Chlorocebus pygerythrus) groups in South Africa using stable isotope analysis. Vervets are omnivores that exploit a wide range of habitats including those that have been anthropogenically-disturbed. As humans encroach upon nonhuman primate landscapes, human-nonhuman primate interconnections become increasingly common, which has led to the rise of the field of ethnoprimatology. To date, many ethnoprimatological studies have examined human-nonhuman primate associations largely in qualitative terms. By using stable carbon (δ13C) and nitrogen (δ15N) isotope analysis, we use quantitative data to understand the degree to which humans impact vervet monkey dietary patterns. Based on initial behavioral observations we placed the eight groups into three categories of anthropogenic disturbance . Using δ13C and δ15N values we estimated the degree to which each group and each anthropogenically-disturbed category was consuming C4 plants . δ13C values were significantly different between groups and categories of anthropogenic-disturbance. δ15N values were significantly different at the group level. The two vervet groups with the highest consumption of C4 plants inhabited small nature reserves, appeared to interact with humans only sporadically, and were initially placed in the mid level of anthropogenic-disturbance. However, further behavioral observations revealed that the high δ13C values exhibited by these groups were linked to previously unseen raiding of C4 crops. By revealing these cryptic feeding patterns, this study illustrates the utility of stable isotopes analysis for some ethnoprimatological questions.This study seeks to understand how humans impact the dietary patterns of eight free-ranging vervet monkey ( Chlorocebus pygerythrus) by analyzing the δ13C and δ15N values of hair samples. In Africa, the northernmost populations of vervet monkeys range from Senegal and Ethiopia while the southernmost populations live in South Africa This study seeks to understand the impact of anthropogenic disturbance on the dietary ecology of free-ranging South African vervet monkeys (13C) and nitrogen (δ15N) isotope analysis is a highly quantitative, and an increasingly cost effective method for studying primate diets 13C and δ15N values of animals are permanently recorded in their tissues and excreta. This allows for the analysis of archived specimens  to better characterize human and nonhuman primate interactions over longer temporal scales. In the areas of South Africa of principle interest here, most trees, bushes, and shrubs use the C3 photosynthetic pathway while most grasses and some sedges use the C4 photosynthetic pathway 3 and C4 plants do not overlap, and stable isotope analysis of a primate’s hair can reveal the relative amount of C3 and C4 plants it consumed 3 vegetation 4 crops are prevalent  and are consumed by crop raiding vervets. Vervets may also consume human foods that include corn products or C4 sugarcane. Thus, δ13C values can be used to understand a population’s reliance on C4 crops or processed human foods. Nitrogen isotopes can also prove useful as they track trophic level Stable carbon (δMacaca sylvanus) inhabiting Gibraltar. They found that macaques living in areas most heavily visited by tourists had significantly higher δ13C values, probably indicating the consumption of human snack foods which often contain C4 sweeteners. The macaques also had lower δ15N values, most likely due to consumption of tourist-provided peanuts, legumes with a relatively low nitrogen isotope composition Although stable isotope data have been used to study the diets of modern 13C and δ15N values of hair from eight South African groups of vervet monkeys with varying degrees of consumption of human foods and utilization of anthropogenically-disturbed habitats. Estimations of the human impacts were broad and based on preliminary observations. An in-depth study of vervet monkey behavioral ecology is required at each field site to truly understand the nature of these human-vervet monkey interplays, but we can use stable isotope analyses to generate hypotheses regarding each group’s feeding patterns and habitat use. For carbon, we predicted that groups of vervet monkeys with high degrees of human contact and consumption of human foods will exhibit higher δ13C values as human foods are frequently processed or sweetened with C4 plants (i.e. corn or sugarcane). We also predicted that the vervet groups with high degrees of human contact that regularly consume human foods or raid crops will exhibit lower δ15N values as foods grown with synthetic agricultural fertilizers tend to be 15N-depleted.Given their omnivorous feeding patterns and varying degrees of overlap with humans, vervet monkeys are excellent candidates to examine ethnoprimatological questions using stable isotope data. As elsewhere throughout the continent, free-ranging South African vervet populations primarily inhabit savanna woodlands and riverine forests Chlorocebus pygerythrus) throughout South Africa  with regard to anthropogenic disturbance. The two groups in the “low” anthropogenic disturbance category (Baviaanskloof/Geelhoutbos and Dronfield) inhabited ranges with little human modification, interacted with humans rarely, and were either rarely or never observed to consume human foods. Three vervet monkey groups that were classified in the “mid” category of anthropogenic disturbance  lived in or used habitats modified by humans and were occasionally observed interacting with humans. These groups were observed begging and/or stealing foods from weekend picnic crowds. Thus, sporadic access to human foods supplemented their natural diets. Three vervet monkey groups were categorized as having “high” degrees of anthropogenic disturbance  because they lived in resorts or city suburbs that were heavily modified by humans and because they consumed large amounts of human foods via provisioning or raiding of discarded human foods.A total of 96 hair samples were collected from eight groups of vervet monkeys  notation in parts per thousand or permil (‰) relative to the PeeDee Belemite (PDB) and atmospheric N2 standards respectively. We estimated the percentage of C4 (%C4) plant consumption for each group using the following formula from Schwarcz et al. Hair samples were washed with ethyl alcohol, cut with a razor blade, weighed (∼700 micrograms), and placed in tin capsules. Samples were combusted in an elemental analyzer  and analyzed for stable carbon and nitrogen isotope abundances using a flow-through inlet system on a continuous flow isotope ratio mass spectrometer . h is the measured value of the hair sample, Δdh is the fractionation factor of 3.2 for hair to diet 3 = −26.7‰  for each vervet monkey group are shown 13C between the groups living in heavily disturbed habitats to those groups inhabiting environments with little anthropogenic disturbance . For age, there was no effect for carbon (P = 0.09) but we found an effect for nitrogen (P<0.05). Since we found a group effect for both carbon and nitrogen we excluded age and sex as variables in subsequent analyses. There were highly significant differences between the eight vervet monkey groups for δ13C . The Oribi Gorge group had the highest δ13C  while Baviaanskloof/Geelhoutbos was the lowest . Baviaanskloof/Geelhoutbos was significantly different from all groups (Games-Howell P<0.0001) and this vervet group inhabits a region that has abundant C3 grasses and very low human impact. Thus, the Baviaanskloof/Geelhoutbos group is close to ideal for having no engagement with C4 foods. When the eight groups were placed in categories of low, mid, and high levels of anthropogenic disturbance we found differences in δ13C . Unexpectedly, Oribi Gorge and Soetdoring had the highest mean for estimated percentage of consumed C4 foods, even though they were placed in the “mid” level of anthropogenic disturbance based on our initial field observations . Pretoria had the lowest δ15N values  while Benfontein  was significantly higher than the other sites (Games-Howell P<0.01). There were also differences in δ15N values for the three categories of anthropogenic disturbance  and the mid level category was higher than the low category (Games-Howell P<0.05). Within group variation for δ15N was highest at Parys (6.4‰), while no other group had a range greater than 2.5‰.We also found highly significant differences for δ13C values of the eight vervet monkey groups. This was also true when the groups were placed in the categories of anthropogenic disturbance. Differences in δ13C values are most likely linked to the consumption of human processed foods enriched with C4 plants or the consumption of C4 crops. Many of the vervet monkey populations that we studied inhabited ecosystems that include of C3, C4, and CAM vegetation. However, behavioral observations revealed no consumption of wild C4 grasses and succulent CAM plants for any of the eight groups. Our prediction that all groups of vervet monkeys with high degrees of human contact and consumption of human foods would exhibit higher δ13C values was not completely supported. For example, the Parys and Pretoria groups both live in urban settings and high levels of C4 consumption were expected. C4 consumption was ∼18% for Parys and ∼20% for Pretoria, which is considerable and consistent with expectations for monkeys with access to human foods. However, C4 consumption for these groups was less than Oribi Gorge (∼43%) and Soetdoring (∼26%) which we placed in the “mid” level of anthropogenic disturbance based on initial behavioral observations  indicating substantial consumption of C4 resources. These initial results were puzzling; however, we later noted that the Oribi Gorge monkeys were leaving the reserve to raid sugarcane crops located at the reserve’s border. We found a similar scenario for the group that inhabits the Soetdoring Nature Reserve located outside of Bloemfontein. The Soetdoring group interacts only sporadically with tourists which is unlikely to account for its high δ13C values (−19.9‰ ±0.4‰) suggesting about 26% of its diet consisted of C4 foods  we found. Given the close proximity between the two sites, the differences in the δ15N values we detected are likely linked the consumption of human foods as each site is characterized by similar habitats. The Benfontein vervet group occasionally interacted with humans and consumed human foods while the Dronfield vervets rarely interacted with humans , higher fecal δ15N values were found in animals that were suspected to raid camp sites compared to animals in more remote areas Lemur catta) groups revealed that the group that consumed left over human foods at the research camp had the highest hair δ15N values 15N enriched insects than the other seven vervet groups and this higher trophic position maybe responsible for the higher δ15N values We also found differences in the δh humans . More in13C and ranged from −15.7‰ to −19.8‰, followed by the Parys group which ranged from −19.8% to −22.4‰ and the Blyde Resort group that ranged from −18.9‰ to −21.4‰ (15N which may be an artifact of the sample size (N = 26) or due to differences in diet and/or individual health. Vervets live in groups with differential access to contested resources and highest ranking individuals may have more access to human processed foods with lower δ15N values due to artificial fertilizers. Alternatively, differences in δ15N may be the result of differences in health. Among free-ranging ring-tailed lemurs, some individuals in poor physical health had the highest δ15N values 15N values, a pattern that has been found among birds 15N values among the Parys group highlights the need for long-term feeding observational data paired with baseline isotope values of the foods that nonhuman primates eat in order to accurately interpret stable isotope values. Our prediction that those vervet monkey groups placed in the high anthropogenic disturbance category  would have lower δ15N values based on the consumption of high levels of human processed foods was not supported. In fact, we found that the groups that were placed in the mid level category of anthropogenic disturbance  had the highest δ15N values. It is difficult to predict what impact human foods would have on the δ15N values we found among these groups since the human foods that were consumed by these vervet groups can be produced at different locations (within South Africa and beyond) potentially driving their δ15N values either up or down.The intragroup level variability we documented may be the result of individual feeding strategies or related to social rank and access to contested foods. The Oribi Gorge vervet monkeys exhibited the greatest variability in δo −21.4‰ . The Par4 plants. This is an important find, as vervet monkeys are considered pests in some South African