PLoS Medicine, volume 2, issue 8, DOI: 10.1371/journal.pmed.0020193In Some of the data presented in the Abstract and Results were incorrect.The third sentence of the Methods and Findings section in the Abstract should read as follows: “Incidence rates rise with age, particularly above the age of 75 y, from 6.7  per 1,000 person years at age 65–69 y to 68.5  per 1,000 person years at age 85 y and above.” The fifth sentence should read as follows: “Hence, it is estimated that approximately 163,000 new cases of dementia occur in England and Wales each year.”In Results, under “Combined Incidence Analysis,” the third sentence should read as follows: “The population burden of these rates equates to approximately 163,000 new occurring dementia cases each year in England and Wales .”


Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5′ untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function.Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical  Drosophila melanogaster enzyme Tan. The gene encoding Tan was originally discovered in the early 20th century as a mutant strain lacking the dark pigmentation of wild-type flies, hence the name tan. Flies lacking Tan function also exhibited mysterious abnormalities in vision, for example, in responses to light. The new findings by True et al. help to explain the vastly different functions of Tan in pigmentation and vision. In the developing epidermal cells that secrete the adult cuticle, the enzyme encoded by tan is required for the production of dopamine, which is needed for dark melanin pigmentation. In the eye, the Tan enzyme converts carcinine, a modified form of the neurotransmitter histamine, back to histamine, which is necessary for the rapid and constant neurotransmission events involved in vision. These two enzyme activities have not been previously characterized in any organism. Surprisingly, Tan appears to be closely related to an enzyme in fungi that is used for production of the antibiotic penicillin.True et al. describe the identification and characterization of the  Drosophila melanogaster ebony and tan genes, mutations in which cause reciprocal pigmentation defects but related neurological phenotypes, have been proposed as such a system (P{g1557})w =XP} CG12120[d07784] (P{d07784}), w1118, t 1, t 2 v 1 f 1 (t 2), t 3, br 1 w e ec 1 rb 1 t 4/FM1 lz 4 (t 4), t 5 v 1 r 1/FM7c (t 5), P{EPgy2}EY04394,w and P{EPgy2}EY05996.765-GAL4  was provier-GAL4  was prov{g1557})  was provUAS-CG12120 rescue and ectopic expression genotypes showed very little variation. Rescue and ectopic expression phenotypes occurred in 100% of the flies that inherited both the GAL4 and UAS-CG12120 elements.All flies were examined at 3–5 d of age, after the full adult pigmentation appeared. 1118, P{XP}CG12120 d07784w females were crossed to 1118; +; P{Δ2–3}, TM3, Sb/TM6Bw males, and dysgenic male F1s were crossed to Pro,os/FM6,B,w females. Female F2 progeny lacking eye color  were crossed to FM6,B,w/ Y males, and their non-FM6 male progeny were inspected for pigmentation phenotype. Four tan-like lines and four “revertant” lines with wild-type pigmentation were chosen for further analysis.tan-like all contained imprecise excisions, two of which, 20A and 37C, contained large deletions  that included the presumptive promoter region. The other two tan-like excisions, 27A and 42A, left small insertions  at the P-element site (data available upon request). All four revertant P excisions were also imprecise, leaving P-element fragments ranging from 12 bp to 1.2 kb in size (data available upon request), but none of these contained deletions of the endogenous CG12120 transcription unit or promoter region.The four excisions classified as tan-like excision lines showed significantly lower CG12120 mRNA levels on average than wild-type lines by quantitative reverse transcriptase PCR assay (data not shown). Revertant excision lines showed no differences on average from wild-type lines in mean CG12120 mRNA expression levels. These results were found at two different developmental stages, 60–75 h after puparium formation and 0–8 h after eclosion. The complete CG12120 mRNA expression dataset is available upon request.The UAS-CG12120, the complete CG12120 cDNA was obtained from the Drosophila Genomics Resource Center (http://dgrc.cgb.indiana.edu/) as clone RH41996 (barcode 17763), consisting of the 2,009-bp CG12120 cDNA in plasmid vector pFLC-1. A 2,133-bp NotI-Acc65I fragment containing the CG12120 cDNA was cloned into the pUAST vector ), and 50 μl of 8 mM NBAD  or N-β-alanyl histamine , prepared in 50 mM phosphate buffer. At different incubation periods, 50 μl of reaction mixture was withdrawn and mixed with an equal volume of 0.8 M formic acid (NBAD reactions) or an equal volume of absolute ethanol  to stop the enzymatic reaction. The formic-acid-treated reaction mixture was centrifuged at 20,000 g for 15 min at 4 °C, and supernatant was analyzed by HPLC with electrochemical detection. Hydrolase activity to NBAD was determined based on the detection of dopamine in the reaction mixture, and hydrolase activity to N-β-alanyl histamine was determined based on the detection of OPT β-alanine and OPT histamine conjugates [Cell lysate supernatant from recombinant njugates .http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the gene products discussed in this paper are A. gambiae CG12120 (AAAB01008846), B. mori CG12120 (BAAB070172112), D. melanogaster CG12120 (NM 132315), and D. pseudoobscura CG12120 (CH379064). The Swiss-Prot (http://www.ebi.ac.uk/swissprot/) accession number for P. chrysogenum IAT is P15802.The GenBank (


The optimal delay length depends on the fitness of resistant viruses and the rate at which target-cell populations recover after therapy is initiated. These observations have implications for both the timing and the kinds of drugs selected for induction–maintenance and therapy-intensification strategies.The tradeoff between the need to suppress drug-resistant viruses and the problem of treatment toxicity has led to the development of various drug-sparing HIV-1 treatment strategies. Here we use a stochastic simulation model for viral dynamics to investigate how the timing and duration of the induction phase of induction–maintenance therapies might be optimized. Our model suggests that under a variety of biologically plausible conditions, 6–10 mo of induction therapy are needed to achieve durable suppression and maximize the probability of eradicating viruses resistant to the maintenance regimen. For induction regimens of more limited duration, a  Clinicians treating HIV infection must balance the need to suppress viral replication against the harmful side effects and significant cost of antiretroviral therapy. Inadequate therapy often results in the emergence of resistant viruses and treatment failure. These difficulties are especially acute in resource-poor settings, where antiretroviral agents are limited. This has prompted an interest in induction–maintenance (IM) treatment strategies, in which brief intensive therapy is used to reduce host viral levels. Induction is followed by a simplified and more easily tolerated maintenance regimen. IM approaches remain an unproven concept in HIV therapy. We have developed a mathematical model to simulate clinical responses to antiretroviral drug therapy. We account for latent infection, partial drug efficacy, cross-resistance, viral recombination, and other factors. This model accurately reflects expected outcomes under single, double, and standard three-drug antiretroviral therapy. When applied to IM therapy, we find that (1) IM is expected to be successful beyond 3 y under a variety of conditions; (2) short-term induction therapy is optimally started several days to weeks after the start of maintenance; and (3) IM therapy may eradicate some preexisting drug-resistant viral strains from the host. Our simulations may help develop new treatment strategies and optimize future clinical trials. The failure of antiretroviral therapies to completely suppress viral replication in some patients represents a major difficulty in the management of HIV infection. In therapy-naive patients without clinically apparent resistance mutations, triple-drug therapy with two nucleoside–analog reverse transcriptase inhibitors and a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor is standard . In thesThe need to minimize drug resistance while reducing treatment-related toxicities has engendered an interest in induction–maintenance (IM) strategies, in which a period of intensified antiretroviral therapy (induction phase) is followed by a simplified long-term regimen (maintenance phase) –25. MostWe have hypothesized that a longer period of a highly suppressive induction therapy that is appropriately timed relative to the start of maintenance therapy may allow minority resistant variants to decay below a stochastic extinction threshold, allowing for successful long-term treatment with simpler and better-tolerated regimens. To explore this hypothesis quantitatively, we constructed a detailed computer simulation model of the dynamics of sensitive and resistant viruses during a hypothetical IM regimen. We show that the timing and duration of induction therapy relative to maintenance therapy can affect the probability that viruses resistant to the maintenance regimen will be eradicated in ways that are somewhat counterintuitive. Under biologically plausible conditions, we find that 6–10 mo of induction therapy are required to maximize the probability of eradicating these resistant viruses. For shorter induction periods, we find that it is optimal to use a “delayed-induction” regimen administered several days to weeks after the start of the intended long-term maintenance therapy.