Obtaining sufficient RNA yield and quality for comprehensive transcriptomic studies is cumbersome for clinical samples in which RNA from the pathogen is present in low numbers relative to the nucleic acids from the host, especially for pathogens, such as yeasts, with a solid cell wall. Therefore, yeast cell lysis including cell wall disruption constitutes an essential first step to maximize RNA yield. Moreover, during the last years, different methods for RNA extraction from yeasts have been developed, ranging from classic hot phenol methods to commercially available specific kits. They offer different RNA yield and quality, also depending on the original storage medium, such as RNAlater.C. albicans cells stored in Tryptic Soy Broth with 15% glycerol, 10 min of bead beating in a horizontal position in RiboPure Lysis Buffer provided complete cell lysis. Cell lysis efficiency was decreased to 73.5% when cells were stored in RNAlater. In addition, the RiboPure Yeast Kit (Ambion) offered the highest RNA yield in comparison with the automated platform NucliSENS easyMAG total nucleic extraction (bioMérieux) and the RNeasy Mini Kit (Qiagen) according to NanoDrop and Fragment Analyzer. Moreover, we showed that, in spite of the decrease of cell lysis efficiency after RNAlater storage, as compared to storage in TSB + 15% glycerol, RNAlater increased RNA yield during RNA extraction with both RiboPure Yeast Kit and easyMAG, as confirmed by Fragment Analyzer analysis and by RT-qPCR of the RNA from the Internal Transcribed Spacer 2.We observed that, for C. albicans cells stored in RNAlater was obtained by horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the RiboPure Yeast Kit.In our hands, the most efficient cell lysis and highest RNA yield from The online version of this article (10.1186/s12866-019-1473-z) contains supplementary material, which is available to authorized users. Candida albicans is the main infectious agent responsible for oral and vaginal candidiasis [Candida spp. frequently disrupt this co-existence [Candida-specific probes, that hybridize with all mRNAs of Candida, including the different splicing variants, to enrich fungal RNA is a promising method to improve our knowledge on this topic [Saccharomyces cerevisiae, methods for maximal cell lysis efficiency, a crucial step prior to RNA extraction, and for RNA extraction, have not been reported in C. albicans so far. Cell lysis itself is not only important to increase yields of nucleic acids (incl. RNA), but also for other downstream applications requiring cell lysis such as those involving proteins such as SDS-PAGE, western blot, ELISA, MALDI-TOF or MS/MS.didiasis . The funxistence . Occasioxistence . The neexistence , 5. Althis topic . In addiis topic , 8, bactis topic , 10, andis topic , 12. Theis topic . WhereasIn this study, we compared different cell lysis methods and evaluated cell lysis efficiency through microscopic visualization. In addition, we compared three commercially available RNA extraction methods and determined RNA yield, RNA purity and RNA integrity. In addition, we studied the effect of storage of cells in RNAlater on the efficiency of cell lysis and RNA extraction. Finally, we assessed amplifiability of the obtained RNA by means of reverse transcription and amplification (RT-qPCR) of the Internal Transcribed Spacer 2 (ITS2) RNA of the obtained RNA-extracts.7C. albicans cells in log growth phase, and stored these at − 80 °C in RNAlater vs Tryptic Soy Broth + 15% Glycerol . We compared lysis of thawed aliquots with Lyticase in Lyticase Lysis Buffer (LLB) without bead beating - as the control method proposed to yield very efficient lysis - vs bead beating in four different lysis buffers. We also carried out bead beating in two different vortexes, a normal vortex in which tubes are oriented vertically and a hands-free vortex that allows bead beating in a horizontal position and thus increases the lysis area. Subsequently, we evaluated microscopically the percentage of cell lysis that could be obtained with the different treatments. Finally, we applied three commercially available RNA-extraction methods that were used after cell lysis with their cognate cell lysis method and determined RNA-yield , RNA-purity (NanoDrop), RNA-integrity  and efficiency of reverse transcription and amplification (RT-qPCR) of the ITS2 RNA of the obtained RNA-extracts. A general outline of the study is depicted in Fig. To evaluate which of different cell lysis methods and commercial RNA extraction kits were the most efficient to obtain high yield and high quality RNA from yeast cells, we prepared 1-ml aliquots, each containing 10β-mercaptoethanol , QIAzol Lysis Reagent and RiboPure Lysis Buffer (RPLB), which were all used in combination with 10 min of horizontal bead beating with 0.5 mm zirconium beads in a vortex fitted with a vortex adapter. This adapter allows hands-free vortexing and also bead beating in a horizontal position, increasing the surface area for shearing and lysis. Using microscopy, we observed 100% cell lysis when Candida cells, stored in TSB-G, were bead beaten with RPLB, which outperformed all other lysis methods tested, since easyMAG, QIAzol and RLT buffers resulted in 60 to 70% of cells lysed  + 1% sed Fig. a and theC. albicans cells stored in TSB-G by means of horizontal bead beating in RPLB, we asked whether vortexing itself, without beads, would be sufficient to lyse yeast cells. Horizontal bead beating and vortexing without beads were compared, resulting in 37% of cell lysis when vortexing in the absence of beads, as shown in Fig. p < 0.05). We concluded that vortexing alone is not sufficient and that bead beating is needed to lyse yeast cells properly.As we obtained 100% cell lysis of Bead beating vs vortexing without beads in RPLB and saline), we wondered about the importance of the horizontal orientation of the tubes for the lysis of Candida cells. To test this, we compared horizontal bead beating with an adapter during 10 min, as described above, with manual vortexing in a vortex mixer in which tubes are bead beaten manually in a vertical position during 10 min, and we did so only for the RPLB and only after storage in TSB-G.As we observed that bead beating in a horizontal position was an essential step for the most effective lysis of yeast cells (see p < 0.05), and indicate that horizontal bead beating with a vortex adapter is more efficient than vertical bead beating.Comparing horizontal vs vertical bead beating, we observed an average of 48% vs 2% cell lysis respectively in saline and 100% vs 83% respectively in RPLB, each for six different replicates .After having shown that horizontal bead beating was more efficient than vertical bead beating, that bead beating was more efficient than vortexing alone and that RPLB was the most efficient lysis buffer, we next asked whether enzymatic treatment with lyticase would be more efficient than bead beating to lyse yeast cells. For this purpose, we incubated the yeast cells in LLB containing 2 U/μl lyticase during 1 h, a standard lysis procedure for yeast cells that e.g. is being used in a commercially available protocol  and that was used in our previous studies . LyticasC. albicans [RNAlater is an RNA protective agent that has been shown to perform equally well as snap-frozen methods to stabilize transcriptomic profiles . In addialbicans . HoweverFor cells stored in RNAlater, we observed a cell lysis efficiency of 73.5%, when horizontal bead beating was carried out in RPLB, compared to cells stored in TSB-G (100%) Fig. e.No such reduction of cell lysis efficiency was observed when horizontal bead beating was carried out in saline (instead of RPLB) in which 44.7 and 48.3% cell lysis was achieved after storage in RNAlater or in TSB-G, respectively. Thus, these results indicate that RNAlater is impairing cell lysis, but only when horizontal bead beating is carried out in RPLB.Candida cells: the NucliSENS easyMAG, the RNeasy Mini Kit and the RiboPure Yeast Kit, following horizontal bead beating in their cognate lysis buffers. These nucleic acid extractions were followed by DNase treatment to degrade chromosomal DNA. Yield was determined with NanoDrop and Fragment Analyzer. The RiboPure Yeast Kit produced 7 and 6 μg of RNA from yeast cells stored in RNAlater and TSB-G, respectively, according to NanoDrop  after RNAlater storage. RQN values were also excellent, i.e., around 10 for all of the methods, except for the RiboPure Yeast Kit after RNAlater storage, i.e., ~ 8 which was significantly lower (p < 0.05) than after storage in TSB-G, i.e., 9.5. More detailed electropherograms , as described above. Cq values that were obtained after RT-qPCR are shown in Table To further validate the results obtained after RNA extraction, quantitative reverse transcription PCR (RT-qPCR) was carried out, amplifying the ITS2 RNA. RNA was extracted from 10Saccharomyces cerevisiae, i.e., 57% cell lysis after bead beating in saline, but using glass beads for a longer period, i.e., 30 min [C. albicans [In this study, different methods to lyse yeast cells were compared: bead beating with different lysis buffers, vortexing without beads, bead beating with vertical and horizontal orientation of the tubes, lyticase treatment, and cell lysis after storage in plain broth (TSB-G) vs storage in RNAlater. We found that RPLB combined with 10 min of horizontal bead beating with 0.5 mm zirconium beads (as indicated in the RiboPure Yeast Kit’s instruction) led to 100% cell lysis, and therefore this method was selected for further analysis. The presence of RPLB was essential, as we found that horizontal bead beating in saline, resulted in only 48% cell lysis. A comparable result has been reported for , 30 min . In addi, 30 min , 17. We albicans . In factFurthermore, cell lysis was also tested after RNAlater storage. RNAlater is an aqueous solution that stabilizes RNA, preventing its degradation as well as avoiding changes in transcriptomic profiles. The main advantage of RNAlater is that it allows to store samples at room temperature for several hours or at 4 °C for up to one week, according to manufacturer’s instructions. This is especially useful when freezing samples in liquid nitrogen is not possible, as is often the case in a clinical environment. However, according to the Qiagen protocol designated “Optimal RNAlater incubation and removal conditions prior to isolation of total RNA from stabilized cell sample”, it is known that tissues after storage in RNAlater become harder and that greater centrifugal forces are required to pellet cells because of the high density of RNAlater. Therefore, we assessed whether RNAlater might impair cell lysis and actually observed that storage in RNAlater reduced cell lysis in RPLB to 73.5% of the cell lysis efficiency obtained after storage in TSB-G.8 cells of S. cerevisiae [7 cells of C. albicans. In addition, a recent study, that used formamide-EDTA for RNA extraction from S. cerevisiae, obtained an RNA yield comparable to that obtained in this study with the RiboPure Yeast Kit, i.e., 22–30 μg RNA from 3 × 107 cells/ml [After assessing the optimal method for cell lysis after storage in RNAlater, we also compared RNA extraction efficiency for three methods: the NucliSENS easyMAG, the