V1.8 is expressed in the peripheral nervous system in primary afferent nociceptive C-fibers and is essential for noxious cold signaling. We utilized functional magnetic resonance imaging on NaV1.8-deficient (NaV1.8−/−) compared with wildtype (WT) mice to identify brain structures decoding noxious cold and/or heat signals. In NaV1.8−/− mice functional activity patterns, activated volumes and BOLD signal amplitudes are significantly reduced upon noxious cold stimulation whereas differences of noxious heat processing are less pronounced. Graph-theoretical analysis of the functional connectivity also shows dramatic alterations in noxious cold sensation in NaV1.8−/− mice and clearly reduced interactions between certain brain structures. In contrast, upon heat stimulation qualitatively quite the same functional connectivity pattern and consequently less prominent connectivity differences were observed between NaV1.8−/− and WT mice. Thus, the fact that NaV1.8−/− mice do not perceive nociceptive aspects of strong cooling in contrast to their WT littermates seems not only to be a pure peripheral phenomenon with diminished peripheral transmission, but also consists of upstream effects leading to altered subsequent nociceptive processing in the central nervous system and consequently altered connectivity between pain-relevant brain structures.The voltage-gated sodium channel subtype Na V1.8 was shown to be responsible for the continued excitability of nociceptors in noxious cold conditions because of its specialized inactivation properties. Its ablation results in a cold resistant phenotype in mice reported and validated from different laboratories2. NaV1.8 is expressed especially in peripheral sensory neurons as well as in small and medium-sized DRG neurons and their axons5 and in at least 75% of slowly conducting C-fibers in the peripheral nervous system6. Thus, high levels of the tetrodotoxin-resistant channel  were detected in sensory but not in central neurons7.Evolutionary pressure requires nociceptive processing functions through the entire range of noxious temperatures from hot to cold to enable protection of the organism from hazardous tissue damage. Previously the sodium channel Na9. Recent research, however, uncovered that the identified areas were not only specifically related to nociceptive processing but process salient signals originating from multisensory input rather than to generate the feeling of pain alone11. A functional magnetic resonance imaging (fMRI) study on human subjects dealing with noxious heat (46 °C) and cold (5 °C) stimulation ruled out that the patterns of brain activation upon noxious heat and cold stimulation were quite common12. Significant differences of activation properties between hot and cold conditions were detected in prefrontal areas12. The impact of the sodium channel NaV1.8 on cold and heat nociception, however, was not addressed in this study. More recently, imaging studies were also performed in rodents, which showed surprisingly consistent results with human brain imaging studies, in regard to quite similar activation patterns14. In particular, a pattern of activated areas in the medial and lateral pain system was detected upon nociceptive processing14. Therefore, these findings demonstrate the potential of functional imaging for translation of findings from mice to humans.In the past, several functional imaging studies addressed the question of which brain regions are uniquely required for and presumably activated during the perception of pain in humans15. Specifically, we focused on the impact of a lack of the voltage gated sodium channel NaV1.8 on the cerebral manifestation of noxious cold and heat temperatures to identify the related brain structures and their interactions contributing to the perception of cold and heat noxious input.In this context we sought to utilize functional magnet resonance imaging (fMRI) in combination with genetically modified mice as a versatile combination to study functions of specific genes/proteins within central processing of noxious input informationV1.8-deficient (NaV1.8−/−) mice had revealed a strongly attenuated sensitivity to cold in the cold plate test compared to the wildtype (WT)2 and a mildly reduced sensitivity to heating in the Hargreaves but slightly increased sensitivity in the hot plate test7. Along this we applied noxious cold (0–20 °C) and heat (40–55 °C) stimulation to the right dorsal hind paws of NaV1.8−/− mice and their WT littermates and simultaneously measured blood oxygenation level dependent (BOLD) fMRI.Previous behavioural thermal pain tests on Nain-vivo experiments in animals was reviewed by the local animal ethics committee  and approved by the local district government .The care and use of animals was conformed to the national guidelines. All experimental protocols were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the relevant guidelines and regulations concerning animal experiments, i.e. the protocol for V1.8−/− and WT-mice of the C57/BL6 strain weighting between 20 and 25 g and bred at the Department of Physiology and Pathophysiology, Erlangen as for the previous paper2, where all details of the mouse strain and details on their behavioural assessments were already published. Therefore, behavioural experiments were not duplicated for the recent approach, particularly in support of the 3R concept of refinement and reduction of animal experiments16. From the initially 13 measured animals per group 10 animal data sets per group were selected for further analysis, 3 animals per group were excluded due to strong motion artifacts caused by irregular breathing. This resulted in n = 10 animals per group. Each mouse was used for both, cold and heat stimulation, at the laboratory of the Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander University Erlangen-Nürnberg (FAU). Delay between experiments was minimum 3 days, 7.4 ± 2,3 days for the NaV1.8−/− group and 5,2 ± 2,8 days for the WT group. A light/dark cycle was pretended simulating natural conditions. Food and water were given ad libitum. The fMRI preparation  took 30–40 min, a time in which also a constant physiological pCO2 level (around 38 mmHg ± 10%) was reached17. The anaesthesia of the mice was induced with medical air plus 5% isoflurane in a vaporisator for 7 min. The animals were placed on a cradle and fixed by a bite bar. A dedicated surface coil  was mounted and the animals were then moved inside the MR scanner . During the whole mounting and the following MRI session a respiration rate around 100 ± 20 BPM and therefore a constant pCO2 level was achieved by slight adjustments of the isoflurane supply (around 1.2%). Respiration rate was measured constantly by a custom made monitoring system using a pressure sensitive breath sensor. Moreover, the body temperature of each animal was maintained at a constant level of 37 °C using a warm water circuit inside the cradle. After the end of the last experiment the animals were killed according to the animal license by isoflurane overdoses and CO2.Two experimental series were performed on 10–14 weeks old male healthy NaThe MRI experiments were performed on a 4.7 T animal MR BRUKER BioSpec scanner 47/40  with a 40 cm horizontal free bore. A whole-body birdcage resonator operating with an actively shielded high-power gradient system (200 mT/m) enabled homogenous excitation. The low-noise, actively RF-decoupled and anatomically shaped 2 × 2 phased array head coil was used to receive the signals. No ear bars were used during the experiment, because ear bars are a confounding painful stimulus, even under light anaesthesia.Firstly, a 1 min single slice echo planar imaging (EPI) MR measurement  was acquired and run as a movie to control the stability of the head fixation. In case of visible movements above 1 pixel the animal was remounted.19 of 55.5 ± 10 for cortex and 53.66 ± 11.84 for thalamus. Thereafter an anatomical reference scan was performed at slice positions identical to the functional ones by a fast spin echo sequence 20.Functional BOLD MRI scans were performed using a T2*-weighted single-shot gradient echo based EPI. A functional scan consisted of 750 acquisitions of 22 axial slices , TEef = 24.4 ms, FOV 15 × 15 mm, in-plane spatial resolution 234 × 234 μm, slice thickness 500 μm). Using this sequence and our optimized phased array coil we were able to achieve a temporal signal-to-noise ratioDuring each of the two 50 min fMRI experiments either a set of only contact cold stimuli of increasing temperature  or only warm/heat stimuli , triggered by the MR, were applied using a customized feed-back controlled Peltier element  attached to the dorsal surface of the right hindpaw . Mixed experiments providing alternating cold and heat stimuli in one experiment were not investigated. The individual stimuli were presented at intervals of 3 min 25 s and the whole set of stimuli was repeated three times. This interval ensured that the baseline was reached between stimuli as validated by the time courses inspected. The stimulation device did not interfere with the magnetic field of the MR scanner. We chose a cross over design, meaning that the chronological order of the complete either heat or cold stimulation experiment was varied through the animals in order to avoid systematic bias due to the stimulation order.21  and MagnAn 2.3,  an IDL6.4 application 22. This 3D atlas was manually registered to the average anatomical template. Via inverting the subject specific registration matrix this atlas was transformed back to the individual first volumes and consequently also to the individual SPMs and the whole time-series data. Next, for each brain structure per animal the significantly activated voxels of the thresholded contrast specific SPMs were labelled using the individually registered digital atlases (see above). A minimum cluster size of 4 voxels was set for defining activated brain volumes. Thus, these significantly activated voxels can be counted as the activated volume per given brain structure.The first volume of the pre-processed time series data was used for segmentation. Segmentation was performed manually in AMIRA  and the resulting binary brain mask was used to cut out the brain. In order to perform a group-wise analysis of significantly activated voxels all animals had to be registered to a reference subject. Registration was performed by an affine transformation scheme (six degrees of freedom) using the grey value information of each individual segmented brain. As reference the subject was chosen which led to the overall lowest variance registering all datasets to that reference. The registered datasets were averaged to create an anatomical template for subsequent labeling of certain brain structures . For that purpose we used a digital 3-dimensional brain atlas which was generated in our lab based on digital outlines of the brain regions from the commercially available mouse brain atlas from Paxinos (third edition)V1.8−/− and WT mice upon either heat (55 °C) or cold (5 °C) stimulation conditions a voxel-wise second order statistic  based on the average SPMs per group was calculated  were multiplied with the binary mask. The resulting data were visualized in AMIRA on top of the corresponding anatomical MRI scans. The negative values, blue-to-green scale, indicate stronger activation in WT and the positive values, red-to-yellow scale, indicate stronger activation in NaV1.8−/−  served as reference values and were subtracted from the values of all the other stimulation temperatures  was performed to show significant differences between WT and NaV1.8-deficient mice for the different parameters.Furthermore, for each brain