+ target cells, free viruses, and three types of infected cells: short-lived infected cells with t1/2 of ∼1 d, moderately long-lived infected cells with t1/2 of ∼2.5 wk, and long-lived infected cells or “latently” infected cells with t1/2 of ∼3.5 y  As found in –37, our y absent A. ThereaAfter initiation of conventional triple-drug therapy, the viral load decays at a rate of 0.6/d (first phase decay) for ∼10 d, then at 0.04/d (second phase decay), until HIV-1 RNA falls below the detection limit of 50 RNA copies (25 virions) per ml of plasma around day 120 B. A popuWe have used this model to investigate two questions about IM therapies. (1) How long should the induction phase be in order to eradicate viruses resistant to the drugs in the maintenance regimen? (2) What is the optimal timing of induction therapy relative to maintenance therapy? Could IM therapies be improved, for example, if the agents that were unique to the induction regimen were started before starting the maintenance drugs? In the simulations below, the maintenance regimen consists of drugs I and II, while drug III is applied only during induction therapy . We defiV1(0) is the density of the resistant virus at the time that therapy is initiated, m is the turnover rate of target cells at steady state, δ is the death rate of infected cells, R0 = psk/cδm, and R1 = psk1/cδm. R0 and R1 are the basic reproductive numbers  for WT and resistant viruses h, where h is the step size, B(x) is the sum of the “birth” terms, and M(x) is the sum of the “mortality” terms. For x < sxminn, we set x(t + h) to x(t) − 1, x(t), or x(t) + 1 according to the probabilities hM(x), 1 − [M(x) + B(x)]h, and hB(x). Use of deterministic equations for x > sxminn strikes a balance between the need to account for stochastic effects at low population densities and the need to reduce computation times at higher densities where stochastic effects are negligible. Since preliminary runs with sn = 25, 50, 100, and 200 gave similar results (but clearly distinct from sn = 1 or sn = 5), we reasoned that sn = 25 would be sufficient to capture most of the stochastic variation that occurs at low density. Probabilities were determined using the random number generator MT19937 [To account for random genetic drift occurring at low population densities, we used stochastic terms similar to those used in  to model MT19937 . Simulat MT19937  yielded 10 viruses, ∼3 × 108 short-lived infected cells, ∼107 moderately long-lived infected cells, and ∼106 latently infected cells per body. Unless otherwise stated, other parameter values used were: s = 2.0 cells/d, m = 0.02 cells/d, k = 0.0008 cells × μl/d, w1 = 0.95, w2 = 0.95, w3 = 0.95, w4 = 0.95, δI = 0.6 cells/d, Mδ = 0.04 cells/d, Lδ = 0.00052 cells/d, fM = 0.07, Lf = 10−6, p = 100 virions/d, pM = 6 virions/d, pL = 2 virions/d, c = 3 d−1, and μ = 1 × 10−4. All three drugs  are set at 20 ng/ml when these drugs are present. The input rate of target cells, s, was set so that the steady state concentration of target cells is 100 cells/μl, or approximately 10% of a typical peripheral blood CD4 T cell count, since not all CD4+ T cells are susceptible to HIV-1 infection. Units for target cells are based on a total estimate of 2 × 1011 CD4 cells per body, of which 2% are in blood. The stochastic cutoff threshold was set at one infected cell per body, or 3 × 10−9 cells/μl. The death rate of latently infected cells of Lδ = 0.00052/d (t1/2 = ∼44 mo) was conservatively set to one of the lower experimental estimates [k12 = k1k2 / k, k13 = k1k3 / k, k23 = k2k3 / k, and k123 = k1k2k3 / k2, as in [m and k, are summarized in the results.To create a realistic simulation of IM therapy, we adjusted the parameters to match the dynamics of viral decay during potent combination therapies ,40,89,90stimates ,89,91–932, as in . The effX is the concentration of immune effectors, sX is the rate of appearance of immune effectors in the absence of immune stimulation, mX is the death rate of immune effectors, kX is the rate at which HIV-1–infected cells activate immune effectors, and sK is a saturation constant describing the negative effect that the immune effectors have on the ability of HIV-1 to initiate infections. The symbols T, I, I1, V, V1, K1, p, c, δ, and μ have the same meanings as in the target-cell limited model above, though when simulating dynamics under this model, we assume that T does not change over time. To extend this immune-control mechanism to the full, stochastic model, we made analogous extensions, setting dX / dt = sX − mXX + kX(I + I1 + … + I1234 + M + … + M1234)X and multiplying the infection rate constants  by Ks / (Ks + X), while keeping T constant. To simulate drug treatment for different rates of turnover of immune effectors without also changing pretherapy viral loads, we increased sX, mX, and kX proportionately. Although we focus on the target-cell limited model described above, we also explored a simple immune-control model to determine how dependent our qualitative results are on the factors that regulate HIV-1 density. In our immune-control model, the virus population expands exponentially without limitation in the absence of immunity. Immune effectors, which increase at a rate proportional to the number of infected cells, interfere with the ability of virus to infect cells . We implemented this initially using the following model with one mutation and one type of infected cell:


Adult females were found to have a significantly larger corpus callosum: brain volume ratio, rostral body, posterior midbody, isthmus, and splenium than adult males. Left-handed individuals had a larger relative overall corpus callosum area than did right-handed individuals. Additionally, a significant sex and handedness interaction was found for anterior midbody, with right-handed males having a significantly smaller area than right-handed females. These results suggest that sex and handedness influences on corpus callosum morphology are not restricted to Homo sapiens.Sex differences have been reported in both overall corpus callosum area and its regional subdivisions in humans. Some have suggested this reflects a unique adaptation in humans, as similar sex differences in corpus callosum morphology have not been reported in any other species of primate examined to date. Furthermore, an association between various measurements of corpus callosum morphology and handedness has been found in humans and chimpanzees. In the current study, we report measurements of corpus callosum cross-sectional area from midsagittal MR images collected  The corpus callosum (CC) is the major white matter tract connecting the left and right cerebral hemispheres, with fibers establishing both homotopic and heterotopic connections along an anterior-posterior gradient. Cross-species data from anthropoid primates suggests that reduced interhemipsheric connectivity via the CC is related to the enhancement of structural asymmetries Sex differences in both overall CC area and its regional subdivisions in humans were first reported by De Lacoste-Utamsing and Holloway Witelson Whether other primates show similar patterns of sex and handedness influences on CC morphology would enhance our understanding of the neurobiological substrates of handedness, as only a few studies have investigated neural correlates associated with hand preference in nonhuman primates. For example, evidence from chimpanzees indicates that hand preferences for non-communicative actions are correlated with asymmetries of the hand knob region of the precentral gyrus, but not language area homologues Here we investigate sex and handedness influences on corpus callosum morphology in capuchin monkeys. The prevailing view is that skilled motor actions are dependent upon left-hemisphere specialization In vivo magnetic resonance images were collected from 18 capuchin monkeys  and behavioral data on handedness was collected from 13  of these subjects. Ages ranged from 1–21 years (M = 10.08±6.65). Of the total subjects, 14 were adults  and four were juveniles  and acetylpromazine (1 mg/kg), and subsequently anaesthetized with propofol (160–330 micrograms/kg/minute). Subjects were placed into the scanner chamber and their heads were fitted inside a 16 cm head coil. Subjects remained anaesthetized throughout the MR procedure and respiration rate, heart rate, and oxygen consumption were continually monitored. T1-weighted images were acquired on a 3.0 T scanner (Siemens Allegra). Images were collected in the sagittal plane using a gradient echo protocol . Subjects were allowed to completely recover from the effects of the anaesthesia before return transport.http://rsb.info.nih.gov/ij/) and followed the methodology of Witelson Morphometric measurements of the CC were performed using ImageJ software version 1.26t  and showed high consistency in hand use across the four trials.Subjects were individually presented with a piece of poly-vinyl-chloride tube 6 cm in length and 1.5 cm in diameter with peanut butter smeared inside. To remove the food, subjects had to hold the tube in one hand and use the fingers of other hand to retrieve the peanut butter. The hand used to retrieve the food from inside the tube was recorded as left or right. Every instance where an individual inserted their fingers into the tube, retrieved peanut butter and brought that hand to the mouth was recorded. Data were recorded until the subject lost interest in the tube as indicated by discarding the tube for at least 10 s. Each subject was tested four times with the task. Subjects performed a mean of 102 responses  was calculated by taking the average HI of all trials for each individual. Z-scores were calculated for MHI to determine if individuals displayed significant hand preferences and to classify subjects as right-handed, left-handed, or ambidextrous. Subjects with z-scores greater than 1.95 or less than −1.95 were classified as unambiguously right- or left-handed. Subjects with z-scores between 1.95 and −1.95 were classified as having no hand preference.Handedness index (HI) scores were determined for each subject by using the hand preference formula (#R−#L)/(#R+#L). The mean handedness index  = .51, p = .03.Individual area measurements of the CC, its subdivisions, total brain volume, body weights, M = 1.84±.06) did not have a significantly different CC:brain ratio than adults (M = 2.01±.17) . However, as age positively correlated with the ratio of CC: total brain volume, r (18) = .57, p = .01, further analyses were conducted on the adult subjects only.Juveniles (M = 1.86±.11) and adult females (M = 2.13±.09) differed significantly in overall CC:brain ratio , posterior midbody , isthmus , and splenium . For all of these subdivisions females had larger areas than males   = 4.02, p = .09; η2 = .68] and splenium . Left-handed individuals (M = 1.01±.11) had a larger genu than did right-handed individuals (M = .87±.10); right-handed individuals (M = 1.03±.09) had a larger splenium than left-handed individuals (M = .89±.10) (see MHI) and cc:brain ratio were not correlated, r (13) = .19, p  = .53.An analysis of variance with handedness classification as the between-subjects factor indicated borderline significant effects of handedness on the genu , and a borderline significant effect was found for the posterior midbody . Right-handed males had a significantly smaller ratio in the anterior midbody than did right-handed females. No differences were found between left-handed males and left-handed females.A multifactorial analysis of variance with sex and handedness classification as the between-subjects factors revealed a significant interaction for the anterior midbody [Several important findings emerged from our study. First, adult female capuchins have a significantly larger overall CC:brain ratio, rostral body, posterior midbody, isthmus and splenium than adult males. Second, we found borderline significant effects of handedness on corpus callosum morphology, with left-handed individuals having a larger relative genu and right-handed individuals having a larger splenium. Finally, a significant sex and handedness interaction was found, with right-handed capuchin males having a smaller anterior midbody than right-handed females. To our knowledge, this is the first demonstration of an interaction between CC morphology, sex and handedness in a nonhuman primate species.Cebus monkeys utilize both arboreal and terrestrial substrates in their locomotor repertoire Spatial-ability differences in males and females have been proposed to explain differences in the shape and size of the corpus callosum in humans, particularly in the posterior regions of the isthmus and splenium The relationship between the direction of hand preference and CC morphology is not consistent across primate taxa. In humans, numerous studies have consistently found that left-handed and ambidextrous individuals have a larger midsaggital area of the CC than right-handed individuals Our results provide support of the role of handedness influences on corpus callosum morphology, and thus hemispheric specialization in capuchin monkeys. As right-handed male capuchins had a significantly smaller anterior midbody than did right-handed females, our results further support the importance of left-hemispheric specialization in skilled motor actions, as has been proposed by some


