Absence of Slc39a14/Zip14 in mouse pancreatic beta cells results in hyperinsulinemia.

Zinc is an essential component of the insulin protein complex synthesized in ß cells. The intracellular compartmentalization and distribution of zinc are controlled by 24 transmembrane zinc transporters belonging to the ZnT or Zrt/Irt-like protein (ZIP) family. Downregulation of SLC39A14/ZIP14 has been reported in pancreatic islets of patients with type 2 diabetes (T2D) as well as mouse models of high-fat diet (HFD)- or db/db-induced obesity. Our previous studies observed mild hyperinsulinemia in mice with whole body knockout of Slc39a14 (Zip14 KO). Based on our current secondary data analysis from an integrative single-cell RNA-seq dataset of human whole pancreatic tissue, SLC39A14 (coding ZIP14) is the only other zinc transporter expressed abundantly in human ß cells besides well-known zinc transporter SLC30A8 (coding ZnT8). In the present work, using pancreatic ß cell-specific knockout of Slc39a14 (ß-Zip14 KO), we investigated the role of SLC39A14/ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses. Glucose-stimulated insulin secretion, zinc concentrations, and cellular localization of ZIP14 were assessed using in vivo, ex vivo, and in vitro assays using ß-Zip14 KO, isolated islets, and murine cell line MIN6. Metabolic evaluations were done on both chow- and HFD-fed mice using time-domain nuclear magnetic resonance and a comprehensive laboratory animal monitoring system. ZIP14 localizes on the endoplasmic reticulum regulating intracellular zinc trafficking in ß cells and serves as a negative regulator of glucose-stimulated insulin secretion. Deletion of Zip14 resulted in greater glucose-stimulated insulin secretion, increased energy expenditure, and shifted energy metabolism toward fatty acid utilization. HFD caused ß-Zip14 KO mice to develop greater islet hyperplasia, compensatory hyperinsulinemia, and mild insulin resistance and hyperglycemia. This study provided new insights into the contribution of metal transporter ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses.NEW & NOTEWORTHY Metal transporter SLC39A14/ZIP14 is downregulated in pancreatic islets of patients with T2D and mouse models of HFD- or db/db-induced obesity. However, the function of ZIP14-mediated intracellular zinc trafficking in ß cells is unknown. Our analyses revealed that SLC39A14 is the only Zn transporter expressed abundantly in human ß cells besides SLC30A8. Within the ß cells, ZIP14 is localized on the endoplasmic reticulum and serves as a negative regulator of insulin secretion, providing a potential therapeutic target for T2D.

Metal transporter SLC39A14/ZIP14 modulates regulation between the gut microbiome and host metabolism.

Metal transporter SLC39A14/ZIP14 is localized on the basolateral side of the intestine, functioning to transport metals from blood to intestine epithelial cells. Deletion of Slc39a14/Zip14 causes spontaneous intestinal permeability with low-grade chronic inflammation, mild hyperinsulinemia, and greater body fat with insulin resistance in adipose. Importantly, antibiotic treatment reverses the adipocyte phenotype of Slc39a14/Zip14 knockout (KO), suggesting a potential gut microbial role in the metabolic alterations in the Slc39a14/Zip14 KO mice. Here, we investigated the hypothesis that increased intestinal permeability and subsequent metabolic alterations in the absence of Zip14 could be in part due to alterations in gut microbial composition. Dietary metals have been shown to be involved in the regulation of gut microbial diversity and composition. However, studies linking the action of intestinal metal transporters to gut microbial regulation are lacking. We showed the influence of deletion of metal transporter Slc39a14/Zip14 on gut microbiome composition and how ZIP14-linked changes to gut microbiome community composition are correlated with changes in host metabolism. Deletion of Slc39a14/Zip14 generated Zn-deficient epithelial cells and luminal content in the entire intestinal tract, a shift in gut microbial composition that partially overlapped with changes previously associated with obesity and inflammatory bowel disease (IBD), increased the fungi/bacteria ratio in the gut microbiome, altered the host metabolome, and shifted host energy metabolism toward glucose utilization. Collectively, our data suggest a potential predisease microbial susceptibility state dependent on host gene Slc39a14/Zip14 that contributes to intestinal permeability, a common trait of IBD, and metabolic disorders such as obesity and type 2 diabetes.NEW & NOTEWORTHY Metal dyshomeostasis, intestinal permeability, and gut dysbiosis are emerging signatures of chronic disorders, including inflammatory bowel diseases, type-2 diabetes, and obesity. Studies in reciprocal regulations between host intestinal metal transporters genes and gut microbiome are scarce. Our research revealed a potential predisease microbial susceptibility state dependent on the host metal transporter gene, Slc39a14/Zip14, that contributes to intestinal permeability providing new insight into understanding host metal transporter gene-microbiome interactions in developing chronic disease.