structure, animal and contrast, the time series of all activated voxels were averaged and an average time profile (atp) of each stimulus temperature was created. These atp’s were used to determine the stimulus-induced change of the mean BOLD amplitude. Additionally, the mean activation probability (probability of brain structure activation across subjects) was calculated. To provide a whole brain group comparison between WT and NaV1.8−/− and WT) and the two stimulation conditions  were calculated using a homoscedastic Student’s t-test (two-sample). Significant different connections per stimulation condition were determined by thresholding the difference matrices at p < 0.05. The resulting binary adjacency matrices were transformed into graphs with brain structures as nodes and significant differences as edges. The weight of the edges was −1 for negative differences and +1 for positive ones. These networks were visualized in a custom made module in AMIRA. The 3D coordinates of each brain structure – node – were taken from our 3D mouse brain atlas derived from Paxinos and Watson22 and nodes without edges (orphan nodes) were omitted. The node-size represents its number of edges, i.e. significantly different connections with other brain structures across the whole brain. For a better appreciation of the 3D node distribution, a transparent mouse brain surface of an average anatomical standard mouse brain was rendered and used as anatomical reference.For connectivity analysis the average time courses of the significantly activated voxels of each brain structure were corrected for global signal fluctuations by linear regression. The residual time courses of all brain structures were cross-correlated separately for each stimulus predictor and animal. Next, the individual cross-correlation matrices for the cold and heat predictors were averaged over all animals resulting in averaged cross-correlation matrices. In order to compare the number of connections across stimulation condition and genotype the average matrices where binarized using FDR threshold . Significant differences between the matrices of the two genotypes (NaV1.8−/−) was used to calculate significant mean differences between genotypes and temperatures. Assumption for all performed Student’s t-tests: The subjects in each sample were randomly selected from a population, the sample data come from normally distributed populations of observation and the variances from the two samples were shown to be equal . Second-order group analysis on SPMs was performed using voxel-wise Student’s t-test. If appropriate, test statistics were corrected for multiple comparisons by FDR . All data met the assumption of each statistical test applied.Data are presented as mean ± SEM. To check for general effects of temperature and genotype initially a two factor ANOVA was calculated with genotype and temperature range (either cold or heat) as fixed factors and the average BOLD parameter values (either activated volume or mean amplitude) per brain structure (n = 196) as dependent variables. For post hoc testing Student’s t-test   generated from the functional data set of NaV1.8−/− are significantly different and smaller compared to WT . These structures encompassed subcortical brain structures including the zona incerta (ZI), the periaqueductal grey (PAG) and structures belonging to the thalamus , ventral posteromedial thalamic nucleus (thVPM)) and the Hippocampus (Hip) , posterior dorsal hippocampus (hcPD), posterior layers of the dentate gyrus (hcDGp)). Other cortical structures like the retrosplenial cortex (cxRS), the cingulate cortex (cxCg), the parietal association cortex (cxPtA), parts of the somatosensory (cxSens) (primary somatosensory cortex (cxS1)) and of the motor cortex (primary and secondary motor cortex (M1/ M2)) also showed significantly larger SPM values in WT compared with NaV1.8−/−. In contrast, the only brain structures demonstrating higher SPM values in NaV1.8−/− mice under cold stimulation were the amygdala (am) and the secondary somatosensory cortex (cxS2) , hcDGp), the pretectal area (PTA), the ventromedial thalamic nucleus (thVM), the limbic output (outLimb) , dorsomedial hypothalamus (hyDM), lateral hypothalamus (hyL), paraventricular hypothalamic nucleus (hyPV), ZI), and finally in the substantia nigra (SN) . Note the fact, that some structures showing only a single significant voxel  depends on slice choice. Slices detected either before or behind the depicted one show also multiple significant voxels for these structures. Thus, the required minimum cluster size of 4 voxels defining activated brain volumes is also given for these structures, albeit this fact is not visible in one single slice presented.In contrast to cold stimulation, the second order SPM group analysis of Na−18; F = 79.65; mean amplitude: p = 1.20*10−10, F = 42.04) and temperature  as well as significant interactions between the two factors . ANOVA analysis of mean BOLD amplitude due to heat stimulation also shows significant effects for both factors . However the ANOVA analysis of heat stimulation BOLD activated volume revealed a significant effect only for the temperature  and not for the genotype  without interactions . Subsequently, we performed the Student’s t-test as post hoc analysis to compare the total of activated volume and mean BOLD amplitude for cold and heat stimulation. The stimulus-response curves describing the total of activated volume, the mean BOLD amplitude as well as the mean activation probability for both genotypes and for cold and heat stimulation conditions, respectively, are given in  as fixed factors and the average BOLD parameter values (either activated volume or mean amplitude) per brain structure (n = 196) as dependent variables. For both BOLD parameters ANOVA analysis of the cold stimulation revealed significant effects for genotype  and 10 °C , and showed a clear tendency for 0 °C   Fig. . Also th WT Fig. . Additio WT Fig. . Differe WT Fig. . Pure efV1.8−/− and WT pertaining to all parameters   Fig. .V1.8−/− and WT)  and heat (55 °C) stimulation separately for the two genotypes (NaWT) Fig. .Figure 3V1.8−/− by using in all following cases a Student’s t-test  in PAG (p = 0.048), in structures belonging to the sensory input (inSens) (p = 0.046), in the lateral and medial thalamus  and the outLimb (p = 0.048). Beyond, cortical structures like parts of the cxSens (p = 0.012), the association cortex (cxAss) (p = 0.007) and the cxCg (p = 0.019) showed markedly reduced activated volumes  , significantly reduced activated volumes were found between WT and Names Fig. . Moreove19) Fig. .V1.8−/− and WT upon heat stimulation conditions (55 °C), the functional group analysis revealed nearly identical activated volumes   Fig. .Subsequent single structure analysis, however, revealed several significant differences of the activated volume and mean BOLD amplitude in single brain structures for both stimulation conditions (5 °C and 55 °C) . The 3D renderings in Fig. V1.8−/−) for a given comparison at their anatomical position within the mouse brain which is rendered as a transparent surface. Comparing networks for cold perception (i.e. Nav1.8−/− cold/ WT cold) WT mice show a higher number of significantly different connections compared with Nav1.8−/− mice, and therefore altered intraregional connectivity, included brain structures belonging to the thalamus (turquoise), the limbic system , the somatosensory (pink) and association cortex (red) as well as structures belonging to the sensory input ) (left side). Concerning heat stimulation (i.e. Nav1.8−/− heat/ WT heat) Fig. V1.8−/− mice compared with WT show qualitatively quite the same functional connectivity pattern and consequently less prominent significantly different connections  and 3D rendering. Less but still significant effects were noticed in the limbic system (green), the thalamus (turquoise) and the sensory input (blue) (right side). The comparison of significant differences in connectivity upon cold and heat stimulation conditions for the NaV1.8−/− group itself (i.e. Nav1.8−/− cold/ Nav1.8−/− heat) reveals significant differences concerning the number of significant connections especially in the limbic system ), the sensory input (blue), the sensory cortex (pink) and the association cortex (red), whereas the WT group for itself (i.e. WT cold/ WT heat) again showed less prominent differences.Cross-correlation matrices and significant correlations from the analysed brain structures for NaV1.8−/− and WT mice using BOLD fMRI upon noxious cold and heat stimulation conditions, we were able to detect certain brain structures as well as their functional connections, which especially underlay the influence of the voltage gated sodium channel NaV1.8. Thus, functionally, the two genotypes can be separated properly. BOLD fMRI has already been performed in rats24 and could recently be transferred to mice by our and other working groups19. Moreover, several experiments demonstrated the possibility of translating findings recorded in rodent pain imaging studies to humans25. Thus, we conclude, that our data on Nav1.8−/− mice might also offer the opportunity to be translated to humans hinting at important facts about human cold pain processing and Nav1.8 as a potential pharmacological target.By revealing significant changes of brain activation in NaV1.8−/− mice during cold stimulation  impacted structures belonging to the inSens, the medial and lateral pain system, including thalamic regions and upstream many structures of the cxSens and cxAss, which are responsible for sensation, attention and integration of information. These findings demonstrate the importance of NaV1.8 concerning the function and interplay of these structures upon cold perception. Additionally, brainstem structures, responsible for descending pain modulation, like the PAG, which receives information from the nucleus accumbens (Acb) and is linked to the limbic coordination of fear and escape reactions27, were affected. Thus, we conclude, that the lack of NaV1.8 might also modify descending pathways of the antinociceptive pain system.In our experiments, the most resounding differences between WT and NaV1.8 knockout, i.e. the ThM, the cxCg and the cxRS. Thus, nociception in mice seems to be a perceptual experience that depends also on cerebral processing and is also associated with changes in motivational behaviour28.Additionally, structures representing the affective motivational dimensions of pain were significantly affected by the Naet al. also detected changes especially concerning the activated volume in thalamic nuclei, the basal ganglia (BG) and insula (cxIns) upon noxious cold (5 °C) and heat (46 °C) stimulation to the dorsum of the left hand of healthy male subjects, respectively. Cortical areas which were further activated during both stimulation conditions included the cxCg and the cxSens (cxS1 and cxS2 (weaker for noxious cold)), the caudate Putamen (CPu), the premotor and motor cortices as well as prefrontal and inferior parietal cortex12. Mohr et al. also detected the prefrontal cortex, the thalamus as well as the cxS1 as important brain structures involved in cold pain perception29. Moreover, Seifert et al. discovered that the anterior cxCg and the BG were highly impacted by cold allodynia27. In agreement to these human studies we detected significant differences in size and activation strength in exactly these structures (except insula) in the mouse brain of WT and NaV1.8−/− mice in response to noxious cold stimulation as well, pointing to an essential role of NaV1.8 for the coordination and interplay of these structures and their essential function in cold pain