E) between the conformers. It is discovered that such an interaction energy can be absorbed into the pre-partitioned fragment channels so that to affect the interaction energies in a particular conformer of Fc. To avoid this, the present study employs a complete fragment channel—the fragments of ferrocene are individual neutral atoms. It therefore discovers that the major difference between the ferrocene conformers is due to the quantum mechanical Pauli repulsive energy and orbital attractive energy, leading to the eclipsed ferrocene the energy preferred structure. The NEDA scheme further indicates that the sum of attractive (negative) polarization (POL) and charge transfer (CL) energies prefers the eclipsed ferrocene. The repulsive (positive) deformation (DEF) energy, which is dominated by the cyclopentadienyle (Cp) rings, prefers the staggered ferrocene. Again, the cancellation results in a small energy residue in favour of the eclipsed ferrocene, in agreement with the ETS scheme. Further Natural Bond Orbital (NBO) analysis indicates that all NBO energies, total Lewis (no Fe) and lone pair (LP) deletion all prefer the eclipsed Fc conformer. The most significant energy preferring the eclipsed ferrocene without cancellation is the interactions between the donor lone pairs (LP) of the Fe atom and the acceptor antibond (BD*) NBOs of all C–C and C–H bonds in the ligand, LP(Fe)-BD*(C–C & C–H), which strongly stabilizes the eclipsed (D5h) conformation by −457.6 kcal·mol−1.Two very different quantum mechanically based energy decomposition analyses (EDA) schemes are employed to study the dominant energy differences between the eclipsed and staggered ferrocene conformers. One is the extended transition state (ETS) based on the Amsterdam Density Functional (ADF) package and the other is natural EDA (NEDA) based in the General Atomic and Molecular Electronic Structure System (GAMESS) package. It reveals that in addition to the model (theory and basis set), the fragmentation channels more significantly affect the interaction energy terms (Δ The ground-electronic state conformation of Fc due to the orientation of two parallel cyclopentadienyl (Cp) rings of Fc, which gives the eclipsed (E) or staggered (S) conformers, is surprisingly difficult to resolve unambiguously in both theory [ca. 0.9 ± 0.3 kcal·mol−1, using electron diffraction in gas phase [−1 using the B3LYP/m6-31G(d) model [5h and D5d further complexes the issue [Since its discovery, ferrocene , i.e., d η5, FeCp or Fc, hh theory  and expeh theory ,5,6,7,8 h theory . Firstlyas phase , and the η5, FeCp or Fc, hd) model  makes th5h conformer [et al. [et al. [et al. [et al. [Detailed structural understanding of the Fc conformers is very important as Fc derivatives may inherit particular properties that only exist in a particular conformer . For exaonformer ,12,13. Donformer ,15,16 pronformer , Coriani [et al. , Roy et  [et al. , Gryazno [et al.  Frenking [et al. ,22,23,24 [et al.  have welet al. [Structures and properties of the staggered (S) and eclipsed (E) conformers of Fc are markedly similar. As indicated by Coriani et al. , Fc is aet al.  and usinet al.  and BPW9et al.  models, et al. ,18,20,27et al. .etc. are almost identical [−1 serves as the signature region for E-Fc conformer in gas phase [The differences between most molecular properties of the Fc conformers, such as energetics, Fe-Cp bond length, rotational constants and total electronic energies dentical , in addidentical . Hence, dentical ,30,31. Aas phase . It was as phase ,8,32, alThe high thermal stability of ferrocene (upto 400 °C) indicates that the chemical bonding of the complex is special as transition metal-carbon bonds are usually unstable . In ordeet al. [et al. [etc. also studied ferrocene from different viewpoint, i.e., using different conformers, or different fragmentation schemes or different models and basis sets (either Gaussian basis sets or Slater basis sets), which provide excellent additional information of Fc. However, it seems that a direct comparison can be difficult, as the calculated energy components depend on multiple factors. Firstly, the energies are fragmentation channel dependent, such as heterolytic and homolytic [etc. are very different, as are the basis sets. Thirdly, most of such EDA studies concentrate on the D5d staggered conformer [Dividing the total molecular energy of Fc into orbital energies using molecular orbital theory is an important method of studying a molecule. There are other ways to study energy contributions of a molecule. It is analogous to a number of manners of cake-cutting. The present study intends to decompose the energy contributions of the conformers using the energy decomposition analysis (EDA) ,34,35. Set al.  Swart [3et al. , Zhang e [et al. , etc. alomolytic . The comonformer ,22,36,38onformer ,38,39. Fonformer ,35 basedonformer ,35 basedonformer  and the onformer ,42,43 baonformer  are emplAny decomposition of the interaction energy into separate terms is artificial, so in principle one cannot go wrong when choosing a particular decomposition scheme . ElectroEnergy decomposition analysis (EDA) is a very useful tool to dissect the physical origins of intra- or intermolecular interactions of molecules ,54,55,56PrepE, is the preparation energy and corresponds to the energy required to deform the separated fragments from their equilibrium geometry to the one they assume in the supermolecule. The second term is a stabilizing term called interaction energy, ΔIntE, which includes the instantaneous interaction between the fragments. It can be considered as the difference between the energy of the molecule and the energies of the prepared fragments [IntE term itself can be decomposed into three quantities that have a direct physical meaning [PauliE, is computed by enforcing the Kohn-Sham determinant of the molecule, which results from superimposing fragments to obey the Pauli principle by ortho-normalizing and anti-symmetrizing the fragment spin-orbitals of one moiety with the fragment spin-orbitals of the second moiety at the supermolecule equilibrium position. This step ensures that same spin electrons do not occupy the same region of space. Usually this term is responsible for the increase in kinetic energy upon formation of a chemical bond and is always positive. The second term, ΔElstatE, is computed as the electrostatic interaction between the unperturbed fragments at the equilibrium position. While ΔPauliE is repulsive and ΔEElstat is usually attractive, superposition of these two terms can be called steric energy [OrbE, in Equation (1) is computed by relaxing the ortho-normalized density to the fully optimized electron density of the entire supermolecule. This contribution includes the charge transfer between the occupied orbitals of one fragment and the unoccupied orbitals of the other fragments. It also includes charge transfer within the occupied and unoccupied orbitals of the same fragment, i.e., the intra-fragment polarization. The ΔOrbE term is always attractive as the total wave function is optimized during its calculation. More specifically, it allows the virtual orbitals on the fragments to be mixed in, and therefore includes highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) interactions [OrbE term further into contributions from orbitals, which belong to different irreducible representations of the point group of the molecule.In the ETS method ,35, the ragments . The ΔEI meaning ,40,45:c energy ,63,64,65a priori manner. Thus, NBO operates by well-known concepts in chemistry such as hybridization, conjugation, hyper-conjugation, charge transfer, and orbital interactions. Natural energy decomposition analysis (NEDA) can be used for analysing intermolecular interactions between host–guest species [ESE is an electrostatic (ES) contribution. It describes the interaction of the unperturbed monomer charge densities and therefore the interaction of the permanent multipoles of the monomer units [POLE is a polarization component (POL) and it describes the extra electrostatic interaction on polarizing the charge densities of the separated fragments in the field of the other fragments when in complex [CTE, is a “charge transfer” (CT) component and the forth term ΔXCE is a “steric exchange” (EX) component. It is considered to represent Pauli exchange-type repulsions between filled orbitals . Natural steric analysis expresses steric exchange repulsion as the energy difference due to orbital orthogonalization, in accordance with a well-established physical picture of “steric repulsions” [DEFE term is the "deformation" energy (DEF), which represents the difference between the energies of the perturbed and relaxed monomer densities.In the natural bond orbital (NBO) concept, it constructs a unique set of atomic hybrids and bond orbitals for a given molecule. It uses the information contained in the (exact or approximate) first-order density matrix , thereby species . The later units . The nex complex . The thiulsions” ,42,43 Foulsions” ,42,43. TThe optimized geometries of Fc (both E and S conformers) were the same as those in gas phase , which w5h) ferrocene (E-Fc) exhibits a slightly lower total energy than the staggered ferrocene (D5d) [5d conformer of ferrocene has been employed for analyses [5d) ferrocene [It is accepted that the eclipsed (Dne (D5d) ,71,72,73analyses ,21,32, aerrocene , which terrocene .2+ cation with the valence electron configuration (a1g)2(e2g)4(e1g)° and (Cp−)2 anion [+ cation and singly charged Cp− anion [2 as interacting fragments. In the homolytic channel, Fe is in a valence electron configuration triplet (a1g)2(e2g)4(e1g)αα, and the ligand Cp2 has the valence occupation (a1g)2(a2u)2(e1u)4(e1g)ββ [PauliE, ΔElstatE and ΔOrbE, of the same ferrocene conformer are different, although the other conditions are the same, e.g., BP86/TZP.Although in principle one cannot go wrong when choosing a particular decomposition scheme in EDA  the inte)2 anion ; (ii) thp− anion ; and (ii4(e1g)ββ  The threPauliE term (staggered Fc) is calculated using the BP86/TZP model as 272.2 kcal·mol−1 by Lein et al. [−1 by Frunzke et al. [−1 by Rayón and Frenking [ElstatE term is calculated as −598.9 kcal·mol−1 by Lein et al. [−1 by Frunzke et al. [−1 by Rayón and Frenking [OrbE term in Equation (2) is calculated as −567.5 kcal·mol−1 by Lein et al. [−1 by Frunzke et al. [−1 by Rayón and Frenking [PauliE term is positive, whereas the ΔEElstat and ΔEOrb terms are both negative. As indicated by Swart [The fragmentation channel dependant differences of the energy components of ferrocene are nowhere close to each other. For example, the Δn et al. , 172.4 ke et al. , and 409Frenking  for the n et al.  −238.5 ke et al.  and −307Frenking  for the n et al. , −171.5 e et al.  and −376Frenking , respecti.e., ferrous Fe2+ cation and (Cp−)2 anion (i) and the same basis set of triple zeta polarized (TZP) in ADF, but OPBE (combination of Handy's optimized exchange (OPTX) with the PBE correlation) rather than BP86 for the eclipsed ferrocene, the calculated energy components are apparently different [In addition to fragmentation channels, the quantum mechanical models employed also impose large component energy differences, which make the EDA quite difficult for comparison and explanation purposes. For example, Swart  using thifferent . As a re5h) and staggered (D5d) conformers, one needs a consistent study based on the same conditions including the fragmentation channels and models. From the results summarized in 2 → Fe ((3d)6, singlet) + 10 C ((2p)2, singlet) + 10 H ((1s)1, doublet), that is, the complex fragments into the smallest possible neutral atoms. Note that the atomic states are not necessarily the ground states of the atoms.In order to obtain a quantitative and consistent analysis for the interaction energies of ferrocene eclipsed (DE column (ΔΔE = ΔE(D5h) − ΔE(D5d)) is the energy differences between the eclipsed and staggered conformers of ferrocene. The results from Zhang et al. [2+ + Cp22−) using the PW91/TZ2P model.g et al.  are alsoE between the eclipsed and staggered ferrocene conformers do not vary apparently with respect to the basis sets. The calculated electrostatic energies, ΔEstatE for D5h, are basically the same using the same theory (BP86) among the basis sets, i.e., both are −2794.94 kcal·mol−1, for TZP and TZ2P, respectively, but a slightly less negative energy of −2793.10 kcal·mol−1 is found when the TZ2P+ basis set is used for the same conformer. Similar trends are found for the ΔPauliE but repulsive, i.e., 12679.83 kcal·mol−1 when both TZP and TZ2P basis sets are used. As pointed by Bickelhaupt and Baerends [Pauli and ΔEElstat [Baerends , the basΔEElstat .OrbE, as this energy term is responsible for orbital interaction or relaxation [iE, for the electrostatic energy and the orbital energy between the eclipsed and staggered ferrocene conformers exhibit negative values, which are balanced but the almost twice as large Pauli positive energy values, leading to the total interaction energy change, ΔΔIntE a very small energy value. For example, using the BP86/TZ2P+ model, the ΔΔE components for ΔΔPauliE, ΔΔElstatE and ΔΔOrbE, are given by 13.41 kcal·mol−1, −5.25 kcal·mol−1 and −9.04 kcal·mol−1, respectively, which results in the interaction energy ΔΔIntE (See Equation (2)) of only −0.89 kcal·mol−1. The results in i.e., BP86, the larger the basis set, the slightly less negative the total interaction energy difference between the eclipsed (E-Fc) and staggered (S-Fc) ferrocene conformers. For example, the absolute value of ΔΔEInt changes from −0.97 kcal·mol−1 (BP86/TZP), to −0.91 kcal·mol−1 (BP86/TZ2P) and to −0.89 kcal·mol−1 (BP86/TZ2P+). Hence, under the same fragmentation scheme , the energy difference of the electrostatic energy between E-Fc and S-Fc remains at −5.25 kcal·mol−1 across the basis sets, followed by the energy term of orbital interaction changes of ΔΔOrbE with −9.04 kcal·mol−1 when TZ2P+ basis set is used. The largest energy change between the conformers is the repulsive Pauli (or steric) energy component, which is as large as 13.41 kcal·mol−1 using the same model of BP86/TZ2P+.The small basis set effects on energy components show in the orbital energy term, ΔΔlaxation . This telaxation . The eneElstatE (more negative), ΔΔPauliE, and ΔΔSterE, but decrease the ΔΔOrbE. As a result, the total interaction energy, ΔΔIntE changes its sign from preferring the eclipsed Fc (in BP86/TZ2P) to preferring the staggered Fc (HF/TZ2P). In addition, the heterolytic fragmentation channel of Zhang et al. [2+ + Cp22−) in the PW91/TZ2P model indicates that the pre-partitioned fragments of the ferrocene conformers, although largely changed the preference of individual energy component, the total interaction energy ΔΔIntE, again, prefers the eclipsed conformer. It also indicated that the energies have already been absorbed into the pre-partitioned fragments of the conformers.It is also interesting to compare the ΔΔEs among BP86/TZ2P, HF/TZ2P and PW91/TZ2P models. As the BP86/TZ2P and HF/TZ2P models are based on the same fragment channel, the energy differences can be attributed to inclusion of electron correlation of the BP86 method. The missing electron correlation energy contributes to increase the ΔΔg et al.   are given by 1.60 kcal·mol−1, 0.76 kcal·mol−1, −0.09 kcal·mol−1 and −0.89 kcal·mol−1 from the HF, M06-2X, B3LYP and BP86, respectively. The HF/TZ2P+ model gives the ΔΔPauliE, ΔΔElstatE and ΔΔOrbE terms as 14.62 kcal·mol−1, −5.25 kcal·mol−1 and −7.76 kcal·mol−1, respectively. The HF results under the same conditions reveal that the largest relative energy changes with respect to the BP86 model between the E-Fc and S-Fc is the ΔΔOrbE term with 16.5%, and followed by the ΔΔPauliE term with −8.3% and the ΔΔElstatE term remain unchanged. The total steric energy between the E-Fc and S-Fc conformers becomes smaller when electron correlation effect is taken into account, as this energy term ΔΔStericE is reduced from 9.36 kcal·mol−1 in the HF/TZ2P+ model to 8.40 kcal·mol−1 in the B3LYP/TZ2P+ model.The decomposed energy terms of a molecule also depends on the theory employed . Table 3PauliE, ΔΔElstatE and ΔΔOrbE, are 13.66 kcal·mol−1, −5.25 kcal·mol−1, and −8.48 kcal·mol−1, respectively. As pointed out by Bickelhaupt and Baerends [PauliE =13.66 kcal·mol−1 higher energy than the staggered conformer. The electrostatic interaction is more attractive in the eclipsed conformer  is superior in not only producing an accurate result in IR spectrum  and geomBaerends  in theirrmer see  as ΔΔEEleraction . In geneeraction . The orbractions .IntE, is a small residue of the balance between Pauli repulsive term ΔΔPauliE and the attractive electrostatic and orbital energies of ΔΔElstatE + ΔΔOrbE. Interestingly, the electrostatic energy term in this table ΔΔElstatE is independent of the level of theory, as the HF and the DFT models produce the same electrostatic energy. As a result, the major difference between the Fc conformers is due to the quantum mechanical Pauli repulsive energy and orbital attractive energy, in agreement with chemical intuition.Conformational isomers such as n-butane  and etha−1, respectively. We may notice a relatively large contribution of the polarization interaction  into the higher relative stability of the eclipsed conformer. These strong electric and charge transfer (CT) interactions are necessary to overcome the strong CORE repulsion, which is 9.73 kcal·mol−1 higher than the staggered conformation.To better understand the nature of interactions in ferrocene conformers in terms of meaningful physical components we applied the natural energy decomposition analysis (NEDA) scheme using the B3LYP/m6-31G(d) optimized geometries , which hNLE, will be expressed asE(full) is the original energy and LE is the Lewis-type localized energy.The NBO 6.0 program  makes itLE) energy which prefers the eclipsed Fc and the non-Lewis (NLE) energy which favours staggered Fc, the total energy between the eclipsed and staggered Fc largely cancels out with a relatively small residue. As one can see in Lewis Contribution) the localized EL contribution strongly favours the eclipsed (D5h) configuration by 564.4 kcal·mol−1, while the delocalization contribution NLE favours the staggered (D5d) conformer by a similar amount of 563.9 kcal·mol−1, leading to nearly equal total energies for the two conformers with a small residue of −0.52 kcal·mol−1. Thus, the stability of the staggered conformer can therefore be attributed to the electronic delocalization energy NLE. In the absence of the Fe atom (section Lewis Contribution (NoFe)) in LE and NLE contributions are much smaller , with the difference between two conformations being also smaller . However, the Lewis contributions with and without Fe in −1 of such energies are due to the Fe atom, which counts for less 5% of the (non-)Lewis contributions.The NBO analysis further indicates that while the Lewis  of the iron atom and the acceptor antibond (BD*) NBOs of all C–C and C–H bonds, which is given in section LP(Fe) − BD*(C-C & C-H) of 5d) conformation by 457.6 kcal·mol−1. It indicates that a significant energy difference between the eclipsed and staggered ferrocene is caused by the interactions between the lone pair of Fe and the antibond electrons of the orientation of the Cp rings, i.e., the conformers. On the other hand, the donor–acceptor interactions involving the donor lone pairs (LP) of Fe atom and the unoccupied Rydberg-type (RY*) acceptor NBOs of the carbon and hydrogen atoms lead to a relatively small destabilization of the staggered (D5d) conformer by 6.4 kcal·mol−1, as given in section LP(Fe) − RY*(C & H) of We can further dissect the delocalizing (non-Lewis) contribution, Quantum mechanically based energy decomposition analyses (EDA) are employed to study interaction energies of ferrocene conformers. Two very different EDA schemes are employed with one based on the extended transition state (ETS) using the ADF package and the other on natural EDA (NEDA) using the GAMESS package. The study reveals that the interaction energy terms of ferrocene depend on a number of factors, not only the model (theory and basis set) but also more significantly, the fragmentation schemes in the EDA. It is discovered that interaction energy can be absorbed into the pre-partitioned fragments such as the heterolytic fragmentation, which may affect the interaction energies in a particular conformer of Fc. The present study employs a complete individual atomic fragment scheme—the fragments of ferrocene are neutral atoms rather than pre-partitioned fragments. In the ETS scheme, it discovers that unlike the alkane rotational conformers such as n-butane and ethane, which the steric hindrance results in the staggered conformer as the energy favoured structure, the major difference between the ferrocene conformers is due to the quantum mechanical Pauli repulsive energy and orbital attractive energy, leading to the eclipsed ferrocene the energy preferred structure.LE contribution strongly favours the eclipsed (D5h) configuration by 564.4 kcal·mol−1, while the delocalization non-Lewis contribution NLE favours the staggered (D5d) conformer by a similar amount of 563.9 kcal·mol−1 leading to nearly equal total energies for the two conformers with a small residue of −0.52 kcal·mol−1, in favour of the eclipsed conformer. The most significant energy between the eclipsed and staggered ferrocene which is not cancelled out is the deletion of all interactions between the donor lone pairs (LP) of the iron atom and the acceptor antibond (BD*) NBOs of all C-C and C-H bonds (LP(Fe) − BD*(C-C & C-H)), which strongly destabilizes the staggered (D5d) conformation by 457.6 kcal·mol−1.The NEDA scheme further indicates the attractive (negative) polarization (POL) and charge transfer (CL) energies prefer the eclipsed ferrocene. The repulsive (positive) deformation (DEF) energy, which is dominated by the cyclopentadienyle (Cp) rings, prefers the staggered ferrocene. The polarization (POL) and charge transfer (CL) energies are nearly balanced out by the deformation (DEF) energy, leading to small attractive total interaction energy residue preferring the eclipsed Fc conformer. Further NBO analysis indicates that the localized Lewis


