We now show that the activity of TRPM3 expressed in mouse dorsal root ganglion (DRG) neurons is inhibited by agonists of the Gi-coupled µ opioid, GABA-B and NPY receptors. These agonist effects are mediated by direct inhibition of TRPM3 by Gβγ subunits, rather than by a canonical cAMP mediated mechanism. The activity of TRPM3 in DRG neurons is also negatively modulated by tonic, constitutive GPCR activity as TRPM3 responses can be potentiated by GPCR inverse agonists. GPCR regulation of TRPM3 is also seen in vivo where Gi/o GPCRs agonists inhibited and inverse agonists potentiated TRPM3 mediated nociceptive behavioural responses.Transient receptor potential (TRP) ion channels in peripheral sensory neurons are functionally regulated by hydrolysis of the phosphoinositide PIPDOI:http://dx.doi.org/10.7554/eLife.26138.001 TRPM3 belongs to a family of channel proteins that allow sodium and calcium ions to enter cells by forming pores in cell membranes. TRPM3 is found on the cell membranes of nerve cells; when ions flow into the nerves through the TRPM3 pores it triggers an electrical impulse. TRPM3 is responsible for helping us to detect heat, and mice without this protein find it difficult to sense painfully hot temperatures. Mice lacking TRPM3 also respond to other kinds of pain differently. Normally, a mouse with an injured paw becomes more sensitive to warm and hot temperatures, but this does not happen in mice that do not have TRPM3.When activated, other proteins called G-protein coupled receptors (or GPCRs for short) can make some members of this family of channel proteins more or less likely to open their pore. This in turn increases or decreases the flow of ions through the pore, respectively. Yet it was not clear if GPCRs also affect TRPM3 channels on the membranes of nerve cells.Quallo et al. have now discovered that “switching on” different GPCR proteins in sensory nerve cells from mice greatly reduces the flow of calcium ions though TRPM3 channels. The experiments made use of two pain-killing drugs, namely morphine and baclofen, and a molecule called neuropeptide Y to activate different GPCRs.GPCRs interact with a group of small proteins called G-proteins that, when activated by the receptor, split into two subunits, known as the α subunit and the βγ subunit. Once detached these subunits are free to act as messengers and interact with other proteins in the cell membrane. Quallo et al. found that TRPM3 is one of a small group of proteins that interact with the βγ subunits of the G-protein, which can explain how “switching on” GPCRs reduces the activity of TRPM3. Two independent studies by Dembla, Behrendt et al. and Badheka, Yudin et al. also report similar findings.There is currently a need to find more effective treatments for people suffering from long-term pain conditions and it has become clear that TRPM3 channels are involved in sensing both pain and temperature. These new findings show that drugs already used in the treatment of pain can dramatically change how TRPM3 works. These results might help scientists to find drugs that work in a similar way to dial down the activity of TRPM3 and to combat pain. Though first it will be important to confirm these new findings in human nerve cells.DOI:http://dx.doi.org/10.7554/eLife.26138.002 Proteins encoded by the TRPM3 gene form non-selective cation channels which are widely expressed in mammalian tissues. The discovery that TRPM3 can be activated by the endogenous neurosteroid pregnenolone sulphate (PS), has facilitated the study of this widely-expressed TRP channel and PS has been utilised as a pharmacological tool for channel characterisation and as a probe for TRPM3 expression . TRPM3 is and Gαq- coupled receptors can sensitise the heat sensitive nociceptor TRPV1 through protein kinase-dependent mechanisms (2) and other phosphoinositides as loss or hydrolysis of PIP2 leads to a reduction in TRPM3 activity that can be restored by application of exogenous PIP2 . For example, activation of both Gαchanisms . Like otIP2 . These feceptors . Howevereceptors , suggest channel  and TRPM channel .i/o coupled GPCRs can modulate TRPM3 mediated nociceptive responses in vivo. For these experiments we have studied three different receptors  which are known to modulate sensory neuron activity   . Therefo2+]i-responses in isolated DRG neurons. This concentration of PS typically activates about 30% of cells in DRG cultures. The first application of PS was used to identify TRPM3 expressing neurons and the second to assess the effects of pharmacological treatments. We refer to the response amplitude of the second PS challenge as R (relative % response). In control experiments, the second PS challenge evoked [Ca2+]i-responses (R) that were 63 ± 2% of the first PS response amplitude (2+]i responses (R < 5%).We used two consecutive applications of a submaximal concentration of pregnenolone sulphate  to investigate the effect of morphine on TRPM3-mediated i (R < 5%). The relative amplitude of the second PS-evoked [Ca2+]i responses was reduced from 66 ± 3% in control experiments to 11 ± 1% in the presence of PYY (p<0.001). Like opioid receptors, Y2 receptors have been reported to possess constitutive activity , baclofen (100 µM) and PYY (100 nM) and challenged with PS (20 µM) in the presence and absence of these drugs . A largei/o-coupled receptors are able to effectively modulate TRPM3 activity. L-AP4 (5 µM) activation of group III metabotropic glutamate receptors (mGLUR4/6/7/8), which are expressed by DRG neurons  compared to control . Cannabinoid CB1 receptors are expressed by a subset of DRG neurons  which reduces TRPV1 desensitization . DRG neui subunits leads to inhibition of adenylate cyclase, the enzyme responsible for producing cAMP . Activation of protein kinase A (PKA) by cAMP and the resulting regulation of ion channel functions by PKA are well characterised. In order to examine whether opioid mediated inhibition of TRPM3 is driven by reduced levels of cAMP, we investigated whether morphine could still exert its inhibitory effects in the presence of a membrane permeable cAMP analogue, 8-bromo cAMP. 8-bromo cAMP was unable to compensate for the morphine-induced inhibition of PS responses. In experiments where 8-bromo cAMP (1 mM) was co-administered with morphine (10 µM) the response amplitude evoked by the second PS challenge was 11 ± 2%  and baclofen (100 µM) were present during the second PS application, the maximum response amplitude was significantly higher than when baclofen was present alone ; the maximum response amplitude in the presence of both gallein and PYY was significantly higher than the maximum response amplitude in the presence of PYY alone . Intriguingly, the scatter plots suggest that the inhibitory effect of gallein on baclofen and PYY mediated inhibition only occurred in a subset of neurons, unlike the strong effect noted for morphine inhibition in almost all cells. Nevertheless, these results indicate that TRPM3 activity can be regulated by a βγ-dependent mechanism after activation of opioid, GABAB and NPY receptors.We next examined whether gallein also prevents baclofen-induced inhibition of PS-evoked i-measurements (see above). HEK293 and CHO cells were studied under Ca2+-free conditions . Excised inside-out membrane patch recordings were made using TRPM3 expressing HEK293 cells. The solution bathing the intracellular membrane face of excised membrane patches contained either (mM) 140 CsCl, 10 EGTA, 10 HEPES or 70 CsCl, 70NMDG-Cl, 10 EGTA, 10 HEPES pH7.3. For some experiments current-voltage relationships were obtained from voltage ramps (300 ms duration) from −100 mV to voltages up to +200 mV. The NMDG containing intracellular solution was used for voltage ramp studies to reduce the amplitude of the outward current.DRG neurons and TRPM3 expressing HEK293 or CHO cell lines were studied under voltage-clamp conditions using an Axopatch 200B amplifier and pClamp 10.0 software  were dissolved in intracellular solution at a final concentration of 50 nM. For some experiment the βγ subunits were inactivated by heating to 100°C for 10 min. βγ subunits (plus CIM0216) were applied by pressure ejection from a blunt pipette positioned close to the excised membrane patch.All behavioural experiments were approved by the King’s College London Animal Welfare and Ethical Review Board and conducted under the UK Home Office Project Licence PPL 70/7510. PS, or a combination of PS/CIM0216 (25 µl) and morphine, baclofen and PYY (10 µl) were injected subcutaneously into the plantar surface  of one of the hind paws using a 50 µl Luer-syringe (Hamilton Co.) fitted with a 26-gauge x 3/8 inch needle. Morphine, baclofen and PYY were injected 5 min before PS/CIM0216. Naloxone (2.5 mg/kg) and BIIE 0246 3 mg/kg were injected intraperitoneally 30 min prior to intraplantar injection of PS. Mice were habituated to the experimental Perspex chambers before the experiment and placed in the chambers immediately after injection of TRPM3 agonists. The duration of pain-related behaviours (licking and biting or flinching and shaking of the injected paw) was recorded using a digital stop-watch. Total pain response times over the first 2 min were used for analysis as the pain behaviours were largely restricted to this period. Groups of 6–11 animals were used for each agent. Pregnenolone sulphate : a 200 µM solution in PBS was prepared from a 60 mM DMSO stock solution. 