RNeasy Mini Kit and the RiboPure Yeast Kit from samples lysed with easyMAG lysis buffer, RLT buffer + 143 mM β-mercaptoethanol and RPLB, respectively. Since QIAzol Lysis Reagent is also used in combination with the RNeasy Mini Kit, it was omitted from the RNA extraction comparison experiments. We found that the RiboPure Yeast Kit was the most efficient regarding RNA yield. As this kit uses RPLB, this result was not unexpected since the total RNA yield harvested is proportional to the degree of cell lysis, that had been shown to be the highest in RPLB . Furtherrevisiae . This recells/ml .7 cells of S. cerevisiae with silica-membrane columns (Qiagen or Invitrogen), we only obtained 2.89 μg NA from 107 cells of C. albicans, not previously frozen in TSB-G or RNAlater, when we applied their method  higher yields  compared to both other methods. However, when RNA integrity is considered, RNeasy Mini Kit and easyMAG showed better quality in samples stored in RNAlater.C. albicans cells were obtained with the RiboPure Yeast RNA Extraction Protocol. This is especially relevant in samples that are strongly biased against yeasts, e.g., in infected hosts for which the RNA content from Candida constitutes less than 1% of the total RNA content. However, for other purposes, it may be more convenient to sacrifice RNA yield, for example in in vitro experiments in which RNA amounts can be raised simply by using larger amount of cells.Overall, although all three RNA extraction methods tested here provided high quality RNA, whether or not samples had been stored in RNAlater, we found that the most efficient cell lysis and the highest RNA yield from Candida albicans reference strain ATCC 90028 was grown on Sabouraud Glucose Agar plates with Chloramphenicol (50 μg/ml) (Sigma-Aldrich) and transferred to Yeast extract - Peptone Dextrose broth (Sigma-Aldrich). Cell suspensions were subcultured at 32 °C in static conditions overnight until the logarithmic growth phase was reached. Cells were counted in the microscope by using a hemocytometer (Bürker chamber), adjusted to 107 cells/ml, centrifuged at 8000 g for 10 min and pellets were resuspended in 1-ml aliquots in TSB  + 15% glycerol  and stored at − 80 °C.C. albicans cells from logarithmic growth phase were harvested by centrifugation during 10 min at 8000 g, adjusted to 107 cells/ml with a hemocytometer, resuspended in 1-ml aliquots in RNAlater , stored overnight at 4 °C to enable the solvent to penetrate into the cells, and subsequently stored at − 80 °C.g and resuspended in 1-ml aliquots of each of four different lysis buffers: easyMAG Lysis Buffer , RLT Buffer  + 143 mM β-mercaptoethanol (Sigma-Aldrich), QIAzol Lysis Reagent (Qiagen), and RPLB . All 1-ml aliquots were transferred to prefilled tubes with 0.5 mm zirconium beads and further bead beaten for 10 min. Two different bead mills were compared, a manual vortex mixer   and a hands-free vortex genie-2 , fitted with a vortex adapter, holding microfuge tubes in a horizontal position .A total of 24 × 1-ml aliquots of yeast cells in TSB-G and another 24 in RNAlater, that had all been stored at − 80 °C, were thawed, centrifuged during 10 min at 20000 7 yeast cells in TSB-G and another six in RNAlater, all stored at − 80 °C, were thawed, centrifuged during 10 min at 20000 g, resuspended in 250 μl of Lyticase Lysis Buffer, i.e., a lysis buffer ) and incubated for 1 h at 37 °C [A total of six 1-ml aliquots, containing 10at 37 °C , in ordeg and the pellets were resuspended in 1 ml of saline. Ten μl of this saline cell suspension was loaded into a hemocytometer. Yeast cells could be observed with light microscopy and were counted in 25 small squares (together representing a 0.1 μl volume) at a magnification of 400x. The resulting average number of cells/square was used to calculate the total cell number/ml, as follows: cells/ml = average cell number/square × 2 (dilution) × 104.After the different cell lysis treatments, the cell suspensions were centrifuged for 10 min at 8000 β-mercaptoethanol, and the RiboPure Yeast Kit was used for cells lysed with RPLB, according to manufacturer’s instructions. All nucleic acid extracts were treated with 8 units of DNase I (Invitrogen) for 1 h at 37 °C, in order to remove contaminating chromosomal DNA. Resulting RNA was analyzed for quantity and quality with a NanoDrop spectrophotometer ND-1000  and a Fragment Analyzer .RNA from lysates after the cell lysis procedure, as optimized in preceding experiments, was isolated using the RNA extraction method of the cognate lysis buffer that had been used: the NucliSENS easyMAG semi-automated extraction platform was used for samples lysed with the easyMAG Lysis Buffer, the RNeasy Mini Kit was used for cells lysed with RLT buffer + 143 mM Reverse transcription was performed for 5 min at 25 °C followed by 60 min at 42 °C with random hexamer primers and with 0.5 μg of total RNA according to the instructions of the manufacturer of the RevertAid First Strand cDNA Synthesis Kit . qPCR was performed in duplicate with a LightCycler 480  to quantify the ITS-2 region as described previously , 22. UniFor statistical comparisons of cell lysis data, independent experiments performed with six replicates were considered. Data were analyzed using the statistical test Friedman and the statistical test Wilcoxon for paired samples with IBM SPSS Statistics v 25.0 .Additional file 1:Figure S1. (TIF). Fragment Analyzer electropherograms of total RNA obtained with different RNA extraction methods from 107Candida cells, stored in TSB-G and RNAlater. (TIF 160 kb)Additional file 2:Table S1. RNA yield and quality for different RNA extraction methodsa. (PDF 178 kb)Additional file 3:Table S2. Comparison between RNeasy Mini Kit and RNASwift RNA purification. (PDF 176 kb)


Prescriptions for Benzodiazepines (BZDs) have risen continually. According to national statistics, the combination of BZDs with opioids has increased since 1999. BZDs  work to calm or sedate a person by raising the level of the inhibitory neurotransmitter GABA in the brain. In terms of neurochemistry, BZDs act at the GABAA receptors to inhibit excitatory neurons, reducing VTA glutaminergic drive to reduce dopamine release at the Nucleus accumbens. Benzodiazepine Use Disorder (BUD) is very difficult to treat, partly because BZDs are used to reduce anxiety which paradoxically induces hypodopaminergia. Considering this, we are proposing a paradigm shift. Instead of simply targeting chloride channel direct GABAA receptors for replacement or substitution therapy, we propose the induction of dopamine homeostasis. Our rationale is supported by the well-established notion that the root cause of drug and non-drug addictions (i.e. Reward Deficiency Syndrome [RDS]), at least in adults, involve dopaminergic dysfunction and heightened stress. This proposition involves coupling the Genetic Addiction Risk Score (GARS) with a subsequent polymorphic matched genetic customized Pro-Dopamine Regulator known as KB220ZPBM . Induction of dopamine homeostasis will be clinically beneficial in attempts to combat BUD for at least three reasons: 1) During detoxification of alcoholism, the potential induction of dopamine regulation reduces the need for BZDs; 2) A major reason for BZD abuse is because people want to achieve stress reduction and subsequently, the potential induction of dopamine regulation acts as an anti-stress factor; and 3) BUD and OUD are known to reduce resting state functional connectivity, and as such, potential induction of dopamine regulation enhances resting state functional connectivity. Future randomized placebo-controlled studies will investigate this forward thinking proposed novel modality. Psych Central reports that in 2013, Xanax, a benzodiazepine, was the most commonly prescribed drug ), at least in adults, involve dopaminergic dysfunction and heightened stress; an epigenetic phenomenon.While Benzodiazepine Use Disorder (BUD) is very difficult to treat, partly because BZD are used to reduce anxiety and paradoxically induce a hypodopaminergia, we are proposing a paradigm shift. Instead of simply targeting chloride channel direct GABAThis proposition involves coupling the Genetic Addiction Risk Score (GARS) with a subsequent polymorphic matched genetic customized Pro-Dopamine Regulator known as KB220ZPBM. Induction of dopamine homeostasis will be clinically beneficial in attempts to combat BUD for at least three reasons: 1) During detoxification of alcoholism, the potential induction of dopamine regulation reduces the need for BZDs; 2) A major reason for BZD abuse is because people want to achieve stress reduction and subsequently, potential induction of dopamine regulation acts as an anti-stress factor, and; 3) BUD and OUD are known to reduce resting state functional connectivity, and as such, potential induction of dopamine regulation enhances resting state functional connectivity. The experimental support for the neuropharmacological and neurological basis of KB220 can be gleaned from the work of Blum et al. . Future


Glrb mutant macrophages, we found that the glycine receptor is not required for pyroptotic cytoprotection. Furthermore, protection against pyroptotic lysis is independent of extracellular chloride conductance, arguing against an effect mediated by ligand-gated chloride channels. Finally, we conducted a small-scale, hypothesis-driven small-molecule screen and identified unexpected ion channel modulators that prevent pyroptotic lysis with increased potency compared to glycine. Together, these findings demonstrate that pyroptotic lysis can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.Pyroptosis is a programmed process of proinflammatory cell death mediated by caspase-1-related proteases that cleave the pore-forming protein, gasdermin D, causing cell lysis and release of inflammatory intracellular contents. The amino acid glycine prevents pyroptotic lysis via unknown mechanisms, without affecting caspase-1 activation or pore formation. Pyroptosis plays a critical role in diverse inflammatory diseases, including sepsis. Septic lethality is prevented by glycine treatment, suggesting that glycine-mediated cytoprotection may provide therapeutic benefit. In this study, we systematically examined a panel of small molecules, structurally related to glycine, for their ability to prevent pyroptotic lysis. We found a requirement for the carboxyl group, and limited tolerance for larger amino groups and substitution of the hydrogen R group. Glycine is an agonist for the neuronal glycine receptor, which acts as a ligand-gated chloride channel. The array of cytoprotective small molecules we identified resembles that of known glycine receptor modulators. However, using genetically deficient  Although pyroptosis contributes to pathological inflammation and cell death, it is also an essential protective host response to infection6. Pyroptosis is mediated by proteases in the caspase-1 family, which are activated by the innate immune signaling platforms termed inflammasomes. Inflammasomes respond to microbial or damage-associated stimuli via pattern recognition receptors in the NOD-like receptor (NLR) and AIM2-like receptor families7. Caspase-1 proteolytically converts the proforms of interleukin 1β (IL-1β) and IL-18 to