H-bond acceptor (A) field, and H-bond donor (D) field were also calculated.The PMH compounds were randomly divided into a training set  for constructing the 3D-QSAR models and a test set  for testing the predictive ability of the biological activity of the models. The ICYL-X 1.3 . To set q2. Secondly, according to the ONC, the 3D-QSAR models were established by non-cross validation, and the correlation coefficient r2, standard error for estimate Es, root mean squared error (RMSE) and the F-test values F were obtained. Parameters based on the PLS analyses above can be used to evaluate the predictive ability and stability of the models, and predict the biological activity of the molecules in the test set. The predicted correlation coefficient for the test set The partial least squares (PLS) method was used to analyze the PMH molecules in training set. Firstly, the leave-one-out (LOO) method was used for cross validation (parameters are default) to obtain the optimal numbers of component (ONC) and the determination coefficient In Equation (2), SD represents the sum of the squared deviations of the experimental biological activity of the test set and the average molecular biological activity of the training set, and PRESS indicates the sum of the squared errors of the predictive biological activity values of the test set and their biological activity as determined by the experiment.−3). Different colors are used to represent the distribution of the MESP on the surface of the molecule. For example, dark red represents the region of the most negative electrostatic potential and dark blue represents the region of the most positive electrostatic potential.Gaussian 09  was used to calculate the MESP and Mulliken charges based on DFT. The molecular conformation of the ligand used in the calculation was generated by energy optimization, and the method and criterion of energy optimization were consistent with those of the previous molecular docking. The B3LYP/6-31G  basis set  was appl2+ bond model and the force field parameters were constructed by using 6-31G* basis set. Energy minimization before simulation was completed by two steps. Firstly, with the constraint  of the solute, 5000 steps of the steepest descent method was performed and followed by another 5000 steps of the conjugate gradient method; then unrestrained minimizations of the whole system were executed by the same stages above with the convergence condition that the energy gradient is less than 0.01 kcal·mol−1·Å−2.The structural models of PaLpxC and complex PaLpxC-Cmpd # 290 were further subjected to MD simulations in the solution system for 120 ns, respectively, using the AMBER16 software package  with the−1·Å−2 and the temperature of the system gradually increased from 0 K to 300 K. Then free isothermal MD simulation of the systems was performed for 119 ns. The conformational changes in the whole process of simulations were tracked and observed by VMD, and using the SHAKE algorithm [+ were also added, and the total numbers of atoms in the PaLpxC and PaLpxC-Cmpd # 290 systems were 38,310 and 38,211, respectively.MD simulation consists of two steps: constrained MD simulation was performed for 1 ns with the constraint force constant at 10 kcal·mollgorithm  to restrEnergy decomposition analysis was performed by the molecular mechanics/generalized Born surface area (MM/GBSA) method . The basX stands for the PCs and thus P(X) is the probability distribution of the molecular system along the PCs, and kB and T are the Boltzmann constant and absolute temperature, respectively.FEL  can promOn the basis of the conformations obtained by MD simulation, conformational cluster analysis was performed using MMTSB tools . The speIn the current work, theoretical molecular simulation studies on a broad-spectrum LpxC inhibitor, a type of pyridone methylsulfone hydroxamate compound, were performed to investigate the relationship between its chemical structure and bioactivity. CoMFA and CoMSIA were used to study the 3D-QSAR of PMH LpxC, and QSAR models with good predictive ability were obtained. According to the results of the contour map, the introduction of bulky groups and electron withdrawing groups in PMH LpxC inhibitors can effectively improve the inhibitory activity of the inhibitors. Based on the analyses of the molecular electrostatic potential of the PMH LpxC inhibitors, the effect of substituents on the inhibitory activity was explained at the molecular level.Comparative MD simulations of the PaLpxC and complex PaLpxC-Cmpd # 290 were carried out for 120 ns. The results of binding free energy calculations based on MD trajectories show that the binding free energy calculation results agree well with the experimental values, and the van der Waals interaction is the main driving force for the formation of the complex. The results of H-bond and energy decomposition show that Cmpd # 290 mainly interacts with M62, T190, F191, H264, I197, and K238 of PaLpxC.2+ (the average length of O-Zn is around 2.27 Å), to provide the reagent for the hydrolysis reaction constantly. In the complex structure, Cmpd # 290 chelates Zn2+ by hydroxamate moiety, which causes the destruction of the binding mode of water molecules. It further blocks the entry of the reaction substrate by enclosing the pocket through its hydrophobic chain, so as to exert an inhibitory effect. This work provides some theoretical guidance for the design of drug molecules targeting PaLpxC.Finally, through the simulation of trajectory sampling analyses, the binding mode of key water molecules in PaLpxC was investigated. Without any constraint, the water molecules can freely access the active pocket of PaLpxC; and with the assistance of E77, H264, T190, and M62, it can coordinate and form a tetrahedral configuration with Zn


Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells. This process is monitored by the spindle assembly checkpoint (SAC) that delays sister chromatid separation in the presence of unattached or improperly-attached kinetochores . KinetocThe endosomal sorting complex required for transport (ESCRT) machinery promotes budding and severing of narrow cytosol-filled membrane invaginations from their inner face . Functioin vitro and promotes localization of RZZ and Mad1-Mad2 complexes to prometaphase kinetochores. Furthermore, constitutive Chmp4c kinetochore targeting induces an RZZ-dependent checkpoint metaphase arrest, suggesting that Chmp4c kinetochore localization dictates spindle checkpoint activity. These results show that Chmp4c acts as a loading factor for the RZZ to unattached kinetochores (In a recent study, we identified novel Chmp4c functions at kinetochores that are independent from its membrane-directed activities . Chmp4c tochores . Howevertochores  2,3]. S,3. Sin vChmp4c also promotes cold-stable kinetochore-microtubules and Chmp4c-deficient cells exhibit reduced average centromere distance compared with controls . These rThe work by Petsalaki et al.  clearly


We study conductance in a molecular breadboard junction accommodating up to 61 circuits and demonstrate switching between 4 conductance states. TP1). The TP1 molecule can adopt multiple conformations through relative rotations of 7 aromatic rings and can attach to electrodes in 61 possible single and multi-terminal configurations through 6 pyridyl groups. Despite this complexity, we show that it is possible to extract well defined conductance features for the TP1 breadboard and assign them rigorously to the underlying constituent circuits. Mechanically controllable break-junction (MCBJ) experiments on the TP1 molecular breadboard show an unprecedented 4 conductance states spanning a range 10 –2G0 to 10 –7G0. Quantitative theoretical examination of the conductance of TP1 reveals that combinations of 5 types of single terminal 2–5 ring subcircuits are accessed as a function of electrode separation to produce the distinct conductance steps observed in the MCBJ experiments. We estimate the absolute conductance for each single terminal subcircuit and its percentage contribution to the 4 experimentally observed conductance states. We also provide a detailed analysis of the role of quantum interference and thermal fluctuations in modulating conductance within the subcircuits of the TP1 molecular breadboard. Finally, we discuss the possible development of molecular circuit theory and experimental advances necessary for mapping conductance through complex single molecular breadboard circuits in terms of their constituent subcircuits.Controlling charge flow in single molecule circuits with multiple electrical contacts and conductance pathways is a much sought after goal in molecular electronics. In this joint experimental and theoretical study, we advance the possibility of creating single molecule breadboard circuits through an analysis of the conductance of a bis-terpyridine based molecule ( Under the application of a bias voltage, the current flowing through the molecule, from one electrode to the other is sensitive to the molecular structure ,10 the chemical nature of the linker which connects the molecule to the electrode,13 the relative position of linkers attached to the molecular core,15 the binding geometry of molecule between the electrodes,18 and the effect of external triggers on molecular structure and environment.22 Thus, these experimental techniques when combined with charge transport theory present a unique opportunity to create dynamic circuits with a library of molecules, which can act like wires,25 switches27 and diodes.29The central goal in molecular electronics is to develop molecules as active electronic circuit elements.2), thiol (–SH), isothiocyanide (–SCN), cyanide (–CN), nitro (–NO2), carboxylic acid (–COOH), dimethyl phosphine (–PMe2), methyl