processing. Tracey et al. observed that upon cold and heat stimulation most structures were activated bilaterally with a slight dominance for contralateral activation12. Further fMRI experiments also revealed a widespread bilateral brain activation in rodents25 and humans26 upon one-sided stimulation. The same phenomenon could be observed in our experiments on mice showing that although the stimulation was executed on the right hindpaw, no clear tendency towards any laterality of activation was obvious. Thus, with our data presented here, we were able to show the striking influence of NaV1.8 on cold associated pain states at the level of the CNS. The high comparability of our fMRI data with human data, mentioned above29, gives rise to a possible translation from mouse to humans allowing for presenting and functional validation of Nav1.8 as a potential target for the development of new drugs for cold associated pain states in humans like allodynia.In human studies Tracey V1.8 resulted in a very distinct phenotype in the cold plate test, where genetically modified NaV1.8−/− mice or mice, where NaV1.8 expressing neurons were killed by diphtheria toxin (DTA mice), show negligible responses to noxious cold temperatures as well as diminished responses to mechanical stimulation1.Furthermore, our results also fit well with the previously observed phenomenon that the loss of NaV1.8 is essential for behavioural responses below 10 °C, especially at 5 °C and 0 °C2. Until now, this behaviour was explained by the phenomenon that the normal processes are interrupted and that the inactivation properties of NaV1.8 are largely cold-resistant, keeping the sensitivity for cold pain perception at a functional level2.In previous studies we were also able to show that NaV1.8−/− mice starting with a reduced thalamic information transmission. This directly reflects the mentioned altered hyponociceptive phenotype of NaV1.8−/− mice and linked electrophysiological features2.Our graph-theoretical connectivity analysis revealed significantly reduced interactions and connections between pain relevant structures at cold temperatures, pointing to an altered information flow within the whole brain network in NaV1.8−/− mice presented slightly reduced activated brain volumes as well as reduced BOLD amplitudes linked with a similar but not identical activation pattern compared to WT. These results properly fit with the observations from the Hargreaves test described by Abrahamsen et al., who observed only a slight tendency towards an increased withdrawal latency at noxious thermal stimulation temperatures in DTA mice compared with the WT, i.e. a decrease in sensitivity to noxious heat stimulation1. On the other hand Akopian et al.7 and Abrahamsen et al.1 noticed a tendency towards a decreased withdrawal latency in the hot plate test at 50 °C and 55 °C stimulation temperature in NaV1.8−/− or DTA mice compared with WT and a tendency towards an increased latency at 45 °C. This unsteady heat phenotype was reflected in our experiments by a reduction of the activated volume in NaV1.8−/− mice at 45 °C and higher activated volumes at 50 °C and 55 °C  anesthetized mice as compared to medetomidine and urethane, although the low correlation coefficients obtained for medetomidine and urethane may be largely attributed to the smaller response and should therefore be not over-interpreted31. Schroeter et al. also stated that nevertheless, for medetomidine, difficulties in obtaining reproducible responses from repetitive measurements in the same imaging session have been reported previously32. The relatively strong analgesic component of medetomidine was further in conflict with our experiments, studying noxious cold and heat signaling. An fMRI study by Bosshard et al. in mice demonstrated the feasibility of a robust BOLD fMRI protocol to study nociceptive processing in isoflurane anesthetized mice33. The authors claimed a high reliability of the isoflurane anaesthesia, which allows for detailed analysis of the temporal BOLD profile and for investigation of somatosensory and noxious signal processing in the brain33.Among numerous positive aspects fMRI in general bears also several limitations particular for animals. It is known, that effects of anaesthesia can influence pain perception. Many anaesthetics can function as analgesics and some even affect the neurovascular coupling or the BOLD signal acting as potent vasodilatators34.One further limitation due to the anaesthetic used is that data with low motion can only be acquired if the animal stays in a fixed position inside the scanner. Thus, anaesthesia has to be deep enough to suppress movement, but light enough to pass activation signaling, which requires an elaborated anaesthesia protocol. Thus, in our experiments we used isoflurane which is only slightly analgesic, allows easy administration, controlled dosing and produces highly reproducible BOLD signalsFurther limitations for fMRI scanning are that fMRI BOLD signals have a low time resolution compared to neuronal activity. Moreover, because BOLD signals measure indirect hemodynamic changes the underlying neural activity can not directly be measured and BOLD signals do not allow to differentiate excitatory and inhibitory processes. Additionally, external influences like scanner noise, might interfere. However, these influences remain constant during the whole experiment and also across different subjects. Conventional GLM analysis of the MRI time series ensures to capture real activation besides such confounders, which can be modelled accordingly in the GLM.V1.8 is expressed only in peripheral neurons and is limited to sensory and myenteric neurons including small and medium-sized DRG neurons and their axons5. With our experiments we demonstrate, that besides the peripheral effect of NaV1.8, higher order processes including supraspinal/central mechanisms are involved altering the CNS processing of noxious cold stimuli and lead to changed interconnections in the CNS of NaV1.8−/− mice. Thus, our results add altered profound CNS differences to the previous findings from electrophysiological and behavioural studies on NaV1.8.NaV1.8−/− mice and the fact that NaV1.8−/− mice do not perceive nociceptive aspects of strong cooling in contrast to their WT littermates is not only an expression of a just peripheral phenomenon going along with diminished peripheral transmission, but is also an effect of the upstream processing with reduced afferent input leading to altered subsequent nociceptive processing in the CNS and consequently altered connectivity between pain-relevant brain structures combined with a reorganization of the response to cold noxious stimuli.We assume, that the altered phenotype of NaV1.8 on noxious cold (and to a less extent heat) signaling. NaV1.8 therefore represents an interesting target for the development of new analgesics for the treatment of especially cold pain states like cold allodynia. Along with our study, we assume, that such drugs might not only act in the periphery, where NaV1.8 is mainly located, but also in the central nervous system effectively diminishing cold pain in associated pain states and could thus offer a advantageous new therapeutic approach.Thus, with our present study we were able to show the impact of NaSupplementary information


The ZnO films were prepared by a drop coating deposition method. This was then combined with a thin layer of the redox active material CeO2 to form CeO2-coated ZnO films. Spherical ZnO nanoshell structures and CeO2-coated ZnO nanoshells have been prepared using polystyrene (PS) sphere monolayer templates. The structural properties and morphologies of the nanostructures were analysed by x-ray diffraction (XRD) and scanning electron microscopy (SEM). The nanostructure compositions are studied in more detail using secondary ion mass spectroscopy (SIMS). The optical properties of the nanostructures were measured using ultraviolet-visible (UV-Vis) absorption spectroscopy in order to ascertain the effects of the nanoshell structures and the whispering gallery modes associated with these structures on the optical properties of the deposits. Our data show UV and visible light absorption was very significantly enhanced due to this nanostructuring.In this paper, ZnO, CeO This new method of light management uses low quality factor whispering gallery resonant modes inside spherical nanoshell structures where the geometry of the structure dramatically improves the absorption and reduces adverse directionality effects due to the substantial enhancement of the effective light path in the active material3. Applying this important recent development in conjunction with a redox mediator and a UV absorber, such as CeO2 and ZnO5, can result in enhancement in the UV light absorption and optical properties, which can in turn be used for more effective applications in a variety of technologies, especially given the relatively favourable band alignment for charge transfer, with a likely small contact potential  can result in a material with enhanced/unique UV absorption and high stability at high temperature and high material hardness5.Materials based on CeO17, nanorings18, nanowires19 and nanobelts20. Among these various structures, hollow nanostructures are interesting structures for applications such as photocatalysis21, solar cells22, drug delivery23 and much more26. Synthesising hollow ZnO nanostructures has been recently done using various templates. For example, Sun et al.29 report extraordinary visible-light responses for ZnO hollow microsphere structures synthesised by their recently developed two-step self-assembly concept. These interesting studies were inspired by examples from the biological realm, including the fly compound-eye and fish-scale structures. Jiang et al. synthesised ZnO hollow spheres using ethanol droplets as soft templates while Neves et al. synthesised ZnO hollow spheres by coating polystyrene beads. Others, like Li et al.30, Shen et al.31 and Deng et al.32, synthesised ZnO hollow particles using a template-free solution method, template-free evaporation method and template-free sonochemical fabrication method, respectively. Although these proposed methods are described as simple and inexpensive, the drop coating method  using a template of polystyrene beads, yields samples with a patterned spherical nanoshells of ZnO with improved crystallinity, purity and optical properties.ZnO nanostructures have recently attracted a great attention due to their interesting properties for photonic applications and the variety of morphological structures which can be achieved including nanorods2 film to achieve enhanced optical properties (UV absorption) primarily for solar-thermal-related applications. The samples used in this study are shown in Fig. 2 nanostructures were synthesised by drop coating and pulsed DC magnetron sputtering and characterised by XRD, SEM, SIMS and UV-Vis spectroscopy. To the best of our knowledge, using drop coating of PS sphere templates to engineer patterned spherical nanoshells of ZnO has not been reported previously, and is a very simple and versatile method. Our work provides useful information on the influence of the nanoshell geometry on the absorption properties of various combinations of these two types of materials. The addition of the CeO2 thin film on top of the ZnO hollow nanostructured deposit enhances the UV light absorption further and provides additional functionality such as oxygen storage capacity via changes in stoichiometry. The redox properties of these types of CeO2 films grown in our group were reported in ref. The aim of the present study is to engineer patterned spherical nanoshells