mature inflammatory cytokines. Caspase-1 also cleaves gasdermin D, releasing the N-terminal pore-forming domain, which inserts into the plasma membrane8. Gasdermin D pores mediate osmotic cell swelling, rupture of the plasma membrane, and release of intracellular contents including the enzyme lactate dehydrogenase (LDH)10. The importance of pyroptotic death in the pathogenesis of disease is highlighted by protection of gasdermin D knockout mice from conditions including septic lethality11 and autoinflammatory disease13.Pyroptosis is a programmed process of lytic, proinflammatory cell death14. Glycine does not inhibit upstream caspase-1 activation, pore formation, IL-1β secretion, or loss of mitochondrial membrane potential, but specifically affects the final lytic event, demonstrating that this process can be independently manipulated16. Glycine also has a well-demonstrated cytoprotective effect on cell death resulting from hypoxia and oxidant injury (reviewed in ref. 17). In these models, there is no role for glycine metabolism, ATP preservation, changes in cytosolic calcium, intracellular pH regulation, or cytoskeletal stabilization. The presence of glycine during hypoxic injury prevents loss of viability and allows cells to recover respiratory function and ATP levels upon reoxygenation18. Although the mechanism underlying glycine protection against hypoxia and oxidant injury is incompletely understood, multiple lines of evidence point to glycine acting as a ligand at an unidentified cell surface receptor17. Glycine administration is highly protective in models of sepsis21, suggesting that understanding the mechanism of glycine action may provide novel therapeutic targets for inflammasome-mediated pathology. In this study, we describe specific structural requirements for glycine protection against pyroptotic lysis. We additionally identify novel inhibitors of pyroptotic lysis with increased potency compared to glycine.Glycine is a simple amino acid, which prevents terminal loss of membrane integrity during pyroptosis via unknown mechanismsSalmonella infection and anthrax lethal toxin to trigger pyroptosis in murine bone marrow-derived macrophages (BMDMs) via the NLRC4 and NLRP1b inflammasomes, respectively22. Pyroptotic lysis was assessed by measuring release of the large cytoplasmic enzyme, LDH. Consistent with prior studies14, we observed that glycine prevented LDH release from Salmonella-infected and lethal toxin-treated macrophages  Fig. , withoutect Fig. . These rSalmonella-induced pyroptotic lysis with methylamine, ethylamine, or ethanolamine  and act as an inhibitory neurotransmitter28. The beta subunit interacts with the scaffolding protein gephyrin, and heteropentameric receptors are thought to consist of either two alpha and three beta subunits or three alpha and two beta subunits31. In heterologous expression systems, alpha subunits alone are sufficient to produce functional homomeric glycine-gated channels28. Alpha subunit expression is anatomically and developmentally regulated in the nervous system. Expression of GlyR subunits in nonneuronal cell types has been described, and functional evidence suggests the possibility of GlyR activity in macrophages33.GlyR is a pentameric receptor composed of alpha and beta subunits; four differentially expressed alpha subunits and one beta subunit have been identified34. We therefore tested the hypothesis that propofol might potentiate the cytoprotective ability of β-alanine and taurine against pyroptotic lysis. Propofol alone afforded no protection against pyroptotic lysis, but enhanced the cytoprotective ability of β-alanine  mutant mice. The spa mutation is a 7 kilobase insertion in intron 6 of Glrb, causing aberrant splicing and reducing expression of the GlyR beta subunit to undetectable levels39. Macrophages from spa/spa mice underwent pyroptotic lysis that was inhibited by both glycine and strychnine  and GlyR function and suggest that these small molecules act at another target to prevent pyroptotic lysis.The neuronal GlyR is a heteropentamer comprised of alpha and beta subunits, but in heterologous expression systems, alpha subunits alone are sufficient to produce functional homomeric glycine-gated channelsium Fig. . These f35, but protection from pyroptotic lysis requires micromolar concentrations, potentially consistent with lower affinity binding to a related receptor. To identify novel inhibitors of pyroptotic lysis, we performed a small-scale, hypothesis-driven screen of small molecules with known activities at the GlyR and related ligand-gated ion channels . In these models, glycine protection is reversible and there is no role for glycine metabolism, ATP preservation, intracellular pH regulation, or cytoskeletal stabilization. During pyroptosis, glycine protection is also rapidly reversible15 and cellular ATP is not preserved16. The profile of molecules that protect against ATP depletion and oxidant injury does not match known GlyRs, but does share some similarities with our findings. For example, similarly to pyroptosis, β-alanine is protective against hypoxic death, while taurine is not46. Strychnine also protects against ATP depletion and oxidant injury, and acts at the cell surface49. More limited data also suggests cytoprotection against hypoxia with pregnenolone sulfate and muscimol50. However, in contrast to our findings with pyroptosis, bicuculline also protects against hypoxic cell death48.Glycine cytoprotection has been well documented in diverse cell types in other models of cell death, including ATP depletion and oxidant injury  containing 5% FCS (DMEM-5). For the GlyR β subunit experiment, BMDM were cultured from wild-type C57BL/6 and B6.Cg-Glrbspa/J mice (JAX #000066) (Jackson Labs). To test the importance of GlyR chloride channel function, macrophages were washed with and incubated in NaCl-HBSS or gluconate-HBSS: 138 mM NaCl (or Na gluconate), 5 mM KCl (or K gluconate), 1.8 mM CaCl2 (or Ca gluconate), 4.2 mM NaHCO3, 0.34 mM NaH2PO4-H2O, and 0.44 mM KH2PO4.BMDMs were cultured from wild-type Balb/c mice (Jackson Labs) for 7 days at 37 °C in 5% COSalmonella typhimurium SL1344 grown in L-broth containing 0.3 M NaCl were used to infect macrophages (MOI 10:1) for 90 min, unless otherwise indicated. Cells were treated with anthrax lethal toxin comprised of 1 µg/ml protective antigen and 1 µg/ml lethal factor  for 2 h14. Cells were treated with the detergent-based lysis buffer included in the Cytotox 96 Kit (Promega) for 30 min. Release of cytoplasmic LDH was determined from triplicate samples using the Cytotox 96 Kit and calculated as 100 /(maximum LDH −spontaneous LDH).Macrophages were pretreated for 30 min with glycine or one of the other small-molecule inhibitors at the indicated dose. All chemicals were purchased from Sigma-Aldrich, with the exception of propofol (EMD Millipore). Late-log cultures of 5 cells/well (24-well plate) prior to infection with S. typhimurium or treatment with anthrax lethal toxin. Active caspase-1 was detected using a caspase-1-specific probe23. FAM-YVAD-FMK (Immunochemistry Technologies) was added to a final concentration of 5 μM for the last 60 min of treatment. Cells were washed twice to remove unbound probe, then fixed and stained with the nuclear dye To-Pro-3 (Molecular Probes). Multiple fields were examined using a Leica SL confocal microscope (W.M. Keck Microscopy Center), and the percent of positive cells was determined using 3–10 fields for each experimental condition.Macrophages were seeded onto glass coverslips at a density of 2 × 10t-test. IC50 values were determined using nonlinear regression and the [Inhibitor] vs. response – variable slope equation in Prism software (GraphPad).Data were analyzed by unpaired two-tailed Student’s Supplemental Material


Ocular diseases cause visual impairment and blindness, imposing a devastating impact on quality of life and a substantial societal economic burden. Many such diseases lack universally effective pharmacotherapies. Therefore, understanding the mediators involved in their pathophysiology is necessary for the development of therapeutic strategies. To this end, the hydrolase activity of soluble epoxide hydrolase (sEH) has been explored in the context of several eye diseases, due to its implications in vascular diseases through metabolism of bioactive epoxygenated fatty acids. In this mini-review, we discuss the mounting evidence associating sEH with ocular diseases and its therapeutic value as a target. Substantial data link sEH with the retinal and choroidal neovascularization underlying diseases such as wet age-related macular degeneration, retinopathy of prematurity, and proliferative diabetic retinopathy, although some conflicting results pose challenges for the synthesis of a common mechanism. sEH also shows therapeutic relevance in non-proliferative diabetic retinopathy and diabetic keratopathy, and sEH inhibition has been tested in a uveitis model. Various approaches have been implemented to assess sEH function in the eye, including expression analyses, genetic manipulation, pharmacological targeting of sEH, and modulation of certain lipid metabolites that are upstream and downstream of sEH. On balance, sEH inhibition shows considerable promise for treating multiple eye diseases. The possibility of local delivery of inhibitors makes the eye an appealing target for future sEH drug development initiatives. Visual impairment and blindness from ocular diseases can profoundly compromise patients’ quality of life, and imposes a substantial economic burden of $35.4 billion per year in the United States . The devThere is a growing awareness of the importance of bioactive lipid metabolism to ocular structure, function, and pathology. Especially, the unique lipid profile of the retina gives an outsized role for docosahexaenoic acid  and DHA-derived polyunsaturated fatty acid (PUFA) metabolites in the eye . DHA is EPHX2 gene, has a C-terminal hydrolase function that acts on lipid epoxides, plus a poorly studied N-terminal phosphatase activity  . sEH, enactivity . Inhibitactivity  and analactivity . But theactivity , whereasactivity . Moreoveactivity . Thus, sGenetic manipulation of CYP/sEH expression and small molecule mediated targeting of sEH have allowed investigation of the role of EpFAs in eye diseases, in particular diseases mediated by inflammation and angiogenesis. Through the metabolism of bioactive EpFAs and production of corresponding diols, sEH plays a role in the regulation of angiogenesis and inflammation relevant to the pathogenesis of numerous eye diseases. This mini-review discusses sEH as a therapeutic target for eye diseases and the role of PUFA metabolites of CYP and sEH in ocular neovascularization and other ocular disorders.Ocular neovascularization  is a prominent feature of blinding eye diseases including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and neovascular “wet” age-related macular degeneration (wet AMD) . The newin vitro: all the chemically stable EDP regioisomers inhibited VEGF-induced angiogenesis in a Matrigel plug assay  , a widelEphx2-/- mice. These mice also had increased plasma levels of 17,18-EEQ and 19,20-EDP, since sEH-dependent degradation of these epoxides into corresponding diols was blocked. In