Octyl syringate is preferentially cytotoxic to cancer cells via lysosomal membrane permeabilization and autophagic flux inhibition.

The autophagy-mediated lysosomal pathway plays an important role in conferring stress tolerance to tumor cells during cellular stress such as increased metabolic demands. Thus, targeted disruption of this function and inducing lysosomal cell death have been proved to be a useful cancer therapeutic approach. In this study, we reported that octyl syringate (OS), a novel phenolic derivate, was preferentially cytotoxic to various cancer cells but was significantly less cytotoxic to non-transformed cells. Treatment with OS resulted in non-apoptotic cell death in a caspase-independent manner. Notably, OS not only enhanced accumulation of autophagic substrates, including lapidated LC3 and sequestosome-1, but also inhibited their degradation via an autophagic flux. In addition, OS destabilized the lysosomal function, followed by the intracellular accumulation of the non-digestive autophagic substrates such as bovine serum albumin and stress granules. Furthermore, OS triggered the release of lysosomal enzymes into the cytoplasm that contributed to OS-induced non-apoptotic cell death. Finally, we demonstrated that OS was well tolerated and reduced tumor growth in mouse xenograft models. Taken together, our study identifies OS as a novel anticancer agent that induces lysosomal destabilization and subsequently inhibits autophagic flux and further supports development of OS as a lysosome-targeting compound in cancer therapy. • Octyl syringate, a phenolic derivate, is preferentially cytotoxic to various cancer cells. • Octyl syringate destabilizes the lysosomal function. • Octyl syringate blocks the autophagic flux. • Octyl syringate is a potential candidate compound for cancer therapy.

Suppression of Pro-Inflammatory M1 Polarization of LPS-Stimulated RAW 264.7 Macrophage Cells by Fucoxanthin-Rich Sargassum hemiphyllum.

Macrophages play an important role in managing the onset and progression of chronic inflammatory diseases. The primary objective of this study is to explore the antioxidant potential and anti-inflammatory properties of Sargassum hemiphyllum ethanol extract (SHE) and its fraction. SHE and its five constituent fractions were assessed for overall antioxidant capabilities and inhibitory effects on LPS-induced inflammation by modulating macrophages polarization in both RAW 264.7 macrophages and bone-marrow-derived macrophages (BMDM). Among the organic solvent fractions of SHE, the ethyl acetate fraction displayed the highest total phenolic content and total antioxidant capacity. Notably, the n-hexane (Hex) fraction showed the most substantial suppression of LPS-induced tumor necrosis factor α secretion in BMDM among the five fractions of SHE. The SHE and Hex fraction significantly reduced the heightened expression of pro-inflammatory cytokines and inflammation-inducible enzymes induced by LPS in RAW 264.7 macrophages. In particular, the SHE and Hex fraction inhibited M1 macrophage polarization by reducing the mRNA expression of M1 macrophage markers in macrophages that were polarized toward the M1 phenotype. Furthermore, the SHE and Hex fraction attenuated the induction in nuclear factor E2-related factor 2 and its target genes, which was accompanied by an alteration in antioxidant gene expression in M1-polarized BMDM. The findings suggest that both SHE and its Hex fraction exhibit inhibitory effects on LPS-triggered inflammation and oxidative stress by modulating the polarization of M1 macrophages within macrophage populations.

The thermogenic characteristics of adipocytes are dependent on the regulation of iron homeostasis.