provinces and crop raiding monkeys may be injured or killed. As such, using stable isotope analysis to detect crop raiding by nonhuman primates could be used as an effective tool to develop conservation initiatives and the implementation of deterrents that minimize stress to nonhuman primates. These measures may include the use of fire crackers, water hoses, chemicals that mimic the scent of predators, using sounds to replicate predators, or primate alarm calls The data presented here suggest that stable isotope analysis can be used for augmenting ethnoprimatological datasets but cannot replace traditional techniques in behavioral observations. Stable isotope values from hair provide feeding data over time and hair collected by ethnoprimatologists can reveal broad dietary patterns before the data collection commences or between field seasons. Stable carbon isotope values from hair reflect the dietary values from foods accrued over days or weeks Stable isotope analysis may add to ethnoprimatological studies by confirming or denying people’s perceptions about how local animals are behaving (i.e. consumption of their crops). However, to fully understand people’s attitudes toward the nonhuman primates they live among, conventional ethnoprimatological methods including interviews and questionnaires should not be abandoned. Quantitative and qualitative techniques have their own strengths and weaknesses and combining these methods can be synergistic, resulting in a more appropriate approach to understanding human-nonhuman primate dynamics. It is our hope that combining stable isotope analysis with behavioral observations and ethnographic techniques will not only shed light on human-nonhuman primate interactions in South Africa, but will better develop this technique as a tool for broader ethnoprimatological inquiries specifically related to identifying crop raiding, improving our understanding of nonhuman primate habitat utilization, and documenting anthropogenic effects on nonhuman primate behavioral ecology through time and space.Table S113C and δ15N values for each individual as well as their group affiliation and the degree to which the group was impacted by humans.δ(DOCX)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Cluster of differentiation 93 (CD93) is involved in apoptosis and inflammation and has a suggested role in angiogenesis, and all of which are involved in the development and dissemination of cancer. We evaluated the expression of CD93 and the association with two single nucleotide polymorphisms (SNPs), rs2749812 and rs2749817, as possible biomarkers in colorectal cancer (CRC).Tissue levels and plasma levels of CD93 were measured using an enzyme-linked immunosorbent assay (ELISA). Expression of CD93 was determined by immunohistochemistry, western blot and gene expression analysis. Genotype frequencies were established for the SNPs by real-time polymerase chain reaction (PCR), and the association with tumour stage and survival was analysed.P < 0.001) in tumours compared to matched normal tissues. Mean levels of soluble CD93 in plasma were 30 % lower (P < 0.001) in the patients compared to the controls. The T/T genotype of SNP rs2749817 was more common in stage IV patients, with consequently higher risk of CRC death  = 1.73, 95 % confidence interval (CI) = 1.11–2.67, P = 0.014), and was associated with a higher risk of CRC recurrence after radical operation .Total CD93 levels were 82 % higher (We showed that the T/T genotype of SNP rs2749817 is associated with disseminated cancer at diagnosis and an increased recurrence rate after radical operation. Patients with this genotype may benefit from early identification. Colorectal cancer (CRC) is the fourth most common cause of cancer mortality worldwide . Local iHuman cluster of differentiation 93 (CD93) is an epidermal growth factor (EGF)-like domain containing transmembrane protein –12, whicThe aim of this study was to evaluate the expression of CD93 and the two SNPs of CD93 (rs2749812 and rs2749817) as possible candidate biomarkers of CRC.This study involved analysis of tissue sections, tissue lysates, plasma, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) samples from CRC patients from southeastern Sweden who had undergone surgical resections for primary colorectal adenocarcinomas at the Department of Surgery, County Hospital Ryhov, Jönköping, Sweden, during 1996 through 2012. The clinicopathological characteristics of the patients were obtained from surgical and pathological records. Follow-up was made through the medical records from all hospital departments and the primary care up to December 31, 2013. The date of an eventual recurrence and the date and cause of death as related to colorectal cancer or not were determined from a review of the patient’s files.The patient group comprised 370 patients, including 197 males and 173 females with a mean age of 70 years (range 25–93). The control group comprised 316 blood donors, including 188 males and 128 females, with no known CRC history and from the same geographical region as the patients with a mean age of 61 years (range 41–70). The study was approved by the Local Ethics Committee of Linköping University, Sweden (Dnr. 2013/271-31), and informed consent was obtained from each patient.n = 58) or rectum (n = 43). The tumours were classified according to the American Joint Committee on Cancer (AJCC) classification system; stage I (n = 17), stage II (n = 30), stage III (n = 34) and stage IV (n = 20). During surgery, tumour tissue and adjacent normal mucosa (~5 cm from the tumour) were excised from each patient and immediately frozen at −70 °C until analysis. Frozen normal and tumour tissue were thawed and homogenized in an ice-cold lysis buffer as previously described [Tumour and matched normal tissue samples were analysed from 101 of the 370 patients. The 101 patients consisted of 59 males and 42 females with a mean age of 70 years (range 36–90) with a tumour localized in the colon (escribed . The pron = 68) or rectum (n = 42) and were classified as stage I (n = 17), stage II (n = 30), stage III (n = 42) and stage IV (n = 21).Venous blood samples were collected during surgery and centrifuged within 1 h to separate plasma and blood cells. Plasma was stored at −70 °C until analysis. Plasma was available from 110 patients  with a mean age of 69 years (range 36–90) and from 106 controls  with a mean age of 60 years (range 41–68). The patient’s tumours were localized in the colon  using a standard protocol. In brief, separated proteins were transferred onto polyvinylidene difluoride (PVDF) transfer membranes , blocked for 1 h with 5 % (wt/vol) non-fat dry milk and probed overnight with goat anti-human C1qR1/CD93 antibody . After extensive washing, the blots were re-incubated for 1 h at room temperature with bovine horseradish peroxidase-conjugated anti-goat IgG secondary antibody . Blots were developed using an enhanced chemiluminescence Prime Western Blotting Detection Reagent system (Amersham).CD93 staining was performed using a standard protocol on 4 μm sections from formalin-fixed paraffin-embedded tissue blocks as previously described . SectionCD93 was measured in tissue lysates and plasma using a commercial enzyme-linked immunosorbent assay (ELISA) kit  and performed as previously described . Tissue Total RNA from 14 matched tumour and normal tissue biopsies was isolated using the RNeasy kit . Complementary DNA (cDNA) amplification and real-time polymerase chain reaction (PCR) analysis were performed as previously described . The CD9Whole blood DNA was extracted from 356 patients and from 305 controls for analysis of the SNPs rs2749817 and rs2749812. DNA was extracted using a QiaAmp DNA blood kit (Qiagen), and a TaqMan SNP genotyping assay was used for the analysis of rs2749812 and rs2749817 genotypes (Applied Biosystems/Life Technologies). DNA (10 ng) was mixed with TaqMan Universal PCR Master mix II (Applied Biosystems) and amplified using the 7500 Fast Real-Time PCR system (Applied Biosystems). All the patients were available with registered follow-up regarding survival, stadium  and localization (colon = 189 and rectum = 167).chi-squared test. The Hardy-Weinberg equilibrium was tested for the genotypes. Differences in CD93 expression between tumour and matched normal tissues were examined by the Wilcoxon’s signed rank test, while differences in plasma CD93 levels between patients and controls were tested by the Mann-Whitney U test. These analyses were performed using the SPSS for Windows computer package . Cancer-specific and disease-free survival analysis was performed by Cox’s regression and Kaplan-Meier analysis with the log-rank test, using the STATA 12 software . The results were considered as statistically significant at P < 0.05.Differences in the frequencies of the CD93 SNPs between patients and controls and the association with localization and stage of the tumour were analysed by the P < 0.001)  higher expression of CD93 in tumour tissue compared to matched normal tissue. The expression of total CD93 was not associated with stage, localization, gender or age (data not shown).Expression of CD93 was predominantly seen in endothelial cells around blood vessels in cancerous tissue Fig. . CD93 waP < 0.001)  compared to the controls  (P = 0.009) when compared to stage I–III. No association between tumour site and the genotype distribution of SNP rs2749817 was seen (Table P = 0.013). The T/T genotype had the highest risk of CRC death with a hazard ratio (HR) of 1.73  = 1.11–2.67, P = 0.014) compared with the genotype C/C and C/T (Table P = 0.047) and an increased risk of recurrence in CRC (Table P = 0.007). When comparing the genotype frequencies of SNP rs2749817 to tissue levels of total CD93, the tumour tissue levels were significantly higher (20 %) among patients with T/T genotype  compared with C/C and C/T genotypes  (P = 0.037). There was no association in normal tissue levels (data not shown). We also examined the genotypes of SNP rs2749812, but no difference in genotype frequencies between patients and controls was observed. Among the patients, no association with stage and levels of CD93 was shown, and the genotypes of SNP rs2749812 did not have any effect on the survival of the CRC patients (data not shown).Genotype frequencies of SNP rs2749817 did not differ between the patients and controls  CD93 was increased and expressed in endothelial cells in blood vessels in CRC tissue; (ii) the plasma level of CD93 was lower in the CRC patients compared with the controls; (iii) the T/T genotype of rs2749817, a CD93-related SNP, was associated with disseminated cancer and an increased recurrence rate in patients that had the entire tumour removed at operation, which correlated to higher CD93 levels in the cancer.During cancer development and progression, elevated inflammation is observed in cancer tissue due to the infiltration of leukocytes  and incrIn our study, the levels of CD93 in plasma in the CRC patients were significantly lower compared to the controls. A low level of CD93 in plasma is surprising since CD93 is cleaved by MMPs and released, and MMPs are found in higher levels in CRC . The lowThe development of molecular markers has started to show promising results, but only KRAS mutation status has been accepted as a predictive marker for CRC and the response to drug treatment . DiffereWe investigated the genotype distribution of two SNPs of the CD93 gene to see whether they were of any clinical importance in CRC. We showed that the T/T genotype of SNP rs2749817 is associated with disseminated cancer at diagnosis and increased risk of recurrence after radical operation, suggesting a lower resistance to the dissemination of tumour cells and reduced ability to control metastasis. No association between plasma CD93 levels and the genotypes of SNP rs2749817 in patients or controls was seen. However, this SNP has previously been studied among Korean women, and it was shown to have a correlation with breast cancer risk . Hence, Our data implies that identifying individuals with the T/T genotype at an early stage could be of importance both for outcome, follow-up and individualized treatment for CRC patients. Even though lower plasma levels of CD93 are found in both CRC and CAD patients, SNP rs2749817 is not associated with CAD, which indicates that this SNP will not be influenced by CAD .The present study shows significantly higher CD93 levels in cancerous compared to matched normal tissues. Our observations also suggest that a low plasma concentration of CD93 may be a novel biomarker for CRC, but this must be further evaluated to be able to reveal the biological mechanism of plasma CD93 in CRC patients. Most importantly, a genotype belonging to an SNP of CD93 is correlated with disseminated CRC and may predict outcome for CRC patients.