contrast, dietary ω-3 PUFAs did not confer inhibitory effects on CNV in mice overexpressing sEH.Dietary intake of ω-3 PUFAs in mice substantially enhanced levels of 17,18-epoxyeicosatetraenoic acid (EEQ) and 19,20-EDP in the serum lipid profile. However, it did not increase levels of EDPs in the retinal lipid profile. Interestingly, dietary intake of 17,18-EEQ or 19,20-EDP also suppressed CNV, suggesting that the protective effect of ω-3 PUFAs against CNV could be mediated by its downstream epoxy metabolites that are generated by CYP. In addition, dietary ω-3 PUFAs interfered with leukocyte invasion into the CNV lesions, while ω-6 PUFA did not . These eEphx2-/- mice. Intravitreal injection of 19,20-DHDP rescued impaired retinal angiogenesis in Ephx2-/- mice. 19,20-DHDP was also found to be a signaling molecule, downregulating the endothelial Notch signaling pathway by inhibiting presenilin-1 dependent γ-secretase activity, which is required for release of the Notch intracellular domain  and “compound 7” (10–30 μM) also suppressed L-CNV lesions  from 1 to 4 days had significantly reduced retinal vascularization    may be bEphx2-/- mice  expression that were downregulated by hyperglycemic conditions. Similarly, sEH inhibition with subconjunctival 10 nM t-AUCB or clinical candidate inhibitor GSK2256294A  , inducing acute inflammation. Contrary to the hypothesis, treatment with UC1728 (3 mg/kg) was not efficacious in attenuating this uveitis. However, it is perhaps premature to conclude that sEH does not play a role in uveitis. Analysis of sEH expression and lipid profiles in the affected site may yet reveal sEH involvement that might respond to local treatment.Uveitis refers to numerous intraocular inflammatory conditions often involving the uvea but not limited to this pigmented tissue layer . Inhibitt-TUCB)  had antit-TUCB) , with a t-TUCB) . In thisOverall, there is strong evidence that stabilization of anti-inflammatory and anti-angiogenic EpFAs through sEH inhibition could be promising therapies for eye diseases. The unique anatomical and physiological features of the eye as a self-contained unit pose both advantages and disadvantages in drug discovery and delivery. Unlike other parts of the central nervous system, the eye is clinically accessible, allowing targeted drug delivery via routes of topical eye drops or intraocular injections, thus systemic side effects can be minimized. Conversely, barriers such as the cornea, blood aqueous-barrier and BRB hinder drug transport and absorption . TherefoOver the past decade, intravitreal anti-VEGF therapy has significantly advanced the treatment of neovascular eye diseases. But the only current delivery route for anti-VEGF agents, due to their large molecular weight, is intravitreal injection. This can be associated with intraocular inflammation, infection, hemorrhage, elevation of intraocular pressure, and cataract , as wellt-TUCB) have been tested for target specificity, with minimal non-specific binding to pharmacologically important proteins, rendering unexpected adverse effects unlikely (Of course, adverse effects are still possible with sEH inhibitors if administered systemically, or if local delivery results in systemic exposure. Some of the compounds discussed here (such as TPPU and unlikely  Nonethelunlikely . UtilizaBP and TC wrote the manuscript, edited the manuscript, and approved the final version of the manuscript.TC is a named inventor on patent applications related to this topic and has received related research support from Inclera Therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N = 356). Study 1 revealed that videos of cute targets evoked significantly more kama muta than videos of targets that were not particularly cute. Study 2, pre-registered, found that, as hypothesized, when cute targets interacted affectionately they evoked more kama muta and were humanized more than when they were not interacting. We measured the level of kama muta by self-reports of sensations and signs and of feelings labeled heartwarming, being moved, and being touched. Participants’ ratings of kama muta were positively correlated with reported cuteness. In addition, as in our previous research on kama muta elicited by other types of stimuli, trait empathic concern predicted kama muta responses and perceived cuteness. The studies thus provide first evidence that cute stimuli evoke the heartwarming emotion of kama muta.A configuration of infantile attributes including a large head, large eyes, with a small nose and mouth low on the head comprise the visual baby schema or Kindchenschema that English speakers call “cute.” In contrast to the stimulus gestalt that evokes it, the evoked emotional response to cuteness has been little studied, perhaps because the emotion has no specific name in English, Norwegian, or German. We hypothesize that cuteness typically evokes kama muta, a social-relational emotion that in other contexts is often labeled in English as being moved or touched, heartwarming, nostalgia, patriotic feeling, being touched by the Spirit, the feels, etcetera. What evokes kama muta is sudden intensification of a communal sharing (CS) relationship, either CS between the person and another, or CS between observed others. In accord with kama muta theory, we hypothesize that a kama muta response to cuteness results from a sudden feeling of CS with the cute target. In colloquial terms, the perceiver adores the cute kittens and their heart goes out to them. When a person perceives cute targets interacting affectionately – that is, intensifying CS between them – this should strengthen a kama muta response. We experimentally investigated these predictions in two studies ( Cuteness overload: An overload of cuteness; when something or someone is so super cute that there is no word for it.Urban Dictionary, 2008Cute attack: A sensational response incited by the witnessing of something cute, precious, fuzzy, or otherwise snuggly. Symptoms include chills traveling up the spine and through the fingertips, impulsive smiling and jerking of the limbs. Severe cases of cute attacks can cause high-pitched squeals and temporary spasms of the entire nervous system, forcing its victim to crumble helplessly to the ground.Urban Dictionary, 2009elérzékenyült in Hungarian, heldinud in Estonian, heltyä in Finnish1. An emotional response to cuteness is widely recognized (if not named) by marketing professionals and utilized in commercial and charity advertising  relationship suddenly intensifies (moved). Kama muta is a positive emotion that people actively seek out, like to evoke in other people, and want to experience together with others. Like other emotions, it varies in intensity.Kama muta theory postulates that a specific emotion, kama muta – which English speakers may label The kama muta construct is based on relational models theory RMT; , 2004. Rbeing moved, touched, heartwarming, tenderness, nostalgia, team pride, patriotism, rapture, being touched by the Spirit, the feels, feeling stirred, and other terms occurs when a CS relationship suddenly intensifies. This conceptualization has been confirmed by robust cross-correlational findings using the appraisal of increased social closeness as a measure of CS  was r = 0.54 . Additionally, the same study by Kama muta theory ,c positsre of CS . In addidicators . For exaAn increase in CS can be recognized subjectively as an increase in trust and feelings of unity with an interaction partner or a relationship partner, or it can be observed. Cues indicating increased CS include commensalism , touch, bodily proximity, synchrony and need-based giving . The sudKama muta theory further posits, and several studies show, that the emotion is characterized by certain sensations and signs. Such experiences typically involve a warm or other feeling in the center of the chest, goosebumps or chills, moist eyes or tears, a lump in the throat, feeling buoyant, being exhilarated, and sometimes also putting a hand to the chest, and saying something like “awww” or corresponding vocalizations in other languages . Being iempathic concern measured by self-reports of being sympathetic, compassionate, tender, softhearted, warm, and moved. Given the similar conceptualizations and operationalizations of empathic concern and kama muta, being moved or touched, correlated 0.35  with trait empathic concern. In a subsequent 19-national study the overall correlation was 0.32  , and be motivated to engage in a CS relation with these protagonists ,b.sudden change.Accordingly, we expect stimuli depicting individual cute animals to evoke first person kama muta (Study 1), and stimuli depicting animals interacting in a loving way to also evoke third person kama muta (Study 2). In this case, the interacting animals should also be perceived as cuter than non-interacting animals specifically because they evoke more kama muta. Our theory also predicts that the change in CS should be experienced as sudden in order to evoke kama muta. Therefore, our appraisal items  tap intosuddenly increased CS, is evoked either by the person’s ‘heart going out to the cute animal’ (first person) or by appraising the loving care that cute animals and their interaction partners display for each other (third person). The cues to increased CS relevant for the third person case that we manipulated in Study 2 are bodily proximity  and feeding. These cues are universal signs of CS  and CS interactions among animals (Study 2) evoke the characteristic components of kama muta, including the typical labels, sensations and signs, motivations, and positive valence. To measure these components, we used a scale highly similar to the validated KAMMUS scale . In a wiKama muta is evoked by sudden intensification of CS, and is indexed by affectionate touching and feeding . Hence, In both studies we tested whether trait empathic concern predicts kama muta responses to cuteness, just as it predicts kama muta responses to the other sudden intensifications of CS we have employed as stimuli.4.The studies were approved by the internal review board of the Department of Psychology, University of Oslo. As recommended by 5The main objective of the first study was to experimentally investigate whether cute animals evoke the kama muta emotion more than animals that are minimally cute. The study tested the following two main hypotheses:H1: Viewing videos of cute animals, compared to videos of minimally cute animals, will evoke stronger kama muta ratings across four components of the emotion: vernacular labels, motivation to form or strengthen CS-relationships, emotional valence, and sensations and signs.H2: Participants higher on trait empathic concern will rate the animals as cuter and will have higher ratings of kama muta in the four components.N = 121 participants through Amazon Mechanical Turk, requesting workers from the United States, and N = 176 Norwegian participants through convenience sampling on Facebook.6 Participants were excluded from the primary analyses based on the following a priori criteria; having more than 20% missing responses, not watching the videos, and being under the age of 18. Of the remaining N = 2177, N = 121 indicated that they were female (N = 3 indicated “other” or skipped that question), N = 105 