The development of thermogenic adipocytes concurs with mitochondrial biogenesis, an iron-dependent pathway. Iron regulatory proteins (IRP) 1 and 2 are RNA-binding proteins that regulate intracellular iron homeostasis. IRPs bind to the iron-response element (IRE) of their target mRNAs, balancing iron uptake and deposition at the posttranscriptional levels. However, IRP/IRE-dependent iron regulation in adipocytes is largely unknown. We hypothesized that iron demands are higher in brown/beige adipocytes than white adipocytes to maintain the thermogenic mitochondrial capacity. To test this hypothesis, we investigated the IRP/IRE regulatory system in different depots of adipose tissue. Our results revealed that 1) IRP/IRE interaction was increased in proportional to the thermogenic function of the adipose depot, 2) adipose iron content was increased in adipose tissue browning upon ß3-adrenoceptor stimulation, while decreased in thermoneutral conditions, and 3) modulation of iron content was linked with mitochondrial biogenesis. Moreover, the iron requirement was higher in HIB1B brown adipocytes than 3T3-L1 white adipocytes during differentiation. The reduction of the labile iron pool (LIP) suppressed the differentiation of brown/beige adipocytes and mitochondrial biogenesis. Using the 59Fe-Tf, we also demonstrated that thermogenic stimuli triggered cell-autonomous iron uptake and mitochondrial compartmentalization as well as enhanced mitochondrial respiration. Collectively, our work demonstrated that IRP/IRE signaling and subsequent adaptation in iron metabolism are a critical determinant for the thermogenic function of adipocytes.

Transfer and biological effects of arsenate from soil through a plant-aphid system to the parasitoid wasp, Aphidius colemani.

The accumulation of metalloid elements during transfer from contaminated soil to higher trophic levels may potentially result in the exposure of parasitic arthropods to toxic concentrations of these elements. This study examined the transfer of arsenate (As(V)) to aphids (Myzus persicae) from pepper plants cultivated in As(V) contaminated soils of two concentrations (2 and 6 mg As(V)/kg dry soil), and the subsequent biological effects on the aphid parasitoid, Aphidius colemani. Results showed that considerable quantities of As(V) were transferred to the plant in a concentration-dependent manner and were partitioned in the plant parts in the order of roots > stems > leaves. The accumulation of As(V) in the aphids increased with the concentrations in the plants; however, the transfer coefficient of As(V) from leaf to aphid was relatively similar and constant (0.07-0.08) at both soil As(V) concentration levels. Increased levels of As(V) significantly affected fecundity and honeydew production in aphids, but survival and developmental time were unaffected. Fecundity (mummification rate) of the parasitoid was not impaired by host As(V) contamination; however, vitality (eclosion rate) was significantly affected. Results are discussed in relation to possible ecological risks posed by the transfer of soil As(V) via the plant-arthropod system to parasitoid arthropods in agroecosystems.

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.

Rbfox family proteins make the homo- and hetero-oligomeric complexes.

Rbfox family of proteins that consists of Rbfox1, Rbfox2, and Rbfox3 in mammals regulates alternative pre-mRNA splicing in various tissues via direct binding to their RNA binding element. Although many studies have indicated the splicing activity of each member of the Rbfox family, the interactions of Rbfox family proteins are largely unknown. Here, we have investigated interactions among Rbfox family proteins. Co-immunoprecipitation (Co-IP) and GST-pull down assays confirmed that Rbfox proteins form homo and hetero complexes. Moreover, in vivo crosslinking using disuccinimidyl suberate treatment indicated that the Rbfox proteins form a dimer which then assembles with other proteins to form a large multiprotein complex. Duolink in situ proximity ligation (PLA) assay revealed that neuron specific Rbfox3 protein interacts with other Rbfox family proteins. This study is the first to provide an evidence that Rbfox family proteins form homo- and hetero-oligomeric complexes in vivo.

Toxicity effects and biomarkers of tebufenozide exposure in Yuukianura szeptyckii (Collembola: Neanuridae).

Tebufenozide is an insect growth regulator used to control pest caterpillar populations. As an ecdysone agonist, tebufenozide is equally toxic to several non-target arthropod species, binding the receptor sites of the molting hormone 20-hydroxyecdysone and causing premature and lethal molting. In this study, the toxic effects of tebufenozide were assessed, and biomarkers of tebufenozide exposure were identified, in the non-target soil collembolan species Yuukianura szeptyckii. Adult mortality and reproduction in Y. szeptyckii exposed to tebufenozide were evaluated after 28 days of exposure and were used to calculate LC50 and EC50, respectively. The LC50 could not be determined, because the mortality values observed were below 50%, even when exposed to the highest concentration tested (700 mg/kg), but the EC50 was 95.5 mg/kg. Effects on hatching and molting rates were evaluated using compressed soils, to prevent experimental individuals from burrowing; thus, all eggs and exuviae were detectable on the soil surface. Significant negative effects of tebufenozide exposure on the hatching rate and molting frequency were observed only at the highest concentration tested (700 mg/kg). Proteomic analyses were conducted to detect the cryptic effects of toxicity in adult collembolans exposed for 28 days to 43.8 mg/kg of tebufenozide, a concentration at which no toxicity effects were observed. The production rates of two ribosomal proteins, as well as proteins involved in apoptotic cell signaling, were higher in collembolans exposed to tebufenozide than in the control group. However, the production of proteins involved in glycolysis and energy production was downregulated. Therefore, the ecotoxicoproteomic approach is a promising tool for measuring the cryptic effects of tebufenozide exposure in Y. szeptyckii at low concentrations.

