Ailuropoda melanoleuca) is an iconic bear native to China, famous for eating almost exclusively bamboo. This unusual dietary behavior for a carnivore is enabled by several key adaptations including low physical activity, reduced organ sizes and hypothyroidism leading to lowered energy expenditure. These adaptive phenotypes have been hypothesized to arise from a panda-unique single-nucleotide mutation in the dual-oxidase 2 (DUOX2) gene, involved in thyroid hormone synthesis. To test this hypothesis, we created genome-edited mice carrying the same point mutation as the panda and investigated its effect on metabolic phenotype. Homozygous mice were 27% smaller than heterozygous and wild-type ones, had 13% lower body mass-adjusted food intake, 55% decreased physical activity, lower mass of kidneys (11%) and brain (5%), lower serum thyroxine (T4: 36%), decreased absolute (12%) and mass-adjusted (5%) daily energy expenditure, and altered gut microbiota. Supplementation with T4 reversed the effects of the mutation. This work uses a state-of-the-art genome editing approach to demonstrate the link between a single-nucleotide mutation in a key endocrine-related gene and profound adaptive changes in the metabolic phenotype, with great importance in ecology and evolution.The giant panda ( A key aim in ecology is to understand the factors that underlie metabolic phenotypes and their ecological consequences, also known as the metabolic theory of ecology . A paralAiluropoda melanoleuca) is an iconic species of wildlife conservation, characterized by its unique biology. Pandas are bears that are endemic to China and feed almost exclusively on bamboo, which has very low nutritional value [DUOX2 gene [DUOX2 gene. It is not yet known whether this premature stop codon results in no translation of the gene or whether a truncated version of the protein is produced that may have biological functions.The giant panda  mouse model carrying the same mutation as the one found in the giant panda , i.e. substitution of C with T, 625 Arginine to Termination codon in the 15th exon of the mouse DUOX2 gene. We investigated growth rate and traits associated with energy balance, such as metabolic rate, spontaneous physical activity and food consumption, body composition, energy assimilation, water consumption and mass of the vital organs, as well as circulating levels of thyroid hormones and composition of the fecal microbiota. We further demonstrate that the phenotypic consequences of this mutation do not stem from the microbiota changes, and can be reversed by supplementation with exogenous T4.ynthesis . Some pr in mice ,10 and h in mice ,9,11–14  in mice  and phys in mice ,19. DUOX in mice ,21. Howeribution . We aimeDuox2 mutation in mice led to changes in phenotype that mirrored the unique biology of the panda. The Duox2 mutant homozygote animals (Duox2A625T/A625T) were much smaller than heterozygous (Duox2+/A625T) and wild-type (Duox2+/+) animals between the ages of 4 and 10 weeks, i.e. during growth (mean body mass 12.2 (Duox2A625T/A625T) vs. 19.1 (Duox2+/A625T) and 19.3 (Duox2+/+) g SE ± 0.4, F2,707 = 534.79, P < 0.001; Fig. Duox2A625T/A625T mice were 27% dwarfed at 10–11 weeks old (16.0 (Duox2A625T/A625T), 21.4 (Duox2+/A625T), 21.9 (Duox2+/+) g SE ± 0.5, F2,57 = 53.77, P < 0.001; Tables S5 and S7), during which time we made physiological measurements. During seven days of measurements, the Duox2A625T/A625T mice were on average 55% less active (moving 3903 (Duox2A625T/A625T) vs. 7851 (Duox2+/A625T) and 8579 (Duox2+/+) m SE ± 514, F2,399 = 23.92, P < 0.001; Fig. Duox2A625T/A625T), 54.3 (Duox2+/A625T), 56.8 (Duox2+/+) kJ/day SE ± 1.1, F2,397 = 45.51, P < 0.001; Tables S1 and S2) and body mass-adjusted daily food intake was 13% lower (47.3 (Duox2A625T/A625T) vs. 52.3 (Duox2+/A625T) and 54.2 (Duox2+/+) kJ/day SE ± 1.4, F2,396 = 3.98, P = 0.020; Fig. Duox2A625T/A625T mice had a lower respiratory exchange ratio (0.78 (Duox2A625T/A625T) vs. 0.83 (Duox2+/A625T) and 0.82 (Duox2+/+) SE ± 0.01, F2,399 = 45.44, P < 0.001; Tables S1 and S2). The daily energy expenditure (DEE) was decreased in Duox2A625T/A625T mice by 12% (40.6 (Duox2A625T/A625T), 44.9 (Duox2+/A625T), 46.3 (Duox2+/+) kJ/day SE ± 0.4, F2,399 = 70.02, P < 0.001), as well as daily resting energy expenditure (REE) by 10% (29.4 (Duox2A625T/A625T), 31.6 (Duox2+/A625T), 32.8 (Duox2+/+) kJ/day SE ± 0.4, F2,399 = 24.02, P < 0.001; Tables S1 and S2). These differences remained significant but were lower after adjustment for body size (5% for DEE: 42.9 (Duox2A625T/A625T) vs. 43.9 (Duox2+/A625T) and 44.9 (Duox2+/+) kJ/day SE ± 0.4, F2,398 = 4.13, P = 0.017; Fig. Duox2A625T/A625T) vs. 31.2 (Duox2+/A625T) and 32.2 (Duox2+/+) kJ/day SE ± 0.4, F2,398 = 2.95, P = 0.053; Tables S1 and S2). The females were 14% smaller than males between 4–10 weeks old , and 14% smaller at 10–11 weeks old . The body mass-adjusted DEE , REE  or RER  did not differ between the sexes. However, females had 7% higher mass-adjusted food intake  and 28% higher activity  than males (Tables S3 and S4).The panda-specific Duox2A625T/A625T mice than in Duox2+/A625T and Duox2+/+ mice (90.8% (Duox2A625T/A625T) vs. 87.8% (Duox2+/A625T) and 87.8% (Duox2+/+) SE ± 053, F2,56 = 3.53, P = 0.036), but the percent of fat was not significantly different . Adjusted daily water intake in metabolic cages was lower in Duox2A625T/A625T mice (3.4 (Duox2A625T/A625T) vs. 6.4 (Duox2+/A625T) and 7.3 (Duox2+/+) g SE ± 0.5, F2,53 = 10.47, P < 0.001), and the daily urine production tended to be higher, but was not significant (2.1 (Duox2A625T/A625T), 1.3 (Duox2+/A625T), 1.4 (Duox2+/+) g SE ± 0.2, F2,53 = 2.87, P = 0.066; Tables S5 and S7). The energy assimilation did not differ between genotypes (90.9% (Duox2A625T/A625T), 90.8% (Duox2+/A625T), 90.9% (Duox2+/+) SE ± 0.2, F2,57 = 0.14, P = 0.873; Tables S5 and S7). After adjustment for body mass , the Duox2A625T/A625T mice had smaller kidneys  compared to Duox2+/A625T and Duox2+/+, but adjusted liver size was not significantly different  and had smaller tails (P < 0.001), skin (P = 0.029), less mesenteric white adipose tissue (P < 0.001), but more subcutaneous white adipose tissue (P = 0.001) and bigger stomachs . Masses of the other organs did not differ between the three genotypes (Tables S6 and S8).The average percent of lean mass corrected for body mass was higher in Duox2A625T/A625T mice than in Duox2+/A625T and Duox2+/+ mice (22.4 (Duox2A625T/A625T) vs. 34.3 (Duox2+/A625T) and 35.1 (Duox2+/+) nmol/L SE ± 2.2, F2,56 = 9.98, P < 0.001; Fig. 2,56 = 0.28, P = 0.757; Fig. 2,58 = 13.48, P < 0.001; Tables S10 and S11), indicating that the Duox2A625T/A625T mice had a lower abundance of commensal Bifidobacterium (correlation coefficient: 0.903) and slightly lower Akkermansia (0.123), but slightly higher Desulfovibrio  was 36% lower in Duox2 mutation were a direct result of the mutation, or rather a consequence of changes in the gut microbiota, 4-week-old Duox2+/+ mice were exposed to an antibiotic mix in drinking water [Duox2+/+, Duox2A625T/A625T or giant panda feces. The mice did not adopt the panda microbiome, but the microbiome from Duox2+/+- and Duox2A625T/A625T-treated mice was not distinct from the donors . In the Duox2A625T/A625T-treated mice, commensal Akkermansia were lower and Lachnospiraceae slightly higher . The treatment did not affect the body mass (22.8 (Duox2+/+) vs. 22.7 (Duox2A625T/A625T) g SE ± 0.3, F1,72 = 0.01, P = 0.922), activity , mass-adjusted DEE  or REE , however, adjusted food intake  was lower in the mice with the Duox2A625T/A625T microbiota (Tables S12 and S13).To show if the effects of the ng water  and thenDuox2 mutation, Duox2A625T/A625T mice were exposed to 5 μg/mL of T4 in drinking water starting from 4 weeks old [1,70 = 46.06, P < 0.001; Fig. 1,63 = 56.07, P < 0.001; Fig. 1,62 = 7.72, P = 0.007; Fig. 1,62 = 32.57, P < 0.001; Fig. 1,62 = 18.59, P < 0.001; Tables S18 and S19). The mass of their kidneys was higher . Under supplementation serum, thyroid hormones were both increased  on bone and soft tissue growth, particularly effects on the hepatic expression of growth hormone receptor and consequent IGF1 production [Duox2A625T/A625T mice were much smaller than their littermates as pups, and they remained dwarfed as adults. Interestingly, in evolutionary history the giant panda seems to be dwarfed as well. The current giant panda, A.melanoleuca, evolved in the Holocene and shrunk in size relative to the extinct panda, Ailuropoda baconi, which was considerably larger and which was preceded by the small Ailuropoda