Escherichia coli cells carrying New Delhi metallo-β-lactamase subclass 1 (NDM-1) can be monitored in real time by NMR spectroscopy, we designed a cell-based NMR screening platform. A strong NDM-1 inhibitor was identified with cellular IC50 of 0.51 μm, which is over 300-fold more potent than captopril, a known NDM-1 inhibitor. This new screening approach has great potential to be applied to targets in other cell types, such as mammalian cells, and to targets that are only stable or functionally competent in the cellular environment.An NMR-based approach marries the two traditional screening technologies (phenotypic and target-based screening) to find compounds inhibiting a specific enzymatic reaction in bacterial cells. Building on a previous study in which it was demonstrated that hydrolytic decomposition of meropenem in living  Drug discovery often begins with screening a collection of compounds in the hope that some active molecules can be identified and later developed into drugs. PhenotypThe concept of “target-based” whole cell screening has been introduced recently to overcome some of these limitations whilst retaining the merits of both of these screening procedures. Approaches exploiting antisense silencing technology or arrays of genetically-altered bacterial strains with inducible control of target expression have been used to screen for and identify inhibitors with antibacterial activity against pathogenic organisms.–7] Howe Howe7] HE. coli cells carrying New Delhi metallo-β-lactamase subclass 1 (NDM-1) can be readily monitored by 1H nuclear magnetic resonance spectroscopy (NMR).DMSO) and ethylenediaminetetraacetic acid (EDTA). All compounds are dissolved in [D6]DMSO to give a final stock concentration of 100 mm. The screening experiment comprises three simple steps  addition of meropenem; and 3) NDM1-catalyzed hydrolysis reaction, quenched by addition of 10 mm EDTA. The final concentrations for NMR detection are 500 μm compound, 100 μm meropenem, 0.5 % [D6]DMSO, and 10 mm EDTA. Under the conditions prior to the addition of 10 mm EDTA, the cells appear to be healthy . The experimental procedure of screening setup is detailed in the Supporting Information. After quenching the reaction, the plate was transferred to an automated NMR screening system to acquire 1D 1H NMR spectra of each well on the plate.To implement a 96-well plate format to screen NDM-1 inhibitors, we selected 92 compounds that are either known metallo-β-lactamase inhibitors or derivatives of them. The firs1H NMR spectra from each well in a 96-well plate format. For compounds with signals overlapping with the 0.9 ppm signal . In Figure 6]DMSO was added. Examination of signals at 1.1 ppm indicates that meropenem was completely hydrolyzed. Signals of well A2 and H11 were from samples with 500 μm EDTA. Since EDTA is a potent inhibitor of NDM-1 in E. coli cells ,[Figure  1.6 μm), hardly a50 values against NDM-1 enzymatic activity in bacterial cells of three screening hits  that were ranked as weak, medium, and strong, following our previously reported procedure , respectively.[The potency of screening hits was ranked as strong >80 %), medium (50–80 %), and weak (20–50 %) based on the percentage inhibition . As most compounds in the 96-well plate are either known Class B β-lactamase inhibitors or their derivatives, it is not surprising that a high hit rate of 34 % was observed. Many compounds showed potencies similar to EDTA. We then determined by NMR spectroscopy the IC0 %, mediE. coli cells. Surprisingly, only 18 % of the hits from this screen were confirmed to inhibit meropenem hydrolysis in our whole-cell NMR assay (data not shown). This illustrates clearly the ability of the whole cell-based NMR assay to directly detect hits by the desired mechanism of action, without the onerous requirement for additional assays to eliminate false positive hits, and further underlines the potential for our method to be integrated into drug discovery programs.Using the same setup, we evaluated 92 hits from a conventional high-throughput phenotypic antibacterial screen of one million compounds run against the NDM-1 m. In contrast to other biophysical methods such as surface plasmon resonance and mass spectrometry, no immobilization of cells or post-screen cell lysis are required. It is cost-effective and time-efficient as protein over-expression and purification steps are avoided. It takes approximately 5 min to record data for one sample. If a mixture of 5 compounds were screened instead of the singlets as shown here, the throughput would potentially be over 10 000 compounds per week. Furthermore, based on coarse ranking of compound potency, hits can be followed up by cellular IC50 measurement using NMR spectroscopy, which can also be handled in an automated fashion.Although NMR spectroscopy is commonly applied in vitro to screen compound  libraries against certain targets, to the b1H, other nuclei such as 13P, 19F, and 13C can also be used as NMR probes for screening.Besides β-lactamases in bacteria, many other targets should be amenable to this approach. For example, further to monitoring enzymatic reactions, several types of binding interactions on cellular surfaces, such as those of small protein growth factors, hormones, or neurotransmitters to their receptors on cancer, endocrine, or neuronal cells, should be observable by NMR spectroscopy. It should in principle be straightforward to design NMR screens aiming to find molecules disrupting these interactions. As well as In summary, we have developed a novel NMR screening method for discovering inhibitors against a specific target in cells where a diffusible substrate or product can be monitored. This approach enjoys the merits of both target-based and whole-cell screening approaches. The outcome of the screen is more predictive of a biologically relevant effect because targets reside in the physiological environment in which enzymatic reactions and inhibition occur. The labo


Furthermore, climate change will cause greater water losses in these peatlands and subject even deeper peat layers to wildfire combustion. However, the rewetting of drained peatlands and the restoration of mined peatlands can effectively lower the risk of these deep burns, especially if a new peat moss layer successfully establishes and raises peat moisture content. We argue that restoration efforts are a necessary measure to mitigate the risk of carbon loss in managed peatlands under climate change.Northern peatlands can emit large amounts of carbon and harmful smoke pollution during a wildfire. Of particular concern are drained and mined peatlands, where management practices destabilize an array of ecohydrological feedbacks, moss traits and peat properties that moderate water and carbon losses in natural peatlands. Our results demonstrate that drained and mined peatlands in Canada and northern Europe can experience catastrophic deep burns (>200 t C ha Sphagnum) with ecophysiological traits that enable long-term accumulation of peat by limiting decomposition3−2 589−2 1313Throughout the Holocene, northern peatlands have been persistent terrestrial carbon sinks that are estimated to contain approximately one-third of global soil carbon−2 −2 The high burn severity of drained tropical/temperate peatland fires suggests that large-scale peatland drainage and mining in northern peatlands over the last century has also likely made managed northern peatlands more vulnerable to wildfire than natural (undrained) peatlands. Recent research showed that an Alberta peatland drained for forestry and an Ontario peatland drained and mined for agriculture and horticulture experienced wildfire carbon losses of 16.8 kg C mρb) profiles and mean seasonal WT position data in natural, drained, mined and restored peatlands to model WT position under various peatland management scenarios  for both historical and future water deficit scenarios. Water table data and ρb data were then used to parameterize the Peat Smouldering and Ignition (PSI) model25To achieve our goals, we compare historical and future water availability  in managed peatlands and synthesize the current knowledge on the causes of contemporary deep burning. We collected data  on bulk density  across Fennoscandia, the Baltic countries, European Russia, Poland, and Belarus , where tρb) is a key peat property that affects peat moisture retention and WT response to water deficit. We found a clear difference in ρb between drained and natural peatlands in both North America and Northern Europe (ρb near 125 kg m−3 throughout the top peat layer (0–60 cm). The natural peatlands have a much lower ρb near the surface (25 kg m−3), which increases with depth and may reach 125 kg m−3 40 cm below the surface. However, such high ρb at 40 cm are associated with a boreal-subarctic continental climate. The mined peatland profile has a surface ρb of 125 kg m−3, while the restored peatland has a surface ρb similar to natural peatlands. The natural peatland profiles retrieved from a databaseρb above 100 kg m−3 in the top 0–20 cm.Bulk density  and the subjacent (Hignition) peat layer  Salteaux, AB, drained for forestry The PSI model relies on  ρb data , we prese the WT . Deep buSphagnum) that function to moderate WT drawdowns during droughtWildfire activity in the boreal is expected to increase in the near future in part due to the drying of previously wet peatlands as a direct consequence of climate changem) and low surface bulk density (ρb) . As such, natural peatlands have Hcomb/Hign quotients of less than one  . These wity (ρb) . Previout mosses , making than one  explainim levels  outcompete Sphagnum. Declining Sphagnum moss cover reduces the ability of surface peat to retain moisture and increases the risk of desiccation in the top moss layer (ρb) due to their higher decay rates compared to most Sphagnum species , but in the top layers drained peatlands have a much higher ρb. Mined peatlands have the highest ρb because of the surface moss and peat removed, exposing deeper and denser peat. High ρb is also associated with a decrease in specific yield (Sy), (i.e. describes the amount of WT decline per unit of water loss) and, therefore, a similar water deficit will cause a greater WT decline in drained peatlands compared to natural peatlands. Hence, differences in peat properties lead to large differences in WT responses to water deficits, as observed in our WT response modelling (When peatland drainage is performed to enhance tree growth (forestry), to permit the growth of crops (agriculture), or to permit the usage of heavy machinery to extract surface peat (mining), peatland WTs are substantially lowered. In our literature survey, we found that the average WT depth in drained peatlands was 41 cm which is 24 cm lower than in natural peatlands. The lowest WTs (60–80 cm) were found in dry boreal continental peatlands. A WT depth of 40–50 cm is considered optimal for tree production and is normally targeted when peatlands are drained for forestryss layer . Over tin Europe . Drainedodelling .m causes a decrease in Hign, while the increase in ρb increases Hcomb resulting in an increase in Hcomb/Hign quotients and an increase in the potential for deep burningThe decrease in surface −2 (or circa 200 t C ha−1) assuming a ρb of 125 kg m−3  peatland back to a system dominated by negative feedbacksewetting  and/or tecessary . Rewettiecessary , may notSphagnum  is necesSphagnum .Sphagnum dominated peatlands and does not include rich fens and blanket peatlands that are primarily comprised of brown mosses and sedges/graminoids, respectively. When subjected to drainage their characteristic flora is often lost and it can be a challenge to successfully restore these habitats. In blanket peatlands, a slow recovery of the hydrological function is observed after ditch blockingSphagnum mosses are likely to invade after ditch-blocking, but