Bortezomib, a proteasome inhibitor, alleviates atopic dermatitis by increasing claudin 1 protein expression.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Many studies investigating AD pathogenesis and its therapy have been conducted but none have been successful. One of the causes of AD is dysfunction of tight junctions through reduction of claudin 1 expression in the epidermal barrier of the skin. In the present study, we investigated the role of bortezomib (BTZ) in the restoration of the reduced expression of claudin 1. Immunoblot and immunofluorescence analyses revealed that BTZ increased the protein expression level of claudin 1 in the human keratinocyte cell line HaCaT, thereby forming paracellular barriers. Furthermore, repeated application of BTZ alleviated atopic symptoms on the backs and ears of 2, 4-dinitrochlorobenzene (DNCB)-induced AD mice, and led to the formation of normal tight junctions in the epidermal barrier of DNCB-induced mice skin. Taken together, these results demonstrate that BTZ-induced claudin 1 expression may be a valuable therapeutic approach for AD.

Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes.

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals. We previously reported that γ-tocotrienol (γT3) attenuated adipose tissue inflammation and insulin resistance in diet-induced obesity, but the underlying mechanism remained elusive. Here, we investigated the effects of γT3 on NLRP3 inflammasome activation and attendant consequences on type 2 diabetes. γT3 repressed inflammasome activation, caspase-1 cleavage, and interleukin (IL) 1ß secretion in murine macrophages, implicating the inhibition of NLRP3 inflammasome in the anti-inflammatory and antipyroptotic properties of γT3. Furthermore, supplementation of leptin-receptor KO mice with γT3 attenuated immune cell infiltration into adipose tissue, decreased circulating IL-18 levels, preserved pancreatic ß-cells, and improved insulin sensitivity. Mechanistically, γT3 regulated the NLRP3 inflammasome via a two-pronged mechanism: 1) the induction of A20/TNF-α interacting protein 3 leading to the inhibition of the TNF receptor-associated factor 6/nuclear factor κB pathway and 2) the activation of AMP-activated protein kinase/autophagy axis leading to the attenuation of caspase-1 cleavage. Collectively, we demonstrated, for the first time, that γT3 inhibits the NLRP3 inflammasome thereby delaying the progression of type 2 diabetes. This study also provides an insight into the novel therapeutic values of γT3 for treating NLRP3 inflammasome-associated chronic diseases.

Joint toxic action of binary metal mixtures of copper, manganese and nickel to Paronychiurus kimi (Collembola).

The joint toxic effects of binary metal mixtures of copper (Cu), manganese (Mn) and nickel (Ni) on reproduction of Paronhchiurus kimi (Lee) was evaluated using a toxic unit (TU) approach by judging additivity across a range of effect levels (10-90%). For all metal mixtures, the joint toxic effects of metal mixtures on reproduction of P. kimi decreased in a TU-dependent manner. The joint toxic effects of metal mixtures also changed from less than additive to more than additive at an effect level lower than or equal to 50%, while a more than additive toxic effects were apparent at higher effect levels. These results indicate that the joint toxicity of metal mixtures is substantially different from that of individual metals based on additivity. Moreover, the close relationship of toxicity to effect level suggests that it is necessary to encompass a whole range of effect levels rather than a specific effect level when judging mixture toxicity. In conclusion, the less than additive toxicity at low effect levels suggests that the additivity assumption is sufficiently conservative to warrant predicting joint toxicity of metal mixtures, which may give an additional margin of safety when setting soil quality standards for ecological risk assessment.

Modulating intestinal health: Impact of chitooligosaccharide molecular weight on suppressing RAGE expression and inflammatory response in methylglyoxal-induced advanced glycation end-products.