25 µl was injected i.pl. into the left hind paw. CIM0216: a 20 µM solution was prepared from a 5 mM DMSO stock solution. A combined PS/CIM solution was prepared by combining 1 ml 400 µM PS with 1 ml 40 µM CIM and 25 µl was injected i.pl. into the left hind paw. Naloxone HCl was administered at a dose of 2.5 mg/kg i.p. 30 min prior to PS. Morphine, 100 µg in 10 µl was injected i.pl. 5 min prior to CIM/PREGS. Baclofen, 60 µg in 20 µl was injected i.pl. 5 min prior to CIM/PS. PYY, 1 µg in 10 µl was injected i.pl. 5 min prior to CIM/PS. BIIE 0246 was made up in saline from a 10 mM DMSO stock and dosed at 3 mg/kg i.p. 60 min prior to PS.2O and DAMGO , SB205607 , U50488  and PYY , were made in physiological extracellular solution. A stock solution of 8-bromo cAMP was made in H2O titrated with NaOH. Stock solutions of L-AP4  and (RS)-Baclofen  were made in molar equivalent solutions of NaOH. Stock solutions were aliquoted and stored at −20°C. Stock solutions were diluted in physiological extracellular solution for their use in experiments. PTX (lyophilised powder) was reconstituted in H2O and was stored at 4°C. PTX was added to cells at a concentration of 200 ng/ml.All compounds were from Sigma-Aldrich, Poole, UK unless otherwise stated. Stock solutions of pregnenolone sulphate, CIM0216 , WIN 55212–2 , AM251 , Gallein  and BIIE 0246  were made in DMSO . Stock solutions of morphine and naloxone were made in HSCR_002865).Data are presented as box and whisker plots showing the mean (square symbol), median , interquartile range (box) and 5% and 95% percentile points (whiskers). For the microfluorimetry and electrophysiology experiments, ‘n’ values represent the number of PS responding neurons or cells studied except where indicated otherwise in the text. For behavioural experiments, ‘n’ values represent the number of animals in each group. No statistical methods were used to predetermine sample sizes, however our sample sizes are similar to, or greater than, those generally employed in other studies in the field. Normality of data was tested using the Shapiro-Wilk Test. Differences in normally distributed data means between two groups were analysed using an independent samples t-test. Differences in normally distributed data means between three groups or more were analysed using a one-way ANOVA, followed by a Tukey’s HSD post-hoc test. Differences in non-normally distributed data means between two groups were analysed using a Mann-Whitney U test. Differences in non-normally distributed data means between three groups or more were analysed using a Kruskal-Wallis test. All statistical analyses were made using IBM SPSS statistics, version 22 ; László Csanády (Reviewer #2); and Alexander Chesler (Reviewer #3).Thank you for submitting your article "Gβγ mediate Gi/o coupled GPCR inhibition of TRPM3 in sensory neurons" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript is one of three that reports exciting new findings on the mechanism of inhibition of TRPM3 channel activity in dorsal root ganglion (DRG) sensory neurons by stimulation of G protein-coupled receptors (GPCRs). Although all three manuscripts received favorable reviews, the essential revisions will require different amounts of time to address, and we encourage the authors to coordinate submission of their revised manuscripts.2+ imaging on dissociated dorsal root ganglion (DRG) neurons, except for a few whole-cell patch-clamp recordings, and some behavioral experiments in live animals. This study was less mechanistically oriented than the companion submissions by two other groups, and some conclusions rely on faith in generally accepted phenomena. These include the potential effects on PIP2 levels that are not directly addressed (the assumption is that those remain unaffected by the Gi/o pathway), and the involvement of G-α is excluded simply based on the lack of effect of a known G-α inhibitor. On the other hand, the results of this study complement those of the co-submissions in several ways:The general conclusion of this manuscript is that TRPM3 channel currents are directly inhibited by G-β-γ subunits released upon stimulation of various GPCRs coupled to heterotrimeric Gi/o proteins, and this inhibition is independent of any effects on cAMP concentrations. The majority of experiments reflect Cai) TRPM3 inhibition through a different subset of GPCRs present in DRG neurons is investigated here. Stimulation of opioid μ,GABA-B, neuropeptide Y, and cannabinoid CB1 receptors are shown to inhibit TRPM3 currents in subpopulations of DRG neurons.ii) Stimulation of submaximally activated TRPM3 channels by inverse agonists of either opioid μ or NPY receptors suggests some tonic inhibition of TRPM3 currents by constitutive activity of these GPCRs (or perhaps the presence of low levels of endogenous ligands). The relevance of this tonic inhibition is also demonstrated through in vivo experiments that show increased sensitivity to TRPM3 agonist-induced pain in the presence of opioid μ or NPY receptor blockers.Essential revisions:1) What is missing in this manuscript is an indication of the selectivity of the receptor activation towards TRPM3-dependent pain signals. To correlate the reduced pain response in the presence of morphine or baclofen to the inhibition of TRPM3 function, it is essential to show that other TRPM3-independent pain responses are not affected. This would involve demonstration that neuronal calcium signals and pain evoked by e.g. capsaicin or mechanical stimuli are not affected by these agonists.2) This study would be greatly strengthened if the authors showed that activation of one of their GPCRs could inhibit TRPM3 currents in HEK cells. It would also be good to demonstrate that G-β/γ applied to inside out patches can inhibit TRPM3 to strengthen the claim of a direct interaction with the channel. It is also not clear what the control condition is for 3) To fully evaluate the quality and robustness of calcium imaging data it is helpful to have representative images as well as traces. This would allow the reader to appreciate the density and health of the cells and neurons as well as the robustness of the responses. Also, glia vs. neurons in the culture should not be highlighted. Absence of KCl response is a poor proxy for glial identification – if this is the only criteria, then calling those cells KCl-negative cells is more appropriate.4) The scatter of data used to generate bar graphs is unclear. These should be included on all summary plots throughout the manuscript to provide a better sense of the effect size and variability. The number of tested cells is quite low for the experiments in 5) There are no positive controls in 6) Gallein is known to inhibit other targets, the short comings of this drug should at least be discussed, and it would be ideal if orthogonal approaches to interfere with GBG signaling were added to strengthen the findings (such as PTX or BARK-ct). Essential revisions:1) What is missing in this manuscript is an indication of the selectivity of the receptor activation towards TRPM3-dependent pain signals. To correlate the reduced pain response in the presence of morphine or baclofen to the inhibition of TRPM3 function, it is essential to show that other TRPM3-independent pain responses are not affected. This would involve demonstration that neuronal calcium signals and pain evoked by e.g. capsaicin or mechanical stimuli are not affected by these agonists.2+ responses in DRG neurons and capsaicin-evoked pain responses in vivo. For the former experiments we used conditions where TRPV1 desensitization was greatly reduced by the presence of cyclosporin to inhibit calcineurin. Under these conditions, morphine did not inhibit TRPV1 (new We have added experiments examining the effects of morphine on capsaicin-evoked CaRPV1 new . In vivoRPV1 new . The inh2) This study would be greatly strengthened if the authors showed that activation of one of their GPCRs could inhibit TRPM3 currents in HEK cells. It would also be good to demonstrate that G-β/γ applied to inside out patches can inhibit TRPM3 to strengthen the claim of a direct interaction with the channel. It is also not clear what the control condition is for We have added the results of experiments where we applied G-β/γ subunits directly to inside-out membrane patches from TRPM3 expressing HEK293 cells. These results complement the whole cell experiments in the original submission. G-β/γ subunits inhibited CIM0216 currents in the excised membrane patches by ~50% similar to the inhibition noted in whole cell experiments. As a control we used heat inactivated (boiled) G-β/γ subunits as suggested. The latter had no inhibitory effect on the CIM0216 evoked currents in inside-out membrane patches.-negative cells is more appropriate.3) To fully evaluate the quality and robustness of calcium imaging data it is helpful to have representative images as well as traces. This would allow the reader to appreciate the density and health of the cells and neurons as well as the robustness of the responses. Also, glia vs. neurons in the culture should not be highlighted. Absence of KCl response is a poor proxy for glial identification – if this is the only criteria, then calling those cells KCl+ solution with a robust increase in intracellular calcium concentration. However, we have replaced the term ‘neuron’ with ‘cells’ in many places as a positive response to the reviewers’ comment.We have added representative images of the cells and Fura-2 fluorescence signals in 4) The scatter of data used to generate bar graphs is unclear. These should be included on all summary plots throughout the manuscript to provide a better sense of the effect size and variability. The number of tested cells is quite low for the experiments in We have replaced the bar graphs with box and whisker plots and show all data points for all experiments. Data from additional experiments have now been added to the original 5) There are no positive controls in 2+ responses at the concentration used in the current experiment . We have not therefore added experimental data on the ability of this compound to stimulate cAMP pathways.The ability of 8-bromo-cAMP to stimulate PKA has been well established in numerous published experiments and one of the authors has published showing that it affects histamine evoked DRG CaWe acknowledge that the action of NF023 in experiments in the original submission was questionable as the compound was applied to intact cells in calcium imaging experiments. Although the compound has been reported to affect intact cells, we believe that it will not cross the plasma membrane easily. We have therefore now included experiments where it is included in the pipette solution at a high concentration (100µM) in whole cell recordings experiments on TRPM3-expressing CHO cells new . This co6) Gallein is known to inhibit other targets, the short comings of this drug should at least be discussed, and it would be ideal if orthogonal approaches to interfere with GBG signaling were added to strengthen the findings (such as PTX or BARK-ct).Experiments showing that PTX inhibits the effect of morphine were included in the study. We have made the wording slightly more prominent to emphasize this.We have also added a caveat that gallein could have ‘off target’ non-G-β/γ subunit effects. This comment could probably be applied to almost any inhibitor used. We could not find published data that gallein inhibited other targets other than those where the effects were interpreted as being via a G-β/γ pathway. We would appreciate any information from the reviewers on this point.