of ZnO coated with a CeOet al.36 and further developed by Byrne et al.37. This method involves drop coating a mixture of zinc acetate (5 mM) in anhydrous ethanol solution onto the substrate surface for a period of 20–25 seconds before being rinsed with fresh ethanol and dried with a nitrogen stream. This process was repeated approximately 60 times (1 time yields approximately a film thickness of 2 nm). The substrates were then annealed at 350 °C for 20 minutes, yielding a uniform textured nanocrystalline ZnO film with a film thickness of 120 ± 10 nm. Patterned spherical ZnO nanoshells were fabricated on a PS sphere template using the same deposition method for the ZnO films. A monolayer of the PS spheres with a diameter of ca. 600 nm  were generated by a self-assembly process on the surface of DI water, at room temperature, and then transferred onto bare substrates33. The deposited close-packed PS sphere monolayer was then heated at 90 °C for 30 seconds to cause them to adhere better to the substrate surface without significantly deforming the spheres. The PS spheres were then O2 plasma treated  at a power of 300 W, a pressure of 100 mbar, an oxygen flow rate of 50 sccm for 25 seconds to reduce the sphere diameter from 600 nm to ~520 nm allowing enough space between the spheres for a connected and mechanically stable ZnO nanoshell structure post-deposition. The nanostructured CeO2 films are prepared on previously cleaned substrates by pulsed DC magnetron sputtering using the same deposition procedures to the ones described in refs 2 thickness of 120 ± 10 nm.Prior to deposition, Si(100) and quartz substrates were cleaved to the desired size (2 × 2 cm) and the substrates were ultrasonically cleaned using acetone and a decontamination solution (30905 Aldrich) and then rinsed with deionised water and blown dry with a nitrogen stream. The ZnO layers used to generate ZnO films were prepared by a method initially proposed and demonstrated by Greene 2-coated ZnO films, the substrate is coated with approximately 40 layers of the 5 mM zinc acetate solution to yield a uniform ZnO film thickness of 80 ± 10 nm. The sample is then transferred to the sputtering chamber to deposit an approximately 50 ± 10 nm of nanostructured CeO2 film on top of the ZnO. This resulted in a 120 ± 10 nm thick CeO2-coated ZnO films to allow for a straight comparison study. The same process was repeated to fabricate the CeO2-coated ZnO spherical nanoshells. A process flow chart is provided in Supplementary Figure 2 films were also grown without ZnO, for the purposes of optical studies as mentioned below. These latter nanostructured CeO2 films are prepared on previously cleaned substrates by pulsed DC magnetron sputtering using the same deposition procedures to the ones described in refs 2 thickness of 120 ± 10 nm. Table For the CeO2-coated ZnO samples were measured using a Bruker D8 Advance X-ray Diffractometer system with CuKα radiation of wavelength λ = 1.5418 Å. The XRD measurements were carried out in locked coupled mode in a 2θ range from 20° to 60°. Sample morphology was studied using SEM (Karl-Zeiss EVO series and Hitachi S-5500 field emission (FE) SEM). The sample composition was studied using SIMS (Millbrook MiniSIMS Alpha). The optical absorption properties of the samples were studied at room temperature (RT) using a Perkin Elmer Lambda 40 UV-Vis spectrometer in the range from 200 to 800 nm with a resolution of 4 nm.The structural properties of the pure and CeO2-coated ZnO film (C_Z_F) and CeO2-coated ZnO nanoshell (C_Z_NS), as-deposited and annealed at 500 °C and 800 °C in air for 30 minutes are shown in Supplementary Figures 2 (111) and ZnO (002) XRD peaks are used as an indicator of the crystallinity quality of the CeO2 and ZnO deposits. The FWHM data is provided in Supplementary Table XRD patterns of the ZnO film (Z_F) and ZnO nanoshell (Z_NS); and, the CeO2 plasma treatment. As shown in Fig. 2 plasma treatment, allowing enough space around the spheres for the Z_NS structures to fully interconnect during growth to ensure mechanical stability. There are some examples of slight movements of spheres, or of a sphere detaching, during the etch despite prior heating at 110 °C for 30 seconds to ensure good adherence, but these are very occasional and do not compromise the overall nanostructure integrity.Figure 2-coated ZnO sample, as shown in the cross sectional view of the fractured nanostructures. Other views of the spherical nanostructures, after a complete removal of the PS spheres by carbonisation  and the addition of the thin CeO2 film (~50 ± 10 nm thick), are also shown in Fig. After the deposition of the zinc acetate films on the PS spheres, the structural morphology of the ZnO deposits depended on the post-deposition annealing temperature. Figure 2 SIMS spectra showed secondary ion peaks of Ce+, CeO+ and CeO2+ 34. As more scans are performed and the probing depth increased due to surface sputtering by the Ga ion beam, Zn+ and ZnO+ peaks start to appear and their intensity increased with the increase in the number of scans. Three different Zn isotopes are observed for the Zn ions, 64Zn, 66Zn and 68Zn. These SIMS data clearly shows evidence of an abrupt interface between the ZnO and CeO2 materials. Supplementary Figure 64Zn+, Ce+, CeO+ and CeO2+) as a function of depth at the boundary of the CeO2-coated ZnO composite layers grown on Si(100) substrate. Further details on the SIMS measurements are provided in Supplementary section Information on the chemical composition and impurity content in the samples were obtained by making SIMS measurements at different locations throughout the deposit. Supplementary Figure 2 film (C_F), Z_F, C_Z_F, Z_NS and C_Z_NS) were investigated by spectroscopic measurements. As mentioned above, the optical absorbance spectra of the films deposited on quartz substrates are recorded in the wavelength range from 200 to 800 nm. Typical absorbance curves for the films grown on quartz are shown in Fig. 2 film on top of the ZnO film is clearly observed in these absorbance spectra. The C_Z_F have high absorption in the UV and visible regions followed by a fall-off in the absorption at wavelengths greater than approximately 380 nm. Both pure C_F and Z_F (with approximately the same film thickness of 120 ± 10 nm) have lower absorbance in the visible region and the absorbance spectra of the composite C_Z_F seems to be due to the joint effects of the two constituent oxides. Adding CeO2 to the ZnO films clearly increases the absorption in the UV spectral region.The optical properties of the as-deposited samples  with the same physical thicknesses of the materials in both the film and nanoshell morphologies.2-coated ZnO thin films and functional nanoshell nanostructures on Si (100) and quartz substrates. The ZnO films and nanostructures were grown by a facile drop coating method using zinc acetate in anhydrous ethanol solution as a starting material while the CeO2 deposits were produced by a pulsed DC magnetron sputtering method. XRD, SEM and SIMS measurements were used to confirm the structural, morphological and compositional properties of the deposited materials. In particular XRD data indicated the poorly crystalline nature of the as-deposited ZnO and CeO2 nanostructures and showed that the crystalline quality improved after post-deposition annealing at higher temperatures. SEM images showed the successful engineering of the spherical nanoshell structures with a clear indication of the central voids. SIMS analysis of the chemical composition showed the presence of the Ce+, CeO+, CeO2+, Zn+ and ZnO+ ionic species in the various relevant samples as well as the three different Zn isotopes , and depth profiling showed the location of the ZnO/CeO2 interface in relevant samples. UV and visible light absorption was very significantly enhanced through the engineering of spherical nanoshells on a PS monolayer template, most likely due to the whispering gallery modes in such nanoshell cavities, as well as the addition of the CeO2 layer. Our results and analysis clearly show that key materials properties such as the UV and visible light absorption can be significantly enhanced by nanostructure engineering of the deposits to create spherical nanoshell cavities. These results may prove very useful in terms of enabling future materials and device developments, with the aim of controlling key deposit parameters for technologically important applications, in particular in the areas of solar-thermal fuel generation and catalysis, where the combination of the nanostructure engineering possible with ZnO and the oxygen storage and variable stoichiometry properties of CeO2 provides a unique set of advantageous deposit properties.In this paper, we have demonstrated a simple and reproducible method to fabricate ZnO and CeOEnhanced Optical Properties of ZnO and CeO2-coated ZnO Nanostructures Achieved Via Spherical Nanoshells Growth On A Polystyrene Template


Organic interfaces that exist at the sea surface microlayer or as surfactant coatings on cloud droplets are highly concentrated and chemically distinct from the underlying bulk or overlying gas phase. Therefore, they may be potentially unique locations for chemical or photochemical reactions. Recently, photochemical production of volatile organic compounds (VOCs) was reported at a nonanoic acid interface however, subsequent secondary organic aerosol (SOA) particle production was incapable of being observed. We investigated SOA particle formation due to photochemical reactions occurring at an air-water interface in presence of model saturated long chain fatty acid and alcohol surfactants, nonanoic acid and nonanol, respectively. Ozonolysis of the gas phase photochemical products in the dark or under continued UV irradiation both resulted in nucleation and growth of SOA particles. Irradiation of nonanol did not yield detectable VOC or SOA production. Organic carbon functionalities of the SOA were probed using X-ray microspectroscopy and compared with other laboratory generated and field collected particles. Carbon-carbon double bonds were identified in the condensed phase which survived ozonolysis during new particle formation and growth. The implications of photochemical processes occurring at organic coated surfaces are discussed in the context of marine SOA particle atmospheric fluxes. A major component of aerosol particles is organic material, which can be directly emitted or formed in situ by secondary processes5. The latter contributes about 60 and 95% of organic aerosol mass in urban and rural air, respectively7, and significantly impacts global estimates of aerosol optical depth9. Predicting organic aerosol concentrations remains difficult, especially in marine environments due to a lack of observations and the possibility of unknown sources of volatile organic compounds (VOCs) as potential secondary organic aerosol (SOA) precursors10. It remains undecided if SOA particles pose a potential warming or cooling of the Earth and the uncertainty in their radiative forcing is comparable with other aerosol particle types i.e., sulfate, nitrate and biomass burning aerosols11. Although well recognized as important, SOA particles are not yet included when estimating anthropogenic radiative forcing changes between modern day and pre-industrial conditions due to an incomplete understanding of their formation9.Aerosol particles reside in the atmosphere where they scatter and absorb light and impact the global radiative balance between the Earth and Sun, thereby influencing the climate12. Organic surfactant compounds reside in the atmosphere on air-water interfaces such as surfaces of aqueous aerosol particles and