G, the volume fraction of solute atoms fv, and the size misfit degree δb. A simple hardening correlation in KCl-KBr solid-solution is proposed as ∆τFcg = 0.27 Gx = 0.55–0.85 in BNxPx1−, where BNxPx1− solid solutions are far harder than cubic BN. Because the prediction is quantitative, it sets the stage for a broad range of applications.We demonstrate that a hardening rule exists in cubic solid solutions with various combinations of ionic, covalent and metallic bonding. It is revealed that the hardening stress ∆τFcg is determined by three factors: shear modulus  High-hardness materials are widely used for wear-resistant coatings and cutting tools. In engineering practice, hardening processes, such as Hall-Petch strengthening, work hardening, precipitation hardening, and alloy hardening, are commonly used to increase the hardness of a material123et al.678Eh. When normal sites are replaced by other atoms or vacancies in a crystal structure, the local electronic states or new energy levers are introduced, leading to a Fm between a dislocation and a single solute atom is characterized by a critical breaking angle θc: Fm = 2Edsinθc, where Ed is the dislocation line energy. Furthermore, the critical stress of solid solutions can be estimated by Gδbf1/2, where G is shear modulus, f is the volume fraction of solute atoms intersecting the unit area of the glide plane, in the isotropic case the size misfit parameter δb  = (da/dc)/a, a is the lattice constant, c is the atomic solute concentrationIn recent years, computational materials science based on first-principles calculations has emerged as an effective supplement to experimentation, especially because it enables powerful prediction of material properties that depend only on electronic density or atomic bonding. Because hardness is the resistance to localized fracture or permanent plastic deformation, it relies strongly on the motion of dislocations in addition to atom bonding1Hk of a single-crystal material relies strongly on the resolved shear stress on the glide plane due to indentation. We proposed a hardness formula. The Knoop hardness of (hkl)[h’k’l’] direction in single crystals can be expressed as followsFrom the perspective of dislocation, the hardness Θ is the orientation factor of a glide plane, ϕ is the angle between a glide-plane spacing vector and the section of indenter along the short edge, and ψ is the angle between the projection of a glide-plane spacing vector on the section of indenter along the short edge and the short edge of the indenter, φ is the angle between the facet and the crystal surface, Λf is a constant, Nbg is the bond electron density in the glide region, fc is covalency, d is bond length. Parameter n is taken as 0 and 1 for brittle and semi-brittle materials, respectively. In solid-solution single crystals, the total hardness Hk of solid-solution single crystals can be expressed as follows:where G, the volume fraction of solute atoms fv, and the size misfit degree δb. The hardening stress p, q are constants, where A, et al.xClx1− solid solution:The most of alkali halides and metals are semi-brittle materials. The solubilities for the alkali halide solid solution are often limited, but some are complete. In the KCl-KBr system, the two compounds are entirely soluble in each other. Thus, it provide a very suitable model system for studying details of hardening of solid solutions. Some data points for the system were determined by Subba fv  = x < 0.5). xCl1−x solid solution. Where the values of shear modulus and the lattice parameters are obtained via linear interpolation of the values for the end members. The Ag-Au alloy also is a semi-brittle material. And it is a classical example of the existence of a entire series of fcc solid solutions. Both bulk and film samples are single-phase alloys for any xwhere we take n. Therefore, we may employ n = 0, p = In addition to semi-brittle materials, a nonlinear increase of microhardness vs composition was observed on brittle solid solution single crystals of the InP-GaP. However, the hardening degrees are not significant compared to those of semi-brittle materials abovementioned. For semi-brittle materials the dislocations are mobile at a slip plane, In brittle materials the dislocations do not move at room temperatures, they tend to fracture along a slip plane before yield can occur. et al.et al.et al.multilayer transition metal carbide/nitride coatings. Such type of hardening is attributed to a large number of different glide-systems suppressing the propagation of dislocations, which is consistent with the theory of hardnesset al. and the corresponding experimental values are great although they try to reveal composition dependence of hardness of TiNxC1−x. In contrast, our scheme reproduce the hardness trend with x correctly. It should be noted that the deviation still exist using A, p, q) in Transition-metal carbides and nitrides are important bulk and thin-film materials and widely used for cutting tools and wear-resistant coatings15x = 0.55–0.85 in BNxPx1− is predicted. As evident in 0.75P0.25 (B4N3P) are hardest among BN-BP solid solutions. Its hardness value, 61 GPa is far greater than that of cubic BN, 46 GPa. Thus, it is a candidate for new superhard materials. To confirm structure and structural stability of BN0.75P0.25 (B4N3P), we performed first-principles calculations using DFT code and the Vienna Ab-initio Simulation Package (VASP)202122234N3P exhibit a zinc-blende structure. The calculated lattice parameters of cubic B4N3P were 3.896 Å. The calculated elastic constants for B4N3P were C11 = C22 = C33 = 565 GPa, C44 = 297 GPa, and C12 = 132 GPa. The mechanical stability criteria in a cubic crystal are given by C11 > 0, C44 > 0, C11 > |C12|, (C11 + 2C12) > 0. The elastic constants of B4N3P clearly satisfy all of the mechanical stability criteria, indicating their elastic stability. The calculated bulk modulus and shear modulus values for B4N3P were 339 GPa and 265 GPa, respectively. The phonon dispersion curves of B4N3P at ambient pressure are presented in 4N3P showed that the band gap is 1.7 eV under ambient conditions, indicating a good semiconductivity. Using modern high-pressure–high-temperature technique, the B4N3P could be fabricated if a suitable synthesis route could be developed.The search for novel hard materials would benefit from the advance in the theoretical treatment of solid solutions. We take BN-BP solid solutions as example. The hardness of binary BN-BP solid solutions depends strongly on their composition. 4N3P solid solution single crystal possesses a superhardness that exceeds that of cubic BN. The successful application of this method would contribute to the continuing search for novel hard solid solutions for industrial applications.In conclusion, we demonstrated the origin of hardening of cubic solid solutions. A hardness formula was developed. The theoretical prediction for BN-BP solid solution indicated that the BBr, the suggested radius of the Br atom is RBr = rBrd/(rK + rBr)fi + fcd/2, where rBr is the effective ionic radius of the Br atom, d is the bond length, and fi and fc are the ionicity and covalencyThe total energies are calculated by using a plane-wave pseudpotential method based on density functional theory (DFT), implemented in Vienna Ab initio Simulation Package (VASP)20How to cite this article: Gao, F. Hardness of cubic solid solutions. Sci. Rep.7, 40276; doi: 10.1038/srep40276 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


According to the World Health Organization recommendation, the optimal fluoride concentration levels in drinking water have to be in the range of 0.5 and 1.5 mg/L since this permissible range is essential for normal mineralization of bones and teeth as well as for dental enamel formation in human's body Bell and budwig, 1970;Adimalla and Venkatayogi, 2017;Narsimha and Sudarshan, 2013,2016;2017 Specifications TableValue of the Data•Data can be helpful as an indicator for fluoride concentration in different geological terrains  groundwater.•The data set is very useful to hydrologist, geochemists, environmental researchers and scientists as an indicator for further take necessary steps to execute the groundwater quality research and its vulnerability estimation.•The distribution pattern of fluoride level/concentration will act as a geochemical baseline, to delineate the groundwater quality and health implications.•Since the dataset is geo-referenced, it can be used for geospatial modelling in GIS.13-, 2+ versus fluoride cross plots are shown in Geologically, in the Medak region majorly occupied by granitic, basaltic, and laterite rocks as depicted in 2Medak district is located in the central part of Telangana, South India. It lies between North Latitudes 17° 27′ and 18° 18′ and East longitude 77° 28′ and 79 °10′ falling in topographical sheet nos. 56 F, G, J and K of the Survey of India, and covers an area of 9,699 Sq. Km .3, total dissolved solids (TDS), calcium (Ca2+), magnesium (Mg2+), sodium (Na+), potassium (K+), chloride (Cl-), sulphate 3-). Using pH/EC/TDS meter (Hanna HI 9811-5), the EC, pH and TDS of water samples were measured in the field immediately after the collection of the samples. Calcium, magnesium, chloride, carbonate and bicarbonate were analyzed by a titration method using the standard procedure as given in APHA (1995). TH was measured by a titration method using a standard hydrochloric acid solution and a standard EDTA solution. Sodium and potassium concentrations were determined using a flame photometer . Sulphate and nitrate were determined using a UV-visible spectrophotometer . The fluoride concentration in water was determined electrochemically, using a fluoride ion-selective electrode (ISE) with an Orion 4 star meter benchtop pH/ISE meter One hundred and ninety four groundwater samples were collected, from bore wells/hand pumps in November 2015 in pre-cleaned 1 L polyethylene bottles