Broiler chickens are compulsive feeders that become obese as juveniles and are thus a unique model for metabolic disorders in humans. However, little is known about the relationship between dietary composition, fasting and refeeding and adipose tissue physiology in chicks. Our objective was to determine how dietary macronutrient composition and fasting and refeeding affect chick adipose physiology during the early post-hatch period.n = 6–10 per group).Chicks were fed one of three isocaloric diets after hatch: high-carbohydrate , high-fat  or high-protein . At 4 days post-hatch, chicks were fed (continuous ad libitum access to food), fasted , or refed (fasted for 3 h and refed for 1 h). Subcutaneous, clavicular, and abdominal adipose tissue was collected for histological analysis and to measure gene expression, and plasma to measure non-esterified fatty acid (NEFA) concentrations (Adipose tissue weights were reduced in chicks that were fed the HP diet and adipocyte diameter was greater in the adipose tissue of chicks that ate the HF diet. Consumption of diets differing in protein and fat content also affected gene expression; mRNAs encoding fatty acid binding protein 4 and a lipolytic enzyme, monoglyceride lipase, were greater in chicks fed the HC and HF than HP diet in all three adipose tissue depots. Fasting influenced gene expression in a depot-dependent manner, where most fasting and refeeding-induced changes were observed in the clavicular fat of chicks that consumed the HC diet. Fasting increased plasma NEFA concentrations in chicks fed the HC and HP diets.The decreased adipose tissue deposition in chicks fed the HP diet is likely explained by decreased rates of adipogenesis. Consumption of the HF diet was associated with greater adipose tissue deposition and larger adipocytes, likely as a result of greater rates of adipocyte hypertrophy. The depot-dependent effects of diet and fasting on gene expression may help explain mechanisms underlying metabolic distinctions among subcutaneous and visceral fat depots in humans. Intensive selection for growth rate and meat yield in broiler chickens has led to correlated increases in voluntary food consumption, fat deposition and incidence of metabolic disorders in breeders . Thus, cDietary macronutrient composition not only affects appetite in birds and mammals but also regulates adipose tissue physiology. When mice are fed a high-fat (HF) diet, hypertrophy occurs in visceral fat whereas hyperplasia dominates in subcutaneous fat as both depots expand in response to excess caloric intake . HoweverIn response to food deprivation and refeeding, a complex array of adaptive metabolic changes are triggered that mediate dynamic alterations in appetite regulation, energy storage and expenditure in peripheral tissues in response to the changes in energy availability. The effects of fasting and refeeding on the gene expression of metabolism-associated genes in different tissues has been extensively studied in both birds and mammals, however, there is little known about the effects of diet on molecular changes in the adipose tissue of chicks. Therefore, the purpose of this study was to evaluate effects of dietary macronutrient composition and fasting and refeeding on adipose tissue physiology in broilers chicks during the first 4 days post-hatch.The Institutional Animal Care and Use Committee at Virginia Tech reviewed and approved all animal protocols and chicks were cared for in accordance with the Guide for the Care and Use of Laboratory Animals. Cobb-500 broiler chicks were transported from a local hatchery on the morning of hatch and were housed in electrically heated and thermostatically controlled cages. The temperature was 30 ± 2 °C with 50 ± 5% relative humidity and 24 h of light. Chicks were randomly assigned to receive one of three diets, with free access to food and water. Diets were formulated to be isocaloric  as shown in Table n = 10 chicks per group. Chicks were euthanized, sexed by visual inspection of gonads, and tissues collected as described below.At 4 days post-hatch, chicks from each diet were randomly assigned to one of three treatments: fasting (3 h), refeeding (1 h of ad libitum access to food after 3 h of fasting) and feeding (continuous ad libitum access to food), with n = 10 chicks as described [2. Size distributions in each image were also analyzed.Subcutaneous, clavicular, and abdominal adipose tissue samples were collected from escribed , 7, fromescribed . Samplesescribed  using NIn = 10 chicks) via capillary blood collection tubes (Microvette®) immediately following euthanasia and decapitation. After collection, samples were centrifuged at 2,000 x g at room temperature and plasma isolated. Plasma NEFA concentrations were measured using the NEFA-HR2 kit (Wako Diagnostics) according to the manufacturer’s instructions. Absorbance was measured at 550 nm using an Infinite M200 Pro multi-mode plate reader (Tecan). Sample concentration was calculated using the following formula: Sample Concentration = Standard Concentration × (Sample Absorbance)/(Standard Absorbance). Units for the concentrations are reported as mEq/L.Approximately 200 μL of blood was collected from the trunk (n = 10 per group) and submerged in RNAlater (Qiagen) and processed for gene expression analyses as described [Adipose tissue samples were collected  was performed for adipose depot weight, percent weights, adipocyte area and diameter, NEFA concentrations, and RQ values using the Fit Model platform of JMP Pro11 . Because sex and interactions between sex and diet did not significantly influence any of the traits measured in this study, sex was excluded from the statistical models. For weights and morphometric measurements, the statistical model included the main effects of diet, adipose tissue depot, and their interaction. Post hoc pairwise comparisons were carried out with Tukey’s test. For RQ data  and NEFAs, the model included the main effects of diet and fasting treatment and the interaction between them. The interaction on NEFAs was separated with Tukey’s test. Significant interactions from real time PCR data were separated with the Slice function of JMP and effects sliced within dietary group for each gene. Significant dietary effects were further analyzed by second ANOVAs with Tukey’s test to separate the means. All data are presented as means ± SEM. Differences were considered significant at P < 0.05.The real time PCR data were analyzed using the ΔΔCT method, where ΔCT = CT target gene − CT actin, and ΔΔCT = ΔCT target sample − ΔCT calibrator . The aveP = 0.0001) than chicks fed the HP diet (Table P = 0.0002). The subcutaneous fat was heaviest, clavicular fat intermediate, and abdominal fat the lightest (P = 0.0001). This was also observed when depots were expressed as a percentage of body weight (P = 0.0001). Adipocyte diameter was greater (P < 0.05) in chicks fed the HF diet than in chicks fed the HC or HP diet (Table P < 0.0001).At day 4 post-hatch, body weights were not different among groups and chicks fed HC and HF diets had greater fat depot weights . Plasma NEFAs were greater in fasted than fed or refed chicks that consumed the HC and HP diets  and NPY receptor 2 (NPYR2) in subcutaneous adipose tissue  was greater in chicks that consumed the HC than the HP diet , peroxisome proliferator-activated receptor gamma (PPARγ), and sterol regulatory element-binding transcription factor 1 (SREBP1) was greater in the subcutaneous adipose tissue of fed than fasted or refed chicks (P < 0.05). C/EBPβ mRNA was greater in fasted than fed or refed chicks (P < 0.05), while Krüppel-like factor 7 (KLF7) was greatest in fasted, intermediate in fed, and lowest in the subcutaneous fat of refed chicks (P < 0.05).There were also main effects of diet and feeding condition. Expression of fatty acid binding protein 4 , and NPYR2 mRNA quantities were affected by the interaction of feeding treatment and diet . The quantities of PPARγ .In clavicular fat, iet Fig. . ExpressAT9 Fig. , SREBP1 BP1 Fig. , and ATGTGL Fig.  showed sARγ Fig.  and NPYRYR2 Fig.  mRNA shoPLIN1), FABP4, and monoglyceride lipase (MGLL) mRNAs were greater in the clavicular fat of chicks that consumed the HC and HF than HP diet , while abundance of C/EBPβ and acyl-CoA dehydrogenase long chain (ACADL) mRNA was greater in fasted than fed chicks (P < 0.05). There was greater expression of comparative gene identification-58 (CGI-58) in fed chicks than fasted or refed chicks (P < 0.05). The expression of Krüppel-like factor 7 (KLF7) and NPY mRNA was greater in fasted than fed or refed chicks (P < 0.05).There were multiple main effects of diet and feeding state in clavicular fat. Perilipin1 . There were also many main effects of feeding treatment on mRNA abundance in abdominal fat. Expression of AGPAT9 was greater in fed than refed chicks (P < 0.05). Expression of C/EBPα was greatest in fed, lowest in fasted, and intermediate in refed chicks, while C/EBPβ was only greater in fasted than fed chicks (P < 0.05). The abundance of KLF7 mRNA was greater in fasted than fed or refed chicks (P < 0.05). Expression of PPARγ was greatest in fed, intermediate in refed, and lowest in the abdominal fat of fasted chicks (P < 0.05). Expression of MGLL was greater in fed and fasted than refed chicks (P < 0.05). The abundance of SREBP1 mRNA was greatest in fed, intermediate in fasted, and lowest in refed chicks and NPY was greater in fed than fasted or refed chicks (P < 0.05).Similar to the other depots, there were multiple main effects in abdominal fat. The abundance of The current obesity epidemic and projected increases in the prevalence of obesity worldwide necessitate the development of novel research models that provide insights on the pathogenesis of obesity at the cellular and molecular level. This research revealed that not only do different anatomical regions of fat develop differently in chicks, but that dietary fat and protein quantity dramatically influence adipocyte development and lipid remodeling. Three hours of fasting was sufficient to induce a rise in plasma non-esterified fatty acids (NEFAs), however the effect was diet-dependent, and gene expression changes were detected in a diet and adipose tissue depot-dependent manner, further illustrating the complex relationship between diet composition, nutritional state, and anatomical depot-specific physiology. These findings highlight the importance of understanding metabolic differences among subcutaneous and visceral adipose tissue depots and the utility of chick as a model to explore the molecular basis for such differences in humans.At 4 days post-hatch, there was less adipose tissue deposition in chicks that consumed the HP diet. Feeding more dietary protein from 18 to 28%) for the first week post-hatch did not affect body fat content in 7 day-old broiler chicks , althoug to 28% fFABP4 (subcutaneous and clavicular), SREBP1 (clavicular) and C/EBPβ  in comparison with the HC diet, and FABP4 (clavicular) and KLF7  compared with the HF diet. The SREBP1 and C/EBPβ are key transcription factors during the early stages of adipogenesis that coordinate the transcriptional regulation of a variety of adipocyte metabolism-associated genes [Differences in adipose tissue weight can be partly explained by the gene expression results. In the adipose tissue of chicks consuming the HP diet, there was reduced expression of ed genes –21. The ed genes . The KLFed genes . CollectIn mice more than 6 weeks old, 60 days of feeding a HF diet revealed that visceral fat expanded predominantly by adipocyte hypertrophy, whereas subcutaneous fat expanded by adipocyte hyperplasia . IncreasPPARγ, SREBP1, and C/EBPα (except in the clavicular fat) and upregulated C/EBPβ and KLF7 mRNA in all three adipose tissue depots. Similarly, 4 h of fasting decreased adipose tissue PPARγ and SREBP1 mRNA in 13 day-old chicks [PPARγ and SREBP1 mRNA in the adipose tissue of rats [FAS and acetyl-coenzyme A carboxylase [de novo lipogenesis, de novo lipogenesis is considered