were US American, N = 101 Norwegian, N = 11 from other countries or missing. Age varied from 19 to 63, M = 31.80, SD = 10.73; two participants did not provide demographic information.We recruited A within- and between-participants design was employed. Condition was a within-participants factor; participants saw both a video of a cute animal and a video of a non-cute animal. The order in which these videos were presented was randomized between participants. After each video, the participants were asked to rate the cuteness of the video and asked about their kama muta labels, valence, communal sharing motivation, and sensations and signs. Lastly, participants responded to the trait empathic concern measure, and provided demographic information.The video stimuli comprised of eight pretested 20- to 40-s video clips depicting either very cute  or minimally cute animals . In each condition, participants saw one video randomly selected from a pool of four videos  to 6 (a lot).The first and last author wrote a cuteness scale of nine items  to measure perceived cuteness of the animals in the videos. The scale was constructed based on a review of the literature, while attempting to identify the most distinctive and prevalent vernacular lexemes colloquially used to denote visual Kindchenschema cuteness. The cuteness scale included distractor items (not included in the number of items), and responses were assessed on a seven-point Likert scale from 0  to 6 (a lot). This measure was an earlier version of the kama muta scale (KAMMUS) later validated in The experience of kama muta was assessed through four subscales, specifically: vernacular labels ; sensations and signs ; motivation to form or strengthen CS-relationships ; and emotional valence . The kama muta scale included distractor items (not included in the number of items), and offered response alternatives on a seven-point Likert scale from 0 (does not describe me well) to 5 (describes me very well). Item-level descriptive statistics and Norwegian translations for all measures can be found in the Empathic concern was measured with a subscale of the interpersonal reactivity index IRI, , 1983. PLastly, participants were asked to indicate whether they listened to the sound of the video  and provide demographic information, outlined in the participants section above.8 (2) three kama muta vernacular labels items , (3) 12 items of sensations and signs , (4) four items of motivation , and (5) the seven items of empathic concern (α = 0.88). The kama muta scores were constructed based on a subset of items validated in Zickfeld et al. (2019)9. We combined all sensation and sign items into one score. Analyses employing the separate sensation and sign factors are presented in the We created five average scores from (1) six cuteness scale items ,rs between 0.48 and 0.79) and in the non-cute condition (rs between 0.45 and 0.77). The consistently strong correlations among the kama muta components support the validity of the kama muta construct, suggesting that these four components tap into the same construct.We first assessed the co-occurrence among the four kama muta components, and the association of these aspects of kama muta with cuteness and empathic concern. In order to do so, we calculated intercorrelations of the cuteness scale, empathic concern, and the four kama muta components  for the cute and non-cute conditions separately see . Intercors between 0.34 and 0.68) in the cute condition. This correlational test of Hypothesis 1 supports the hypothesis that the emotion evoked by seeing cute animals is, in fact, kama muta. The four kama muta scores also correlated with empathic concern in the cute condition, supporting H2.In addition, all four kama muta components correlated strongly with perceived cuteness  for all main effects. We tested the hypotheses that cute animals would evoke more of all four components of kama muta than non-cute animals (H1), and that empathic concern would moderate the effect of condition (cute vs. non-cute) on kama muta ratings and cuteness ratings (H2). We did this by fitting mixed models using the First, we observed a main effect of condition for all five models, as seen in 12 We did not detect a significant interaction effect between empathic concern and order for any of the models.Supporting our second hypothesis, trait empathic concern positively predicted ratings of cuteness, and ratings of all four components of kama muta. For all models we observed an interaction effect of empathic concern with condition: the effects of trait empathic concern were stronger for the high cuteness videos :13Study 1 established that images of cute animals evoke kama muta. Study 2 tested whether adding well-established signs of CS to these stimuli, namely affiliative contact between the cute animals or feeding them results in greater cuteness perception and stronger kama muta reactions, compared to videos of cute animals not interacting and not being fed. A second objective of this study was to investigate whether kama muta responses mediated the effect of the touching and feeding CS manipulations on cuteness perception. Hence, we preregistered the following six hypotheses  videos will be judged as cuter than low CS videos.H2: Compared to low CS videos, high CS videos will evoke more kama muta, as measured by (a) the kama muta sensations and signs, and (b) labels.H3: The effect of CS on cuteness ratings will be mediated by kama muta, as measured by (a) the kama muta labels and (b) sensations and signs.H4: Trait empathic concern positively predicts cuteness ratings.H5: High CS videos will lead to more perceived humanness of the animal protagonists than the low CS videos.H6: Humanness ratings of the animals and kama muta evoked will correlate.a priori power analysis based on an effect size of f = 0.15 , which suggested a total sample size of 148 participants14. We recruited N = 201 participants in Norway through convenience sampling on Facebook and a student research participation pool at the University of Oslo where students were invited to participate in a study investigating emotional responses to video stimuli. As pre-registered, participants were excluded from the primary analyses if they indicated participating for their personal educational purposes only , having more than 20% missing values, and not watching the whole video. Of the remaining N = 139,15N = 107 indicated that they were female (N = 1 indicated “other”), N = 130 were Norwegian, N = 9 from other countries. Age varied from 16 to 63, M = 24.28, SD = 7.58.We conducted an 16) engaging in an affectionate interaction like cuddling, liking, and feeding one another (high CS), and a video of two subjects interacting minimally or not at all (low CS). Another within-subjects factor was type of animal: participants saw one video featuring dogs and one video featuring cats. The between-subjects factors were the video version (Video A or B of a particular stimulus set), and the order in which the videos were presented (high CS first or high CS second).A mixed design was employed. Condition was a within-subjects factor where participants saw a video of two subjects  with two videos each , and five aspects of their own kama muta emotion. Finally, participants responded to the trait empathic concern items, and provided demographic information.not at all) to 6 (a lot). The same empathic concern measure and demographic questions used in Study 1 were again presented in Study 2.A revised six-item scale from Study 1 measured perception of cuteness; 3 negatively worded items  and 3 positively worded items . The experience of kama muta was assessed through five subscales using an earlier version of the KAMMUS that has since been further validated in We created six average scores (1) from the six cuteness scale items ; (2) from the three kama muta vernacular labels ; (3) the 12 items of sensations and signs ; (4) the four items of CS intensification appraisals ; (5) four items of motivation , and (6) the seven items of empathic concern (α = 0.76). As in Study 1, the KAMMUS subscales were constructed based on rs between 0.28 and 0.52), supporting H6. As in Study 1, all of the kama muta factors correlated with all of the others in both the low and high CS condition (rs between 0.42 and 0.79). We also observed positive correlations between the cuteness scale and all five kama muta indicators. Finally, as in Study 1, empathic concern correlated more with all other variables in the experimental  condition, compared to the low CS condition.Correlations among the main variables are presented in 17 The final dataset consisted of a total of 278 video reactions. For all models, intercepts were allowed to vary randomly across participants. We regressed each dependent variable  in a separate model on the same set of predictors: cuteness condition, type of animal presented, order of video, and video version, as well as interactions between condition and order, and between animal type and version. For the cuteness model, we added trait empathic concern as a covariate. All factors were contrast coded  for all main effects. We used a series of mixed models to test the hypotheses.oded see  and empaSeen in Model 1 of 18 There was also a significant main effect of animal type (cats were rated as cuter than dogs).Seen in Model 2, cuteness ratings were higher in the high-CS videos in contrast to the low-CS videos, supporting H1. Supporting H4, we also observed a positive effect of trait empathic concern on the cuteness ratings. Seen in Model 3, participants’ ratings on the kama muta labels were higher in the high CS condition than in the low CS condition see . The samSeen in Model 5, participants rated the animals in the high CS condition as more human than animals in the low CS condition, therefore supporting H5.Finally, we also explored whether condition influenced CS motivation and positive valence ratings. We observed that motivation ratings in Model 6 were higher in the high CS condition compared to the low CS condition. We also observed in Model 7 that ratings of positive valence were higher in the high CS condition than in the low CS condition.In Models 2, 3, 6, and 7 we also found an order effect where the first video had higher ratings than the second video on each of the kama muta components and on cuteness. Order did not interact with condition in any of the models, thus, the order effects do not invalidate the conclusions from the hypothesis tests.Mediation analyses were conducted to test H3, that the effect of high or low CS (video condition) on cuteness ratings  was mediated by kama muta, as measured by the sensations and signs (Model 1) and labels . This finding complements that of moved. Going beyond Batson and colleagues findings, the present findings indicate that one cuteness response is kama muta, by providing evidence for the various components typical of an emotional episode – not only a label but also sensations and signs, an appraisal, and a motivational tendency  – something very much like kama muta – as an outgrowth of the human maternal instinct to care for their own babies, extended to an emotion experienced in a vast array of eliciting situations:Indeed, precisely because of this, we speculate that the phylogenetic