hydrometeors20. A concentrated layer of organic and biogenic material also resides at the sea surface, referred to as the sea surface microlayer (SML); this includes surfactants, lipids, proteins, polysaccharides, fatty acids, alcohols, and fragments of cellular materials, all of which can be aerosolized and have been detected on marine derived airborne particles28. Recent laboratory studies have linked aerosol production29 and fatty acids composition (8–18 carbon atoms per molecule)27 with marine biological activity supporting the notion that aerosol bound surfactants from ocean waters may be pervasive. When irradiated by sunlight, surfactants on water, aerosol particles and droplets may undergo unique photochemical reactions primarily due to the distinctly different physical and chemical properties of interfaces compared with the underlying bulk or overlying gas phases30.Our study suggests a new source of SOA particles due to the emission of VOCs photochemically produced by fatty acids at the air-water interface36. Photosensitized reactions, in particular, occur when chromophores such as humic acids or chlorophyll absorb light and then react with neighboring molecules. They are important under conditions where direct photolysis of organic compounds, nitrogen oxide or O3 is not efficient. Light can enhance the loss of gas phase oxidants such as O3 to chlorophyll in seawater, resulting in production of other radicals such as OH and Cl35. In the presence of the photosensitizer imidazole-2-carboxaldehyde (IC) and light, oxidation of limonene was observed which has implications for photosensitized SOA formation in aqueous aerosol36 and production of HO2 radicals37. Recent work details evidence that photosensitized chemistry between IC and octanol under irradiation occurs at the organic-coated aqueous interface and results in the production of unsaturated carbon bonds and carbonyl and carboxyl functionalities34. Production of saturated and unsaturated VOCs was observed due to photosensitized reactions at the air-water interface of a fatty acid and humic acid solution32. Subsequent ozonolysis of these compounds led to new particle formation and growth31.Photochemical reactions at organic interfaces have received increasing attention, as they are suspected to affect atmospheric trace gas and aerosol budgets12. Experiments were performed in a flow reactor with a short residence time on the order of seconds and thus, observations of SOA nucleation which typically occurs on longer time scales was not possible12. Although a new source of SOA particles due to fatty acid photochemistry was suggested, observational evidence and evaluation of its importance to aerosol nucleation was lacking.Photoactivity of organic surfactants without the presence of a previously known photosensitizer, i.e. IC, was recently observed and could be explained by a triplet state excitation from a spin forbidden electronic transition31, in which a filled basin of water resided with one of two organic surfactants, nonanoic acid or nonanol. Concentrations of VOCs over time were monitored while the interface was illuminated with UV radiation having a wavelength range similar to that of UV light from the sun hitting the Earth’s surface. The nucleation and subsequent growth of SOA particles in the chamber was observed following dark ozonolysis reactions initiated immediately after UV lights were switched off. Additional experiments were performed to investigate UV light exposure and simultaneous UV and ozone exposure on SOA particle production. The organic carbon composition of the SOA was quantified with discrimination of various functionalities including unsaturations, hydroxyl, carbonyl and carboxyl functionalities using scanning transmission X-ray microscopy coupled with near-edge X-ray absorption fine structure spectroscopy (STXM/NEXAFS). A comparison between measured carbon spectral signatures of SOA particles formed in our chamber and previous laboratory and field studies is presented. The atmospheric implications of our results are discussed in the context of particle production rates and fluxes derived from experimental data.Here, an investigation of SOA particle formation due to photochemistry occurring at surfactant interfaces is presented. Experiments were conducted in a multiphase atmospheric simulation chamberNp, in the atmospheric simulation chamber over time, t, for an experiment in which nonanoic acid at an air-water interface inside the chamber was illuminated with UV light followed by the introduction of gas phase ozone in the dark. Nucleation of SOA particles up to a maximum concentration, Np,max ~200 cm−3 was observed after ozone was introduced. New particle formation did not occur after chamber irradiation when nonanol was used as a surfactant or when only water was present as shown in Fig. −3 in experiments, as outlined in the Supplementary Information. SOA particle formation above 10 cm−3 was considered significant and observed for all irradiation followed by ozone exposure experiments using nonanoic acid  interface, but only in the presence of a photosensitizing compound34. In accord with our results, the authors concluded that octanol alone does not photochemically react when exposed to UV light34.Figure 9 carboxylic acid was, in fact, photoactive. Rossignol et al.12 discussed several possibilities for why the nonanoic acid-coated water surface shows photoreactivity, whereas the alcohol-coated surface does not. Our observations point to a specific role of the terminating functional group of the surfactant for possible photochemical SOA formation pathways at an air-water interface i.e., the acid group may play a role in photochemical properties of surfactant molecules. Neat fatty acid liquids can undergo singlet-triplet state excitation centered at 272 nm39 however, the reason why this absorption band is present at high concentrations and not observed for single molecules (e.g. in the gas phase) remains unknown. We speculate that fatty acids undergo unique intermolecular interactions when closely spaced, i.e. at a highly concentrated interface. Shorter chain fatty acids, C1–C4, can form stabilized hydrogen bonded dimers in solution with a parallel orientation, in which the distance between carbon atoms of two monomer head groups is 5–6 Å40. The surface pressure of a nonanoic acid monolayer is approximately 35 dynes cm−1 measured at about 1 mM concentration41 which corresponds to a range of 5–10 Å2 per molecule42, a closer head group distance (~3 Å) than expected for dimer formation in solution. This may influence the hydrogen bonding network and delocalize carbonyl bonds in nonanoic acid giving rise to >300 nm UV light absorption. In turn, absorption can allow for triplet state excitation, which is accessible at high concentrations of the acid, i.e. in the neat liquid12.In the absence of a photosensitizer we found that photochemistry of nonanol did not occur while the corresponding Cet al.12, followed by reaction with ozone yielding SOA particles. The concentrations of selected VOCs measured in the chamber using PTR-MS detection are shown in Fig. 3O+ reagent ions shown are octenal (C8H14O detected at m/z = 127.11) and octanal (C8H16O detected at m/z = 129.13), which are unsaturated and saturated aldehydes, respectively. Also shown is C8H15+ (detected at m/z = 111.12), a common fragment of octanal38 and C5H9+ (detected at m/z = 69.07) having contributions due to isoprene, (C5H8 + H)+, and fragmentation products of other parent ions32. These VOCs and other detected alkanes, alkenes, and saturated and unsaturated aldehydes were identified using H3O+ and NO+ reagent ions38. These same compounds were previously shown to be prominent photochemical products in solution12. Figure The formation of particles can be explained by the photochemical production of VOCs in the chamber via the mechanism proposed in Rossignol et al.12 proposed that photolytic cleavage is one mechanism among others leading to the formation of radicals, including in this case the OH radical. Subsequent reactions with neighboring organic molecules should result in aldehyde formation. The presence of OH and excited nonanoic acid can result in further radical chemistry, unsaturated aldehyde formation and H2O elimination, the latter being favorable in the organic rich layer. Reactions between unsaturated compounds and O3 results in products with low volatility capable of SOA particle nucleation as previously discussed. Figure A reaction mechanism for VOC production and SOA formation is shown in Fig. Ana, on the Teflon walls was on the order of 5–10 Å2 as was present at the air-water interface42. The vapor pressure of nonanoic acid is on the order of 2 ppm at 25 °C (~14 µg m−3)43, which is about 3–5 times higher than observed in chamber experiments where gas phase nonanoic acid concentrations ranged 0.5–0.8 ppm. To our knowledge, estimates of nonanoic acid molecular surface adsorption and Ana on Teflon films at subsaturated conditions have not been made. Previous investigations of oxidized organic vapor partitioning on Teflon walls was done by Zhang et al.44, however their formulation is driven by vapor concentrations exceeding supersaturation, which is not the case in our experiments. Previous research has shown that time scales for organic monoacids to equilibrate with Teflon chamber walls occurs on the order of minutes45. Therefore, our experimental time scales on the order of 1000 min between introduction of surfactants and UV irradiation should have allowed for adsorption-desorption equilibrium conditions, such as Ana or wall concentration, Cw, to be met. To estimate Ana, we first assume that nonanoic acid partitioned to Teflon walls following Henry’s law as a rough estimate. This assumption neglects that other organic molecules (in addition to water) may be present on the walls due to the previous experimental chamber history, although at concentrations which we have shown to be negligible for measurements of SOA or VOC production. For an observed nonanoic acid partial pressure of 8.1 × 10−2 Pa and a Henry’s law constant46, H = 6.9 × 10−3 mol L−1 Pa−1, Cw = 5.6 × 10−4 mol L−1. Following Seidl41, Cw equates to a spreading pressure of about 27 mN m−1. Finally, the pressure-area isotherm for nonanoic acid measured by Gilman et al.42 yields Ana = 9 Å2 for our conditions which is slightly greater than what is calculated for the water surface as previously discussed. Therefore, a surface layer of nonanoic acid on the walls at these concentrations may have been responsible for photochemical VOC production. We note that these estimates are uncertain and thus, more investigation must be done to better understand organic vapor wall partitioning and the effect on surfactant photochemical reactions.Experiments were performed to evaluate the role of an air-water interface on nonanoic acid surfactant photochemistry by injecting gas phase nonanoic acid into the chamber without the presence of water in the basin Figs  and S9. via proton transfer reaction and may contribute to different background VOCs detection prior to UV illumination. Concentrations of VOCs shown in Fig. When nonanol was used as a model surfactant, UV irradiation did not yield any VOC production and instead, concentrations were perturbed toward decreasing values as seen in Fig. The chemical composition of single SOA particles collected from two different chamber experiments were investigated using STXM/NEXAFS. Figure 12. The presence of carbon double bonds is surprising since ozonolysis should have depleted any carbon unsaturation in VOCs produced in our experiments. Ozonolysis of VOCs ultimately led to the formation of condensed phase