to take place almost exclusively in the liver of avian species, similar to humans [PPARγ and SREBP1 mRNA expression in chicken adipose tissue after fasting does not necessarily suggest reduced lipogenesis in response to fasting, but may reflect a downregulation of triacylglycerol synthesis in the absence of available substrate.Expression of genes involved in the early stage of adipogenesis, the commitment of mesenchymal stem cells to form new adipocytes, was downregulated in the abdominal fat of broiler chickens after 5 h of fasting . In the d chicks . These e of rats , 28. In  of rats . SREBP1 boxylase , 31. Howo humans . TherefoFABP4, LPL and others [Except for its roles in lipogenesis, the activation of PPARγ also promotes terminal differentiation, which is achieved through the induction of a variety of differentiation-dependent genes that are crucial for fatty acid uptake and storage, such as d others . In addid others . As a crd others .PPARγ and SREBP1 had similar expression patterns in each adipose depot in response to fasting, there were some depot-dependent changes. Fasting decreased AGPAT9 mRNA in subcutaneous fat, decreased NPY in abdominal fat, while it increased NPY expression in clavicular fat, and decreased NPYR2 in clavicular and subcutaneous fat. The AGPAT9 encodes an enzyme that catalyzes the initial step of de novo triacylglycerol synthesis [NPYR2 resulted in reduced body weight gain and adiposity [Although ynthesis . Sympathynthesis , and knodiposity . IncreasATGL mRNA was increased in retroperitoneal WAT after 24 but not 8 h of fasting [ATGL mRNA was not upregulated after 3 h fasting in any of the three adipose depots, indicating that lipolysis might be promoted by an upregulation of the enzyme at the protein level, similar to mammalian studies, although it should be noted that 3 h of fasting increased plasma NEFA concentrations only in chicks that consumed the HC and HP diets.In rats, circulating NEFAs increased after 4 h of fasting and peaked at 8 h, however,  fasting . Thus, t fasting . Adipose fasting . However fasting , suggestMGLL was not upregulated after fasting in any adipose tissue depot. Fasting increased NPY and ACADL mRNA and decreased CGI-58 mRNA expression in the clavicular fat of chicks. ACADL is responsible for the first step of the β-oxidation of long chain fatty acids in the mitochondria [Under basal conditions, PLN1 may inhibit lipolysis by blocking the binding of lipase to triacylglycerols and/or by sequestering CGI-58 . In the chondria . The difAccording to Nguyen et al. (2014), there was a greater sympathetic drive, triggered by food deprivation, to subcutaneous  WAT than visceral (mesenteric) WAT depots, with increased sympathetic nervous system drive only being observed in the subcutaneous  WAT after the lipolytic stimulus of food deprivation in Siberian hamsters . In genePPARγ and SREBP1 to control levels after 8 h of fasting, however, this time did not allow for the recovery of FAS [PPARγ and SREBP1.Similar to fasting, a cascade of events occurs during refeeding after fasting to allow tissues such as adipose tissue to adapt metabolically in order to restore nutritional homeostasis. In the WAT of rats, 3 h of refeeding was sufficient to restore the mRNA expression of y of FAS . In the In conclusion, reduced adipose tissue deposition in chicks fed a HP diet might be explained by decreased rates of adipogenesis and associated fatty acid incorporation into triacylglycerol synthesis. Conversely, chicks that consumed the HF diet had heavier fat pads and larger adipocytes, likely as a result of greater rates of adipocyte hypertrophy. In response to fasting, transcriptional regulation of adipogenesis-associated factors is induced, the net effect likely being an inhibition of preadipocyte differentiation and adipocyte hypertrophy, in a depot-specific manner. The lipolytic response to 3 h of fasting in chicks that consumed the HP diet also supports that the balance between adipogenesis/hypertrophy and lipid catabolism is shifted to favor a reduced rate of fat deposition. These results may provide implications for understanding how dietary macronutrients affect the development of different adipose tissue depots during the early life period and mechanisms underlying depot-dependent differences in metabolism across species.


The rhizosphere is the zone of soil influenced by a plant root and is critical for plant health and nutrient acquisition. All below ground resources must pass through this dynamic zone prior to their capture by plant roots. However, researching the undisturbed rhizosphere has proved very challenging. Here we compare the temporal changes to the intact rhizosphere pore structure during the emergence of a developing root system in different soils. High resolution X-ray Computed Tomography (CT) was used to quantify the impact of root development on soil structural change, at scales relevant to individual micro-pores and aggregates (µm). A comparison of micro-scale structural evolution in homogenously packed soils highlighted the impacts of a penetrating root system in changing the surrounding porous architecture and morphology. Results indicate the structural zone of influence of a root can be more localised than previously reported . With time, growing roots significantly alter the soil physical environment in their immediate vicinity through reducing root-soil contact and crucially increasing porosity at the root-soil interface and not the converse as has often been postulated. This ‘rhizosphere pore structure’ and its impact on associated dynamics are discussed. The zone of soil around the root that is altered by plant activity is defined as the rhizosphere. Many studies have reported distinct and unique differences with soil from the root-soil interface, including soil structure4, hydraulic properties5 and mechanical stability6. Likewise the role of exudates and associated microorganisms in affecting rhizosphere biochemistry2 and biophysics9 is well reported and widely considered beneficial to root proliferation.The physical characteristics of soil surrounding roots regulate many key plant processes and interactions including water uptake, nutrient acquisition, gaseous exchange and microbial proliferation. Soil immediately adjacent to the root has very different physical, chemical and biological properties when compared with the bulk soil10 and significantly reduce soil porosity. Aravena et al.11 showed root induced soil compaction can increase root-soil contact which has key implications for hydrological behaviour which they demonstrated by modelling approaches. Whether the physical structure of the rhizosphere is changed by natural processes such as particle rearrangement during root expansion3; shrinkage during water extraction14; mucilage exudation influencing soil water affinity17; or anthropogenic impacts such as agricultural seedbed preparation18 and vehicular traffic19, there are generally important implications for root-soil processes.Previous empirical models and experimental work have predicted root growth can cause densification in the rhizosphere due to radial expansionet al.20, have attempted to study the undisturbed rhizosphere as an entire entity i.e. in-situ without attempting to disentangle isolated processes. Thin-section microscopy techniques have enabled the exploration of rhizosphere physical21 and hydraulic properties22. North and Nobel23 successfully used field impregnated thin sections to measure root-soil contact in Opuntia ficus-indica under drought conditions to assess the impact of the rhizosheath on hydraulic conductivity. More recent advances in nano-scale secondary ion mass spectrometry have enabled the extensive exploration and imaging of nitrogen use in the rhizosphere25. However both techniques are to an extent laborious, require substantial root and soil disturbance, and do not allow for the dynamic study of living roots over time. Non-destructive imaging such as X-ray radiography26, X-ray Computed Tomography (CT)28, and Neutron Radiography17 are enabling us to overcome these limitations by providing undisturbed, rapid visualisations of the soil and root environment at a wide range of rhizosphere relevant spatial resolutions (µm to mm).To date relatively few direct investigations, such as Schmidt in-situ in unstructured (e.g. sieved) soil collected from the field; ii) assess how early-stage root development influences the physical architecture of the rhizosphere over multiple time-points; and iii) investigate the effect of soil texture i.e. soil particle size on rhizosphere development and assess specifically how the pore morphology around a growing root system relates to known functions of different parts of root axis. We used X-ray micro CT to image the development of soil micro-structure surrounding living roots in three dimensions (3-D), offering new insights into the speed and scale at which the architectural arrangement of the rhizosphere takes shape.This study focused on the biophysical impact of root growth on the micro-scale structural development in aggregating soil and creating pore space during the early stages of rhizosphere formation. Specifically, we aimed to: i) image the pore structure of the rhizosphere −3 with air dried soil sieved to ≤2 mm. The soils used were a Newport series loamy sand  and Worcester series clay loam  soil from the University of Nottingham farm at Bunny, Nottinghamshire, UK . The water retention curves and selected physical properties for these soils are provided as supplementary information , and then the matric potential lowered to −8 kPa for three days. Following this microcosms were returned to −5 kPa (over c. two days) and maintained at this tension through seedling establishment and growth. This process of wetting-drying-rewetting took approximately one week and was important to stabilise the soil structure and minimise slumping so we could isolate changes associated with root growth, without inducing any noticeable cracking due to soil shrinkage. Our pilot investigations showed one wetting and drying cycle as described above was optimal for stabilising soil structure and further wet-dry cycles had no significant effect on soil porosity measurements. Surgical micropore tape  was placed over the columns during soil preparation to reduce soil surface evaporation and prevent sample contamination from the atmosphere, whilst still enabling gaseous exchange. Seeds of tomato (Solanum lycopersicum) cv. ‘Ailsa Craig’ were germinated on moist filter paper for 48 hours before being planted 5 mm below the soil surface. Plants were grown in a controlled environment room (27 °C day/22 °C night); 40% relative humidity; a 12 hour photoperiod with a photosynthetic photon flux density at plant level of 330 µmol m−2 s−1 in a climate chamber. The mass of each sample was recorded daily to ensure the moisture content was maintained.Four replicate columns (70 mm height × 25 mm diameter), per soil texture, were uniformly packed to a bulk density of 1.2 Mg m30 and equating to a total dose of 37.8 Gy over the eight day incubation. All images were cropped 3 mm from the column edge after tomographic reconstruction to remove soil near the container sides to minimise potential edge effects  and subsequently processed in VG StudioMax® 2.1 software.All samples were scanned using a Phoenix Nanotom 180NF X-ray micro-CT scanner . The source had a 3 µm focal spot, with the centre of the sample 5.4 cm from the X-ray source and a resultant imaged voxel size of 12 µm. The entire sample was imaged with of field of view 2308 × 2308 pixels, X-ray energy of 115 kV, current of 110 µA and an exposure time of 750 ms. A 0.2 cm Cu filter was used and 1600 image projections (appropriate for this system) were taken, at an averaging of 3 and skip of 2 images per projection. Each scan took 70 minutes to complete. Although faster scan times are possible we chose this duration to optimise the image quality. Each sample was scanned at zero, two, four, six and eight