the biodiversity goal can be missed unless additional treatments such as species introduction and top-soil removal are performedSphagnum-dominated scenario as brown mosses have lower moisture retention and capillary rise compared to Sphagnumρb in brown moss/herbaceous peatThe restoration scenarios described in −1 yr−1 −1) should be a greater driver for improving peatland management practices.Our study explores the underlying processes responsible for the increased risk of deep burns in managed northern peatlands and provides a more comprehensive knowledge to assess and mitigate the risk of deep burns in drained peatlands. We demonstrate that the large area of managed peatlands in Europe will likely be more vulnerable to future wildfire due to climate change. Given the elevated risks of carbon losses, ecosystem shifts24P) and potential evapotranspiration (PET), where PET was calculated according to a version of the Penman equation using the ShuttleworthG) was not included in the calculation of PET, since it was not an archived output variable for CRCM and, at a daily time step, G is a small component of the energy balance in peatlands49G and bulk surface resistance on ET, we applied an empirical coefficient to our water deficit calculation. We calculated maximum annual water deficit as P–0.77PET. The empirical coefficient (0.77) was derived from archived Fluxnet dataET/PET was calculated from several years of eddy covariance data from Mer Bleue bog in south-east Ontario (bog) and Degerö Stormyr in Sweden (poor fen). Although not directly comparable with ET/PET a studyα values of 0.62 and 0.75 for pristine bogs and fens, respectively, in Finland, and reference other studies which range between 0.51–0.98.We used data from the Canadian Regional Climate Model CRCM 4 with a hρb) profiles in natural, drained, mined and restored peatlands from paired studies. These data cover a range of peatland types, from rich/intermediate fens to bogs. Data on ρb profiles in boreal peatlands, representing the Euro-Asia continent, were taken from a countrywide study that measured ρb in drained (N = 651) and natural (N = 360) peatlands at a large spatial scale25455ρb profiles with profiles reported in a peat properties database for northern peatlandsρb profiles from sites meeting the above criteria (25 of 70 sites).In order to estimate the effect of peatland management on WT drawdown for a given water deficit, we collected data  on bulk density (ρb profiles by using the aforementioned near-surface ρb data (0–50 cm) and thereafter, based on the selected ρb profiles fromρb and peat depth down to 1.5 m, where we prescribed a value of 150 kg m−3. For the restored scenarios, we assumed that site restoration was achieved by re-establishing a shallow WT and the re-generation of Sphagnum cover. We prescribed a 12 and 28 cm thick restored layer, with the same upper ρb profile as the natural site, as these new moss/peat thicknesses have been shown to maintain a relatively shallow WT for a range of maximum summer water deficits of 40 to 100 mm at mined peatland in Quebec, Canadaρb profiles from the mined and drained peatlands. In order to estimate WT response to water deficit, the depth-dependence of specific yield (Sy) needs to be known because Sy is the depth of water that must be added to or removed from a soil column in order to cause a unit rise or fall in the WT. We estimated Sy from the ρb data using the equation from Duke (1972), where ρb dependent moisture retention parameters were derived for Sphagnum peatSy values. For comparative purposes, we used water deficits at a continental and maritime location in both North America and Europe where managed peatlands are common. Additional lateral water losses due to drainage were not considered, thus our modelled WT values are a conservative estimate of WT drawdown given our simple treatment of ET.In order to represent natural, drained, mined, and restored peatland sites, we generated A literature review of managed peatlands informed our peat burn severity modelling scenarios. WT depths for drained peatlands were conservatively based on data from the mid-points between drainage ditches. Estimated average WT depths (growing season) in peatlands drained for forestry and natural peatlands were 41 cm  of the peat, and the model is supported by detailed physics-based models58m throughout the peat profiles were estimated using peat moisture retention curves as a function of ρbHcomb) and energy required for ignition (Hign) between successive peat layers (5 cm thick in this study) in a peat profile  was estimated using the Peat Smouldering and Ignition model25How to cite this article: Granath, G. et al. Mitigating wildfire carbon loss in managed northern peatlands through restoration. Sci. Rep.6, 28498; doi: 10.1038/srep28498 (2016).


The developed system opens up a host of novel applications for the neuroscience community.Investigation of the transient processes integral to neuronal function demands rapid and high-resolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Here we describe a compact light sheet fluorescence microscope, featuring a 45° inverted geometry and an integrated photolysis laser, that is optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We demonstrate the utility of this design for three-dimensional morphological reconstruction, activation of a single synapse with localized photolysis, and fast imaging of neuronal Ca Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to illuminate the sample, with the fluorescent signal detected perpendicular to the illuminated planeA variety of applications have already been reported for LSFM. For example, its fast imaging speed has facilitated real-time monitoring of neuronal responses within a live Zebrafish brain with single-cell resolution2356Generating a light sheet requires independent illumination and detection arms, which is classically accomplished by a pair of objectives perpendicular to each other. These two objectives can be on either a horizontal plane or a vertical plane, depending on the specimen to be imaged. The horizontal geometry is suitable for translating and especially rotating large samples, and is more popular. The vertical geometry is termed inverted selective plane illumination microscopy (iSPIM)Since LSFM typically requires two perpendicular objectives and other beam shaping optics, it requires a generous laboratory space, which restricts its utilization. The openSPIMA typical neuron in the brain of a mammal such as rat or human has a dense arbour of tree-like processes called dendrites which can extend hundreds of microns from the cell bodyHere we report the development of a light sheet fluorescence microscope of novel compact inverted design. Our prototype instrument is capable of imaging a wide area (300 × 300 μm) of the dendritic or axonal arbour of a dye-filled neuron in hippocampal slice at 100 Hz (or faster if the FOV is reduced). To the best of our knowledge this is the first time that compact LSFM has been applied to living mammalian brain slices.LSFM instruments optimized for neuroscience experiments are likely to require other features integrated into the setup, such as optical trapping122+ influx during photolytic stimulation of a single spine; a time series showing action potential-evoked Ca2+ influx into presynaptic terminals over a substantial length of axon; and a time series showing spontaneous neurotransmitter release evoking Ca2+ influx in dendritic spines. Thus we demonstrate that our approach to LSFM offers a powerful new functionality to the neuroscience community that is not achievable with traditional imaging methods.We demonstrate several applications of this novel, compact LSFM, including: the acquisition of a 2-channel Z-stack image of a living, large pyramidal neuron filled with two different fluorescent dyes; a time series acquired at 200 Hz showing Ca2+ influx) over a considerable length of dendrite or axon with high temporal resolution  and transferred to the LSFM  image stack acquired at 200 Hz. At each of 4 regions of interest  line-scan on a confocal microscope – with LSFM there is no need to decide ahead of time exactly where to set up the line scan. LSFM can acquire data at 200 Hz or faster over a considerable length of neuronal dendrite or axon, so that post-hoc analysis can explore the XYT stack, potentially revealing transient events that might have been missed by an XT line scan. For example in 2+ influx occurs starting about 200 ms after the initial response. This late-phase response can probably be interpreted as Ca2+-induced Ca2+ release (CICR) from intracellular Ca2+ stores such as the endoplasmic reticulum (ER)14We also demonstrated photolytic release of the excitatory neurotransmitter glutamate using the 405 nm photolysis laser, which is integrated into our prototype LSFM. A living neuron was filled with dyes AF594  and Oregon Green BAPTA-1  in the axonal membrane2+ transients such as confocal line scan: with LSFM data can be collected from many points across the image plane simultaneously.  in a customized sample chamber which is held on a XYZ translation stage . Automatic scanning of the sample was achieved by translating the perfusion chamber using a automatic linear actuator .A 405 nm laser  was introduced with fibre and adjustable fibre collimator  to the back aperture of the detection objective for photolysis. The adjustable collimator, along with fiber which acts as a spatial filter, produces a diffraction-limited spot for photolysis on the image focal plane with diameter less than 1 μm.  with 594 nm wavelength was added. Currently it is mounted outside the breadboard (not shown in μ Manager2+ transient data as in System control and image acquisition were achieved with open-source software 2) on Millicell culture inserts (Millipore) in culture medium for 7–21 days before use. The culture medium was composed of 79% Minimum Essential Media, 20% heat-inactivated horse serum, 1% B-27 (Invitrogen), 30 mM HEPES, 26 mM glucose, 5.8 mM NaHCO3, 1 mM CaCl2, and 2 mM MgSO4. Culture medium was exchanged 3 times weekly.Experiments were conducted in accordance with current United Kingdom (UK) legislation regulating welfare of animals used for scientific procedures. Organotypic slices were prepared as previously described14Pyramidal neurons in the CA1 or CA3 region of the hippocampus were filled with synthetic dyes, including AF488 , AF594 , and OGB-1  through a patch pipette using patch-clamp electrophysiology, or with genetically encoded fluorescent protein GCaMP6s by viral transfection−1 with carbogenated (95% O2/5% CO2) ACSF containing (mM) 145 NaCl, 16 NaHCO3, 11 glucose, 2.5 KCl, 1.2 NaH2PO4, 3 CaCl2, and 1 MgCl2, supplemented with 1 mM Trolox to minimize phototoxic damage during imaging. For experiments requiring low-Mg ACSF, 4 CaCl2 and 0 MgCl2 (mM) was substituted. For experiments where spontaneous release was imaged, ACSF containing 4 mM Ca2+ and 0 Mg2+ was maintained at 33 °C and supplemented with 20 μM D-serine in order to augment NMDAR-mediated Ca2+ influx, and 1 μM tetrodotoxin (TTX).An organotypic slice containing one or more neurons filled with fluorescent dye as described above was transferred to the LSFM’s recording chamber and perfused at 2 mL min2+ traces, regions of interest (ROIs) were selected at appropriate locations on spine, dendrite or axon, as well as a nearby larger ROI to represent the background fluorescence. For each ROI, the mean intensity at each time point, with background subtracted, was divided by the mean baseline intensity and plotted as %ΔF/F against time.Analysis was performed using image processing software FIJIhttp://dx.doi.org/10.17630/8392656d-6249-4027-8831-53a3328f8acb).The research data  supporting this publication can be accessed at: “Data underpinning - A compact light-sheet microscope for the study of the mammalian central nervous system” .