oxidized compounds observed as absorption peaks in Fig. 47. As expected, the main feature of the X-ray absorption spectra of a reference fatty acid, stearic acid (red spectrum), was a peak observed at 288.6 eV due to the carboxyl functionality, but lacking other oxygenated functional groups. It may be possible that continued heterogeneous reaction of ozone or OH in the presence of UV light with SOA particle surfaces yielded aged compounds with carbon double bonds. Alternatively, we suggest that oxidized VOCs with different volatilities initially having multiple carbon unsaturations partitioned to the particle phase with carbon double bonds that survived ozonolysis. The latter explanation points toward increasing evidence for the photochemical production of unsaturated compounds at the interface38. Once in the condensed phase, these organic functionalities and unsaturated compounds may be buried by continual condensation of SOA and protected from surface O3 reaction surviving for long time scales48.Figure et al.49 and used here for comparison with chamber-formed SOA particles. Among those reported, the most similar spectra are of particles referred to as “type i” and “type j” characterized as multiple-transition aerosol particles, i.e. particle exhibiting a variety of carbon functionalities49. The spectral shapes of the field collected and chamber-formed SOA particles are qualitatively similar, i.e. the main feature is the carboxyl functionality, a secondary C=C peak and a broad absorption shoulder occurring between 286.0-288.2 eV, indicating hydroxyl and carbonyl functionalities. Takahama et al. suspected a variety of potential compounds exhibit these features, such as humic and fulvic acids50 and others stemming from soil and biomass burning sources47, however these particular features were seen in every marine, terrestrial and polluted environment investigated49. We suggest that these highly functionalized particles found in the atmosphere by Takahama et al.49 may have a contribution due to secondary photochemical sources stemming from SOA precursors such as those produced from photochemical reactions at a surfactant interface.Absorption spectra of field collected particles from a variety of sources, including marine, terrestrial and urban were reported in Takahama From the observations described above, surfactant fatty acid photochemistry may impact the budget of SOA particles in the atmosphere, and especially at low wind speeds over the oceans where the organic and surfactant rich SML is irradiated by sunlight. While our experimental design allows an understanding of the photochemical process at the air-water interface, it nevertheless cannot catch the full complexity of marine boundary layer processes. Nevertheless, we attempt to estimate the potential importance of SOA formation from our results.Np(t), for all chamber experiments were reproduced as a continuous function by fitting to an exponentially decaying sigmoidal equation,b1, b2 and b3 are fitting constants and k = 6.2 × 10−3 min−1 is the particle loss rate independently determined from the decay of Np over time after nucleation was over. Losses were due to chamber walls and the sampling air flow out of the chamber. This sigmoidal function was arbitrarily chosen to capture the initial increase in Np as a function of t, the change in concavity as Np approached maximum values and the decay of Np after new particle formation stopped. Figure b1 = 260 cm−3, b2 = 0.25 min−1 and b3 = 21.5 min. Particle production rates, P(t), were then determined bykNp(t), and the time rate of change of particle concentrations, ΔNp(t)/Δt, calculated from Eq (Fp(t) was derived asP(t) is multiplied by the ratio between the chamber volume, Vch, the air-water interfacial area, Aint, and finally by r = 11.3, which is the ratio between the integrated solar irradiance (91.7 W m−2) and chamber irradiance (8.1 W m−2) at 300–420 nm in wavelength assuming particle production is linear in irradiance. Average Fp values from all experiments employing nonanoic acid at the air-water interface  m−2 s−1 with an order of magnitude uncertainty determined from the maximum and minimum average Fp values over time  losses of oxidized VOCs to chamber walls which tends toward under prediction of SOA formation, ii) that high ozone concentrations not typical for the atmosphere were employed in experiments, iii) experimental variability in relative humidity and temperature, iv) that pH and surface tension values are not within a range of ambient oceanic conditions, v) that seed aerosol or pre-existing particles in ambient air will scavenge VOCs and vi) that only pure water was employed. Nevertheless, our data do suggest that further attention should be given to marine surface photochemistry.In addition to our derived flux of SOA particles, Fig. et al.54. We assume that extrapolated SOA particle flux scales with a 12-hour diurnal solar irradiance cycle at the Earth’s surface  = −Fp°sin(πt/12 + 8), where Fp° = 1.47 × 106 m−2 s−1 with an uncertainty taken from Fig. Fp(t) values and corresponding particle production rates, P(t), are shown in Fig. 54, simulated aerosol particle concentrations, Np(t), shown in Fig. −3 on average with an uncertainty range between about 60–6000 cm−3 during the daytime hours. During the night when Fp = 0 m−2 s−1, Np decreases over time. By comparison, aerosol particle concentrations in clean marine regions typically are <300 cm−3 54.This can also be highlighted by simulating the aerosol particle concentrations in a column of air with a height of 0.5 km (representing a column integrated boundary layer), to investigate the potential importance of our experimentally derived flux on marine aerosol particle concentrations following de Leeuw ace Fig.  i.e., F10. We note that this is an abiotic photochemical source of SOA precursors as opposed to direct emissions of biogenic SOA precursor gases. Increasing particle concentrations and particle sizes due to photochemical SOA formation may also impact atmospheric budgets of cloud condensation nuclei, which are especially important in clean marine environments where cloud forming particles can be relatively scarce compared with polluted environments. Certainly, future studies should aim to better constrain estimates and uncertainties of this abiotic photochemical SOA formation pathway for atmospheric application.This discussion indicates that SOA formation due to fatty acid photochemistry at the SML can play a potentially significant role in determining total aerosol particle concentrations in the marine boundary layer. Many soluble, insoluble and particulate organic components reside at the SML and are concentrated with respect to bulk ocean water. Carboxyl functionalities which can be common in marine organic matter are suggested to be photochemically active. If so, the SML may be a location for photochemical reactions similar to what has been observed here resulting in an increase in VOC and SOA particle loading in the boundary layer. In turn, surfactant photochemical SOA particle production may be an additional source of particulate organic matter, which can help close the gap for calculating organic aerosol concentrations that are consistently under-predicted in marine environments57. This new surfactant photochemical process may also then contribute to in-cloud SOA nucleation. We suggest that interfacial organic photochemistry is ubiquitous, occurs in a variety of environments under a wide range of conditions, results in the emission of VOCs and SOA nucleation and growth and constitutes a condensed phase radical source important for organic aerosol and trace gas budgets affecting the oxidizing capacity of the atmosphere and air quality.We demonstrated SOA nucleation and growth due to dark ozonolysis of unsaturated organic gas phase products arising from photochemistry at a nonanoic acid-coated aqueous interface. Although the potential for SOA production was suggested in previous studies, limited chamber volumes and short residence times were not optimal for detecting new particle formation. The SOA produced had unique carbon bonding signatures and include carbon unsaturation and oxygenated functionalities. The X-ray spectra measured here may be used as a reference for comparison against other laboratory and field collected organic aerosol probed with the same technique. We have found that a nonanoic acid surface may be necessary to explain photochemical production of VOCs in our experiments, but this can be either an air-water or air-solid interface. Intermolecular interactions may occur at a nonanoic acid surfactant interface due to the fact that nonanoic acid molecules are so highly concentrated there. Thus, light absorption and photochemistry may be altered compared to purely gas phase processes, which can explain observed VOCs and subsequent SOA formation. The surfactant nonanol did not yield any photochemical products under the same conditions as experiments using nonanoic acid highlighting the potential influence of closely spaced acid groups at an interface on light absorption. Therefore, photochemistry at interfaces in the presence of saturated fatty acids of various carbon chain lengths or other organic components with carboxyl functions may also result in saturated and unsaturated VOCs and SOA precursors. Fatty acids and other organic surfactants and materials from a variety of sources are frequently observed not only in the sea surface microlayer, but also in marine and terrestrial aerosol particles. Cloud droplets are known to possess surfactant compounds, and photochemistry occurring on their surface may therefore be a source of SOA precursors. We have shown that SOA particle nucleation can occur even under high relative humidity (~75%). Previous studies have observed ultra-fine and accumulation mode particle concentrations within and at the borders of clouds which may have been the result of new particle formation over spatial scales of 100 m3 multiphase atmospheric simulation chamber following Bernard et al.31. Note that the chamber used here has been significantly improved over the study by Bernard et al.31 by lowering its particle background levels, thereby increasing its sensitivity to new particle formation. A basin holding 30 L of water (resistivity of 18.2 MΩ cm) was placed at the bottom of the Teflon chamber to which nonanoic acid  or nonanol (99% Alfa Aesar) was added at 2 mM resulting in a pH of about 432. These covered the 0.6 m2 water surface in excess such that surfactant lenses were in equilibrium with a monolayer at its respective equilibrium spreading pressure32. When desired, humic acid was added to the water at a concentration of 3 mg L−1. Visible and UV fluorescent light irradiated the chamber volume and the surfactant at the air-water interface31. Figure 58. Gas phase concentration of volatile organic compounds (VOCs) was monitored with a selective reagent ion time of flight mass spectrometer  using either H3O+ or NO+ as source reagent ions. For all experiments  up to 80% experienced in the chamber yielded (H2O)H3O+ ion clusters amounting to about 20% of total ions. In the NO+ mode, reactions with VOCs undergo charge transfer, hydride ion transfer, hydroxide ion transfer or clustering. Instrument settings used in NO+ mode were 5.5 mA source current, 24 V source voltage, 70 V source outlet voltage and a 5.4 and 2.8 sccm O2 and N2 flow, respectively. These settings yielded total source impurities O2+, NO2+ and H3O+ at <10% of the reagent ion signal. For both ionization modes, drift tube pressure, temperature and overall applied voltage was 2.24 mbar, 60 °C and 600 V, respectively, which led to an E/N ratio of 135 Td  chosen