days after planting , exposing each plant to a calculated dose of 7.55 Gy per scanning sessionAll data were analysed in GenStat Release 15.1  using repeated measures single-variate mixed models (REML), to isolate the effects of sampling period on X-ray CT measures of the rhizosphere structure over the eight day incubation, with a combination of sample and distance from root surface as a random effect. Standard residual plots were examined in GenStat to check data normality.The datasets generated and analysed during this study are available from the corresponding author on request.31. To assist with separating solid material from surrounding pore space, the greyscale histogram was calibrated against the soil pore space and a common aluminium reference object present in all scans using the surface calibration algorithm within VGStudioMAX (version 2.1) software . This tool uses a global thesholding approach by applying a surface at the grayscale value equal to 50% of the range between background (air space) and a material  sampled regions. The aluminium reference object was 5 mm × 3 mm × 0.5 mm and chosen as it had a similar grayscale value to the soil matrix. Its use enabled a common threshold value to be applied relative to specific X-ray attenuation across all scans at all time points. A region of interest was subsequently generated from this surface and defined as soil matrix. This soil matrix region was then inverted and the segmented root material subtracted, in order to isolate soil pore space and root material. Roots were segmented from the soil matrix using an adaptive region growing algorithm in VGStudioMax software, which starts from a fixed point containing known properties  and includes all common surrounding voxels within a specified selection. Whilst the observed image quality seemed appropriate for the study, and image blur, an important source of error32, was minimal, we chose as a precaution to exclude all data within one voxel of the root surface to compensate for any partial volume effects or mis-segmentation of the root system itself , root material and air and water filled pore space (hereafter referred to as pore space). Qualitative visual inspection of the CT images showed clear feature boundaries which aided the segmentation of phases and regions of interest Fig. . Segment32. The volume of pore and soil material within each of the dilated regions was calculated from which the 3-D porosity of each region moving away from the root could be ascertained.In order to assess soil structural changes with increasing distance away from the root surface, a new imaging protocol was developed. The segmented root region was dilated in 3-D with increasing two voxel (24 µm side length) iterations to a total of twenty voxels (240 µm) dilations, and then every five voxels (60 µm) up to fifty voxels (600 µm) Fig. . A furthIn order to assess how the porosity of each of the seventeen dilated regions altered with increasing depth down the root axis, images were false-coloured so that the pore space of the dilated region was ‘black’, with all other image features  shown as ‘white’. A top-down (XY) image stack of 1050 images for each scan was exported, with each image corresponding to a 24 µm increase in depth. This process was repeated, but with solid material within each dilated region false coloured as ‘black’ and all other features ‘white’. Coupled with the pore image stack, this gave an independent map of the pore and solid material within each dilated region. A total of 34 image stacks were exported per sample per day of growth (one pore and one soil stack per dilated region), generating 142,800 images for analysis. Each binarized image stack was approximately 4 GB in size. The total and individual area of all pore and solid material for each image was calculated, from which the porosity along the root axis at 24 µm intervals was also calculated using the particle analyser function in ImageJ.33  pore characteristics within discreet regions were analysed individually  and 2) pore characteristics within compounded (i.e. cumulative) regions moving away from the root surface  were analysed. Data were also presented as two discreet distances from the root surface, defined as 24–120 µm (‘root surface’) and 480–600 µm (‘bulk soil’) . Furthermore, roots grown in the clay loam soil exhibited higher numbers of lateral roots  soils. However the degree of structural change at the root surface was governed by soil texture and time Fig. . The loaThere was a strong relationship with the rhizosphere pore structure measured along the whole root axis and with distance from the root surface, which was soil texture dependent. There was a significant effect of distance from the root (P < 0.001) and soil texture (P < 0.001) on soil porosity as an individual factor, and a significant interaction between time and texture (P < 0.001). The loamy sand soil exhibited a larger root influenced structural zone (0.72 mm) after day two, decreasing in size with time (0.24 mm) from the root surface by day eight Fig. . This deMeasurement of soil pore thickness offers a unique way to characterise changes to the 3-D rhizosphere pore structure through space and time and across treatments, by accounting for the entire sample. Changes to pore thickness relate to the zones of influence within the rhizosphere observed in the overall mean porosity plots, with large increases in pore thickness in both the clay loam and loamy sand Fig.  confined3 of soil increased from 1.01 (±0.11) to 2.27 (±0.18) under the same conditions. This corresponded to a 700% increase in total macropore volume/mm3 clay loam soil over the eight day incubation, increasing from 5 × 10−3 (±2 × 10−3) to 4 × 10−2 (±9 × 10−3) mm3/mm3. Likewise mesopore characteristics changed over the incubation period in the clay loam soil. Mean total number of mesopores increased from 34 (±2.7) to 349 (±72.3) between days two and eight respectively at the clay soil root surface, whilst the number of mesopores/mm3 increased from 4.47 (±0.52) to 13.99 (±0.83) over the same time period. This corresponded to a 225% increase in total mesopore volume/mm3 between days two and eight, changing from 4 × 10−4 (±6.3 × 10−5) to 1.3 × 10−3 (±5.6 × 10−5) mm3/mm3. There was no significant change in the total number of macropores or macropores/mm3 in the sandy loam soil at the immediate root surface over time, but mean macropore volume/mm3 increased by 145% from 1.1 × 10−2 (±2.2 × 10−3) to 2.7 × 10−2 (±4.3 × 10−3) mm3/mm3 between days two and eight. Furthermore the total number of mesopores at the sandy loam soil root surface increased from 3 (±1.3) to 23 (±9.6), and corresponded to an increase in mesopore number/mm3 from 0.30 (±0.12) to 1.17 (±0.34) over the incubation. Mean mesopore volume/mm3 in the sandy loam soil increased by 276% from 2.5 × 10−5 (±1.3 × 10−5) to 6.9 × 10−5 (±1.9 × 10−5) mm3/mm3 between days two and eight respectively  and 3-D pore volume (P < 0.05) between day two and day eight in the clay soil Fig. , and dem and 3-D Crucially we have shown the physical architecture of the rhizosphere soil is different at selected positions along a root axis, which has not previously been measured non-invasively. We noted variations in pore thickness at discreet zones along the root axis Fig. . In part34, where a reduction in porosity was found immediately adjacent to the root using radiographic methods. They argued that the decrease in porosity was due to soil compression as the root expanded. One possible reason as to why we observed this zone of increased porosity at the root interface is the high resolution we employed (12 µm) compared to other studies such as 20. The increase in porosity adjacent to the root we found is consistent with localised particle/micro-aggregate rearrangement following the combined influence of shear and compressive forces generated by the mechanisms of root growth35. Supplementary Figure 34 equilibrated the soil to −10 kPa compared to −5kPa in this work; hence it is possible differences in matric potential which affect shear and compressive strength may account for contrasting observations.In both soils the porosity adjacent to the root was significantly higher than in the bulk soil Fig. . Within 36. Although the initial (up to day two) increase in porosity in the clay loam soil was much smaller than in the sandy loam , porosity decreased between days two and eight. This is consistent with the idea of soil compression by an expanding rootoam Fig. . The poroam Fig. . In the oam Fig. . The radet al.37 found lateral roots in a clay soil exhibited a clear root growth strategy proliferating through existing pore channels in search of water and nutrients. We also observed early root proliferation associated with pore structure in the soil, which were subsequently beneficially utilised as sites for lateral root emergence. Agar plate and rhizotron studies have reported lateral root initiation depends on localised nutrient supply39. However the proliferation of lateral roots into soil pores suggests a potential thigmotropic response, which may be missed in agar, hydroponic and possibly even sand culture experiments. The temporal changes to pore thickness mapped in Fig. 41. Hence, we hypothesise that we are observing the effects of localised wetting and drying cycles in the rhizosphere even though soil moisture content was maintained at a constant value throughout our experiment. Under moist conditions such as those in this investigation, it is widely acknowledged the clay constituents in soil swell42, increasing inter-aggregate contacts and creating increased hydraulic conductivity among aggregates43. Hence, this decrease in contact surrounding the root would be expected to have important implications for water and solute movement, by reducing the water capillary potential of the surrounding aggregates, but maintaining the pore continuity within the rhizosphere and improving oxygen and carbon dioxide movement to and from the root system respectively. Both factors are important for plant growth, and require further study if modelling approaches are to be correctly utilised.We observed that the pore structural changes in the rhizosphere were not uniformly distributed around the root Figs , 4, 6, wc. 25 GB per sample). Furthermore the imaging resolution (12–24 µm) prevented the quantification of root hairs (c. 15–18 µm diameter), which are known to extend beyond the rhizosphere pore structure reported here and impact on aggregate stability44 and nutrient acquisition45.Constraints of the present imaging approach include the comparatively small column size, limiting the investigation of young plants as the root systems beyond this become pot bound, and the significant computing hardware limitations in handling such large datasets  creating a highly porous region at the root:soil interface and further matures over time. In addition, we found the physical architecture of the porosity within the rhizosphere has a number of structurally distinct zones some of which are far smaller than known biological and chemical gradients surrounding growing plant roots, i.e. within the order of μm as opposed to mm. We have shown that initially the porosity of the rhizosphere soil adjacent to the roots increased in contrasting soils but thereafter, it decreased in a sandy loam soil whereas it increased in a clay loam soil. The temporal changes in this rhizosphere pore structure were consistent with expected differences considering in the conventional physical behaviour associated with these soils. These results highlight the importance of the representing the microscopic soil physical environment, and in particular, the different soil structural regions that exist within the rhizosphere in future modelling efforts concerning the behavioural dynamics of the rhizosphere particularly those concerned with water, nutrient and gas flows around plant roots.Supplementary Figures