The general profiles of differentially expressed genes were visualized as volcano plots and heatmaps. Detailed analyzes consisted of identifying significantly enriched gene ontological groups relevant to adrenal physiology, by means of DAVID and GOplot bioinformatics tools. The results of our studies showed that compensatory adrenal growth induced by unilateral adrenalectomy exerts a limited influence on the global transcriptome profile of the rat adrenal gland; nevertheless, it leads to significant changes in the expression of key genes regulating the circadian rhythm. Our results confirm also that regulation of compensatory adrenal growth is under complex and multifactorial control with a pivotal role of neural regulatory mechanisms and a supportive role of other components.Compensatory adrenal growth evoked by unilateral adrenalectomy  constitutes one of the most frequently studied in vivo models of adrenocortical enlargement. This type of growth has been quite well characterized for its morphological, biochemical, and morphometric parameters. However, the molecular basis of compensatory adrenal growth is poorly understood. Therefore, the aim of this study was to investigate the rat adrenal transcriptome profile during the time of two previously described adrenocortical proliferation waves at 24 and 72 h after unilateral adrenalectomy. Surgical removal of the left adrenal or a sham operation was accomplished via the classic dorsal approach. As expected, the weight of the remaining right adrenal glands collected at 24 and 72 h after hemiadrenalectomy increased significantly. The transcriptome profile was identified by means of Affymetrix Adrenal growth, particularly its cortical compartment, has been the subject of numerous studies for years. The specific reactivity of this gland to various endogenous and exogenous factors ensures maintenance of the organism homeostasis, for which the adrenocorticosteroid hormones are responsible. Maintaining proper homeostasis, especially in the case of longer periods, is usually associated with changes in adrenal mass, mainly its enlargement. These changes are caused by the proliferation (hyperplasia) and/or hypertrophy of adrenocortical cells. As known, the main factor regulating adrenal cortex growth in vivo is ACTH. However, in addition to ACTH, a number of other humoral factors, as well as the nervous system, are involved in the regulation of gland growth. It should be underlined that adrenal cortex growth disturbances resulting in hypertrophy or hyperplasia of the gland may lead to the development of adrenal tumors.In 1984, Dallman, in her excellent monograph, described various types of adrenal cortex growth in vivo and described the main factors regulating them [p-values are shown in The average weight of the right adrenal glands of the control group was 21.1 ± 2.5 mg. As expected, the weights of the right adrenal glands of rats tested 24 and 72 h after hemiadrenalectomy increased statistically significantly. It amounted to 23.1 ± 1.5 and 25.2 ± 1.9 mg, respectively . The difp-value with FDR correction 10%), expression of only 38 genes  was regulated in the 24-h vs. control group comparison , as well as correlation coefficient analysis, was performed on a whole set of differentially expressed genes from all studied comparisons A,B. PCA p-value  was grouped using a hierarchical clusterization algorithm. The results of such analysis are presented as a heatmap . Clustern = 14, p-value = 3.83 × 10−2); “GO:0071383—cellular response to steroid hormone stimulus“ ; “GO:0051726—regulation of cell cycle” ; “GO:0071383—cellular response to steroid hormone stimulus” ; “GO:0048545—response to steroid hormone” ; “GO:0048511—rhythmic process” ; “GO:0042752—regulation of circadian rhythm” ; “GO:0033993—response to lipid” ; “GO:0071396—cellular response to lipid” ; “GO:0033554—cellular response to stress” ; “GO:0032922—circadian regulation of gene expression” ; “GO:0008202—steroid metabolic process” ; “GO:0042752—regulation of circadian rhythm” ; “GO:0007623—circadian rhythm” ; “GO:0006950—response to stress” ; and “GO:0033554—cellular response to stress .Due to the limited number of differentially expressed genes, assignment to the relevant gene ontological (GO) group was performed on all differentially expressed genes simultaneously (both from 24 h and 72 h groups vs. control). For this purpose, 158 differentially expressed gene names (38 from 24 h vs. control and 120 from 72 h vs. control) were used as a query for DAVID GOTERM BP database, searching with the assignment of differentially expressed genes to appropriate GO groups. Twelve processes relevant to adrenal gland physiology were isolated from them and are presented in Cyp 51, Slfn13, Aox1), different genes forming specific GO categories were derived from 24 h vs. control and 72 h vs. control groups. In addition, the majority of genes from 24 h vs. control underwent positive stimulation whilst the expression of genes from 72 h vs. control was mostly down-regulated.Due to the high similarities of certain GO groups, they were classified into four categories: (A) “circadian rhythm”; (B) “cell cycle”; (C) “stress”; and (D) “lipid and steroid” and presented as circos  plots , Per2 , Per3 , Arntl , and Nr1d1 .The expression of genes belonging to the “circadian rhythm” category show high dynamics of change. In comparison of 24 h/c five genes were up-regulated and two down-regulated whilst in the 72 h vs. control groups five genes were down-regulated A. IntereKi-67 (mki67) was not significantly changed in the 24-h to control comparison but it was up-regulated after 72 h of compensatory adrenal growth. Ccna2 (Cyclin A2) gene was regulated in a similar manner while Cdkn1c (cyclin-dependent kinase inhibitor 1C) was up-regulated after 24 h of adrenal compensatory growth and remained unchanged at 72 h of the experiment.The “cell cycle” category consisted of only one gene ontological group—“regulation of cell cycle” B. As befThe gene expression pattern of the “stress” category was notably changed during the experiment C. In thiStAR, Cyp11a1, Cyp11b1, Cyp11b2); however, we observed changes in the expression of genes involved indirectly in steroidogenesis, such as hormone-sensitive lipase (Lipe), whose expression was lowered at 72 h of compensatory growth.In the “lipid and steroid” GO category D, the DEUts2 (urotensin 2) gene, which is listed in the “circadian rhythm,” “stress,” and “lipid and steroid” GO categories.Due to the ambiguous nature of the gene ontology structure, it is worth noticing that there were genes that belong to several described GO categories. Among others, this concerns As mentioned in the introduction, the genetic basis of adrenal growth regulation in vivo is not well known and only a few publications have been devoted to this issue. Normal adrenal growth with age ensures that the adrenal mass and its secretory function is maintained during the changing body mass and changing demand for corticosteroids. As Dallman  points oThe unilateral adrenalectomy-induced compensatory adrenal gland growth has been known for almost 100 years . This typ-value with FDR correction: 10%). Using the Affymetrix microarray system, in the remaining adrenal glands at 24 and 72 h of the experiment, we showed only 155 significantly regulated genes. After bioinformatics analyses, these genes could be grouped into four significantly enriched GO categories. These categories are “circadian rhythm,” “cell cycle,” “stress,” and “lipid and steroid”. It is worth noticing that neural adrenal stimulation affects all of them. Splanchnic nerve stimulation enhances the production of glucocorticoids in response to ACTH, as well as stimulates basic adrenocortical steroidogenesis [In the available literature, we have not found a publication on the global expression of adrenal genes in the case of compensatory growth induced by hemiadrenalectomy. The present study revealed that in the rat, compensatory adrenal growth induced by unilateral adrenalectomy exerts a limited influence on adrenal gland gene expression profiles, even with the assumption of relatively low cut-off values , Cry1, Per1, Per2, Per3, and Nr1d1 —undergo rhythmical changes in the mice adrenal gland. Clock and Cry2 transcripts were also detected but did not show obvious circadian rhythm changes. The expression of these genes also did not change in our studies. Our studies do not confirm the expression changes in Cry1; however, expression of this gene was slightly elevated at 72 h (fold = 1.4), but did not reach statistical significance (adj. p = 0.37). Considering the above-described results, we assume that unilateral adrenalectomy-induced compensatory adrenal growth, through the splanchnic nerve activation, causes a shift in the expression of core genes of the circadian clock. Circadian pattern of expression may affect approximately 5% of the mice adrenal transcriptome, including genes involved in cholesterol uptake, regulators of steroidogenesis, proliferation, and cell cycle regulation [In this study, the most significant changes in gene expression during compensatory adrenal growth were observed in the GO category “circadian rhythm.” We found that unilateral adrenalectomy significantly affected core genes regulating the circadian rhythm, which are responsible for the daily rhythmical secretion of adrenal hormones. Currently, it is well known that adrenal circadian rhythms are controlled by three factors: (I) hypothalamic–pituitary–adrenal (HPA) axis, (II) hypothalamic suprachiasmatic nucleus (SCN) acting via the autonomic nervous system, and (III) local intra-adrenal circadian clocks . It is gan cycle . Our curr et al.  who revegulation . In our gulation . The infMtnr1a: fold 24 h/c = −1.10, fold 72 h/c = −1.05, adj. p 24 h/c = 0.92, adj. p 72 h/c = 0.90; Mtnr1b: fold 24 h/c = −1.14, fold 72 h/c = −1.09, adj. p 24 h/c = 0.91, adj. p 72 h/c = 0.86). Rhythmic expression of melatonin and several clock genes in the mouse, rat, and primate adrenal glands, as well as in adrenal explants, has previously been reported [Our observations are also in line with the data reported and reviewed by Dallman . She shoreported ,27,28,29reported . These oKi-67 (mki67) was not changed at 24 h of compensation but increased at 72 h of the experiment.Unilateral adrenalectomy-induced compensatory adrenal growth is characterized by a prompt increase in weight and DNA and RNA in the remaining adrenal; however, the RNA/DNA ratio does not change ,32,33. SUts2)—coding one of the potent vasoconstrictive peptides—was significantly elevated at 24 h of compensatory adrenocortical growth. Intra-adrenal blood flow rate is highly controlled by a number of different neural and hormonal stimuli in which activation of the splanchnic nerve, as well as ACTH, plays a crucial role [Uts2 expression is strongly up-regulated by ACTH. Therefore, we can assume that increased Uts2 expression during the early stages of adrenal compensatory growth can be exerted by ACTH.Several studies point to the supportive role of ACTH or other POMC-derived peptides in the early stage of the compensatory adrenal growth. It has been shown that pro-gamma MSH stimulates proliferation of the remaining adrenal at 24 h of compensatory growth . Moreoveial role . The resThe gene expression pattern of the “stress” category was notably changed in the course of compensatory adrenal growth. As expected, the expression of most of the stress category genes was up-regulated at 24 h of the experiment while down-regulated at 72 h.StAR, Cyp11a1, Cyp11b1, Cyp11b2) did not change in the enlarged adrenal gland. Unlike these genes, genes indirectly involved in steroidogenesis, such as hormone-sensitive lipase (Lipe), were reduced at 72 h in the experiments. Lipe is a major cholesterol hydrolase of the adrenal glands. It is also defined as a key enzyme involved in steroid hormone synthesis. Down-regulation of Lipe gene indicates that the efficiency of adrenal steroidogenesis may be reduced at 72 h of adrenal compensatory growth. [In our research, rather surprising results are evident concerning genes belonging to the “lipid and steroid” GO group. Unilateral adrenalectomy changed the expression of only a small number of these genes. It is interesting that the expression of genes directly involved in the classical pathway of steroidogenesis  were obtained from the Laboratory Animals Breeding Center, Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland. Animals were kept under the standard conditions of 14:10 h light–dark cycle (illumination onset at 6:00) at 23 °C constant temperature. The animals had free access to standard diet and water. All procedures described below were approved by the Local Ethics Committee for Animal Research , permission number: LKE-11/2015. Maximum efforts were made to minimize the number of animals and their suffering.If not otherwise stated, all reagents were obtained from Sigma-Aldrich  or Avantor Performance Materials Poland S.A. .