Inflammation constitutes one of the important components of colorectal cancer (CRC) pathogenesis. Tumor necrosis factor-α (TNF-α), a cytokine and an important inflammatory mediator plays a pivotal role in the malignant cellular proliferation, angiogenesis, tissue invasion and metastasis in CRC. The studies on association of various polymorphisms in human TNF-α gene including TNF-α-308G/A single nucleotide polymorphism (SNP) are limited, mixed and inconclusive.The aim of this study was to analyze the association of TNF-α-308G/A promoter SNP with colorectal cancer (CRC) susceptibility and development risk and also to evaluate the modifying effects of possible TNF-α-308G/A genotypes on different risk factors of CRC in ethnic population of Kashmir, India through a case–control setup. The genotype frequencies of TNF-α-308G/A promoter SNP were compared between 142 CRC patients and 184 individually matched healthy controls by using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. The associations between the TNF-α-308G/A SNP and CRC risk were examined through conditional logistic regression models adjusted for multiple possible confounding (third) variables. Further, the associations between this SNP and various clinico-pathological parameters, demographic variables and environmental factors within the case group subjects with regard to CRC risk were also evaluated.The association between the TNF-α-308G/A SNP and the modulation of risk of CRC was not found to be significant . The effect of less common TNF-α-308A allele on the risk of colorectal cancer was also not found to be significant . The variant genotype (AA) was nonexistent in the study population. Further, we found no significant effect modulation of CRC risk by wild and heterozygous TNF-α-308G/A SNP genotypes in presence of different possible risk factors (p > 0.05). We also found no significant association of TNF-α-308G/A SNP with the subsets of various characteristics of the case group subjects under study (p > 0.05).This study indicates that there is no significant association between the TNF-α-308G/A promoter SNP and the risk of developing CRC in ethnic Kashmiri population. However, in order to substantiate our findings, this study needs to be replicated with bigger sample size and should involve other ethnically defined populations with high CRC risk. GloballThe CRC development is influenced by or contributed to by multiple factors that include dietary and lifestyle habits, demographic factors, various pathological conditions and genetic predispositions . Each ofCytokines play a key role in the mediation and regulation of immune response including inflammation. Several of these cytokines including pro-inflammatory and anti-inflammatory have been implicated in the process of tumorigenesis . Tumor nSeveral studies have reported the involvement of TNF-α and the important role it plays in the pathogenesis and progression of various cancers . TNF-α hThe functional significance of many of these SNPs such as those at positions − 238, − 308, − 376, − 857, − 863 and − 1031 has been reported and elucidated and have been related to the modulation of TNF-α production mostly through their effect on transcriptional activity that in turn results in differential gene expression . Among tVarious meta-analyses and other studies have reported the association of TNF-α-308G/A polymorphism with differences in susceptibility between different individuals and on a larger scale between different populations to several diseases including various types of neoplastic diseases . SeveralIn this study, we systematically conducted a case–control evaluation of the possible association between TNF-α-308G/A SNP and susceptibility to CRC in Kashmiri population. We also evaluated the possible effect modulation of CRC risk by age, gender and smoking status. Further, we investigated the possible relationship of this SNP with various clinico-pathological parameters, demographic variables and environmental factors including smoking habit and hence their role in modulating the risk of colorectal cancer in the population under study.22.1The present study involved two subject groups: case and control. The case group included 142 consecutively recruited patients with histopathologically confirmed primary colorectal cancer who underwent surgical resection for primary CRC tumors at the Department of General Surgery, Sher-I-Kashmir Institute of Medical Sciences, the largest and the only tertiary care hospital in the Kashmir valley. The tumor stage and tumor grade were classified according to the 8th edition of TNM classification of Union International Control of Cancer (UICC). Only those cases who had not received any chemo or radiotherapy were chosen for this study. All the cases were more than 18 years old and had no prior history of any malignancy. Blood and tissue samples were obtained from these CRC patients. The control group included 184 healthy individuals with no history or prior diagnosis of any malignant disorder or any other serious disease from whom blood was collected and used as control for the present study. The control group included both population based subjects and hospital-based subjects. The control group subjects were matched to the case group subjects individually for age (± 5 years), sex, place of residence  and smoking habit. Both the case and control subjects chosen for this study were ethnic Kashmiris.2.2The data relevant to the study concerning all the CRC patients including various clinico-pathological parameters, demographic variables and environmental factors including smoking habit were obtained and evaluated from patient medical records (files), pathology reports and also from the personal interviews with patients and/or their guardians (for those who were illiterate). The data collected included tumor location, Dukes Stage, lymph node status, sex, age, place of residence, smoking habits among several other relevant parameters. The relevant data was also obtained for each of the recruited controls mostly through personal interviews. All the patients and/or their guardians were informed about the study and their willingness to participate in this study was documented using a predesigned questionnaire. Same procedure was followed for the controls. The study was approved by the Institutional Ethics Committee. The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments in humans.2.3The tumor tissue samples collected after surgical resection were immediately snap frozen in liquid nitrogen and then stored at − 80 °C until further use for DNA extraction and other research purposes. Prior to DNA extraction, tumor tissue samples were washed 2–3 times in phosphate buffered saline (PBS) and adipose and connective tissue portions if any were dissected away. Peripheral blood sample, 3–5 mL from each case and control group individual was collected into EDTA-blood vacutainer collection tubes and stored at − 80 °C until further use. Genomic DNA was extracted from both the tumor tissue and blood specimens using DNeasy Blood and Tissue Kit  and Quick-gDNA™ MiniPrep kit  according to the manufacturers' instructions. The extracted DNA was stored at − 20 °C until further use. The qualitative and the quantitative assessments of the extracted genomic DNA samples were carried out by absorbance measurements at 260 nm and 280 nm using UV–visible spectrophotometeric analysis and also by agarose gel electrophoresis. The DNA extracted from blood samples of case and control group subjects was used for this study.2.4The TNF-α-308G/A (rs1800629) SNP was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.2.52; 0.28 mM deoxynucleotide triphosphate mix ; 0.56 μM forward and revere oligonucleotide primers  and nuclease-protease free water  added up to a final volume of 25 μL.The PCR for TNF-α-308G/A SNP was carried out in a total volume of 25 μL containing 100 ng–1 μg of genomic DNA, 0.7-1 U Taq DNA polymerase with lX Standard Taq reaction buffer , 2.1 mM MgClCCAT-3′ (forward) and 5′-CTGTCT-CGGTTTCTTCTCCATGGCG-3′ (reverse). The underlined base at the 3′-end of the forward primer was incorporated to generate an NcoI restriction site in the polymorphic region under study . The digestion was carried out according to the manufacturers' instructions in a 30 μL reaction volume containing 10 μL of PCR product and 10 U of NcoI enzyme and incubated at 37 °C overnight. NcoI enzyme cleaved the TNF-α-308G (TNF1) allele into two fragments of 173 bp and 22 bp but not the TNF-α-308A (TNF2) allele that gave a single undigested fragment of 195 bp. The digestion products were separated on 3.6–4.0% agarose gel stained with ethidium bromide . The statistical power of 80% is used widely to avoid false negative associations and to determine a cost-effective sample size under the assumption of 25% variant allele frequency, 1:1 case-to-control ratio, and 5% type I error rate (α).The effective sample size and statistical power were computed by using “Genetic Power Calculator” developed by Purcell et al. , median and inter-quartile range for continuous variables. Conditional logistic regression analysis was carried out to calculate unadjusted and adjusted odds ratios (ORs) and corresponding 95% confidence intervals (CIs) to assess the possible association of relevant TNF-α-308G/A SNP genotypes with CRC risk and to assess the possible gene-environment interactions. In order to eliminate the possible confounding (third) variables, the conditional logistic regression models were adjusted for known risk factors like gender, age and smoking habit and also with place of residence. The comparison of genotype and allele distributions of TNF-α-308G/A SNP between the CRC patient group and control group using conditional logistic regression involved two-sided chi-square test. The correlation between genotypes and clinico-pathological parameters, demographic variables and environmental factors including smoking habit within the case group was analyzed by Fisher exact test. The fitness of the genotype distributions to Hardy-Weinberg equilibrium for allele and genotype frequencies in the population under study was tested using the chi–square test. A two sided probability value of or less than 5% (p ≤ 0.05) was considered statistically significant for all types of analyses. All statistical analyses were performed using IBM SPSS Statistics v21 software.4A total of 142 primary CRC patients and 184 control subjects were included in this study with prior consent of each individual. The frequencies of various clinico-pathological parameters, demographic variables and environmental factors in colorectal cancer case subjects and relevant parameters in control subjects are listed in 2 = 0.65; p value = 0.420 and controls: χ2 = 1.91; p value = 0.167).The frequencies of the genotypes for TNF-α-308G/A SNP for both the case and the control groups are listed in The possible effect modulation of CRC risk by age, gender and smoking status is summarized in The number and the frequencies of the subsets of various characteristics of the case group subjects under study i.e. age, gender, dwelling, smoking status, tumor location, tumor grade and lymph node status for the TNF-α-308G/A SNP are listed in 5CRC is one of the most prevalent cancers worldwide and is associated with a high degree of cancer related mortality and morbidity. CRC is one of the few cancers in which inflammation is mostly evident at the earliest stages of tumor progression as a basic pathological incident and is capable of transforming the incipient tumors into full blown malignancy . CRC devTumor necrosis factor-α (TNF-α) gene is located on short arm of chromosome 6 (region p21.33) and is expressed as a 26 kDa membrane bound protein which through proteolytic cleavage by the enzyme metalloproteinase disintegrin called TNF-α converting enzyme (TACE) produces the final 17 kDa soluble active TNF-α molecule. The regulation of TNF-α expression mostly occurs at the transcriptional level . The 5′-Several meta-analyses studies have shown a moderate to strong association of TNF-α-308G/A SNP with an increased risk of and in many cases the progression of various cancers including hepatocellular carcinoma , prostathaploinsufficiency effect whereby the elevated CRC risk associated with homozygous variant (AA) genotype may be significantly decreased when only heterozygous (GA) genotype is present. At the protein level this means that TNF-α expression associated with heterozygous (GA) genotype though will be high compared to wild genotype (GG) but this increased expression resulting from a single TNF-α-308A allele might be insufficient to influence the CRC risk. It should be noted, however, that this haploinsufficiency effect though seems plausible here does not stand true for this SNP or any other SNP in every population studied as the final outcome may be influenced by several other intricate factors. Another plausible explanation of this lack of association with CRC risk is related to the location of TNF-α gene within the human leukocyte antigen (HLA) gene complex. TNF-α-308G/A SNP is located in the major histocompatibility complex (MHC) class III, telemetric to the MHC class II and centrometric to MHC class I gene. This is the most polymorphic region of the genome and there is strong linkage disequilibrium (LD) between the alleles across the HLA gene complex including that between TNF-α and other HLA genes. The direct effect of this linkage disequilibrium is that TNF-α gene and the polymorphisms within may also be linked to another gene, region or haplotype. As a result, the association or disassociation of TNF-α haplotypes itself and of other HLA haplotypes with TNF-α phenotypes and further with various pathological