sulphide (–SMe), hydroxyl (–OH), and carbodithiolate based linkers.30 These studies have helped deconvolute the effect of the linkers from that of the molecular core on the single molecular junction conductance. Several experimental and theoretical attempts have been made to understand and/or predict the possible quantum interference patterns which govern the conductance properties of conductance pathways across the molecular core.37 These studies compared the relative conductance of molecules which differed in terms of either the chemical structure of the molecular core or the placement of anchoring groups within the molecule. In this context, the introduction of molecular breadboard circuits would be a significant advance, enabling the comparison of multiple conductance pathways within a single molecular scaffold. Indeed, steps have been taken towards realizing multiple circuits within a single molecular junction. Kiguchi and co-workers have recently demonstrated a conductance switch based on control of the molecule-electrode contacts within a quarterthiopene molecular junction with 4 thiophene anchoring groups.17 The authors captured 3 distinct conductance states and assigned it to bithiophene, trithiophene, and quarterthiophene circuits.17 Miguel and co-workers recently demonstrated 2 stable conductance pathways within a para-oligophenylene ethylene (p-OPE) molecular scaffold. By substituting the central benzene ring with sulfur and nitrogen heterocycles, the authors were able to demonstrate a new high conductance channel formed by the linking of the central pyrimidine ring to the electrodes.18 More recently, Kiguchi and co-workers have also shown the existence of 3 conductance states within a tripyridyl–triazine molecular junctions.38 In the studies above 38 single circuits were assigned to each conductance state observed in the experiments and upto 3 circuits across the molecular scaffold were proposed to be accessible by the experiments.In conventional electrical and electronics engineering, solderless breadboard scaffolds are routinely used for quickly creating/testing a range of electrical circuits of varied complexity . AdaptinTP1), wherein multiple electrode anchoring pyridyl groups create 61 possible breadboard circuits within a single molecular scaffold bis, scaffold . FurtherDetails of the synthesis and characterization of all molecules examined in this manuscript are provided in Section S.1 of ESI.39 All the measurements were done in solution at room temperature. We provide more details in Section S.2 of the ESI.Single molecular conductance measurements were performed by employing home built mechanically controllable break junction set up. Further technical details of the MCBJ setup and data analysis procedures were reported in our previous publications.TP1 molecule. The NEGF calculations are based on an INDO/s Hamiltonian and DFT optimized geometries of the molecule. This computational framework has been described in detail previously and shown to be effective in several previous studies of charge transport properties of small organic molecules.42 We outline the procedure for studying TP1 below. A more detailed description is provided in Sections S.3–S.5 of ESI.We use the framework of Non-Equilibrium Greens Functions (NEGF) to compute single molecule conductance of the TP1 molecule is expected to exhibit significant conformational flexibility in terms of ring rotation. We therefore considered multiple molecular geometries which differ in terms of their relative orientations of the pyridine rings in our study. Different conformations of TP1 were manually drawn wherein each aromatic ring was allowed to adopt one of two conformations, either in plane or orthogonal, relative to its neighboring rings. This procedure resulting in 24 distinct starting geometries, which were optimized in Gaussian 09 using DFT with a B3LYP exchange correlation functional and a 6-31G* basis set.43 The optimization yielded 18 unique geometries  is captured for the TP1 breadboard potential energy landscape. Further, as all conformations are thermally accessible at room temperature, additional sampling through molecular dynamics simulations around each local minimum is expected to refine  but not alter any of the conclusions presented here.The 46 we assumed that the TP1 molecule is connected to the electrodes through the nitrogen atoms of the flanking terpyridine arms  in the atomic basis with non-zero elements corresponding to the coupling of specific atoms to the electrode (eqn (1)). We assume that only nitrogen atoms are electronically coupled to the electrodes (ΓN = 0.1 eV). However, nitrogen atoms on the peripheral pyridine rings of the terpyridine units are more accessible to the electrodes than those on the central pyridine rings. We thus constructed a model wherein the broadening matrix includes different electrode