microta [DUOX2 mutations in the evolution of panda body size remains unknown.Among other hypothyroid mouse strains, a spontaneous mutation in oduction . Moreoveoduction , but alsoduction . In factoduction , which sDuox2A625T/A625T mice were also significantly decreased, even though the effect was reduced after correction for their small body mass. Therefore, it seems that the mutation in the DUOX2 gene may have contributed to this unusual metabolic trait of the pandas. However, the impact of the mutation on DEE in mice was much lower than that observed in pandas [Duox2A625T/A625T mice was decreased, but pandas also have lowered T3 levels, which was not observed in the Duox2A625T/A625T mice. The mutation in DUOX2 affects both T4 and T3 synthesis, but T3 is largely generated by conversion of T4 to T3 by deiodinases (DIO) [DIO genes are unlikely to influence T4 to T3 conversion in the giant panda. Hence, in addition to the DUOX2 mutation, the full metabolic phenotype of giant pandas likely depends on yet-unknown genetic mechanisms affecting T4 to T3 conversion, or TSH levels. Other possible mutations affecting panda metabolic phenotype may include genomic mitochondrial genes. Two such mutations have been identified in pandas, one in cytochrome c oxidase (COX), a rate-limiting enzyme of the electron transport chain [ATP8, encoding and affecting post-translational modification of ATP synthase [Duox2 mutation reported by Johnson et al. [Duox2A625T/A625T mice. However, after recalculating the units used in both studies, the T4 of wild-type mice in the Johnson study is twice as high as those in our study. Also, the methodology used to assess T4 levels was different, as well as the age of the mice. Moreover, the T4 level in pandas is ∼50% of that expected for similar-sized mammals, which is close to the level we found. However, it is also possible that the panda-unique mutation leads to translation of a truncated protein, which still retains some biological functions.Our previous work indicated that giant pandas have lower than expected DEE , and then pandas . Similares (DIO) . We therrt chain , and anosynthase . The effDuox2A625T/A625T mice and the other two genotypes was very strong, which indicates that the DUOX2 gene mutation may be largely responsible for the panda's low physical activity. The mutation may also affect the range of panda distribution, as pandas tend to choose parts of the forest with gentle slopes, allowing more energetically efficient travel [DUOX2 mutation may have been a key innovation in the panda lineage, enabling survival on their nutritionally poor bamboo diet.Physical activity data for captive and wild pandas indicate that they spend only ∼30%–50% of their time on physical activity, which is lower than in other bears . The dift travel . The mutDuox2A625T/A625T mice were smaller. It is not known whether the spleen and lungs are also smaller in pandas, like in the Duox2A625T/A625T mice. However, the vital organs account for ∼60% of resting energy expenditure, even though their weight is only ∼5% of total body mass [Duox2A625T/A625T mice, despite the liver mass being unchanged. The Duox2A625T/A625T mice were overall leaner, and adipose tissue has low energy expenditure [Duox2A625T/A625T mice and giant pandas are smaller. The smaller kidneys of Duox2A625T/A625T mice seem to be associated with their increased urination. This may be due to retarded kidneys, or a direct effect of hypothyroidism leading to kidney injury [Duox2A625T/A625T mice had enlarged stomachs, the other sections of their gut remained unchanged.In giant pandas, some organs are smaller compared to similar-sized mammals , and simody mass . Therefoenditure . Brain senditure , and cony injury . Pandas Duox2 mutation had a significant impact on the fecal microbiota. The fecal microbiota of Duox2A625T/A625T mice was depleted in Bifidobacterium and Akkermensia. Bifidobacterium can be detected in adult pandas [Bifidobacterium affects health, immunity and metabolism, due to its role in milk digestion and defense against pathogenic microbes [Bifidobacterium is gradually replaced by Firmicutes/Bacteroidetes. After panda cubs begin to eat bamboo the Bifidobacterium is replaced with other communities too. So, the Duox2A625T/A625T mice microbiota was