conditions might be due to the variation in a linked gene that regulates the expression of this cytokine rather than due to polymorphism within the TNF-α gene itself. Further, due to the wide differences in the distribution of HLA alleles across different populations in different geographical areas, the associations between TNF-α polymorphisms including TNF-α-308G/A SNP and various pathological conditions including cancer shows a considerable variation which makes it important to evaluate and provide a direct functional link of any polymorphism with the disease or condition under study so as to nullify the possibility of observed association or disassociation due to the existence of linkage disequilibrium with the actual causative gene. Therefore, further mechanistic studies are required to fully evaluate and validate the effect of this SNP on CRC risk in our population. The nonexistence of variant genotype (AA) in our population can be explained through the wide variation in the distribution of TNF-α-308A allele across different ethnically defined populations and geographical areas worldwide. The frequency of TNF-α-308A allele is significantly lower in the Asian population compared to most other world populations. The TNF-α-308A is present in 30% of white Caucasians in the United Kingdom, 10–23% of Europeans, 8–10% of South Americans, 5% of South Africans and 2–9% of East Asians  variables. The major limitations of this study are modest sample size of the study to detect gene-gene and gene-environment interactions which usually requires much larger sample size. Further studies incorporating a larger sample size and/or another ethnic population in our study are needed to confirm in depth the role of TNF-α-308G/A SNP in relation CRC susceptibility. However, these limitations are unlikely to affect the final outcome of this study.6We have demonstrated through this study that there is no significant association between the TNF-α-308G/A promoter single-nucleotide polymorphism and the risk of developing CRC in our ethnic Kashmiri population. Further, we have also shown that there is no significant association between this SNP and various clinico-pathological parameters, demographic variables and environmental factors. However, it is important to emphasize that this study does not nullify the role of TNF-α itself in the pathogenesis of CRC but only indicates that this particular and important TNF-α SNP may not be associated with CRC risk in the population studied. Further, our findings need to be replicated with bigger sample size and should involve other ethnically defined populations with high CRC risk.None exist between any parties.MZB acknowledges University Grants Commission (UGC), GOI for UGC-BSR fellowship.


In Australia Cane toads have invaded further into high latitude regions than expected. Comparison of high and low latitude toads reveals that high latitude toads have an increased ability to raise their metabolic rate when acclimated to low temperatures. This increased thermal plasticity may be facilitating the ongoing invasion into high latitude regions. Temperature has pervasive effects on physiological processes and is critical in setting species distribution limits. Since invading Australia, cane toads have spread rapidly across low latitudes, but slowly into higher latitudes. Low temperature is the likely factor limiting high-latitude advancement. Several previous attempts have been made to predict future cane toad distributions in Australia, but understanding the potential contribution of phenotypic plasticity and adaptation to future range expansion remains challenging. Previous research demonstrates the considerable thermal metabolic plasticity of the cane toad, but suggests limited thermal plasticity of locomotor performance. Additionally, the oxygen-limited thermal tolerance hypothesis predicts that reduced aerobic scope sets thermal limits for ectotherm performance. Metabolic plasticity, locomotor performance and aerobic scope are therefore predicted targets of natural selection as cane toads invade colder regions. We measured these traits at temperatures of 10, 15, 22.5 and 30°C in low- and high-latitude toads acclimated to 15 and 30°C, to test the hypothesis that cane toads have adapted to cooler temperatures. High-latitude toads show increased metabolic plasticity and higher resting metabolic rates at lower temperatures. Burst locomotor performance was worse for high-latitude toads. Other traits showed no regional differences. We conclude that increased metabolic plasticity may facilitate invasion into higher latitudes by maintaining critical physiological functions at lower temperatures. Rhinella marina) within Australia, where this species has had a significant negative ecological impact. The question of whether adaptation will facilitate invasion beyond current predictions is critical for informing management strategies.Recent evidence indicates that biodiversity is declining on a global scale . The traR. marina) is a large anuran species native to Central and South America  hypothesis . SeveralAn interesting observation to emerge from the studies by o2rest; a proxy for the metabolic cost of basic physiological maintenance), peak post-exercise rate of oxygen consumption , aerobic scope  and locomotor performance were measured at 10, 15, 22.5 and 30°C to test the following hypotheses: (i) based on the considerable thermal metabolic plasticity of cane toads  and low (northern) latitudes were acclimated for a minimum of 8 weeks to temperatures of 15 or 30°C. The resting rate of oxygen consumption  per week, while those at 15°C were fed two adult cockroaches per week. This was chosen based on the reported energy density of this cockroach  were divided equally and randomly across two acclimation temperatures of 15 and 30°C. Toads were maintained in groups of five in plastic containers 65 cm long × 35 cm wide × 35 cm deep containing water and damp bark chips deep enough for burrowing. Containers were housed inside two temperature-controlled rooms, in which the acclimation temperatures were regulated to ±1.5°C, and maintained a 12 h light–12 h dark photoperiod. All toads were acclimated to these conditions for a minimum of 8 weeks and a maximum of 14 weeks before measurements. Animals were collected at different times during the year to reduce the time over which measurements were taken and to align with the later emergence of high-latitude toads, thus the collection and acclimation of high-latitude toads began as experimental procedures on low-latitude toads commenced. Toads maintained at 30°C were fed an average of four adult cockroaches . The V.o2max and locomotor performance were measured during the active circadian phase (night). A stratified random-order regimen was used for all measurements.The physiological traits of V.o2rest (in millilitres of O2 per hour) of an individual toad was measured using positive-pressure flow-through respirometry. Atmospheric air was drawn from outside using a pump  and scrubbed of CO2 using soda lime and water vapour using Drierite before passing through a mass flow controller  that regulated the flow rate to a nominal value of 50 ml min−1 at 10, 15 and 22.5°C and 100 ml min−1 at 30°C. Mass flow controllers were calibrated using a NIST-traceable bubble film flowmeter . After the mass flow controller, air was rehumidified using a 1 litre gas-washing bottle  before passing through the respirometry chamber (460 ml plastic container). The humidifying gas-washing bottle and respirometry chamber were housed inside a temperature-controlled cabinet  that regulated the test temperature to ±0.5°C. After the respirometry chamber, air was scrubbed of water vapour using Drierite before passing through a CO2 analyser  and an O2 analyser . The CO2 and O2 analysers were interfaced with a PowerLab 8/30 A/D convertor , which recorded fractional concentrations of CO2 and O2 in the excurrent air at a frequency of 10 Hz. The CO2 analyser was calibrated with dry CO2-free air and a certified gas mixture , and the O2 analyser was calibrated using dry CO2-free air.The V.o2) and rate of carbon dioxide production (V.o2) were calculated according to standard equations  differed between high- and low-latitude toads. Low-latitude toads were tested on consecutive nights after all V.o2rest measurements were complete, with 20 toads measured per night at a single test temperature each night until all toads had been tested at all test temperatures. As with V.o2rest measurements, all toads in a container were tested consecutively in a random order, with the order of containers from both acclimation treatments also randomized. For high-latitude toads, every 5 days following a block of V.o2rest measurements on 20 toads at a given test temperature, those toads underwent the V.o2max measurements the following evening at the same test temperature and in the same random order in which they had undergone V.o2rest measurements. Both high- and low-latitude toads were placed in a temperature-controlled cabinet at the test temperature for the night prior to measurements in enclosures similar to those in which they normally lived.Logistical constraints dictated that the measurement regimen for V.o2max, toads were exercised in a swimming flume  at a water velocity of 0.35 m s−1 until exhaustion, and then oxygen consumption was immediately measured using the same respirometry system described for V.o2rest measurements. Similar methods have previously been shown to provide values of V.o2 that approximate, if not exceed, V.o2max during exhaustive exercise and are thus a good indication of maximal respiratory capacity  mounted above the swim chamber and connected to a PC was used to record each swimming session at 60 frames s−1 using VirtualDub v1.9.11 . The time for toads to reach exhaustion was used as a measurement of locomotor endurance. The largest distance that they were able to propel themselves with a single jump from the back of the chamber against the water current was used as a measurement of burst locomotor performance. Jumps were considered for analysis if the toad was not prodded or touched, did not push off from the sides and did not leave the water. Jump distance was measured from the video footage with Kinovea v0.8.15 (www.kinovea.org).Measurements of both locomotor endurance and burst locomotor performance were made during the exercise phase that was used to obtain estimates of V.o2rest from V.o2max. Absolute aerobic scope was used as opposed to factorial aerobic scope (ratio of V.o2max to V.o2rest) because factorial scope was not considered to be informative  and log(mass) (P < 0.001), and there was a significant three-way interaction between test temperature, acclimation temperature and region (P = 0.006), indicating that between high- and low-latitude regions the effect of acclimation temperature on V.o2rest varied with test temperature  (P < 0.001), and there was a significant two-way interaction between test temperature and acclimation temperature (P = 0.012). There was no effect of region, indicating that high- and low-latitude toads exhibit the same response, which is an effect of acclimation temperature that varies with test temperature , but no effect of log(mass) (P = 0.12). Both high- and low-latitude toads showed very similar patterns of endurance, with cold-acclimated animals becoming exhausted in approximately half the time at 22.5 and 30°C , and there was a significant two-way interaction between test temperature and region (P = 0.004), indicating that there was no effect of acclimation temperature, but that the effect of test temperature on performance varied with region  (h region . Post hoP < 0.001) and a significant two-way interaction between test temperature and acclimation temperature (P = 0.008). The pattern seen here is almost identical to that of V.o2max because values of V.o2rest are so small compared with V.o2max that they make little difference when subtracted. A post hoc analysis showed significantly higher absolute aerobic scope for cold-acclimated toads at 10°C   between 1964 and 1966 . It is nThe cane toad has already gained a considerable foothold within Australia. While the damage is already done in some regions of the continent, there are areas yet untouched by this species. Management decisions regarding the spread of this species depend critically upon whether it will invade pristine ecosystems further or halt at the currently understood limits of its environmental tolerance. The importance of assessing whether adaptation may facilitate the invasion of regions beyond what has been predicted previously is obvious. Here, we add to the growing body of evidence to suggest that the cane toad is in fact evolving rapidly since its introduction to Australia. If the cane toad exceeds invasion predictions by a significant and unexpected margin then not only will the current impacts upon biodiversity intensify, but new species, as yet unaffected, may be impacted by this uniquely successful invader as predator, prey or competitor. This is particularly important as the cane toad invades higher latitudes and different thermal environments, where it will increasingly coexist with a growing list of native inhabitants. These concerns apply not only to this situation but to all instances of invasive species worldwide and illustrate the importance of assessing adaptation of invaders as they move into novel environments. Such assessments will be valuable in ensuring that appropriate decisions can be made to manage this growing threat to global biodiversity.This research was supported by University of Queensland and ARC funding to C.R.W. and C.E.F.Supplementary DataClick here for additional data file.