electronic couplings for peripheral (end pyridine rings) and core  nitrogen atoms on the terpyridine units of TP1:Γcore is exponentially screened relative to Γperipheral: Γperipheral/Γcore = exp(β × deff), where β = 3.0 Å–1 is the decay of the electronic coupling through vacuum. The effective screening length deff was defined as the distance between the central (core) nitrogen atom and the centre of mass of the peripheral nitrogen atoms for each terpyridine arm of TP1. From a structural analysis of the 18 optimized geometries of TP1 . The potential drop is assumed to be symmetric across the electrodes. The small biases applied here are not expected to cause significant changes in molecular geometry or energies, and no potential drop is assumed across the molecule. For the INDO/s band gaps calculated for TP1 geometries, the electrode Fermi level (set to –5.1 eV) lies closer to the HOMO (hole transport barrier of ∼2.9 eV) than the LUMO. DFT calculated band gaps are lower with the electrode Fermi level again lying closer to the HOMO (hole transport barrier of ∼1.4 eV) than the LUMO. However, recent experiments on bis-terpyridine poly-p-phenyl molecules and molecules with pyridyl anchors have suggested that transport in these systems is in fact electron dominated.48 Thus, we also carried out calculations in the electron dominated regime by setting the Fermi level to 2.9 eV below the LUMO  that the single molecule break-junction experiment might sample in individual traces to build the conductance statistics for the junction. In these calculations, the conductance is primarily averaged over dihedral degrees of freedom and neglects inelastic contributions to charge transport. Inelastic contributions arise due to the coupling of the electronic transport to molecular vibrational degrees of freedom and become prominent when the dwell/transit times of the electron inside the molecular barrier become comparable to the vibrational timescale. For the TP1 molecule, the length of sub-circuits lies between 4–16 Å and barriers for tunneling are >1 eV. Thus, for these systems the Landauer–Buttiker barrier transit time50 is of the order of a few fs and shorter than the timescale of vibrational motions. The fastest motions which might compete are the vibrations of bonds between hydrogens and heavy atoms , which do not significantly influence the dominant π transport channels in these systems. These considerations allow us to neglect inelastic contributions to charge transport in our calculations.Our computational protocol considers multiple thermally accessible 52 The neglect of correlated electron injection thus allows a simple decomposition of multi-terminal circuits into a superposition of single terminal circuits. Several considerations justify our choice of neglecting electron correlation between electrodes coupling to the TP1 molecule in the present study:Our calculations neglect electronic correlations for metal electrodes which couple to the molecules. Such correlations can lead to interesting interference effects boosting or suppressing multi-terminal circuit currents relative to that obtained by a simple addition of currents from the constituent single terminal circuits.TP1 nor the shape of electrode tips interacting with the molecule is precisely known. Further, for the TP1 molecule the pyridine ring rotational degrees of freedom alter the relative positions of nitrogen atoms  Neither the gold coordination sphere around the nitrogen atoms of TP1 geometries. However, in this case, the multi-terminal circuits are composed of asymmetric (in terms of number of aromatic rings) single terminal circuits with significantly different conductance values. For instance, in (2) It is possible, of course, that nitrogen atoms on adjacent pyridine rings may share their gold coordination sphere for some (3) Finally, for multi-terminal circuits with symmetric single terminal conductance pathways such as L–R or L–R(S2), the relevant terminal nitrogen atoms (S1 and S3) are well separated (∼8 Å), and a model assuming independent electronic coupling at these positions is reasonable.C62 = 15 (where Ckn = n!/k!(n – k)! denotes the number of ways to pick k sockets from a set of n sockets). If we allow each of the two alligator clips to contact more than one socket, the breadboard can potentially offer up to 301 single and multi-terminal circuits (see Section S.4 in ESITP1 molecular breadboard (IL→R(Vbias) = IR→L(Vbias)), and distinct sets of nitrogen atoms contacted by each electrode. Expressed in terms of the number of contacts  made by the two (L/R) electrodes, contact configurations  can be symmetric (M = N) or asymmetric (M ≠ N). Here, we assume that a single electrode cannot contact pyridyl groups on both terpyridine arms of TP1 simultaneously. Further, in break junction experiments (e.g. MCBJ) the smallest electrode separation formed in a MCBJ experiment