more similar to bamboo-eating adult pandas, yet not enriched with any cellulose-digesting genus, such as Bacillus or Pseudomonas [Duox2 expression is regulated by two signaling pathways, induced by normal gut microbiota, and is further upregulated in a state of dysbiosis [Duox2 and several other antimicrobial genes was increased in the gut epithelium of mice that were fed a low-protein diet, previously subjected to microbiota transplant from mice fed a high-fat diet [Bifidobacterium and Akkermensia are not reported to induce Duox2 expression as a defense mechanism of the gut [Duox2A625T/A625T mice we observed a mild increase of pathogenic Desulfovibrio. The microbiota of Duox2+/+ mice gavaged with microbiota from Duox2A625T/A625T mice was poorer in Akkermensia, which, like Bifidobacterium, is common in healthy mice. This treatment led to decreased food intake. Thus, the Duox2 mutation may have induced changes in the gut microbiome contributing to an overall reduction in food intake, but did not contribute to the other phenotypes observed.The gut microbiota plays a critical role in health and physiology ,37, and t pandas , but is udomonas . Howeverysbiosis ,38. Expr the gut ,38, theyDuox2 mutation on serum T4, the main phenotypic effects on growth, metabolism, activity, food intake and kidney mass were reversed and the magnitude of reversal almost exactly mirrored the deficit caused by the mutation. This confirms that physiological consequences of the single-nucleotide panda-specific mutation come almost exclusively from its effects on the endocrine system.Treatment with T4 at doses previously used in the literature ,39 causeDuox2 gene led to several changes in the metabolic phenotype, similar to those observed in the giant panda, and these changes were reversible by T4 supplementation. Hence, the mutation of this single nucleotide in a key endocrine gene resulted in disproportional changes to various adaptive physiological and anatomical traits. The mutation appears to underlie the low energetic requirements of the panda, which likely played an important role in the evolution of the panda-unique metabolic phenotype, affecting its ability to exploit bamboo, its life history strategy, aspects of its behavior and relation with its environment. Moreover, we demonstrated how links between genetic factors and adaptive changes in phenotypic characteristics of wild protected species can be investigated using laboratory model animals modified using state-of-the-art genome editing techniques.In summary, we demonstrated here that the decrease in T4 levels directly caused by the panda-unique mutation in the Duox2 mutation, analogous to one discovered in the giant panda [Duox2+/A625T mice) has been genetically engineered using a conditional strategy CRISPR-Cas9. The mouse was based on the C57BL/6 background and contained heterozygous mutation to the Duox2 gene, where the 625th Arginine was changed to a Termination codon (A625T). Next, the heterozygous population (Duox2+/A625T) was expanded to achieve the mutant homozygous (Duox2A625T/A625T) and wild-type homozygous (Duox2+/+) genotypes.All experimental procedures were approved by ethical committee numbers HP2018007, HP2019021 and HP2019030. To investigate the effects of a Duox2+/+, Duox2+/A625T and Duox2A625T/A625T. To measure energy balance, the animals were maintained in individual cages and fed with baseline diet . After 1 week, the resting metabolic rate, spontaneous activity and food consumption were measured using the TSE PhenoMaster system  for seven subsequent days. The body composition was measured before and after the metabolic rate measurement by magnetic resonance spectroscopy (EchoMRI-3N1-100TM). Next, energy assimilation, water consumption and urine production were measured in metabolic cages  for three days. Finally, the animals were killed by exposure to CO2 and dissected. The mass of various organs was measured and the blood was collected for analyses of thyroid hormones T4 and T3 levels. The analysis was performed using radioimmunoassay. The fecal samples were collected for analysis of the composition of microorganisms. The composition was investigated using DNA sequencing of the 16S rRNA gene.All newborn animals were genotyped before the experimental procedures using