to minimize the formation of water clusters due to high RH38. Temperature and RH in the chamber were recorded  and the concentrations of NO and NO2 (Eco Physics CLD88p coupled with PLC860) and O3 (Thermo Scientific 49i) were measured. Chamber experiments were performed in a N2 atmosphere with a slight positive operating differential pressure >3.5 Pa using a flow of ~7.5 L min−1 to compensate a total sampling flow of ~4.5 L min−1 and any chamber leaks. Ozone was generated using O2 gas at 0.5 L min−1 which passed either through a corona discharge or a UV light generator (Jelight Model 600) and then injected into the chamber.Experiments were conducted in a 2 mnts Figs –S9, sett−1 of N2 for more than 48 hours after which RH <5% was maintained. After flushing, background checks were performed using a 7.5 L min−1 flow of N2 and turning on and off visible and UV light and with and without the presence of 0.6–5.0 ppm of O3. Clean chamber conditions were met if particle concentrations remained below 1 cm−3 and the sum of NO and NO2 concentrations (NOx) was maintained at <0.6 ppb. After cleaning, 30 L of water was introduced into the basin at a liquid flow rate of ~1 L min−1 using a peristaltic pump and Teflon plumbing lines31. Background checks were again performed using UV irradiation and ozone injection into a chamber before and after filling the basin with pure water. Concentration of NOx always remained <1 ppb and particle concentrations <10 cm−3 during water injection. After all background levels were established, the basin was emptied, dried and then refilled while injecting 10.5 mL of nonanoic acid inline using a syringe through a septum installed immediately before the basin31. The chamber was then flushed (40 L min−1 N2) for hours or days and returned to experimental conditions (7.5 L min−1 N2) for another day before commencing UV irradiation of the surfactant interface. When VOC concentrations reached an approximate steady state concentration while irradiated with UV, ozone was injected in a short burst (between 20 s and 2.5 min depending on the generator used) reaching a maximum chamber concentration of about 600 ppb. Before and after ozone injection, aerosol particle concentrations, Np, with particle diameters dp > 3 nm were monitored  and aerosol particle size distributions for dp > 10 nm were measured .The experimental chamber and water basin was scrubbed using ethanol, then rinsed with water and dried thoroughly before each experiment. After cleaning, the chamber was flushed with 40 L minNp as a function of time after ozone injection in a chamber experiment having the lowest total observed particle numbers. The new particle formation event is clear from the steady increase of Np that reached a maximum after 40 min, similar in time to other SOA particle nucleation events from experiments presented here. An example of an instance in which particle concentrations sharply increased immediately after ozone injection in a chamber containing pure water is shown as blue symbols. Particles inadvertently introduced into the chamber were likely due to contamination from the corona discharge ozone generator. Small particles were removed prior to entering the chamber by directing the O2/O3 flow through 10 m of ¼ inch Teflon tubing and 2 HEPA filters. Despite these measures, instances of particle contamination did occur, although infrequently and easily identifiable. These were not considered as new particle formation events. Particle contamination using the UV light generator did not occur.Figure 58 located at a fixed point at the center of the chamber 10 cm above the air-water interface. Light produced from the UV fluorescent tubes used in chamber experiments largely had wavelengths, λ, between 300 to 400 nm. We note that measurements for λ < 300 nm yielded detection limit values on the order of 10−3 W m−2 nm−1, thus we assume that total light output in this range is negligible, but shown in Fig. http://cprm.acom.ucar.edu/Models/ TUV/Interactive_TUV/). Rossignol et al.12 irradiated a 15 mL reactor cell half filled with a solution of 2 mM nonanoic acid and water using a Xenon lamp with an irradiance spectrum shown in Fig. The absolute irradiance Fig.  was measet al.59 and brought to the PolLux beamline (X07DA) of the Swiss Light Source located at the Paul Scherrer Institute for analysis using STXM/NEXAXS60. A more detailed overview of this technique can also be found in previous literature49 and is only briefly described here. STXM/NEXAFS was used to explore the organic carbon functionality of particles using monochromatic X-rays with energy between 278–320 eV, which is the range of electron binding energies for ground state electron orbitals of the carbon atom, also known as the carbon K-edge. Absorption spectra of X-ray light at the carbon K-edge were converted to optical density (OD) as a function of X-ray energy. Energy calibration was performed by comparing the measured lowest energy peak of polystyrene with its literature value61. Series of particle OD images at closely spaced energy steps were taken with a spatial resolution of 35 × 35 nm. Reported spectra used a 2 ms pixel dwell time scanning over the entire particle and imaging at 98 energies or energy points. The OD averaged over the 2-D projected particle area is reported, where OD = −ln(I/Io), and I and Io are the transmitted and initial light intensities, respectively.STXM/NEXAFS was used to investigate the organic carbon composition of SOA particles by irradiating them with X-rays and measuring absorption with high energy resolution. Nucleated SOA particles were collected on copper TEM grids with a quantifoil film (Agar Scientific) following R’mili If particles were damaged by the X-ray beam, then it would be expected that the carbon absorption spectra would change significantly upon repeat exposure of the same particle. Figure Supplementary Information


Ultrathin two-dimensional palladium nanosheets with selectively exposed surface facets were controllably synthesized by designed functional surfactants. e.g., COOH, pyridyl and quaternary ammonium) and halide counter ions  in the long-chain surfactants onto different Pd planes results in the epitaxial growth of {100}, {110} and {111}-exposed surface facets of ultrathin PdNSs. Our synthetic approach is a general, powerful and scalable method to precisely control the surface facets of ultrathin 2D PdNSs, thus providing an opportunity to evaluate facet-dependent catalytic performance of Pd nanocrystals. Ultrathin PdNSs have been examined as the electrocatalysts for hydrogen evolution reactions (HERs). We show that {100}-exposed PdNSs display superior catalytic activity and stability for HERs, compared to that of {110} and {111}-exposed ones as well as their bulk counterparts. Conceivably, our findings will offer a general guideline in rational design of surfactant templates for other 2D metal nanosheets.We report a facile bottom-up synthetic approach to preparing ultrathin two-dimensional (2D) palladium nanosheets (PdNSs) with selectively exposed surface facets. Our synthetic strategy is based on the utilization of the nanoconfined lamellar mesophases of amphiphilic functional surfactants to template the growth of PdNSs in aqueous solution. Preferential adsorption of functional groups ( C22) that direct the self-assembly into lamellar mesophases, (ii) the hydrophilic functional heads which stabilize the lamellar mesophases, bind/confine metal precursors with specific planes, and drive the epitaxial growth along plane directions into ultrathin 2D nanosheets, and (iii) the halide counter ions that assist the control of the exposed surface facets of 2D nanosheets  between functional head groups of the surfactants and different Pd crystal planes 2CH2COOH (Br–) (CnN–COOH (Br–), n = 14–22) as templates. COOH group of surfactants thermodynamically favors the adsorption onto Pd{100} planes.2PdBr4, obtained by the rapid ligand exchange between PdCl42– and Br–)22N–COOH (Br–) in aqueous solution, to direct the self-assembly into lamellar mesophases at 35 °C  simultaneously as the templates and capping agents.The nanostructures of as-resultant PdNSs{100} are revealed by electron microscopy. As shown in anosheet . The frianosheet , slightlanosheet  and the 22–Py (Br–)). The as-designed functional surfactant of C22–Py (Br–) thus directed the growth of ultrathin PdNSs{110}. On the other hand, the surfactants with only QA groups and Cl– as counter ion (C22–QA (Cl–)) were designed to decrease the chemisorption interactions with the Pd{100} or {110} planes, and to expose the Pd{111} planes with low surface energy.To synthesize PdNSs with {110} and {111}-exposed surface facets, a set of functional surfactants were further examined by varying binding interactions of hydrophilic head groups with Pd crystal planes. Surfactants having functional Py groups that favor to interact the Pd{110} crystal planes,Low-magnification TEM images show that PdNSs{110} and PdNSs{111} are ultrathin with similar sheet nanostructures as PdNSs{100} . PdNSs{1We extended our investigations to understand whether the alkyl lengths and reaction temperatures affect the nanostructures of as-resultant PdNSs. As shown in 22 were used to form stable lamellar mesophases; while the head groups and halide counter ions were varied  led to the formation of PdNSs with the mixed surface facets of {100} and {110} planes with a ratio of 39 : 61, confirming the preferential interaction of Br– ions with Pd{100} or {110} planes  and C22–Py (Cl–), PdNSs{111} mixed with PdNSs{100} ({111} : {100} = 46 : 54) and PdNSs{110} ({111} : {110} = 32 : 68) were simultaneously observed  at a scan rate of 5 mV s–1. PdNSs with different exposed surface facets exhibited completely different HER activities, following an order of PdNSs{100} > PdNSs{110} > PdNSs{111}. The lowest overpotential of 67 mV was seen for PdNSs{100} at a current density of 10 mV cm–2, which is 91 and 160 mV lower than that of PdNSs{110} and PdNSs{111}, respectively (–2). The similar trends of HER activities were also observed at current densities at 50 and 100 mV cm–2  and Pd black (PdB)  chronoamperometric response was also recorded by a continuous potential of 42 mV at the current density of 50 mA cm–2 , C22–Py (Br–) and C22–QA (Cl–), respectively. Lastly, the surface facet-dependent electrocatalytic activity of ultrathin PdNSs was also examined by HERs. We found that the PdNS{100} exhibited much better electrocatalytic activity and stability, compared to the PdNS{110} and PdNS{111}. This work represents the first successful example for facet-controlled synthesis of ultrathin PdNSs. We believe that the concept of 2D-templated strategy deriving from the self-assembly of novel designed functional surfactants could provide new insight in the prospective construction of other kinds of ultrathin noble metals or related inorganic nanomaterials.In summary, we reported a novel but facile surfactant-assisted synthetic approach for epitaxial growth of ultrathin 2D nanosheets with well-controlled exposed surface facets, for the first time. The key to the synthesis of ultrathin PdNSs with specific exposed facets is the utilization of surfactants having functional hydrophilic head groups as the templates and capping agents. The change in hydrophobic carbon chain lengths, functional head groups and halide counter ions of the surfactants resulted in specifically exposed surface facets of ultrafine metal nanosheets. As a result, all three low-index exposed crystal facets of {100}, {110} and {111} in ultrathin PdNSs were synthesized using the surfactant of CThere are no conflicts to declare.Supplementary informationClick here for additional data file.