after breaking gold–gold contact (snap-back distance) is estimated to be ∼0.4–0.6 nm,12 which allows both symmetric and asymmetric circuits within each of the terpyridine arms of TP1. Under these constraints, 61 distinct circuits can be formed across the TP1 breadboard, which are enumerated in L–2R/2L–1R circuits can be decomposed . The 18 optimized TP1 geometries thus obtained were separated energetically by ∼3 kcal mol–1 /tetrahydrofuran (THF) . The breaking of atomic Au–Au contacts appears as plateaus observed in the conductance-distance traces in the range 1 ≤ G/G0 ≤ 10, where G0 = 2e2/h is the quantum of conductance. Additional plateaus appearing in the range 10–1G0 ≥ G/G0 ≥ 10–7.5G0. The noise limit of our MCBJ setup under the current experimental conditions is reached at 10–8G0. TP1. The corresponding two-dimensional (2D) histogram of TP1 is shown in z = 0 at G = 0.7 G0, which is characterized by a sharp drop in conductance immediately after breaking the last Au–Au monatomic contact upon pulling the leads apart. The quantitative analysis of the 1D and 2D histograms  upon breaking of the monatomic Au–Au contact,53 we estimate an absolute most probable gap distance  in the range of 0.45 nm to 0.65 nm. The second highest conductance state “G2” is observed in the range 10–4.5G0 ≤ G2/G0 ≤ 10–3G0 with the most probable value , and corresponding absolute gap distance . Similarly, the next two conductance states G3 and G4 are seen in the range 10–6.5G0 ≤ G3/G0 ≤ 10–4.2G0 and 10–7.8G0 ≤ G4/G0 ≤ 10–6G0 and ,  respectively. The corresponding absolute most probable gaps for G2 and G3 conductance states are  and  respectively. These data clearly demonstrate that the single molecular junction conductance of TP1 decreases in distinct steps upon pulling the gold leads apart and exhibits four conductance states.The presence of four distinct conductance features in the histograms of TP1 molecular scaffold (vide supra) and their thermally accessible conformations. We thus carried out electronic structure calculations on the optimized TP1 geometries and Green's function based tunneling transport calculations (see Methods) for each single and multi-terminal molecular circuit enumerated in TP1 geometries. The conductance of the single and multi-terminal circuits in the TP1 breadboard spans almost five orders of conductance values which matches the span of the experimental conductance range (10–2G0 to 10–7G0) in The origin of the conductance features produced in the experiments  should bML–NR; M, N = 1, 2, 3) in vs. multi-terminal) made by each electrode. In fact, multi-terminal circuit currents are only slightly higher (within a few fold) of corresponding single terminal currents within each conductance band. It can be shown  rings as shown in TP1 (e.g. L(S1)–R(S2) and L(S1)–R(S3) from e.g. (L(S1)–R(S2′) from e.g. (L(S1)–R(S1′) or (L(S1)–R(S3′) from Each of the three conductance bands in TP1  which spans the junction gap as a function of electrode separation. Contributions from the specific single and multi-terminal circuits considered in TP1 are accessible to MCBJ experiments at short electrode separations (Δz < 0.4 nm). As the electrode separation increases from 0.4 nm to the full molecular length of 1.6 nm, the number of circuits contributing to the conductance of TP1 decreases as the terminal N–N atom separations for some circuits cannot span the junction gap. For instance, the highest conductance single terminal (1L–1R) circuit does not contribute to the TP1 conductance at electrode separations of 0.5 nm and beyond. Thus, as circuits drop out, the total conductance is expected to reduce in discrete steps as a function of electrode separation. The MCBJ experiment measures only the total conductance as a function of electrode separation (In paration  which caTP1 molecule contains a large number (61) of possible circuits, there are degeneracies in many conductance values (see vs. electrode separation traces (4 peaks observed for TP1). In order to develop the concept of a molecular breadboard further, it is critical to map the experimental conductance features to the underlying contributing circuits. Note that a molecular breadboard is expected to have features distinct from a macroscopic electrical breadboard. First, molecules such as TP1 exhibit thermally driven conformational flexibility which can vary the conductance of individual circuits by an order of magnitude . In G1, G2, G3, and G4 and their correlation with electrode separation. The computational results suggest that there may be a fine structure to the G3 conductance profile near 1 nm which may not be resolved in the experiments. Further, the computational analysis indicates that the MCBJ experiments contact all 61 circuits and probe the full range of conductance values of the TP1 breadboard circuit within the experimentally accessible electrode separations (0.4–1.7 