The accumulation of methylglyoxal (MGO) produced in high-temperature processed foods and excessive production in the body contributes to intestinal barrier dysfunction. In this study, we investigated the effects of chitooligosaccharides (COSs) of different molecular weights (<1 kDa, 1-3 kDa, 3-5 kDa, 5-10 kDa, and >10 kDa) on MGO-induced intestinal barrier dysfunction. We investigated the effect of COSs on inhibiting intracellular MGO accumulation/MGO-derived AGEs production and regulating the receptor for AGE (RAGE)-mediated downstream protein expression, including proteins related to apoptosis and inflammation, intestinal barrier integrity, and paracellular permeability. Pretreatment with COSs ameliorated MGO-induced increased RAGE protein expression, activation of apoptotic cascade/inflammatory response, loss of intestinal epithelial barrier integrity, and increased paracellular permeability, ameliorating intestinal dysfunction through MGO scavenging. 1-3 kDa COSs most effectively ameliorated MGO-induced intestinal dysfunction. Our results suggest the potential of COSs in improving intestinal health by ameliorating intestinal barrier dysfunction by acting as an MGO scavenger and highlighting the need for the optimization of the molecular weight of COSs to optimize its protective effects.

Potential range shift of a long-distance migratory rice pest, Nilaparvata lugens, under climate change.

The biogeographical range shift of insect pests is primarily governed by temperature. However, the range shift of seasonal long-distance migratory insects may be very different from that of sedentary insects. Nilaparvata lugens (BPH), a serious rice pest, can only overwinter in tropical-to-subtropical regions, and some populations migrate seasonally to temperate zones with the aid of low-level jet stream air currents. This study utilized the CLIMEX model to project the overwintering area under the climate change scenarios of RCP2.6 and RCP8.5, both in 2030s and 2080s. The overwintering boundary is predicted to expand poleward and new overwintering areas are predicted in the mid-latitude regions of central-to-eastern China and mid-to-southern Australia. With climate change, the habitable areas remained similar, but suitability decreased substantially, especially in the near-equatorial regions, owing to increasing heat stress. The range shift is similar between RCP2.6-2030s, RCP2.6-2080s, and RCP8.5-2030s, but extreme changes are projected under RCP8.5-2080s with marginal areas increasing from 27.2 to 38.8% and very favorable areas dropping from 27.5 to 3.6% compared to the current climate. These findings indicate that climate change will drive range shifts in BPH and alter regional risks differently. Therefore, international monitoring programs are needed to effectively manage these emerging challenges.

Effect of RNF113A deficiency on oxidative stress-induced NRF2 pathway.

The ring finger protein 113A (RNF113A) serves as an E3 ubiquitin ligase and a subunit of the spliceosome. Mutations in the RNF113A gene are associated with X-linked trichothiodystrophy (TTD). However, the cellular roles of RNF113A remain largely unknown. In this study, we performed transcriptome profiling of RNF113A knockout (KO) HeLa cells using RNA sequencing and revealed the upregulation of NRF2 pathway-associated genes. Further analysis confirmed that the KO of RNF113A promotes nuclear localization of the NRF2 protein and elevates the mRNA levels of NRF2 target genes. RNF113A KO cells showed high levels of intracellular reactive oxygen species (ROS) and decreased resistance to cell death following H2O2 treatment. Additionally, RNF113A KO cells more sensitively formed stress granules (SGs) under arsenite-induced oxidative stress. Moreover, RNF113A KO cells exhibited a decrease in glutathione levels, which could be attributed to a reduction in GLUT1 expression levels, leading to decreased glucose uptake reactions and lower intracellular glucose levels. These alterations potentially caused a reduction in ROS scavenging activity. Taken together, our findings suggest that the loss of RNF113A promotes oxidative stress-mediated activation of the NRF2 pathway, providing novel insights into RNF113A-associated human diseases.

3,5-Dicaffeoylquinic acid attenuates microglial activation-mediated inflammatory pain by enhancing autophagy through the suppression of MCP3/JAK2/STAT3 signaling.