source of kama muta is maternal bonding. Mothers must instantly form intense CS bonds to offspring at the moment of birth. In the small percent of species that form pair bonds and the smaller percentage in which siblings and other kin contribute to care of the infant, the father and those kin, too, must instantaneously form CS bonds with the infant. Thus we concur with McDougall who described the In the human being, just as is the case in some degree with all the instinctive responses... there takes place a vast extension of the field of application of the maternal instinct. The similarity of various objects to the primary or natively given object, similarities which in many cases can only be operative for a highly developed mind, enables them to evoke tender emotion and its protective impulse directly.McDougall, 1923moving and heart-warming in itself, even when the protagonists are not cute . In addition, the inter-correlations in The results of Study 2 showed that cute animals interacting communally were seen as more human than cute animals not interacting, albeit with a small effect size , but they do suggest that the sample may not have been representative on relevant dimensions of the Norwegian population especially. For example, people sensitive to cuteness may have been more likely to actually complete the whole study. This was less of a problem for the United States sample in Study 1, in which participants were paid for their time. In this light, the convergence of the findings for Norway and the United States bolsters the central conclusions.A statistical issue in the current studies was the high skew of some of the measures. For example, the sensations and signs of tears and goosebumps were rarely reported, skewing the distributions. To check the robustness of the findings for such measures, these analyses were therefore repeated using non-parametric models. The main results did not differ substantially from the multilevel models see , 17. Thumoved by cute videos seems difficult to reconcile with being moved consists of the experience of a positive, transcendentally significant core value. They write that being moved (or être ému) “is the experience of a positive core value... perceived by the moved subject as standing out” , a round face, and a large head comprise the In sum, the evidence of kama muta responses to cute kittens and puppies poses intriguing challenges to existing understandings of emotions. If these experiments are not persuasive, one only needs to open a browser and search for “cute images and videos.” The enormous amount of cute content on the Internet, the number of views and likes, and the responses that people post in response to them provide overwhelming evidence for the ubiquity and impact of kama muta responses to cuteness.https://osf.io/bjuva/.All datasets, stimulus material and procedures are available at our OSF project page: The current research was carried out in accordance with the recommendations out forward by the Norwegian Centre for Research Data and the Declaration of Helsinki where all subjects gave informed consent. Both studies described in this article received ethical approval from the Department of Psychology’s Research Ethics Committee at the University of Oslo, Norway.KS, BS, and AF designed the studies. KS produced the stimulus material, programmed, and conducted the studies. KS and BS wrote the introduction. JB and JZ analyzed the data, wrote up the methods and results, and created the tables, figures, and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chemical signaling underlies both temporally phasic and extended activity in the brain. Phasic activity can be monitored by implanted sensors, but chronic recording of such chemical signals has been difficult because the capacity to measure them degrades over time. This degradation has been attributed to tissue damage progressively produced by the sensors and failure of the sensors themselves. We report methods that surmount these problems through the development of sensors having diameters as small as individual neuronal cell bodies (<10 µm). These micro-invasive probes (µIPs) markedly reduced expression of detectable markers of inflammation and tissue damage in a rodent test model. The chronically implanted µIPs provided stable operation in monitoring sub-second fluctuations in stimulation-evoked dopamine in anesthetized rats for over a year. These findings demonstrate that monitoring of chemical activity patterns in the brain over at least year-long periods, long a goal of both basic and clinical neuroscience, is achievable. Helen Schwerdt et al. report micro-invasive probes capable of monitoring chemical signaling in the rat brain for over a year. The probes have diameters as small as single neuronal cell bodies and can monitor sub-second fluctuations in chemical signaling without significant induction of inflammation or tissue damage. A major test case for chronic application of neurochemical sensors has been in the use of microscale carbon fiber electrodes (CFEs) to detect redox current of dopamine by fast-scan cyclic voltammetry (FSCV)4. Implantable interfaces have enabled measurement of dopamine at highly localized (microns) and rapid (milliseconds) dynamic scales at which these molecules operate in the brains of rodents and primates17. The capacity of currently existing neurochemical sensors to provide reliable signals over extended time-frames approaching a year has, however, been extremely difficult to achieve because the signals degrade over time-frames tested18. Neurochemical sensors have displayed consistent decline of the signal amplitude over time6 and temporal distortion 12. This apparent degradation brings into question the accuracy of extended measurements and thus lessens the possibility of tracking dopamine over slowly developing behaviors and disease conditions, including Parkinson’s disease.Prolonged changes in chemical signaling and neurotransmission in the brain are thought to underlie cognition, learning, and fluctuations in mental state. Methods for long-term, stable monitoring of the molecular status of individual brain cells, circuits and networks are particularly critical. With these, advances could be made not only in understanding the normal functions of molecular species over extended time-periods, but also in studying the etiology of debilitating neural disorders15 to create micro-invasive probes (µIPs) to address these barriers to chronic implantation. A 7 µm diameter carbon fiber is normally used as it has been shown to provide the optimal electrochemical sensing interface for sensitive detection of dopamine and other electroactive compounds17. Furthermore, the cellular-scale carbon fiber is small enough on its own to produce negligible perturbation and tissue response in the brain20. Chronically applied CFEs, typically constructed with a 90 µm diameter silica shaft12, occupy implant footprints large enough to disrupt normal neuronal circuitry and astrocytic configurations that can extend up to 150 µm and farther from their implanted path and structure24. These inflammatory processes could thus alter extracellular chemical gradients and lessen the physiologic accuracy of sampled molecules at the carbon fiber sensing interface26. The induced trauma further limits practical, safe use in humans. The µIPs that we developed have cross-sectional diameters on the order of individual neuronal cell bodies (<10 µm diameter)15, which are the smallest of any implantable neurochemical sensor available. These sensors gave accurate signals and minimum tissue damage, but were not tested for long-term use. Here, we report modifications of the sensors and their implantation that allow stable, reliable dopamine monitoring for periods of over a year, promising for clinical application.Here, we attempted to resolve these problems in order to provide at least year-long accurate measurements of dopamine release in rats. We first built on our initial attempt27, was applied onto bare carbon fiber (7 µm diameter and 5–7 mm long) to provide a highly conformal and impermeable insulation  built from fused-silica capillaries (90 µm diameter)12. The smaller size resulted in a flexural rigidity that is over three orders of magnitude lower than that of CFEs . The small areal extent of the implanted µIPs minimized tissue response31, while the resulting increased compliance attenuates micromotion-induced inflammation of the indwelling implant33  and compliance of all implanted parts of the devices.The extremely small and flexible features of the µIPs required temporary encasement in a rigid polyethylene glycol (PEG) shuttle 0.5–1 mm thick)  . The µIPs and CFEs were tested, in parallel, in a flow cell containing aCSF to determine how responsive they were to physiologic concentrations of dopamine . Overall background noise levels (Methods) were also measured to determine the minimum distinguishable concentrations  of the sensors. We used FSCV in all reported measurements to record electrochemical current generated at the carbon fiber in response to the reduction and oxidation (redox) of dopamine99) Fig. .15. The measured oxidation current was normalized to the apparent capacitance of the sensor to account for differences in the final exposed lengths of carbon fiber across probes. The normalized sensitivity was virtually unchanged  for both µIPs (5.75 ± 1.22 × 10–5 nA nM–1 nA–1) and CFEs (4.09 ± 0.67 × 10–5 nA nM–1 nA–1)   Fig. . The curnA) Fig. .P = 0.92, two-tailed t-test) before treatment (5.75 ± 1.22 × 10–5 nA nM–1 nA–1) and after PEG treatment (5.52 ± 1.8 × 10–5 nA nM–1 nA–1)  before (0.06 ± 0.01 nA) and after PEG application (0.05 ± 0.01 nA)  Fig. . The noinA) Fig. . These m12. Our goal in creating smaller implants was to reduce the induced inflammatory response and tissue damage surrounding the probes, which can restrict neurochemical diffusion12 and can compromise the long-term safety of the implant. We compared tissue response surrounding the implanted shafts of both µIPs (<10 µm diameter) and CFEs (90 µm diameter) after 74–361 days of chronic implantation in the brains of rats. This assessment was performed around the shafts to evaluate the trauma representative of the majority of the implanted device. The histochemical analyses were performed on successive horizontal brain sections to identify the vertical tracks of implanted devices through the tissue of the striatum. Selected sections were ~0.5–1 mm away from the probe tip and >2 mm from the dural surface to focus on regions around shafts, away from lesioned tips and away from any surgically induced trauma at the cortical surface. Selective markers of activated microglia (Iba1), astrocytes , and blood brain barrier permeation  were used to assess the tissue inflammatory response . The CFEs produced extensive responses of 6.73 for GFAP and 10.5 for IgG . The minimal neuroimmunological response induced by µIPs could provide less restricted diffusion of targeted dopamine molecules to the sensor, thus reducing temporal distortion, a well-known limitation of CFE-based dopamine measurements38.The µIPs induced remarkably negligible tissue response, in contrast to the response to CFEs  around the center of implanted sites. The radius of this protrusion