nm). In To derive the computed conductance  gap see  and S6A†G1, G2, G3, and G4 conductance states respectively. Note that we obtain a range of circuit combinations for the G3 and G4 conductance states as these states show a much stronger drop in conductance with electrode separation than the G1, and G2 conductance states  there are only 5 types of single terminal circuits  from the 5 single terminal circuits shown in TP1 at an electrode separation of 0.4 nm into single terminal 2-ring and meta-3-ring circuits. We applied the decomposition procedure shown in G1, G2, G3, and G4) are presented in G1 conductance, we can estimate the 2-ring circuit conductance to be: G(2-ring) = (0.81 × G1(experiment))/12. On the other hand the 3-ring circuit conductance can be estimated in two ways, (1) either using the experimental G1 value: (meta-3-ring) = (0.18 × G1(experiment))/6, or using the measured G2 conductance: G(meta-3-ring) = (0.90 × G2(experiment))/2. Estimates of absolute conductance values for all 5 single terminal circuits  obtained by this procedure are listed in the last row of From the data in see also . Circuituits see  present βExperiment = 3.4 nm–1 (Section S.9 of ESIβTheory = 3.8 nm–1 (Section S.9 of ESI–1), OPE (2.0–3.4 nm–1), and oligophenyleimine (OPI: 3 nm–1).6 However, we stress that the interpretations of tunneling decay constants for circuits embedded within a molecular scaffold is different and more complex than that for isolated circuits. For instance, the drop in TP1 junction conductance occurs not only due to a change in the dominant circuit (increasing number of rings) with electrode separation, but also due to a drop in the number of contributing circuits (see TP1: (1) are composed of mixture of pyridine and phenyl rings, (2) do not have linear ring connectivity, and (3) differ in their ring connectivity positions (ortho/meta/para) to the electrodes. The next section sheds further light on the relative conductance values of the dominant circuits assigned in TP1.The conductance plateaus in uits see . Further58 Xing et al. found that phase cancellation of currents through molecular orbitals of opposite symmetry diminished the contribution of frontier orbitals to both electron and hole transport in OPE molecules.41 Arroyo et al. found interference effects between charge transport pathways through HOMO and LUMO of a benzene ring coupled to thienyl anchoring units through ethynyl spacers.57 Further, anchoring group placement at ortho-, meta-, or para-positions of six membered rings have been shown to significantly modulate the conductance of conjugated systems.58Several studies have reported quantum interference effects in molecular junctions.TP1 junction conductance at different electrode separations show diverse terminal nitrogen placements, and in this section we examine interference effects in these circuits. For each 2–5 ring embedded fragment of TP1, we computed conductance for electrode electronic couplings with atoms at para-, meta-, and ortho-positions of the pyridine rings of the molecule . In order to cover the three para-, meta-, and ortho-connectivities for each 2–5 ring fragment, we considered carbon atom terminated circuits in addition to the dominant circuits assigned in the last section. While, previous studies have suggested that the electrode can couple to the carbon backbone through the π orbitals.61 It is not our intent to explore the through space coupling effect here. Rather, the carbon terminated circuits serve as surrogates of nitrogen terminated circuits to assess the effect of ring-electrode connectivity on the conductance of each 2–5 ring embedded fragment of TP1. In TP1.The dominant 2–5 ring circuits assigned to the Gavg(2-ring) ∼ Gavg(para-3-ring) ≫ Gavg(meta-3-ring) > Gavg(4-ring) ≫ Gavg(5-ring). Clearly, the conductance drop with electrode separation does not correlate with the increase in the tunneling length (number of aromatic rings) of the dominant circuits contributing to the conductance at increasing electrode separations. The data in para anchors produce the highest possible para-3-ring conductance  in Methods) an effective ∼100 fold reduction in the electronic coupling for the core nitrogen atom relative to the peripheral nitrogen atoms of each terpyridine ring. In this case, the G2 conductance primarily arising from the 3-ring circuit  in G1 conductance primarily arising from the 2-ring circuit  as observed in the experiments  across the panels, we find ductance , while tductance . Thus, ice plots  or 6. Theriments . We presmeta-anchoring group position and the highest currents involve an ortho-/para-anchor. For instance, in 2-ring circuits (Gavg(YP/YO) ≫ Gavg(ZM). In the 3-ring circuits of Gavg(PP) ≫ Gavg(MM) and Gavg(YY) ≫ Gavg(XX/ZZ/XZ) respectively. We find that the effect of the anchoring group ring position on circuit conductance is preserved even when the terminal