Microglial activation in the spinal cord contributes to the development of inflammatory pain. Monocyte chemotactic protein 3 (MCP3) can induce microglial activation, resulting in increased pain sensitivity; however, the underlying mechanism remains poorly understood. 3,5-dicaffeoylquinic acid (3,5-DCQA) has shown protective effects against inflammation-related diseases, but the effect of 3,5-DCQA on microglial activation and inflammatory pain is not evaluated. This study aimed to investigate the effects of 3,5-DCQA on microglial activation-induced inflammatory pain. Furthermore, the underlying mechanism inhibited by 3,5-DCQA via MCP3 suppression was studied. To induce microglial activation, LPS was treated in BV2 microglial cells. The LPS-induced microglial activation and pro-inflammatory cytokines production were significantly reduced by 3,5-DCQA treatment in BV2 cells. Moreover, 3,5-DCQA suppressed LPS-induced MCP3 expression, resulting in reduced phosphorylation of JAK2/STAT3. Interestingly, the suppressed JAK2/STAT3 signaling enhanced autophagy induction in BV2 cells. The increased autophagy by 3,5-DCQA and knockout of MCP3 inhibited LPS-induced inflammatory response in BV2 cells. To establish the inflammatory pain, CFA was injected into the right paw of mice. The CFA-induced pain hypersensitivity and foot swelling were attenuated by the oral administration of 3,5-DCQA. Moreover, CFA-induced microglial activation was reduced and the autophagy markers were recovered in the spinal cord of 3,5-DCQA-administered mice. Similar results were observed in cultured primary microglia. Our findings indicate that 3,5-DCQA attenuates inflammation-mediated pain hypersensitivity by enhancing autophagy through inhibition of MCP3-induced JAK2/STAT3 signaling. Therefore, 3,5-DCQA could be a potential therapeutic agent for alleviating inflammatory pain.

Simultaneous analysis of social behaviors and neural responses in mice using round social arena system.

Stable and accurate capturing of detailed social behaviors is essential for studying rodent sociability. Here, we introduce a round social arena (RSA) system that enables close-up monitoring of detailed social behaviors in mice. We describe the steps to build RSA apparatus and set up the wiring for video synchronization. We then detail how to conduct RSA experiment with simultaneous Ca2+ imaging or optogenetics. This protocol also includes a custom MATLAB script for aligning the behavioral dataset to Ca2+ trace data. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).

Neural circuit-specific gene manipulation in mouse brain in vivo using split-intein-mediated split-Cre system.

Neural network studies require efficient genetic tools to analyze individual neural circuit functions in vivo. Thus, we developed an advanced circuit-selective gene manipulating tool utilizing anterograde and retrograde adeno-associated viruses (AAVs) encoding split-intein-mediated split-Cre. This strategy can be applied to visualize a specific neural circuit as well as manipulate multiple genes in the circuit neurons. Here, we describe the production and purification of the AAVs, viral injection to the mouse brain, and imaging analysis for a specific neural circuit. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).

Neural circuit pathology driven by Shank3 mutation disrupts social behaviors.

Dysfunctional sociability is a core symptom in autism spectrum disorder (ASD) that may arise from neural-network dysconnectivity between multiple brain regions. However, pathogenic neural-network mechanisms underlying social dysfunction are largely unknown. Here, we demonstrate that circuit-selective mutation (ctMUT) of ASD-risk Shank3 gene within a unidirectional projection from the prefrontal cortex to the basolateral amygdala alters spine morphology and excitatory-inhibitory balance of the circuit. Shank3 ctMUT mice show reduced sociability as well as elevated neural activity and its amplitude variability, which is consistent with the neuroimaging results from human ASD patients. Moreover, the circuit hyper-activity disrupts the temporal correlation of socially tuned neurons to the events of social interactions. Finally, optogenetic circuit activation in wild-type mice partially recapitulates the reduced sociability of Shank3 ctMUT mice, while circuit inhibition in Shank3 ctMUT mice partially rescues social behavior. Collectively, these results highlight a circuit-level pathogenic mechanism of Shank3 mutation that drives social dysfunction.

By-product of Korean liquor fermented by Saccharomyces cerevisiae exhibits skin whitening activity.

Herein, the skin whitening effect of the fermentation residue of Saccharomyces cerevisiae was investigated. The fermentation residue showed radical scavenging activity and attenuated tyrosinase activity. Furthermore, the fermentation residue of S. cerevisiae significantly suppressed melanin generation in B16F10 cells. Interestingly, the sample-containing formulation exhibited increased skin whitening activity compared with that by the control formulation in a clinical study. Notably, the endogenous tyrosinase expression was not altered by the fermentation residue of S. cerevisiae; however, the enzymatic activity of tyrosinase was inhibited. Furthermore, the sample did not change TRP1 and TRP2 expression in B16F10 cells. Thus, the fermentation residue of S. cerevisiae was assumed to directly suppress the tyrosinase enzyme. It was confirmed that the fermentation residue of S. cerevisiae was a competitive inhibitor of tyrosinase. Taken together, the fermentation residue of S. cerevisiae could be a novel skin whitening agent originating from the traditional Korean liquor production process. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01062-7.