We establish Lyapunov-type inequalities for a system involving one-dimensional  More precisely, he proved that, if u is a nontrivial solution of the problemφ-Laplacian operator:The standard Lyapunov inequality  -(DBC) is established in this section, and some particular cases are discussed.If (S)-(DBC) admits a nontrivial solutionthenv, we obtainu and integrating over v and integrating over Let mentclasspt{minimamentclass2pt{minimmentclass2pt{minimAs a consequence of Theorem Let us assume that there exists a nontrivial solution ofwhereandThenAn application of Theorem p-Laplacian equation.Taking mentclass2pt{minimTaking p-Laplacian systems is due to Nápoli and Pinasco [The concept of generalized eigenvalues was introduced by Protter  for a sy Pinasco . Inspireσ.Let us consider the generalized eigenvalue problemWe assume thatmentclass2pt{minimThe following result provides lower bounds of the generalized eigenvalues of Letbe a generalized eigenvalue ofThenwhereis the function defined bywithLet equality  the funcmentclass2pt{minimAs consequences of the previous obtained result, we deduce the following Protter’s type results for the generalized spectrum.There exists a constantthat depends onaandbsuch that no point of the generalized spectrumσis contained in the ballwhereandis the Chebyshev norm inLet ∈σ. From , we obtamentclass2pt{minimLetbe fixed. There exists an intervalJof sufficiently small measure such that, ifthen there are no nontrivial solutions ofC is defined in Theorem Suppose that ion with . TherefoLyapunov-type inequalities for a system of differential equations involving one-dimensional


The data presented in this article are related to the research article entitled “Hygro-thermal and durability properties of a lightweight mortar made with foamed plastic waste aggregates”   Specifications tableValue of the data•The data indicate the suitability of end-of-waste plastic aggregates for producing a lightweight mortar.•The data can be used as comparison by researchers that are investigating lightweight mortars containing different plastic aggregates.•The provided data can be used for validation or calibration of analytical models.•The experimental data reported a reduced thermal conductivity, water vapour resistance and capillary absorbed water at increasing plastic aggregates content.•Data can be further enriched using other characterization techniques.1In this dataset are listed the results of a study concerning the use of plastic aggregates  according to EN 12390-7 Wab) was determined as the ratio between the amount of absorbed water (difference between the mass of the saturated and dry specimen) and the mass of the dry mortar specimen. Water absorption and water absorption variation, compared to the reference sample, are reported in Water absorption  was calculated according to EN 1015-18 M2 and M1 are the mass of the specimen, in grams, after 90 min and 10 min of immersion, respectively. Prior to test, specimens were oven dried up to constant mass at 60 °C. Then, prismatic samples were immersed in deionized water for about 5 mm and the mass variation was measured. The results are reported in The absorption of water due to the capillary rise was determined on three prismatic specimens for each mixture and the capillary water absorption coefficient . Wvp was measured on three flat cylindrical specimens  was measured according to the following equation:A is air permeability (1.94⋅10-10 kg/(Pa m s)) in test conditions (20 °C and 50% RH) and Wvp is water vapour permeability. The results are summerised in Water vapour permeability, 3 (Thermal conductivity measurements were carried out on mortar specimens of size equal to 30 × 30 × 5 cm3 a, manufa3 b was des


Many challenges must be overcome in order to create reliable electrochemical energy storage devices with not only high energy but also high power densities. Gaps exist in both battery and supercapacitor technologies, with neither one satisfying the need for both large power and energy densities in a single device. To begin addressing these challenges (and others), we report a process to create a self-assembled array of electrochemically active nanoparticles bound directly to a current collector using extremely short (2 nm or less) conductive tethers. The tethered array of nanoparticles, MnO in this case, bound directly to a gold current collector via short conducting linkages eliminates the need for fillers, resulting in a material which achieves 99.9% active material by mass (excluding the current collector). This strategy is expected to be both scalable as well as effective for alternative tethers and metal oxide nanoparticles. Electrochemical energy storage devices are the leading means of storing charge where size and weight are critical12345241782361011122478143x) nanoparticles tethered to a current collector using conductive organic linkers of 2 nm or less. Depending on the specific choice of active MnOx material, this self-assembled structure can be used as an anode for Li-ion batteries (MnO), a cathode for Li-ion and Li metal batteries (MnO2), an electrode for an oxide supercapacitor (MnO2), or a catalyst for oxygen reduction in Li-air batteries (MnO2)1517181920212223242526272829303132333435363738x gives the synthetic route and nanoparticle structure reported on here a wide breadth of potential applications. In the oxide supercapacitor application, MnO2 nanoparticles can exhibit pseudocapacitance two ways: faradaic reactions in aqueous solvents (such as a solution of Na2SO4) and “extrinsic” pseudocapacitance through Li+ intercalation in organic electrolytes at sites located at the surface or near-surface regions9313940414243x based device would be able to support rapid charge and discharge times due to the extremely short intercalation distances (5 nm or less), operating much more like a supercapacitor and with increased power densities. There has even been at least one report of MnO2 in an oxide supercapacitor application where a solvent containing multivalent (in this case Ca2+) ions was usedx based nanoparticle structure extremely versatile in the field of energy storage.While the work presented herein does not address all of the aforementioned issues, it does provide one significant step towards achieving high reliability, energy density, and power density in a single affordable device. Presented here is the development of a synthetic route for manganese oxide , are widely regarded as excellent charge storage materials due to the availability of multiple oxidation states and the structural ability to incorporate cations3233342) and many Li-ion cathode materials, especially those rich in cobalt and nickel (LiCoO2 and Li(NiMnCo)O2). For example, although RuO2 has a very high specific capacitance (~1000 F/g), a RuO2 based capacitor large enough to power an electric vehicle would cost over $1 millionth most abundant element found in the earth’s crust with Ti and Fe being the only transition metals found in higher quantitiesAs discussed above, our choice of active material are the manganese oxides, which in addition to their broad range of potential applications . Several syntheses were identified but a few proved to be irreproducible in our laboratory. A communication published by Seo et al.−1) and CH stretching modes , which are strong indications that the 4-bromoaniline was bound to the surface of the MnO nanoparticles. Similar shifts to lower frequency have been reported upon aniline binding to other transition metals51We hypothesized that it should be feasible to replace the oleylamine ligands used in the synthesis published by Seo ν(NH2) = 3238 cm−1, ν(CH) = 2931 cm−1, νs(SH) = 2549 cm−1, ν(CN) = 1085 cm−1, γ(CH) = 838 cm−1. The presence of the phenyl group stretching mode overlaps with water peaks present in the SAM spectra and due to the weaker absorption signal associated with attenuated total reflectance (ATR) FTIR measurements of the SAM they were not discernable.The next step in preparing a self-assembled array of tethered nanoparticles was to prepare a SAM on a suitable current collector. Gold was chosen as the current collector since thiol based SAMs on gold are extremely well characterized, their preparation straightforward, and several studies completed on the electron transport in thiol based molecular junctions53545556585956The final step in forming a tethered array of MnO nanoparticles was in making a chemical linkage between the nanoparticle ligands and the self-assembled monolayer (SAM). An amination reaction between the bromine termination on the 4-bromoaniline and the 4-aminothiophenol forms a linkage between the particles and the SAM. 58ν(NH2) = 3359 cm−1, ν(CH) = 2929 cm−1, ν(CN) = 1070 cm−1, γ(CH) = 806 cm−1. The increase in the area under the ν(CN) peak gives us additional confidence that a secondary amine linkage was formed and the number of CN bonds for the system doubled.We deliberately avoided the use of catalysts to aid the amination reaction in order to exclude impurities. Nevertheless, it is certainly possible that Pd, Cu, or CsOH based catalysts could make the amination reaction more efficient. We allowed the nanoparticles and SAM on gold to react in a solution of dimethylformamide (DMF) over a period of 48 hrs. We did experiment with elevated temperatures (50 °C) to make the reaction more efficient but there was evidence that either decomposition of the organic ligands, decomposition of the SAM, and/or undesirable side reactions were occurring. A FTIR spectra of the tethered MnO nanoparticles, with the spectra of the 4-aminothiophenol SAM on gold provided as a reference, is displayed in Further characterization of the self-assembled array of nanoparticles (and confirmation of a successful synthesis) was provided through high resolution scanning electron microcrope (SEM) imaging of the nanoparticle SAM. A high resolution SEM image of the surface is shown in A self-assembled array of MnO nanoparticles bound directly to a gold current collector using short (~1.5 nm) conducting organic linkers was synthesized and characterized. This was accomplished by incorporating complementary reactive organic ligands to both MnO nanoparticles and a gold substrate. Dimethylformamide (DMF) proved to be the most effective solvent for this reaction with results confirmed by FTIR spectroscopy. This array of tethered nanoparticles, and new arrays fabricated in a similar fashion, can have an immediate impact as a new route to fabricate oxide supercapacitor electrodes, Li battery electrodes, and Li-air battery catalysts that can operate in both aqueous and organic electrolytes. Furthermore, tethered nanoparticles are a step towards addressing some of the most difficult challenges facing electrochemical energy storage: achieving high energy and power density in the same device, increasing cycle life through a structure more impervious to degradation, decreasing inactive electrode material, and improving charge transfer.3O4, or MnO2 nanoparticles tethered to a current collector through facile modification of the nanoparticle synthesis or by post synthesis electrochemical oxidation of the manganese. Additionally, the approach can be modified to accommodate a wide range of metal and metal oxide nanoparticles, organic linking chemistries, and substrates. As a logical next step, the electrochemical response of our tethered MnO nanoparticle array in both aqueous and organic electrolytes will be characterized. We also hope this work will excite other researchers to build upon these results and begin to address key problems in electrochemical energy storage.The self-assembly synthesis approach presented here is amenable to coating both small and large scale areas up to and beyond a meter. Furthermore, this single synthetic route can easily produce an array of MnO, Mnet al. with a few notable differences. Specifically, the oleylamine surfactant and solvent was replaced with 4-bromoaniline. A manganese(II) acetylacetonate or [Mn(acac)2]:4-bromoaniline:water molar ratio of 1:24:10 (0.9 g Mn(acac), 14.6 g 4-bromoaniline, and 0.630 g water) was used and the