electrode contacting pyridine rings are separated by intervening aromatic rings. For instance, in 4-ring circuits (Gavg(YP) ≫ Gavg(XM/ZM) and in 5-ring circuits (Gavg(PP) ≫ Gavg(MM). In accordance with previous reports,14 we find higher currents for para-3-ring circuits relative to that for meta-3-ring systems (Gavg(PP) in Gavg(YY) in TP1. The largest variations in conductance are seen for 5-ring circuits  and YO (para–ortho) circuits show higher conductance values relative to the other circuits. The meta-3-ring terpyridine circuit in meta-anchor  and high conductance with low fluctuations for ortho-/para-anchors . To summarize, we find that after including the effects of conformational heterogeneity, the modulation of conductance due to quantum interference is cleanly resolved for 2-ring and para-3-ring circuits  = G(2-ring) × G(para-3-ring) and G(5-ring) = G(2-ring) × G(para-3-ring) × G(2-ring). The data in Predicting the conductance through a molecular circuit in terms of transport properties of individual molecular components is an unsolved fundamental challenge in field of molecular electronics. From elastic scattering theory, transport through a molecular super circuit formed by binding two oligomers with transparencies T1 and T2 in series is proportional to T1 × T2.s in TP1  should b62 Venkataraman and co-workers,64 verified the parallel superposition law proposed by Joachim and co-workers,55 and found evidence for constructive quantum interference in parallel superposition of molecular units in single molecular junctions. For TP1, single terminal circuits form channels which combine to yield each of the four conductance states observed for the breadboard. Thus, it is reasonable to ask if the conductance of the breadboard can be predicted from conductance data on isolated single terminal subcircuits. To this end, we measured the conductance of two single terminal circuits in isolation  appears to be in good agreement with the experimentally measured high conductance of the R1 molecule (GR1H = 6.8 × 10–4G0). However, this apparent match is misleading since the R1 molecule and the 2-ring circuit within TP1 differ in two crucial ways: (1) the accessibility of the terminal nitrogen atoms to electrodes in the TP1 2-ring circuit is not the same as in R1, and (2) the electronic structure of the TP1 2-ring system is coupled to that of the TP1 framework, whereas R1 represents an isolated 2-ring system. Contrary to the conventional macroscopic breadboard circuits, where adding one extra branch to the circuit does not influence the electrical properties of the other subcircuits, any new branch added to a molecule effectively creates a new molecule with different electronic properties.62 The differences between the component single terminal circuits in isolation and within the TP1 breadboard is more evident for the para-3-ring circuit wherein the estimated 3-ring circuit conductance in –6G0) is two orders of magnitude lower than the experimentally measured conductance for the R2 molecule (1.35 × 10–4G0). Further, isolated subcircuits may have distinct conductance features which may not show up in the conductance features of their host breadboard. For instance, the R1 circuit has a low conductance feature (GR1L = 1.3 × 10–5) which should not show up in the TP1 breadboard as the most probable junction gap for this configuration (0.9 nm) indicates a dimer configuration .Joachim and co-workers have theoretically examined superposition rules in the context of molecular junctions where multiple molecular units were connected either in series or parallel to form super-circuits. circuit . The absTP1 are necessary to build a reliable circuit theory for breadboard circuits.The observations in this subsection indicate that more studies on differences, in terms of both geometry and electronic structure, of subcircuits in isolation and within larger molecular scaffolds such as –2G0 to 10 –7G0) within a single molecular scaffold (TP1. The multi-anchoring configurations in the TP1 molecule were shown to create 61 single and multi-terminal circuits with conductance features spanning 5 orders of magnitude (TP1 breadboard in the MCBJ experiments originate from distinct combinations of the 61 circuits at different electrode separations (TP1. The combination of theory and experiment presented in this paper provides guidelines for rational molecular design enabling the access to specific circuits and conductance features in future experiments. In this context, development of circuit theory for molecular breadboards through a systematic study of component circuits was discussed.We have experimentally demonstrated for the first time four distinct conductance steps separated over 5 orders of magnitude (10 scaffold . We modeagnitude . Moleculagnitude . The conarations . Based oarations . We detearations . Effectsarations . Howeverarations . We have