Mechanical hyperalgesia is one distressing symptom of neuropathic pain which is explained by central sensitization of the nociceptive system. This sensitization can be induced experimentally with the heat/capsaicin sensitization model. The aim was to investigate and compare spinal and supraspinal activation patterns of identical mechanical stimulation before and after sensitization using functional spinal magnetic resonance imaging . Sixteen healthy subjects  were investigated with mechanical stimulation of the C6 dermatome of the right forearm during spinal fMRI. Testing was always performed in the area outside of capsaicin application . During slightly noxious mechanical stimulation before sensitization, activity was observed in ipsilateral dorsolateral pontine tegmentum (DLPT) which correlated with activity in ipsilateral spinal cord dorsal gray matter (dGM) suggesting activation of descending nociceptive inhibition. During secondary mechanical hyperalgesia, decreased activity was observed in bilateral DLPT, ipsilateral/midline rostral ventromedial medulla (RVM), and contralateral subnucleus reticularis dorsalis, which correlated with activity in ipsilateral dGM. Comparison of voxel-based activation patterns during mechanical stimulation before/after sensitization showed deactivations in RVM and activations in superficial ipsilateral dGM. This study revealed increased spinal activity and decreased activity in supraspinal centers involved in pain modulation  during secondary mechanical hyperalgesia suggesting facilitation of nociception via decreased endogenous inhibition. Results should help prioritize approaches for further in vivo studies on pain processing and modulation in humans. Mechanical pinprick hyperalgesia is a very distressing symptom presented by approximately 29% of neuropathic pain patients Anatomically, the vast majority of the nociceptive second order neurons is located in the dorsal gray matter (dGM) of the spinal cord. From there they project to supraspinal nuclei in the brainstem and the thalamus before these afferent nociceptive impulses are transferred to further subcortical and cortical structures With the emergence of functional magnetic resonance imaging of the brain (fMRI) 2*-weighted gradient echo imaging sequences, which have rather poor field homogeneity In order to address these challenges, recent spinal fMRI studies use a turbo spin-echo sequence with a relatively short echo time rather than the more conventional gradient echo imaging sequence sensitive to the blood oxygenation level-dependent (BOLD) effect However, spinal fMRI studies of human pain processing are still rare Therefore, the aims were (A) to investigate spinal and supraspinal processes occurring as a consequence of mechanical stimulation and (B) to compare activation patterns of identical mechanical stimuli before and after induction of sensitization with the heat/capsaicin model in healthy subjects in order to investigate the specific pain-related components of secondary mechanical hyperalgesia. Within this study we could successfully demonstrate (A) increased activity in ipsilateral dGM after induction of hyperalgesia, (B) decreased activity in nociceptive regions of the brainstem and (C) a correlation between these supraspinal deactivations and activations of ipsilateral dGM during secondary mechanical hyperalgesia suggesting a facilitation of nociception via decreased descending endogenous inhibition.Sixteen right-handed On the right  lateral volar forearm approximately 3 cm distal to the elbow in the C6 dermatome a 3×3 cm square area  and an adjacent 2×5 cm area directly distal to it  were marked .All subjects underwent two identical MRI sessions back-to-back on the same day with mechanical stimulation in the same area before and after sensitization.3. To reduce sources of motion artefacts , a spatial saturation pulse was applied to the region anterior to the vertebral column.All scans were performed in a 3T human MRI scanner (Philips Achieva 3T). For acquisition of functional image data, a half-Fourier, single-shot turbo spin-echo sequence with a phased-array head-neck receiver coil  was used In order to enable the stimulation of a larger dermatomal area  mechanical stimulation was performed using a self-made brush consisting of 15 stiff von-Frey-hairs (166 mN). Care was taken that the site of stimulation was always within the area of secondary hyperalgesia [area B]. The mechanical stimulation paradigms consisted of 8 stimulation periods of 40 seconds alternating with eight baseline periods (64 volumes). After each session, subjects were asked to rate the stimulus intensity on the numerical rating scale  by verbal response.The heat/capsaicin sensitization model was used to induce secondary mechanical hyperalgesia For the analysis of the resulting 3D fMRI image data a general linear model (GLM) was used. The basis set consists of a boxcar model paradigm convolved with the tissue response function Spinal cord segments adjacent to C6 were included in the analysis since (A) primary afferent fibers split up into longitudinal collaterals innervating the bordering segments and (B) slightly individual anatomical dermatome-borders with a coincidental stimulation of surrounding dermatomes  were respected.(v)), signal change (Δ(S)) and % signal change (Δ(S/S)) across all volunteers provided by the fMRI data. P values <0.05 were considered to be statistically significant.Psychophysical data are presented as mean ± standard deviation (SD) unless otherwise specified. Wilcoxon matched pairs test was used for calculation of psychophysical intragroup differences and Spearman rank test for correlation calculations using the numbers of voxels . However, the dimension of secondary mechanical hyperalgesia was stable  and included the area of stimulation [area B] at all times, thus ensuring the stability of the pain model throughout the experiment. Corresponding to mechanical hyperalgesia, the subjects’ mean ratings of pain intensity for the mechanical stimulus were higher compared to those before sensitization  in T1 and deactivations in C7 in ipsilateral superficial dGM. Ventral activations were observed in ipsilateral ventral gray matter (vGM) of C4 before and after sensitization , 4.(S): R = 0.513; p = 0.042) and after sensitization (Δ(S): R = 0.533; p = 0.041), i.e. the higher the perceived pain intensity, the higher were activations in vGM of C7.Activations in ipsilateral vGM of C7 correlated positively to the subjects’ NRS-ratings before  dGM. The observed lateral deviation of superficial dGM-activations might arise from BOLD-contributions in signal change located in superficial draining veins (v): R = 0.611; p = 0.012) and C5 (n(v): R = 0.613; p = 0.012).Prior to sensitization, supraspinal activity was observed in the ipsilateral dorsolateral pontine tegmentum (DLPT) . The act(v): R = 0.546; p = 0.035) and T1 (n(v): R = 0.621; p = 0.012). Deactivations in the ipsilateral pons correlated with activations in ipsilateral dGM of C4 (n(v): R = 0.595; p = 0.019) and T1 (n(v): R = 0.555; p = 0.039; Δ(S/S): R = –0.538; p = 0.047), i.e. the higher the deactivations in the dorsal pons, the higher were activations in ipsilateral dGM (note that R in n(v) is positive because n(deactivated voxels) correlates positively to n(activated voxels) while Δ(S/S) is negative because activations, i.e. positive Δ(S/S)-values are correlating to deactivations, i.e. negative Δ(S/S)-values).In contrast to measurements prior to sensitization, deactivations in contralateral DLPT were observed during secondary mechanical hyperalgesia . These d(v): R = 0.699; p = 0.004; Δ(S): R = –0.827; p<0.001; Δ(S/S): R = –0.770; p = 0.001; C6: Δ(S): R = –0.537; p = 0.039). Deactivations in midline RVM correlated with deactivations in ipsilateral dGM (C5: n(v): R = 0.556; p = 0.031; C7 =  n(v): R = 0.599; p = 0.018; Δ(S): R = 0.608; p = 0.016). Visible deactivations in the caudo-dorsal contralateral medulla corresponded to the localization of the subnucleus reticularis dorsalis : R = –0.560; p = 0.030; C8: Δ(S/S): R = –0.578; p = 0.024; T1 = n(voxels): R = 0.554; p = 0.040).After sensitization, deactivations were also visible in the ipsilateral and median caudal pons/rostral medulla most likely corresponding to the area of the rostral ventromedial medulla RVM, . Deactivlis SRD,  and corrSupraspinal calculations of contrast maps showed deactivations in the RVM .The present study shows spinal and supraspinal signal intensity changes during mechanical stimulation and secondary mechanical hyperalgesia in healthy subjects. The main findings are (A) increased activity in ipsilateral dGM after induction of hyperalgesia, (B) decreased activity in supraspinal centers of pain processing and modulation  and (C) a correlation between supraspinal deactivations and activations of ipsilateral dGM during secondary mechanical hyperalgesia induced by the heat/capsaicin model. Results suggest a facilitation of nociception via decreased descending endogenous inhibition during secondary mechanical hyperalgesia.Anatomically, the vast majority of primary nociceptive afferent fibers projects to the ipsilateral dGM. Therefore, application of noxious stimuli should lead to activation of projection neurons located in the marginal layer of the dorsal horn Mechanical hyperalgesia and increased dGM activity can also be due to deficient inhibitory interneuron (ININ)-mediated inhibition of projection neurons Signal intensity changes were also observed in contralateral dGM during both conditions, consistent with a previous study using tactile stimuli Pain stimuli lead to withdrawal reflexes, i.e. the activation of ipsilateral motor neurons via spinal interneurons or supraspinal reticular nuclei Animal testing shows that the DLPT plays a central role in the modulation of nociception. With its noradrenergic fibers to the dGM, it mediates a negative feedback loop triggered by noxious stimuli to prevent excessive pain sensation An absence of this noradrenergic inhibition by the DLPT is thought to be a mechanism for the development of neuropathic pain Besides decreased inhibition, central sensitization is also thought to be mediated by coexisting excitatory descending facilitation Based on current concepts of pain modulation, OFF-cells, a specific cell-type of the RVM, physiologically mediate antinociception The SRD is believed to play a major role in the mechanism of diffuse noxious inhibitory control (DNIC) Even though spin-echo spinal cord fMRI is a new reliable and non-invasive method to show functional processes in the spinal cord Using spin-echo spinal cord fMRI, it was possible to investigate pain modulatory processes during secondary mechanical hyperalgesia in the brainstem and spinal cord of healthy subjects. This study is the first one to show an increase of ipsilateral dGM-activity during secondary mechanical hyperalgesia in human subjects in vivo. Furthermore, this study succeeds in showing decreased activity in areas of the brainstem that have been proven important for processes of central sensitization in animal experiments . Moreover, those deactivations correlated with dGM-activity. With these findings, this study gives new insights in human pain processing in vivo. Since findings of animal experiments and results of previous fMRI studies could be reproduced, spin-echo spinal cord fMRI has been shown to be a reliable method for the examination of spinal and supraspinal pain processing and modulation. Herewith, it qualifies for further investigations including treatment of neuropathic pain.