®, Pfizer Europe, Sandwich, UK) and xylazine  anesthesia. Hemiadrenalectomy was accomplished via the classic dorsal approach [Surgical removal of the left adrenal gland  or sham operation was performed under standard ketamine  and subsequent purification by RNeasy Mini Elute Cleanup Kit , according to the manufacturer’s guidelines. The concentration of the total RNA was assessed spectrophotometrically (260 nm). The purity of the isolated RNA was determined by applying the 260/280 nm absorption ratio, which was equal to or greater than 1.8 . The quality and integrity of the RNA were also verified in a Bioanalyzer 2100 . The resulting RNA integrity numbers (RINs) ranged from 8.5 to 10 with a mean of 9.2 . Total RNA in a concentration of 50 ng/µL was used for the microarray experiment.The microarray study was carried out according to the previously described procedure ,39,40,41The generated CEL files were subjected to further analysis using the R statistical language and Bioconductor package with the relevant Bioconductor libraries. The Robust Multiarray Average (RMA) normalization algorithm implemented in the “Affy” library was used for normalization, background correction, and calculation of the expression values of all of the examined genes . Assignep-values of selected GO terms were corrected using Benjamini–Hochberg false discovery rate method described as adjusted p-values [p-values below 0.05 were visualized using bubble plots. Details of the genes belonging to particular GO terms with their fold change values were presented as circus plots using “GOplot” library [All differentially expressed genes from both comparisons (24 h to control and 72 h to control) were subjected to functional annotation and clusterization using the DAVID  bioinformatics tools . Gene syp-values . GO grou library .t-test. In the case of data obtained from microarrays, differences were evaluated by statistical programs included in particular bioinformatics analyses.Differences in adrenal mass between individual experimental groups were evaluated by a Student’s The presented study concerns the determination of the adrenal transcriptome profile at two critical stages of adrenal compensatory growth. We found that compensatory adrenal growth induced by unilateral adrenalectomy had a limited influence on the adrenal gland gene expression profile. In our comprehensive approach, we used the whole adrenal gland to define transcriptome profiles and, thereby, pronounced changes in expression may have been underestimated by the predominance of unresponsive cells. Further detailed transcriptome studies are required based on separated adrenocortical zones and medulla. Nevertheless, we identified 155 genes that were significantly regulated both after 24 and 72 h. In the rat, unilateral adrenalectomy-induced compensatory adrenal growth leads to the most significant changes in the expression of key genes regulating the circadian rhythm. Our results confirm also that regulation of compensatory adrenal growth is under complex and multifactorial control, with a pivotal role of neural regulatory mechanisms and a supportive role of other components.


China has high stroke rates despite the population being relatively lean. Uncertainty persists about the relevance of adiposity to risk of stroke types. We aimed to assess the associations of adiposity with incidence of stroke types and effect mediation by blood pressure in Chinese men and women.The China Kadoorie Biobank enrolled 512 891 adults aged 30–79 years from ten areas  during 2004–08. During a median 9 years (IQR 8–10) of follow-up, 32 448 strokes (about 90% confirmed by neuroimaging) were recorded among 489 301 participants without previous cardiovascular disease. Cox regression analysis was used to produce adjusted hazard ratios (HRs) for ischaemic stroke (n=25 210) and intracerebral haemorrhage (n=5380) associated with adiposity.2 (SD 3·2), and 331 723 (67·8%) participants had a BMI of less than 25 kg/m2. Throughout the range examined (mean 17·1 kg/m2 [SD 0·9] to 31·7 kg/m2 [2·0]), each 5 kg/m2 higher BMI was associated with 8·3 mm Hg (SE 0·04) higher systolic blood pressure. BMI was positively associated with ischaemic stroke, with an HR of 1·30 (95% CI 1·28–1·33 per 5 kg/m2 higher BMI), which was generally consistent with that predicted by equivalent differences in systolic blood pressure (1·25 [1·24–1·26]). The HR for intracerebral haemorrhage (1·11 [1·07–1·16] per 5 kg/m2 higher BMI) was less extreme, and much weaker than that predicted by the corresponding difference in systolic blood pressure (1·48 [1·46–1·50]). Other adiposity measures showed similar associations with stroke types. After adjustment for usual systolic blood pressure, the positive associations with ischaemic stroke were attenuated (1·05 [1·03–1·07] per 5 kg/m2 higher BMI); for intracerebral haemorrhage, they were reversed (0·73 [0·70–0·77]). High adiposity (BMI >23 kg/m2) accounted for 14·7% of total stroke .Mean baseline body-mass index (BMI) was 23·6 kg/mIn Chinese adults, adiposity was strongly positively associated with ischaemic stroke, chiefly through its effect on blood pressure. For intracerebral haemorrhage, leanness, either per se or through some other factor (or factors), might increase risk, offsetting the protective effects of lower blood pressure.UK Wellcome Trust, UK Medical Research Council, British Heart Foundation, Cancer Research UK, Kadoorie Charitable Foundation, Chinese Ministry of Science and Technology, Chinese National Natural Science Foundation. The reference lists of relevant articles were reviewed. About 20 prospective cohort studies examining the association between adiposity and risk of stroke types were identified, including five in China, along with a few meta-analyses of cohort studies. With a few exceptions, most of the studies only included body-mass index (BMI) and focused on mortality rather than incidence of stroke, with little information on diagnosis of stroke types. Moreover, none examined the quantitative association between BMI and risk of stroke types in the context of the associations between BMI and blood pressure, and between blood pressure and stroke types. In general, these studies have shown positive associations between ischaemic stroke and total stroke and BMI, but the strength of the associations differed substantially between different populations and, within specific populations, between different studies. For intracerebral haemorrhage, the findings have been conflicting, due partly to the relatively small number of events recorded and lack of neuroimaging for diagnosis of stroke types in many studies.Added value of this study2 [SD 3·2]) and apparently healthy Chinese population, there was a strong positive and log-linear association between general  and central  adiposity and the incidence of ischaemic stroke, with the risk estimates consistent with those predicted by equivalent differences in systolic blood pressure associated with adiposity. By contrast, the risk estimates for intracerebral haemorrhage were less extreme than for ischaemic stroke, and much weaker than predicted by the corresponding difference in systolic blood pressure, especially at a BMI of less than 25 kg/m2, which covers two-thirds of study population. The risk estimates were higher in men than in women, due mainly to stronger associations in men of systolic blood pressure with stroke. After adjustment for usual systolic blood pressure, the positive association of baseline BMI (and other adiposity measures) with ischaemic stroke largely disappeared, whereas for intracerebral haemorrhage it was completely reversed. BMI at the age of 25 years was weakly positively associated with ischaemic stroke and intracerebral haemorrhage, and the association was largely unaffected by adjustment for systolic blood pressure.This study represents the world's largest investigation to date in a single study of the association between adiposity and risk of incident stroke types. In this relative lean  accounted for 15% of total stroke . The positive association between adiposity and ischaemic stroke is chiefly mediated through its effect on blood pressure. However, there was no association between adiposity and intracerebral haemorrhage across a broad normal range , suggesting that leanness, either per se or through some other factor (or factors), might increase risk, thereby offsetting the protective effects of lower blood pressure. Future Mendelian randomisation studies are needed to establish the causal relevance of adiposity for intracerebral haemorrhage. Since ischaemic stroke constitutes the majority of total stroke cases in China and elsewhere, the findings on intracerebral haemorrhage should not diminish the fundamental importance of high adiposity as a major modifiable determinant of overall stroke. Given the substantial risks associated with high blood pressure, the present study further highlights the importance of controlling blood pressure (and other intermediate risk factors) for prevention of both ischaemic and haemorrhagic stroke, irrespective of levels of adiposity.Globally, the mean levels of adiposity have been increasing steadily in recent decades. In this Chinese population, high adiposity , and medical history by trained health workers using interviewer-administered laptop-based questionnaires. Physical measurements  were recorded using standard methods. A non-fasting venous blood sample was collected for storage and on-site random plasma glucose testing using the SureStep Plus system . Two periodic resurveys were done from May 26, to Oct 10, 2008, and from Aug 4, 2013, to Sept 18, 2014, on approximately 5% of randomly selected participants, collecting similar information as at baseline to enable assessment of within-person variation and temporal trends in exposures.2), BMI at age 25 years used self-reported weight at age 25 years and height measured at baseline, and waist-to-hip ratio was the ratio of waist circumference to hip circumference.Anthropometric measurements were recorded once, and usually to the nearest 0·1 cm or 0·1 kg.Blood pressure was measured twice using the UA-779 digital sphygmomanometer  after participants had remained at rest in the seated position for at least 5 min. If the difference between the two measurements was more than 10 mm Hg for systolic blood pressure (in about 5% of participants), a third measurement was made and the mean of the last two measurements was used.The vital status of participants was obtained periodically from local death registries,The main stroke types examined were ischaemic stroke (ICD-10 code I63), intracerebral haemorrhage (I61), subarachnoid haemorrhage (I60), and total stroke . All analyses were restricted to first events of any stroke type occurring between the ages of 35 years and 79 years, with censoring when participants had a stroke or reached 80 years of age (or loss