was measured by computing the distance from the center of the implant track to the position at which DAPI intensity reached background levels. µIPs displaced cells to a radius of 5 µm, whereas CFEs induced 34 µm of displacement. These values correspond closely to the radius of the implanted shaft, as expected. The negligible tissue distortion induced by the µIP could help to retain normal extracellular chemical signaling processes, thus promoting more accurate measurements of neuronal circuit activity.The reduced expression of inflammatory markers, coupled with the smaller disruption of the local cellular infrastructure, illustrates the capacity of the µIPs to induce minimal damage to healthy tissue of the nervous system. We next tested whether these properties could enhance their performance in up to year-long molecular sampling.n = 7), 3–5 µIPs were implanted into the striatum in order to increase the yield of implanted sites situated in active dopamine-releasing volumes (~50% probability)15 15 Fig. . Stimula)15 Fig. . MeasureRecordings with the µIPs demonstrated successful measurement of dopamine release at extended time-points after implantation Fig. . Initialn = 26 recording sessions). These amplitudes, however, varied from day to day . A constant set of stimulation parameters was applied to each rat in an attempt to fix the levels of striatal dopamine overflow sampled by the implanted probe , indicating the lack of a significant relationship with the indwelling periods of the implants. Equal levels of increasing and decreasing trends were observed from the chronically implanted probes, and both types of trends were observed from multiple probes implanted within an individual subject, affirming that these trends were not related to the specific subject.The µIPs individually displayed both increase and decrease in recorded dopamine, but these changes were not correlated with elapsed time since implantation or with implanted subject Table . The treP < 0.0001, one-way ANOVA for all four rats), but not across probes within a given rat . This variability could be related to slight differences in position of the stimulating electrode relative to the topographic MFB connections to the striatal recording sites. The standard score (Z-score) of changes in evoked dopamine was calculated to assess variability over time . These variations and trends were most significant during the first 2 months of indwelling and could be related to an early phase of inflammation induced by acute trauma following surgical implantation31, or to differences in activation of downstream MFB structures.Temporal dynamics of evoked dopamine signals was also computed to characterize the capacity of implanted µIPs to retain subsecond resolution Fig. . The aveWe aggregated all of the evoked signals recorded each month from the implanted probes (for a fixed stimulation parameter per probe) to allow a comprehensive assessment of the distribution of concentrations sampled by the µIPs Fig. . The proR = 0.87). These strong correlations were reproducibly observed across successive measurements made over months  of measured cyclic voltammograms, as well as by their correlation to dopamine standards obtained in vitro. All chronically measured cyclic voltammograms expressed distinctive dopamine redox potentials and were highly correlated with those of dopamine standards   . The noise value represents the practical LOD of the probes: a signal-to-noise ratio of ≥3 is a frequently applied criterion implemented for voltammetric dopamine measurementsst) Fig. , and werst) Fig. .40. PCA was used to characterize how well the recorded signals could dissociate dopamine from pH. Cyclic voltammograms recorded in vivo were combined with cyclic voltammograms of in vitro dopamine and pH standards, and were represented in the principal component space   Fig. . The in es) Fig.  and pH (es) Fig.  standardes) Fig. . PCA wases) Fig. .6. Here, we approached this issue quantitatively. We implemented longitudinal metrics of probe integrity in terms of noise, selectivity, and correlation of the recorded signals to in vitro standards. Our findings not only demonstrate the chronic recording capability of the µIPs, but further corroborate µIP function in recording subsecond dopamine dynamics with preserved sensitivity, accuracy, and selectivity without significant infraction of the tissue environment. The sustained performance of the cellular-scale neurochemical µIPs validates their use in recording rapid chemical fluctuations with the needed reliability over many months.This study introduces, to our knowledge, the first sensor capable of monitoring chemical activity in the brain over year-long time periods. We tested our probes in anesthetized rats using controlled electrical stimulation to assess performance in dopamine chemical monitoring over time. There has been very little prior information about the fidelity of such chronically implanted chemical sensors as a function of time31. The cellular-scale probes reported here markedly minimized tissue disruption and inflammation, which could have helped enable longitudinally stable recording of neurochemicals over chronic timescales.Sensors so far have not been shown to record chemicals without degradation of signals over time. The protocols reported here were developed to quantify comprehensively signal stability and longitudinal performance, parameters critical to ensure measurement accuracy of the implanted chemical sensors. Decreasing both the diameter and the stiffness of the implanted devices could be critical to reducing disruption of the submicron cellular architecture that underlies neurotransmission. Very few differences were observed in the relative intensity of inflammatory markers between intact brain tissue and µIP implanted sites examined in the same brain in ipsilateral and contralateral hemispheres. CFE chemical sensor implants tended to cavitate the tissue and to create widespread damage in the form of scarring, inflammation, blood brain barrier disruption, and infiltration of foreign molecules and toxins. Tissue responses were measured around the implanted shafts 0.5–1 mm dorsal to their sensing tips to evaluate contributions of the bulk of the implant, and these shafts differentiated the geometry of the µIP from the CFE implant. Shaft-induced tissue responses were observed to spread over hundreds of micrometers—distances sufficient to encapsulate some portion of the sensing tip that would restrict diffusion of targeted molecules to the sensing interface and undermine long-term function. In addition to implant size, other factors, including the mechanical and/or material properties of the implanted shaft, could have distinctive influences on tissue response and the progressive performance of neurochemical measurements6 in what remains, to our knowledge, the only existing report of recording stimulation-evoked dopamine over time. Instead, we found that the µIPs collectively displayed no coherent trend in detected dopamine levels, and the cumulative distributions of recorded concentrations were similar from month to month. The lack of a decreasing trend in recorded signals and the similar range of detected concentrations across probes demonstrate the sustained sensitivity and accuracy of the chronically implanted µIPs.The chronically implanted µIPs detected a wide range of dopamine concentrations as evoked by controlled electrical stimulation. We expected a consistent decrease in the amplitudes of detected dopamine, as seen before with CFEs14, and/or changes in stimulation efficacy39. Microscale differences in the location of the stimulation electrode in the MFB can significantly affect the amount of dopamine released at its projections within the striatum15. This variability could result from greater intra-tissue movement, and induced tissue response39, of the stimulation electrode, which is over 200 times larger  and therefore 196 times stiffer  than the striatal µIP. Further, we have not demonstrated here the operation of the µIPs in awake, behaving animals, in which it is likely that day-to-day variations in maximum concentration detected by these probes could be higher based on the state of the animal. Methods to confer uniform detection environments and sensitivities of the implanted probes will thus be needed to make accurate comparisons of amplitude fluctuations between sessions. Nevertheless, an encouraging property of the chronic measurements made with the µIPs is the sustained temporal characteristics of the recorded dopamine signals. This feature could have important consequences in accurately characterizing the wide range of temporal signaling dynamics of dopamine and other neurotransmitters and neuromodulators.The concentrations recorded by the chronic probes did, however, vary from day to day. These variations could be due to changes in the probe’s sensitivity, movement of striatal probes or MFB stimulation electrodes, depth of anesthesia43. The chronically operational devices that we report here allow tracking of chemical activity for periods up to a year, durations suitable for analyzing dopamine release related to learning and to changes in motivational states in animals. Future experiments in non-human primates over even longer periods of recording will be needed to assess the feasibility of the use of these sensors in humans. If successful, such an ability to perform chronic neurochemical measurements could be critical in improving and enabling online diagnostics for adaptive remediation of debilitating conditions, including Parkinson’s disease and other movement and mood disorders.Cellular-sized implants have rapidly emerged over the last few years with the goal of alleviating tissue response and enabling chronic recording of electrical neural activity15   was heated until liquid (hot plate at 80 °C) and applied onto the probes placed above a releasable glass substrate. The liquid was cooled to room temperature to form a rigid 0.5–1 mm thick shuttle that could be used to secure mechanically the ultra-small and compliant probes as they were inserted into the brain.Probes  were fabricated largely as previously reported15 with a noise floor of <0.1 nA, dynamic range of ±2000 nA, and sampling rate of 214 samples per scan. Implanted sensors were defined as nonfunctional if they had current noise greater than 1 nA or current saturation , both of which indicate a perforated or damaged insulation  as those of the CFEs  over a range of physiological dopamine concentrations (0.25–1 µM)  at 0.05 intervals past the testing electrode in a bath of aCSF (pH of 7.4) and by recording subsequent changes in current. pH was adjusted by adding small amounts (<100 µL) of sodium hydroxide or hydrochloric acid to a 2 L aCSF solution and subsequently measured for each titration . Dopamine hydrochloride  was added to the aCSF to measure probe dopamine sensitivity at physiological levels of 0.25, 0.5, and 1 µM. Each device was evaluated three or four times at each condition and analyte concentrations with two separately prepared solutions of analyte. A syringe or gravity pump was used to drive solutions across the sensor. A sample injection valve  or stopcock valve was used to switch flowing solutions. Devices were