We demonstrate, through quantum mechanical calculations, that an origin of this phenomenon is a prevalence of ionic over electronic disorder in stoichiometric materials. Schottky defect formation provides a mechanism to self-regulate the concentration of charge carriers through ionic compensation of charged point defects. The equilibrium charged vacancy concentration is predicted to exceed 0.4 % at room temperature. This behavior, which goes against established defect conventions for inorganic semiconductors, has implications for photovoltaic performance.Hybrid halide perovskites such as methylammonium lead iodide (CH The same structure is adopted by the chloride and bromide perovskites, with solid solutions on the anion sublattice also reported. and “partial” disorder with respect to the methylammonium iodide  or lead iodide [Reaction (3)] sub-lattices.For inorganic perovskites, Schottky disorder is a dominant type of defect, which is associated with the formation of a stoichiometric amount of anion and cation vacancies, which can be randomly distributed in a crystal. It is foµµI=ΔH2 (µPb+2 µI=ΔHReactions (1), (2), and (3) are charge-neutral (self-compensated), for example, [The equilibrium concentration of lattice vacancies arising from Schottky disorder can be calculated by applying the law of mass action to reaction KC represents the fraction of the lattice sites (HS), and contributions from the changes in vibrational entropy are neglected.in which 3 is 2.29 eV per defect,[3NH3 and I sublattice will be vacant (in an open system). Such nonstoichiometric behavior goes beyond the non-interacting point defect limit; hence, inter-defect correlations will be important to consider in future quantitative models.The computed reaction energies—a combination of the formation energy of each of the individual charged point defects—are summarized in Table r defect, which reThe defect chemistry of this hybrid halide perovskite is unusual. It is common for wide-band-gap materials to favor ionic disorder (self-compensated arrangements of charged point defects) and low-band-gap materials to favor electronic disorder . Here, ionic disorder is favored despite the fact that electrons and holes are facile to form, with all of the defects investigated here being shallow donors ( ( and ). The selfq2=4 for oxides (O2−); 1 for halides (I1−). Hence for the metal halides both anion and cation vacancies can form with high probability due to a decrease in the chemical bond strength. Whereas point defects, formed due to configurational entropy, facilitate significant equilibrium hole densities of up to 1018 cm−3 in thin-film photovoltaic absorbers such as Cu2ZnSnS4,[3NH3PbI3 limits their formation as it provides a route to minimize the free energy of the crystal without generating charge carriers.One factor behind this behavior is the lattice energy: in comparison to metal oxide perovskites, for halides the electrostatic potential of all lattice sites is reduced due to the lower formal oxidation states. The elecu2ZnSnS4, Schottky2 V−1 s−1.[It is now well established that carrier diffusion lengths in hybrid perovskite thin films can be extremely long (>1 μm) in high quality materials. The effe V−1 s−1. The contThe behavior reported here has important implications for the application of hybrid perovskites in photovoltaics: 1) The stoichiometric hybrid perovskites can simultaneously be highly defective and electronically benign, with the low carrier concentrations ensuring effective Fermi-level splitting for operation of a p–i–n photovoltaic device. The comb3NH3PbI3 identified here is key to its success as an intrinsic photovoltaic material. The first report by Weber on this material in 1978,[In summary, the unusual defect chemistry of CH3NH3PbI3 was calculated in a 96 atom pseudo-cubic perovskite (2×2×2) supercell with a Γ-centered 3×3×3 Monkhorst-Pack special k-point grid (400 eV plane wave cut-off), using a set-up previously reported.[The total energy of bulk and defective CHreported., 25 LattThe formation energy of the individual charged defects is defined as corrected to accoucorrected which inAll structures and energies were calculated using Kohn–Sham density functional theory (in the code VASP). Interactions between the core and valence electrons is described within the PAW method includinThe PBEsol exchange–correlation functional was employed. PBEsol i


Tumor angiogenesis is measured by counting microvessels in tissue sections at high power magnification as a potential prognostic or predictive biomarker. Until now, regions of interest•Whole slide imaging with Pannoramic SCAN (3DHISTECH)•Computer-assisted sampling in Pannoramic Viewer (3DHISTECH) extended by two self-written AutoHotkey applications (AutoTag and AutoSnap)•® and Bridge® (Adobe Systems)The use of digital grids in PhotoshopThis rapid procedure allows traceability essential for high throughput protein analysis of immunohistochemically stained tissue. The stained slides were scanned with the slide scanner Pannoramic SCAN (3DHISTECH) to obtain a whole slide image  on a Ventana Benchmarkde image . A 20× Pde image A.® environment, the open-source macro-creation and automation software utility AutoHotkey can be used. Semi-automatic naming of point annotations in Pannoramic Viewer during manual annotation was performed with a self-written AutoHotkey script AutoTag running in the background and monitoring the activity on the screen. It names the point annotations with sequentially increasing numbers starting from one (Supplementary Code AutoTag: lines 183–210), allowing the quick generation of point annotations by merely clicking a number of locations in the image.When generating a point annotation in Pannoramic Viewer  at a chosen ROI A, a nameA Q= ΣiNi/ΣiVi,ref with an i value from 1 to 25 ROIs, expressed as number of microvessels per area, with iN the number of counted vessels in ROI i and Vi,ref the number of grid points hitting tissue in ROI i) were calculated 1000 times by resampling from the pool of 25 ROIs. The coefficient of variation (CV) was plotted versus the number of ROIs (f(x) = ax−b was added and the first derivative of this function (f′(x) = −abx−b−1) was used to define the minimum number of ROIs required. The local derivative must be smaller than 0.50%. After creating these graphs (The minimum number of regions of interest (ROIs) required for analysis of microvessel density was calculated by using random sampling with replacement , semi-automatic image generation requires more time on average to construct one image at a given point annotation . The process could be speeded up, but is more robust when pauses (sleep functions) are built-in to handle lag due to software processing and network speed. However, due to the automatic nature of AutoSnap no user input is required and, therefore, the process can be run overnight and frees time for other activities. Importantly, the AutoTag and AutoSnap applications semi-automate a sampling method that allows traceability due to the final results being linked to the exact locations in each tissue section.When all necessary point annotations are created, the magnification is asked by the AutoTag application (Supplementary Code AutoTag: lines 104–142). In general, a 20× magnification is sufficient for the correct identification of microvessels. Finally, AutoTag generates a .txt file that lists the processed samples in column one, the number of semi-automatically created annotations in column two and the magnification in column three (Supplementary Code AutoTag: lines 3–17). This file can be read by a second AutoHotkey application, named AutoSnap, allowing the automatic creation of images of all the ‘AutoTagged’ samples A. The po2, for 40× and 20× magnification, respectively) were created in Adobe Photoshop® . These grids are applied to the images created with AutoSnap in a batch process with Adobe Bridge®  automatically pastes the grid as an extra image layer and saves this as a new image.To allow unbiased counting of the microvessels, two digital grids with a transparent background  and the number of grid points overlapping with vessels per area (AA) AQ was highly associated with biological differences between the samples (78%) and a smaller contribution from inherent methodological factors (17%) or observers (5%) Tumor angiogenesis is most frequently measured by counting the smallest vessels, known as microvessels, in tumor tissue sections viewed at a high magnification (200×– 400×)