to follow-up).Individuals with previous history of stroke or transient ischaemic attack (n=8884) or ischaemic heart disease (n=15 472) at baseline, and those with missing, implausible, or extreme values of anthropometric measurements (n=493) were excluded, leaving 489 301 for our report. Individuals with hypertension or diabetes were retained , as these could be mechanisms whereby adiposity predisposes to stroke.2), standardised by 5-year age groups, sex, and study area. The cut points were chosen to cover different thresholds for defining overweight and obesity in Chinese people and to ensure reasonable participant numbers in each group. Cox proportional hazards models with time since baseline as the timescale were used to estimate hazard ratios (HRs) for incident stroke by adiposity and systolic blood pressure, stratified by 5-year age-at-risk, sex, and area (ten groups), and adjusted for education (five groups), smoking (four groups), alcohol use (five groups), physical activity , and self-reported health status (four groups). Floating variance estimates  were used to estimate group-specific 95% CIs for each log HR to enable comparisons between any two categories (rather than just pairwise comparisons with the reference category).The prevalence or mean values of baseline characteristics were calculated across seven BMI categories , with 331 723 (67·8%) having a BMI of less than 25 kg/m2 and 18 862 (3·9%) having a BMI of 30 kg/m2 or more . Overall mean BMI was slightly higher in women than in men , and higher in urban than in rural areas . BMI was weakly positively associated with education (p=0·0008) and alcohol intake (p=0·0009), especially at a BMI of less than 25 kg/m2, and inversely associated with smoking in both men and women. Individuals with a BMI of less than 18 kg/m2 or more than 25 kg/m2 tended to have lower levels of physical activity and poor self-rated health status than did those with a normal BMI , and the mean BMI was 23·6 kg/mrmal BMI .Table 1BBMI was strongly positively associated with weight (p<0·0001), and weakly positively associated with sitting and standing height . The incidence of ischaemic stroke per 10 000 person-years was higher in urban than in rural areas (82·2 vs 53·3), but the converse was true for intracerebral haemorrhage (7·5 vs 18·7).Incident strokes from enrolment until Jan 1, 2016 (a median of 9 years [IQR 8–10]), are included in these analyses, by which time 37 289 (7·3%) participants had died and 4875 (1·0%) were lost to follow-up. During 3·85 million person-years of follow-up, there were 32 448 incident cases of stroke  at ages 35–79 years, including 25 210 (77·7%) due to ischaemic stroke , 5380 (16·6%) due to intracerebral haemorrhage , 434 (1·3%) due to subarachnoid haemorrhage , and 1424 (4·4%) unspecified strokes . The standardised total stroke incidence per 10 000 person-years was 84·3, and was higher in urban than rural areas (94·6 2 higher BMI was associated with 8·3 mm Hg (SE 0·04) higher systolic blood pressure  for ischaemic stroke, 1·48 (1·46–1·50) for intracerebral haemorrhage, and 1·29 (1·29–1·30) for total stroke.Throughout the distribution, each 5 kg/m2 [SD 0·9] to 31·7 kg/m2 [2·0]), with each 5 kg/m2 higher baseline BMI associated with an adjusted HR of 1·30  for intracerebral haemorrhage, an effect size that was weaker than that for ischaemic stroke, and substantially weaker than predicted from the corresponding difference in systolic blood pressure . Moreover, the shape of the association with intracerebral haemorrhage appeared non-linear (p<0·0001 for test of non-linearity). Among the 68% of participants with a BMI of less than 25 kg/m2, there were no associations between BMI and risk of intracerebral haemorrhage, even though systolic/diastolic blood pressure differed by 11·5/5·3 mm Hg across the four BMI groups studied. Above a BMI of 25 kg/m2, the risk of intracerebral haemorrhage increased with increasing levels of BMI, but the association was still weaker than that predicted by the difference in systolic blood pressure. BMI was unrelated to subarachnoid haemorrhage, but the number of events (n=434) was small (2), as was the case for total stroke . In this population, high adiposity (defined as BMI >23 kg/m2) accounted for 14·7% of total stroke, including 16·5% of ischaemic stroke, and 6·7% of intracerebral haemorrhage, and overweight  accounted for 9·2% of total stroke, including 9·9% of ischaemic stroke and 6·7% of intracerebral haemorrhage , especially at age-at-risk of more than 60 years. For intracerebral haemorrhage, there was a weak inverse association with BMI at ages 70–79 years but, at younger than 70 years, BMI was weakly and positively associated with intracerebral haemorrhage.The HRs for ischaemic stroke per 5 kg/mder ages . The HRsheterogeneity=0·035 for ischaemic stroke and pheterogeneity=0·0038 for intracerebral haemorrhage) and, to a lesser extent, by physical activity . The associations were slightly attenuated by additional exclusion of people with any previous diseases, people with self-reported poor health status at baseline, the first 3 years of follow-up, and ever-regular smokers .The associations were, however, substantially attenuated by additional adjustment for blood pressure. For ischaemic stroke, simultaneous adjustment for usual systolic blood pressure (and diabetes status) reduced the HR from 1·30 (95% CI 1·28–1·33) to 1·05  were generally similar to those for BMI , as wereThis large prospective study showed that, among relatively lean but healthy Chinese adults, adiposity was strongly positively associated with systolic blood pressure, and that systolic blood pressure was strongly positively related to stroke incidence, particularly intracerebral haemorrhage, without evidence of any threshold throughout the range studied. However, despite these two continuous and apparently causal relationships, adiposity showed an expected strong positive association only with ischaemic stroke, but not with intracerebral haemorrhage.vs 1·22, per 5 kg/m2 higher BMI),2 BMI) was similar to that observed in previous east Asian studies. Likewise, a meta-analysis of 97 prospective studies, including 33 from Asia, which examined the associations of BMI with total stroke only, reported pooled RR in Asian cohorts that were similar to those reported in the present study (1·29 vs 1·27 per 5 kg/m2 higher BMI), but stronger than those in high-income populations (1·14).2) accounted for 15% of total stroke, which was less than the 20% previously estimated for China in a recent Global Burden of Disease reportSeveral large prospective studies, and meta-analyses of such studies, have consistently reported positive associations between BMI and risks of ischaemic stroke and total stroke.vs 1·16, per 5 kg/m2 higher BMI). In the high-income cohorts, more than 80% of the haemorrhagic stroke cases were from one UK study, which used self-reported BMI and had a disproportionally large number of subarachnoid haemorrhage cases compared with intracerebral haemorrhage cases (3062 vs 2790). Moreover, this UK study reported no increased risk of intracerebral haemorrhage even among those who were obese, nor a positive association with risk of ischaemic stroke at BMI levels of less than 27·5 kg/m2, in contrast with the findings of this and many other previous studies.16Most previous studies have focused mainly on haemorrhagic stroke rather than intracerebral haemorrhage specifically, and the results have been inconsistent, partly because of the relatively small number of cases studied and the strong possibility of misclassification of stroke types in studies without widespread use of brain imaging. In the most recent meta-analysis, which included more than 17 000 haemorrhagic stroke cases,2.2, there was no association between adiposity and intracerebral haemorrhage. Given the strong positive associations observed between adiposity and systolic blood pressure and between systolic blood pressure and intracerebral haemorrhage, the lack of any apparent association with intracerebral haemorrhage below BMI 25 kg/m2 was unexpected and unexplained. There was a mean difference of at least 11/5 mm Hg in systolic/diastolic blood pressure across the BMI range below 25 kg/m2, and this difference should, other things being equal, correspond to an HR of 1·6 for intracerebral haemorrhage, but it was totally flat. The disparities are too extreme to be accounted for by chance, by known confounding factors, or by reverse causality, although one cannot exclude the possibility that some other unmeasured or unknown risk factors associated with low BMI might offset the protective effects of lower blood pressure. Future Mendelian randomisation studies are needed to establish the causal relevance of adiposity for intracerebral haemorrhage.Consistent with our study, several large prospective studies in east Asian populations have reported little or no clear positive associations between BMI and risk of haemorrhagic stroke at a BMI of less than 25 kg/mFew prospective cohort studies have examined the associations between stroke types and measures of adiposity other than BMI, such as waist circumference, waist-to-hip ratio, and body fat percentage.vs non-lacunar ischaemic stroke) or of intracerebral haemorrhage . Second, information collected on diet was limited and did not include salt intake, so residual confounding might still persist, even though adjustment for fresh fruit and meat consumption did not alter the associations. Third, it was not possible to fully explore the mediating effects of lipids and other blood-related factors, although examination of a subset of data indicated that further adjustment for lipids had little additional impact over and above blood pressure.Our study had several strengths, including a very large study population, availability of different measures of adiposity, completeness of follow-up, and a high proportion of stroke types reliably diagnosed by neuroimaging. Moreover, this study explored the effects of mediators  on the associations of adiposity with stroke types both directly and indirectly. However, the study also had several limitations. First, despite the widespread use of neuroimaging, the study was not able to examine the associations between adiposity and different subtypes of ischaemic stroke , suggesting that leanness, either per se or through some other factor (or factors), might increase risk, thereby offsetting the protective effects associated with lower blood pressure. Since ischaemic stroke constitutes the majority of total stroke cases, the findings on intracerebral haemorrhage should not diminish the fundamental importance of high adiposity as a major modifiable determinant of overall stroke. In view of the substantial risks associated with high blood pressure, this study highlights the importance of controlling blood pressure (and other intermediate risk factors) for prevention of both ischaemic stroke and intracerebral haemorrhage, irrespective of levels of adiposity.In summary, our study shows that, among relatively lean but healthy Chinese adults, adiposity was strongly and positively associated with ischaemic stroke, mainly through its effect on blood pressure. However, there was no association between adiposity and intracerebral haemorrhage across the normal range (ie, BMI <25 kg/m