conditioned at 60 Hz for ~5 min prior to testing. The µIPs were shown to have similar sensitivity and correlations to levels of dopamine oxidation current (51.96 nA µMµM) Fig. . NormaliµM) Fig. 15. The ny-axis) as successive time points (x-axis) to display voltage dependent current changes . Dopamine produces current at selective reduction and oxidation (redox) potentials of –0.2 and 0.6 V for the implemented FSCV parameters18. Current changes also occur outside these redox potentials due to electrochemical reaction of other molecules, including those producing changes in pH40. PCA was implemented to dissociate current contributions of targeted dopamine molecules from pH and background current drift, and to compute the apparent change in concentrations of dopamine based on in vitro flow-cell calibration data18. All analyses were done in MATLAB  as previously reported6. Comparisons of recorded dopamine concentrations between animals and across probes within a subject were made using one-way ANOVA.Background-subtracted data Fig. , 3 and 8–1 oxygen) and administered an analgesic  subcutaneously. The skin overlying the calvarium was incised and retracted. Craniotomies were stereotaxically guided  to place sensors in the right striatum , a reference electrode in the contralateral hemisphere , and 2–3 stimulation electrodes in the MFB ipsilateral to the recording probes . Bone screws  were installed along the perimeter of the opening in the calvarium to secure the implanted devices for chronic use. A bare stainless steel wire  was wound around 2 or 3 screws to serve as an electronic ground connection in some rats. Implant devices were then secured to the skull by cementing  them to their neighboring screws. The dura mater was removed from the tissue above the striatum and MFB with the bent tip of a 32G hypodermic stainless steel needle.All procedures involving animals were approved by the Committee on Animal Care at the Massachusetts Institute of Technology and were conducted in accordance with the U.S. National Research Council Guide for the Care and Use of Laboratory Animals. Nine Sprague-Dawley male rats  were used for probe implantation. Seven were implanted with µIPs, and two were implanted with CFEs. They were pair-caged pre-implantation and were individually caged post-implantation, with ad libitum food and water. Rats were anesthetized  0.9% saline. The probes were lowered incrementally until they reached a depth of dorsoventral (DV) 4.5–5.5 mm. CFE sensors (an array of 4 spaced ~500 µm apart) were implanted without PEG toward the same coordinates and with a similar insertion speed.The µIPs were implanted by lowering non-PEG coated probe tips with a micromanipulator at a rate of ~100–500 µm minReference Ag/AgCl electrodes were made of insulated silver wires  that were exposed 0.5–1 mm at the tip and were chlorinated in bleach overnight. These were placed above the exposed cortical surface (330 µm diameter wires) or penetrated 0.5 mm (178 µm diameter wires) into the contralateral hemisphere. The stimulation electrodes were 2 or 3 Pt/Ir electrodes (75–125 µm diameter) spaced 300–600 µm apart. The tip impedances ranged 2–1000 kΩ. These electrodes were lowered to an initial depth of DV 6.0 mm and then incrementally (100–200 µm) to the optimal depth (DV 6.0–8.7 mm) where stimulation was observed to produce the highest amplitude of dopamine release as measured by implanted striatal sensors. All implanted devices were connected to metal sockets  to provide connection for FSCV on subsequent days of recording. All devices were covered in cement so that only the sockets were exposed.–1 oxygen) and connected to FSCV instrumentation in each recording session. Current was delivered to the implanted MFB electrodes in a train of 24, 48, or 72 biphasic pulses with a fixed frequency of 60 Hz and pulse width of 2 ms) through a stimulus isolator  as triggered by the recording software. These parameters were derived from commonly reported MFB stimulation paradigms in rodents39. A variable number of pulses and amplitude  were used, and these parameters were usually fixed across sessions and for each subject  in 0.1 M phosphate buffer. Brains were then post-fixed by storing them in glass vials in 4% PFA overnight with gentle rocking at 4 °C. PFA solution was replaced by a 25% glycerol cryoprotectant solution in 0.1 M phosphate buffer with 0.1% sodium azide  until the brain sank (overnight) at 4 °C. The brains were stored in the same solution at 4 °C until sectioning. The brains were then frozen in dry ice, and were cut at 30 μm thickness in the horizontal plane on a sliding microtome. Sequential sections were placed individually into adjacent wells of a multi-well plastic tray containing 0.1% sodium azide in 0.1 M phosphate buffer for storage at 4 °C until the immunostaining procedure.Sections were rinsed three times for 2 min in 0.01 M phosphate buffer saline (PBS) containing 0.2% Triton X-100 (Tx)  and then were incubated in PBS-Tx with 0.5% of tyramide signal amplification (TSA) blocking reagent  for 20 min. The sections were incubated with primary antibody solutions containing chicken anti-GFAP  [1:1000], rabbit anti-Iba1  [1:2000] or rabbit anti-tyrosine hydroxylase (TH)  [1:4000], and/or goat anti-mu-opioid receptor 1  [1:500] in PBS-Tx with TSA blocking reagent for 24 h at 4 °C for primary incubation. The sections were then rinsed three times for 2 min in PBS-Tx, and then were incubated for 2 h in the secondary antibody solution containing goat anti-chicken Alexa Fluor (AF) 633  [1:300], goat anti-rat AF 546  [1:300], goat anti-rabbit AF 488  [1:300], and/or donkey anti-goat AF 647  [1:300] in TSA blocking reagent in PBS-Tx. The secondary antibody solution contained donkey anti-chicken FITC  [1:300], donkey anti-rat 550  [1:100], donkey anti-goat 647  [1:300], and/or donkey anti-rabbit AF750  [1:300] for identifying recorded sites inside of striatal defining boundaries  and/or MFB expressing markers . The sections were rinsed three times for 2 min in PBS-Tx, incubated for 2 min in DAPI  [1:1000] in PBS, and then were rinsed three times for 2 min in 0.1 M phosphate buffer, mounted onto glass slides and coverslipped with ProLong Gold antifade reagent .n = 4 CFE and n = 4 µIP type devices). Two-tailed t-test was used to assess differences in normalized intensity values between groups.Histochemical images were obtained on a scanning  confocal microscope  with ×10 air objective. The location of probe tracks was determined in each horizontal slice by identifying holes that matched with expected probe configurations and geometries. Implant identification was straightforward for CFEs that produce a demarcated 70–90 µm diameter hole for most stains . µIPs required more procedures to locate because their appearance was seen to be similar to that of physiological blood vessels. µIP implanted sites were located by first identifying tip locations marked by electrolytic lesions that reproducibly produced pronounced changes in IgG and sometimes Iba1 and GFAP Fig. . These lSupplementary InformationSupplementary Data 1Description of Additional Supplementary Files


Granuloma Multiforme (GM) is a reactive skin disorder with annular lesion and focal necrobiosis. It was first described in Nigeria (Africa), since than it was mostly described from various regions of Africa where leprosy was endemic. Outside Africa, this disease is rarely reported. Till 2016 only five cases were reported from India. This is the second case report from Nepal. A fifty-nine-year-old female, farmer by occupation, presented with annular and polycyclic plaques with elevated beaded border for one year. It was distributed over upper trunk, lower back, and arms, and was mildly pruritic. Histopathologically, loss of elastic fibres at focal area of upper dermis, with elastic fibres engulfed histiocytes were noted. GM was mostly noted from leprosy endemic areas of Africa. Clinically, it is very difficult to distinguish from granuloma annulare. This report adds the number of GM occurrence in Asia especially from area where leprosy is still prevalent. This report also emphasizes in resource poor settings where most diagnosis is made clinically, the granuloma annulare should be looked for very carefully in order not to miss GM. Granuloma Multiforme is a reactive skin disorder of unknown aetiology, characterized clinically by confluent annular lesions and histologically by focal necrobiosis and histiocytic granuloma . It was A fifty-nine-year-old female, from Western Terai region of Nepal, presented with slightly itchy lesion on trunk and arm for one year. The first lesion was noted on chest, then gradually new lesions were seen on the arm and lower back. It started as skin color small palpable lesion that gradually increased in size giving ring shape. There was no history of other disease and drug intake. She was a farmer by occupation.On examination, there were multiple skin color and slightly erythematous annular and polycyclic plaques with elevated, beaded border with central clearing. Lesions were distributed over chest, forearm, upper, and lower back with size ranging from 2 cm to 10 cm in diameter Figures 1. The senThe clinical possibilities considered were granuloma annulare, leprosy, annular sarcoid, and actinic granuloma.Complete haemogram, renal and liver function test, blood glucose level, and routine urine examination were within normal range.The elliptical incisional biopsy was taken from the margin of lesion and sent for histopathological examination. It showed keratinized stratified squamous epithelium with normal maturation. There was loss of elastic fibres at focal area of upper dermis . The perThe patient was prescribed topical steroid, but showed no improvement at follow up after six weeks.Granuloma Multiforme is found among adult over the age group of 40 years with the predilection for the female sex. The sun exposed sites of the upper trunk and arms are predominantly affected. The initial lesions are usually papules, which soon evolve to form annular and polycyclic lesions with papular or nodular edge. The lesion tends to last for months or years. It is pruritic and irritating, especially when new lesions are forming . ScatterSince the time first described, its important differential diagnosis has been leprosy. The absence of loss of sensation and normal peripheral nerves will easily rule out leprosy. Granuloma Annulare (GA) sometime without histopathological support can be very difficult to differentiate. GA has smaller lesion and is seen in children. The presence of palisading granuloma differentiates it from GM. Annular sarcoid will show naked granuloma histologically and infiltrated reddish brown plaque clinically. Actinic granuloma or Annular elastolytic giant cell granuloma can be differentiated by its asymptomatic nature and absence of necrobiosis in histology. Some authors believe Actinic granuloma and GM are the same entity.The exact aetiology of this disease is not known. In past there were reports even suspecting droppings of bat, leaves as the aetiological factors but none were confirmed . It is sThere is no effective therapy for this condition, but recent report has shown some response with dapsone .This is the second reported case from Nepal . Like mo