synthesis was run at 200 °C for 12 hours. The resulting brown suspension was cooled to room temperature and the particles recovered by centrifugation for 15 min. at 4500 rpm and the supernatant removed. A mixture of 10 mL of ethanol and 20 mL of hexanes was added to the brown precipitate, which was vortex mixed and sonicated for 15 min. at 50 °C to redisperse the particles. The particles were pelleted by centrifuging at 4500 rpm for 15 min. and resuspended in hexanes.All chemicals and reagents were purchased from Sigma-Aldrich and used as received. Water used in the synthesis of the MnO nanoparticles was treated by a building wide DI water system and had a measured resistivity of 18.2 MΩ. The MnO nanoparticle synthesis followed the approach as described in the publication by Seo 4-aminothiophenol monolayers were formed on 1 cm square gold coated silicon wafers . The substrates were sonicated in ethanol for 20 min. and then cleaned using UV/ozone for an additional 20 minutes just prior to immersion in a 1 mmol solution of 4-aminothiophenol in ethanol for a period of 48 hrs. At the completion of the 48 hr. soak, the SAM coated substrates were removed from the 4-aminothiophenol solution, rinsed with ethanol, and blown dry with nitrogen. In order to form the layer of tethered MnO nanoparticles, a 100 μL solution of MnO particles dispersed in hexanes were added to 10 mL of dimethylformamide (DMF) and allowed to soak at room temperature for a period of 48 hr. along with the SAM coated gold substrate. After the 48 hr. soak time, the nanoparticle coated substrates were removed from the solution, rinsed with ethanol, and blown dry with nitrogen.Fourier Transform – infrared (FTIR) spectra were obtained with a Bruker IFS 66 S FTIR spectrometer  in a sample chamber purged with nitrogen gas. MnO nanoparticles were pressed into a KBr pellet with a sample concentration of 2% (w/w). Reference spectra of neat 4-aminothiophenol and 4-bromoaniline were collected by depositing the liquid samples between two KBr windows. FTIR spectra of the 4-aminothiophenol monolayers and tethered MnO nanoparticles were collected via the reflectance-absorbance FTIR spectroscopy (RAIRS) technique using the Seagull accessory from Harrick Scientific .Bright field TEM images were acquired with a JEOL 1200 EX  using an acceleration voltage of 120 kV. The instrument has a point to point resolution of approximately 9 Å. Images were collected on an Advanced Microscopy Techniques (AMT) XR280L-A 2.8 Mpixel CMOS camera . Samples for TEM analysis were prepared by depositing the nanoparticles onto a holey carbon coated copper TEM grid . A 3 μL aliquot of MnO nanoparticles suspended in hexanes was withdrawn from a vial immediately after vigorously vortexing and then added to the surface of the TEM grid. Filter paper placed under the TEM grid was used to aid in wicking away excess solvent and to evenly disperse the nanoparticles across the holey carbon surface.in situ 2 minute plasma clean of the sample. In this BD mode, a primary beam energy of 1 kV was emitted from the Field Emitter gun and combined with a beam deceleration of 500 V at the sample, which yielded a 500 V beam on the sample surface. This low energy beam voltage provided high resolution imaging with high surface sensitivity, minimizing sample interference without sacrificing resolution and contrast.SEM images were collected using the FEI Magellan 400 Extreme High Resolution (XHR) SEM. Images displayed in the publication were taken at a 1.6 mm Working Distance with a 500 V/3.1 pA beam in Beam Deceleration (BD) mode after an All plots were generated using Igor Pro software .How to cite this article: Stevens, T. E. et al. Self-Assembled Array of Tethered Manganese Oxide Nanoparticles for the Next Generation of Energy Storage. Sci. Rep.7, 44191; doi: 10.1038/srep44191 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The thiolate compounds are readily prepared via the reaction of metal bases and tertiary-alkyl thiols. The thiolate complexes are soluble in common organic solvents and can be solution processed by spin coating to yield thin films. Upon thermal annealing in the range of 200–400 °C, the tertiary-alkyl thiolates decompose cleanly to yield volatile dialkyl sulfides and metal sulfide films which are free of organic residue. Analysis of the reaction byproducts strongly suggests that the decomposition proceeds via an SN1 mechanism. The composition of the films can be controlled by adjusting the amount of each metal thiolate used in the precursor solution yielding bandgaps in the range of 1.2 to 3.3 eV. The films form functioning p-n junctions when deposited in contact with CdS films prepared by the same method. Functioning solar cells are observed when such p-n junctions are prepared on transparent conducting substrates and finished by depositing electrodes with appropriate work functions. This method enables the fabrication of metal chalcogenide films on a large scale via a simple and chemically clear process.We report the preparation of Cu Since their discovery, solar cells have been recognized as an appealing energy source due to the abundance of solar energy, their simplicity and lack of moving parts and their ability to generate power in remote locations. Although the practical use of solar modules in the past has been confined to niche applications (where their high cost could be justified), the aggregate amount of energy produced by solar modules has increased by more than one thousand times over the past 20 years and power generation from photovoltaics now constitutes approximately 1% of global electrical energy productionRecent years have witnessed the development many new types of solar technologies which can be deposited like inks by low cost solution coating techniques including organic bulk heterojunction (BHJ) solar cells22 materials have been shown to yield PCEs of over 20%8102 cells can be fabricated by processing films from solutions of hydrazinium metal selenides (using hydrazine as a solvent) followed by thermal annealing and selenization of the precursor films2 type solar cells, hydrazine is a toxic, corrosive and chemically reactive solvent. Alternatively, CuSe2 nanoparticles (NPs) can be synthesized and deposited by solution processing1314Another route to accomplish the same goals as BHJ, DSSC, CQD and perovskite approaches is to develop strategies to prepare more traditional inorganic solar cells by solution processing methods. CuInSThe ultimate goal of researching solution-processed solar cells is to enable the manufacture solar cells on a large scale using industrial printing equipment. In the context of large-scale printing operations, inks based on non-toxic and non-polar solvents offer the best performance due to their low reactivity, low surface tension, good wettability, evaporation rates and ability to form smooth and uniform films. Thus, the ability to process solar cells using non-toxic, non-polar solvents offers the best compatibility with industrial-scale printing equipment.2 or CuSe2 solar cells being deposited from precursors using common organic solvents1617181921There are few examples of CuInS1 or E1 mechanisms at elevated temperatures to yield volatile iso-butene and CO2 byproducts2425In this contribution, we have used a rational, organo-chemical approach to design metal complexes which are soluble in non-toxic, non-polar solvents and decompose thermally to yield pure metal sulfide films. The t-butoxy carbonyl (TBOC) group was used as a model and starting point in the design of these complexes; the TBOC moiety is well-known to decompose quantitatively via SN2 and CuS2 films can be readily prepared by annealing TAMT films which are deposited from solutions in common organic solvents, as illustrated in We show that CuInS2 precursors which thermally decompose via a well-understood reaction. The complexes yield volatile by-products with low reactivity  and metal sulfide films which are free of organic residue. The simple reagents used, coupled with the straightforward synthesis and processing of the materials make this approach well-suited for large scale solar cell production.This report marks the first of its kind to utilize rationally designed CuInSThe TAMT materials were conveniently synthesized by reacting metal alkoxides or acetates with tertiary alkane thiols as shown in In order to quantify the thermal decomposition of the precursors into metal sulfides, thermogravimetric analysis (TGA) was performed. The 1:1 molar mixture of CuTDT and InTDT converts to the metal sulfide phase somewhat more gradually over a range of 46.5 °C, losing 62.8% of its weight without showing two distinct decomposition steps. The losses in mass are consistent with the thermal decomposition mechanisms outlined in In order to confirm the decomposition mechanism and identity of the reaction by-products, we decomposed samples of the complexes by heating to 300 °C under vacuum and trapped the volatile decomposition products with a liquid nitrogen trap. The decomposition products were dissolved in chloroform and analyzed by gas chromatography -mass spectrometry MS, see  and Rama2S, i.e. the alkene, indicating that the C-S bond of the thiol was completely fragmented during ionization in the MS instrument. This is consistent with reference MS spectra of t-dodecanthiol (2-methyl-2-undecanethiol) which show no peak at M = 202 corresponding the un-fragmented compoundA control sample consisting of the thiol starting material was analyzed as a reference, however, no peak corresponding to the MW of the parent thiol (M = 202) was observed in the MS spectrum and the largest MW observed in the thiol spectrum (M = 168) corresponded to the MW of the thiol – H1 mechanism as shown in 1 mechanism, , the Raman spectrum becomes much sharper while the relative intensity of the shoulder decreases, indicating that the tetragonal chalcopyrite phase crystallizes and that the crystal quality improves upon annealing at higher temperature. However, it is apparent that the crystallinity of films post-annealed in this way is still inferior to films which are epitaxially grown or sulfurized at high temperatures.The presence of CuInS300 cm−1 . Phase-p2S, In2S3, and different compositions of CuS2 are shown in 2S absorbs light up to 1020 nm optical energy gap (Eg (opt) = 1.22 eV), whereas In2S3 exhibits an absorption onset at 550 nm. (Eg (opt) = 2.25 eV). CuInS2 films were prepared by spin coating a mixed TAMT solution with 1:1 molar ratio, the CuInS2 absorption spectra exhibit a clear absorption onset which is distinct from either Cu2S or In2S3. This implies that a distinct CuInS2 phase is formed, as opposed to a mixture of Cu2S and In2S3 phases.UV-vis absorption spectra of Cu2 phase after annealing at 400 °C and 570 °C was confirmed by X-ray diffraction (XRD)  . Diffrac2 by adding GaTDT to mixed CuTDT/InTDT solutions. This process leads to a widening of Eg in the CuS2 films, as Ga atoms replace In atoms in the structure. A 3:1 molar ratio of In:Ga in CuS2 film exhibits a blue-shifted absorption onset by 60 nm, while a 140 nm blue-shift is observed in the film with 1:1 molar ratio of In:Ga. In contrast, pristine Ga2S3 films show virtually no absorption of visible light due to the wide bandgap of Ga2S3 (3.26 eV). 