The formulation of the nutrient source was important to meet other nutrient deficiencies in most of the soils. Farmers using Legumefix + Sympal require yield increases of 35% for profitability  while farmers using Minjingu + inoculant require a yield increase of at least 68%. Inoculants used alone were most profitable but this is advisable only when farmers are too resource constrained to afford fertilizer. For sustainable yields Legumefix + Sympal or Biofix + Sympal were recommended.Soybean grain yields in sub-Saharan Africa have remained at approximately 50% below those attained in South America despite numerous efforts. A study was conducted in Siaya County (western Kenya) involving 107 farms with soils of different fertility status. The main objective was to test combinations of two inoculants (Legumefix and Biofix) and nutrient sources  to raise soybean grain yields. Inoculation used Legumefix or Biofix with and without Minjingu or Sympal in a factorial design. There was soil acidity and a widespread deficiency of potassium, nitrogen, and phosphorus. Inoculation and nutrient source led to increases in nodulation and nodule occupancy. For grain yields the response varied from nil in some sites to high increases in others. Highest grain yields (3000–4000 kg ha In othed States  and 28 korldwide . To achiorldwide . Nitrogeorldwide . Soil isorldwide . The alticulture . The coniculture .Rhizobia inoculants have proven to be a more viable and sustainable approach to meet the its high N demand estimated at 80 kg N per 1000 kg of soybean grain . Soybean−1  of Siaya County in western Kenya. Siaya County is located at 00 08.468′N, 34°25.378′E, and 1336 m asl. The experimental sites were in the lower midland 1 (LM1), lower midland 2 (LM2), and upper midland 1 (UM1). These agroecological zones experience bimodal rainfall with long rains (LR) from March to July and short rains (SR) from late August to December . Average2O) was determined as described by The farmers who participated in the trials were selected randomly First all the villages in each region were listed and random numbers were generated using Microsoft excel. From each selected village the names of all the farmers in the village were obtained and four were selected using random numbers. In total, 107 randomly selected farmers participated in the trials. Initial soil samples were taken for analysis before treatments were applied. Available P was determined using the Mehlich 3 method ; pH (H2O2.2® soya from Legume technology (UK) containing Bradyrhizobium japonicum strain 532c (® soya from MEA Ltd (Kenya) containing Bradyrhizobium diazoefficiens strain USDA110   was used2.3This was done at 50% podding when nodules are expected to be fully functional. In one of the inner inoculated rows about 50 cm from the beginning of the line, a length of 50 cm was cut and the roots and nodules were dug out. Data on the number of plants and the nodule biomass were taken. The nodules were surface sterilized and stored in glycerol  for dete2.4This was done at physiological maturity of the crop within the effective area, omitting the outer lines and the sampled row. Weights were taken at harvest and after oven drying to determine grain yields2.52 for 2 min, then rinsed three times with sterile distilled water. One nodule was crushed in 150 μl of sterile water and DNA was directly extracted xtracted . Total g2.5.12. The PCR amplification was performed in a Bio-Rad iCycler™ thermal cycler adjusted to the following program: initial denaturation for 5 min at 94 °C, 35 cycles of denaturation (30 s at 94 °C), annealing (30 s at 58 °C) and extension (30 s at 72 °C) and a final extension (7 min at 72 °C).Genetic diversity was determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) amplification and restriction of the 16S-23S rDNA intergenic spacer region. A 930–1100 bp intergenic region between the 16S and 23S rDNA was amplified by PCR using rhizobia-specific primers derived from the 3′ end of the 16S rDNA   and from2EDTA·2H2O, and 0.55% boric acid), pre-stained with 0.033 mg ml−1 of Ethidium Bromide. The gel was photographed under UV illumination with Gel Doc (BIO-RAD) Software (USA). Aliquots (10 μl) of PCR products were digested with the restriction endonucleases MspI and HaeIII (5 U) in a total volume of 15 μl for 2 h at 37 °C. The restriction fragments were separated by horizontal electrophoresis in 1X TBE buffer with 3% agarose gel prestained with 0.033 mg ml−1 of Ethidium Bromide. The gels were run at 100 V for 3 h and photographed under UV illumination with Gel Doc  software. Strains with identical restriction fragment profiles  were classified into the same intergenic spacer (IGS) group.The PCR products were visualized by electrophoresis of 3 μl of the amplified DNA on 2% horizontal agarose gel in TBE buffer  was computed using Eq. The values obtained were then used to draw frequency cumulative curves with grain yield increases and a VCR value of 3 used to determine profitability .33.1−1 of soil) for 87% of the soils and thus it would be necessary for farmers to include a P source for soybean production. Only 8 farms had P in levels that could be considered adequate for the short term. About 60% of the soils had low to very low N (less than 0.12% N). Other soils had moderate N (highest 0.2% N) and therefore a response to inoculation was expected as soil N would not be adequate to support optimal soybean growth.The summary of soil characterization data is shown in −1 of soil) and 29% as low (121–160 mg K kg−1 of soil). Similarly, a deficiency in calcium was common with 103 of the 107 farms having levels rated as low (9500–1000 mg Ca kg−1 soil) and very low (<500 mg Ca kg−1). The data from soil chemical analysis showed low soil pH, N, and P, and widespread deficiency in K and Ca.Organic Carbon was rated as either low or moderate (0.5–3% C) for all the farmers; 64% had a K rating of very low  due to inoculation, P source, and their interaction. The highest nodulation was observed in season 1 of LR2014 for all the treatments and a significant reduction was observed in the next two seasons (SR2014 and LR2015). In each season the non-inoculated controls (Control – None) had the lowest nodulation that was attributed to the low initial population of rhizobia in most soils as indicated by the MPN tests. As expected no differences were observed between the control group using Biofix and those with Legumefix. Similarly, there were no significant differences (P < 0.05) between Minjingu for farmers using Biofix and those using Legumefix.−1) in Wagai Division. There were no significant differences between non-inoculated controls and those receiving either Minjingu or Sympal alone in all the divisions, indicating that inoculation is paramount for increased nodulation and BNF to be observed. It was also noted that there was higher nodulation where Sympal + inoculant was used compared to Minjingu + inoculant and this was probably due to the difference in the formulation of the two products. The trend in nodulation for the three seasons was similar with the lowest being in Controls, slightly higher (even though not significant) in plots receiving P, and highest in P + inoculant plots. Nodulation was highest in season 1 (LR2014) and lower in both SR2014 and LR2015.The highest nodulation was observed when the inoculants were combined with a P source . LegumefApplication of Sympal (Containing 0:23:15[N: P2O5:K2O] + 10CaO + 4S + 1MgO + 0.1Zn) led to a response for both nodulation and grain yields compared to Minjingu (containing P and Ca). This is in agreement with the findings that reported K and S are among the important drivers of BNF . Nodulat3.3P < 0.05) affect nodule occupancy but having a P source was important especially for strain 532c which gave better nodule occupancy with P than without it. This could be related to the N-fixing efficiency of the strains. The more efficient strain 532c is likely to be more sensitive than the strain USDA110 to an adequate P supply for the energy-demanding process of BNF.Nodule occupancy was determined for 33% of the inoculated soybean plots for the LR2014 and SR2014 cropping seasons . InoculaNodule occupancy results indicated that strain USDA110 (Biofix) had a high nodule occupancy rate regardless of P application, suggesting that it could be more adapted to the local environmental condition. The strain USDA100 in Biofix could be less sensitive to the nutrient deficiency-related stress. This may explain the widespread use of USDA110 in inoculum production worldwide and could have made it more competitive than strain 532c under the conditions of the study. The increased nodule occupancy rates in season 2 for Legumefix may seem to support this theory as the residual effect of the season 1 crop would have slightly improved the soil nutrient status as it was also accompanied by a reduction in overall nodule mass. It was also considered a successful inoculation campaign as the 66% threshold of nodule occupancy was met .−1  and non-legumes in mixed stands . Address−1  and comprgentina . This ma3.4−1. Application of Minjingu or Sympal led to a significant yield gain (P < 0.05) above the controls of about 400 kg ha−1. Similar yield increases were obtained when inoculations with Biofix or Legumefix were done, suggesting that P or inoculation alone would lead to only modest yield increases. When inoculant and P source were combined yield increases were about 1000 kg ha−1 above the Control. The yield trends for the three seasons remained the same as in the LR2014 but were significantly higher for all treatments except the non-treated controls and Biofix alone. −1) in some farms in each of the treatments and much higher yields that were more than double the treatment mean observed in all the treatments except for the control, Minjingu + Biofix, and Sympal alone  between the five divisions with the highest mean grain yields obtained in Boro Division and the lowest in Ukwala Division  and all treatments receiving Minjingu in Ukwala Division. Variability in responses to ISFM packages has been reported  based on the negative control were determined and presented against the cumulative frequency of yield increase for Biofix A and LegAlthough impressive yields were observed, yields did not improve in some farms, suggesting that there could be other limiting factors that were not addressed. Minjingu hyper phosphate had better results in soils with lower soil pH and reduced incidences of the no-yield response but the yield gains remained low, probably because of limitations from other missing nutrients essential for optimal yields. Similarly, other key nutrients such as S, Mg, Ca, etc. in the context of full ISFM, may need to be investigated as we seek to improve soybean yields in the wider SSA region. Similarly, varied yields have been reported in Nigeria  in a mulFor adoption of ISFM packages, farmers will often consider both short and long-term benefits of their returns on investment. Traditionally farmers in SSA do not use fertilizers on legumes and even where they do so, levels used are generally much lower than those recommended . The low3.5The value cost ratios (VCR) were variable across treatments . CumulatThe yield responses and profitability of the inoculation and addition of either Sympal or Minjingu also depended on soil conditions in individual farms. Although the use of inoculants was economically very attractive due to their low prices this is only as a starter in a stepwise introduction of inoculants and its accompanying ISFM packages for smallholder farmers to avoid further mining of soil nutrients that would lead to a decline in yields . In the 4−1). Soil analysis showed deficiencies in most of the nutrients tested . Thus, it was possible to raise soybean grain yields up to 4000 kg ha−1 through using rhizobia inoculation and by addressing selected nutrient deficiencies. For a sustainable economic return from using rhizobia inoculants in soybean production in Siaya County of western Kenya, we recommend that their use in the context of ISFM considers other potential limiting factors such as secondary and micro-nutrients, as well as soil organic matter and pH. The geographical spread of the study region which has three agroecological zones demonstrated the need for ISFM packages that can respond to limitations in soil fertility for increased nodulation and grain yields.On average, yields of the TGx1740-2F variety of soybean were relatively low in Siaya County of western Kenya (2000 kg haIITA through the COMPRO-II project funded by the Bill and Melinda Gates Foundation (USA) and the RCT study implemented by the Paris School of Economics and funded by the Conseil National de Recherche Scientifique (France).This work was supported by