2 films. As we confirmed in TGA, TAMTs could be converted to metal sulfides below 200 °C. However, films were annealed at 300 or 400 °C to encourage crystallization and to obtain suitable absorption data; it is clear that films annealed at 400 °C show sharper absorption onsets and higher optical density than films annealed at 300 °C. For comparison, absorption data were plotted as Tauc plots of (αhν)½ vs hν 2 plots showed good fits as well, except for In2S3, which has been shown to have both direct and indirect optical transitions, causing an indistinct absorption onset which is consistent with previous observations31As expected, were able to tune the optical bandgap of CuInS)½ vs hν  and  images were taken of the surface. An SEM image of a CuInS2 film annealed at 300 °C for 10 minutes is shown in The surface morphology of each film was characterized by atomic force microscopy (AFM). AFM images of Cu 400 °C, , the hor2 films and CuInS2/CdS/ZnO trilayer films prepared on glass/ITO substrates were collected as well. These images are shown in 2 films are approximately 80 to 100 nm thick.SEM images were collected for cross-sections of CuInS2S, In2S3 and CuInS2 films  data  of each layer were obtained from the Fermi edge relative to an Au reference. We completed the energy band diagram deducing the level of the conduction band edge (ECB) of each film from the collected UV-vis absorption data. ZnO and CdS layers showed deep EVBs of 7.2 eV and 6.5 eV, respectively, while three different CuS2 films exhibit the same EVB at 5.4 eV. This implies that the different absorption onsets of these films can be ascribed to changes in ECB due to Ga incorporation.X-ray photoelectron spectra (XPS) were collected for CuS2 films  confirmiPS) data  and UV-vPS) data  were use2/MoO3/Au structure. CuInS2 layers were spin cast with 1:1 molar ratio of CuTDT and InTDT and annealed at 300 °C for 45 min. CdS window layers were also fabricated from Cd thiolate precursor following a similar method developed by our group2 solar cells are shown in 2 layer by varying the number of coats. Using an overall concentration of 100 mg/mL in THF and a spin rate of 1500 rpm, a thickness of ~80–100 nm is obtained for each coat. Devices with 2 coats of CuInS2 layer showed better current density (JSC) and open circuit voltage (VOC) compared to devices with 1 coat of CuInS2. However, further increases in the thickness of the CuInS2 layer caused a decrease in performance. We presume that the annealing temperature of 300 °C was not sufficient to fully crystallize the CuInS2 phase, which leads to poor carrier extraction in thick films. A decrease in VOC in the device with more than 2 coats of CuInS2 indirectly verifies an increase in the charge carrier recombination rate in thicker CuInS2 layers.Solar cells were prepared by spin coating and annealing TAMTs onto conductive, transparent ITO substrates with an ITO/ZnO/CdS/CuInS2 include a JSC, VOC, FF and PCE of 5.8 mA/cm2, 0.472 V, 0.408 and 1.11%, respectively. 2 layer would increase the JSC. However, the EQE was significantly reduced after 2 coats of CuInS2. This indicates that the reduced EQE can be attributed to lowered internal quantum efficiency of the device, or poor charge carrier extraction due to the ill-defined crystalline structure confirmed by Raman (2 film (two coats) first onto glass/ITO substrates, followed by deposition of CdS and ZnO layers. A J-V curve corresponding to this device architecture is shown in SC, VOC, FF and PCE of 7.4 mA/cm2, 0.497 V, 0.403 and 1.53%. These PCE values are comparable to other CuInS2 devices prepared by solution processing methods such as NPs or other precursors without a high-temperature annealing or selenization step13142 or CuSe2 devices which are grown epitaxially or selenized at high temperature. Although the films are chemically pure, the relatively low PCE and thin optimum active layer thickness are consistent with a disordered film structure with small crystal domains, where abundant grain boundaries lead to short minority carrier diffusion lengths and limit the active layer thickness. Initial attempts to increase the annealing temperature to 400 °C unfortunately led to a dramatic drop in VOC and high rate of device failure, which we presume may be due to instability between the ITO substrates and deposited chalcogenide layers at high temperatures. Although the band structure of the Ga-doped films indicates a slope in the conduction band which should provide an increased driving force pushing photogenerated electrons towards the p-n junction, initial attempts to incorporate Ga into the device structure yielded low JSC values. We are currently investigating methods to improve the crystallinity of the materials using higher annealing temperatures with alternative substrates such as molybdenum, as well as post-deposition selenization, which we anticipate will allow much higher efficiencies.The solar cell characteristics of the device using an active layer comprising two coats of CuInSby Raman  and XRD by Raman . The dif1 mechanism, analogously to the TBOC group. Binary, ternary, and even quaternary systems of metal sulfide films were solution-processed using mixed TAMT precursor solutions by spin casting followed by mild thermal annealing. The versatility of this method was demonstrated using four different metal complexes. The films were characterized electronically and exhibited band structures consistent with semiconducting Cu2S, In2S3, CuInS2 and CuS2 films. Functional solar cells were fabricated using the same techniques and equipment used for polymer solar cells by successive deposition of metal sulfide layers. This report marks the first of its kind to demonstrate the preparation of environmentally friendly CuInS2 type solar cells from non-polar thiolate precursors which are soluble in common organic solvents. In contrast with the widely used epitaxial growth methods used to prepare metal chalcogenide films, this approach constitutes a rapid, low-cost, strategy to prepare metal chalcogenide thin films, which is compatible with large-scale deposition methods such as printing or roll-to-roll coating. It offers great potential for application in the fabrication of a range of large-area semiconducting devices such as solar cells, LEDs, photodiodes and transistors, as well presenting the possibility for use in conjunction with solution-processed organic and hybrid semiconducting devices.Various metal thiolates  were synthesized as effective metal sulfide precursors through a low-cost and straightforward synthetic strategy. Spin coated films could be easily processed using non-polar and non-toxic solvents and decomposed cleanly at moderate temperatures to yield films free of organic residue. The volatile decomposition products were identified as dialkyl sulfides indicating that the complexes decomposed via a well-defined SNAll reagents were procured from Aldrich or Alfa Aesar and used as received. Unless otherwise noted, all materials were handled in a nitrogen filled glovebox or using standard Schlenk techniques. TAMT materials were stored in a nitrogen filled glovebox and protected from light by wrapping in aluminum foil. It should be noted that the thiols used in this work possess a very strong odor and should be handled carefully.Copper (II) acetate monohydrate  was dissolved in a mixture of 12 mL ethanol and 12 mL water yielding a deep blue solution. A small amount of solid did not dissolve. t-dodecanethiol  was separately dissolved in 8 mL of ethanol. The two solutions were combined in a large vial and shaken for 15 minutes, during which time the liquid phase changed from blue to green to hazy yellow, indicating a reduction of Cu (II) to Cu (I), while the remaining copper acetate dissolved. The mixture was centrifuged at 2000 rpm for 5 minutes and a thick oily phase separated to the bottom. The supernatant liquid was discarded and the oily phase was washed several times with anhydrous methanol. The viscous material was transferred to a Schlenk flask and dried under vacuum overnight.In a nitrogen-filled glovebox, indium isopropoxide  was weighed into a 10 mL vial and dissolved in 4 mL of anhydrous THF. T-dodecanethiol  was added to the solution and shaken vigorously. The mixture was allowed to stand at room temperature for 2 hr. before being filtered through a 0.45 micron PTFE syringe filter into a Schlenk flask. The solvent and excess thiol were removed under vacuum and the resulting viscous clear material was dried under vacuum overnight.The same procedure as used for InTDT was used, with the exception that gallium isopropoxide  was used instead in place of indium isopropoxide.Was prepared from cadmium acetate and t-nonanethiol following a previously reported procedureUnless otherwise noted, films were prepared by dissolving metal TAMTs in THF at a concentration of 100 mg/mL, filtering the solution through a 0.45 μm PTFE filter and spin casting at 1500 rpm. Films were then annealed on digital hotplates, in a nitrogen filed glovebox at temperatures in the range of 200–400 °C for 10 minutes to facilitate conversion of the TAMTs to sulfide films.TGA traces of each sample were collected using a TGA instrument, Q500. To collect each trace, ~20 mg samples were put in aluminium pans and the temperature was raised from 25 to 450 °C at 5 °C/min. XRD patterns were collected using an X-ray diffractometer D/MAX2500V/PC . Powder samples for TGA and XRD were prepared through annealing of TAMT complexes at 300 °C in a vial under vacuum. Gas chromatography-mass spectrometry (GC-MS) spectra were gathered using Bruker 450-GC & 320-MS. For the measurement, samples were prepared as described in the main text. UV-vis spectrometry was performed using a Varian Carry 5000 spectrophotometer in the range 300 to 1200 nm with 1 nm resolution. AFM images were collected using a Veeco Multimode microscope with 300 kHz silicon tips operating in tapping mode.2 film samples prepared as described in the text. Raman spectra were measured using 532 nm excitation wavelength with a WITec alpha300R confocal Raman microscope. Laser beam power was 1 mW and the collected spectra were integrated 10 times.SEM images were gathered using NanoSEM 230, FEI. For Raman measurements, sample was prepared by heating an equimolar mixture of Cu and In thiolates to 300 °C under vacuum and collecting the volatile byproducts using a liquid nitrogen trap and dissolving this material in chloroform. Raman spectra were additionally collected for CuInS−9 Torr. UPS measurements were carried out using a He I (hv = 21.2 eV) source while XPS measurements were performed using a monochromated Al-Kα X-ray source. UPS and XPS spectra were collected for films annealed at both 300 and 400 °C, but did not show significant differences.50 nm thick gold films were deposited on pre-cleaned Si substrates with a thin native oxide. Solutions were spin cast onto the gold films using the same procedures used to fabricate devices, as described in the text and device fabrication sections. UPS and XPS experiments were carried out using a Thermo Fisher Scientific ESCALAB 250XI which was maintained at a base pressure of 1.0 × 10–6 torr; 1 torr ~133 Pa), and 100 nm thick Al electrodes or 80 nm thick Au electrodes were deposited on top of the active layer by thermal evaporation. J-V measurements were carried out using a Keithley 2635A source measurement unit with the solar cells inside a nitrogen filled glove box using a high quality optical fiber to guide the light from a Xenon arc lamp to the devices. An aperture with an area of 13 mm2 defined the active area of the devices. The J-V were measured under AM 1.5G illumination at 100 mWcm−2 calibrated with a Si photodiode  which was standardized at the National Renewable Energy Laboratory in Golden, Colorado (US). Reported J-V curves represent the best devices for each condition tested. EQE measurements were conducted in ambient air using an EQE system (Model QEX7) by PV measurements Inc. . Spectral mismatch factors between the simulator and AM1.5G spectrum were found to be 10% or less.Glass/ITO substrates were cleaned with detergent, then ultra-sonicated in acetone and isopropanol and subsequently dried in an oven overnight at 100 °C. Metal sulfide films were processed from solutions of each TAMT in THF as described above. In the case of CdS, a solution of CdTNT in chloroform (6 mg/mL) was used. ZnO films were deposited by diluting 200 µL of a solution of diethyl zinc in toluene  with 400 µL of anhydrous THF, filtering the diluted solution through a 0.45 um PTFE filter and spin coating this solution in air at 3000 rpm, followed by annealing in air at 110 °C for 10 min. Completed devices were pumped down in vacuum .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.