PCR. A balanced number of animals from both sexes and three genotypes was randomly assigned to the experiment. A series of anatomical and physiological traits were investigated in 10 male and 10 female individuals for each of the three genotypes: Duox2 mutation was a direct result of the mutation, or an indirect effect mediated by changes in gut microbiota, a subpopulation of juvenile Duox2+/+ mice were exposed to an antibiotics mix in the drinking water [Duox2+/+, Duox2A625T/A625T or giant panda feces. Next, the metabolic rate, activity and food intake was measured and the fecal samples were analyzed.To investigate if the effect of the ng water . The gutDuox2 mutation, a separate population of juvenile Duox2A625T/A625T mice was exposed to T4 in drinking water. The animals were subjected to the same measurement protocols as the animals in the main part of the experiment. The experiment was initially performed using a 5 μg/mL concentration of T4 [To investigate if treatment with T4 would reverse the phenotypic effects caused by the on of T4 ,39 and wStatistical analyses were performed with IBM SPSS 23. One-way or two-way ANOVA and ANCOVA tests were used and results were presented as least square means (LSM) ± SE and P values at alpha 0.05. The fecal microbiota was analyzed with PCA, followed by one-way ANOVA.A detailed description of the methods has been included in the Supplementary Data.The data underlying this article are available from the corresponding author John R. Speakman upon request, and are deposited in a public repository, Zenodo, accession number 10.5281/zenodo.4674583.nwab125_Supplemental_FileClick here for additional data file.


However, the mechanism of interaction between iron and copper in recombinant Tegillarca granosa ferritin (TgFer) remains to be investigated. In this study, we investigated the crystal structure of TgFer and examined the effects of Fe2+ and Cu2+ ions on the TgFer structure and catalytic activity. The crystal structure revealed that TgFer presented a typically 4–3–2 symmetry in a cage-like, spherical shell composed of 24 identical subunits, featuring highly conserved organization in both the ferroxidase center and the 3-fold channel. Structural and biochemical analyses indicated that the 4-fold channel of TgFer could be serviced as potential binding sites of metal ions. Cu2+ ions appear to bind preferentially with the 3-fold channel as well as ferroxidase site over Fe2+ ions, possibly inhibiting the ferroxidase activity of TgFer. Our results present a structural and functional characterization of TgFer, providing mechanistic insight into the interactions between TgFer and both Fe2+ and Cu2+ ions.In addition to its role as an iron storage protein, ferritin can function as a major detoxification component in the innate immune defense, and Cu Ferritidroxide) . Estimatdroxide) . Despite2+ ion entry points into the cage cavity, evidence has only been provided to support ion entry through the highly hydrophilic 3-fold channels in animal ferritin (2+) ions, whereas the L-subunit lacks such ferroxidase center but does have a nucleation site , and represents one of the rare invertebrates that possess hemoglobin-containing red hemocytes in the hemolymph  and both iron and copper ions remain unclear. In the present study, we present the first crystal structure for recombinant TgFer. In addition, the crystal structures of Cu2+-loaded TgFer (TgFer + Cu), Fe2+-loaded TgFer (TgFer + Fe), and Fe2+-loaded TgFer + Cu (TgFer + CuFe) were successfully determined. Accompanying biochemical experiments examining different ion-loaded TgFer molecules were also performed in this study. The structural and functional characterization reported here provides a comprehensive overview of the molecular mechanisms of TgFer in T. granosa.The blood clam, emolymph . Both fe granosa ; howeverT. granosa ferritin (GenBank: ADC34696.1). PCR products were digested and subcloned into a pET-28a (+) expression vector (Novagen) containing NdeI and XhoI sites, followed by an N-terminal SUMO tag. The primer sets for TgFer  were designed to provide NdeI and XhoI sites at the 5 and 3′ termini, respectively. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells (Novagen). When the cells reached an optical density at 600 nm (OD600nm) ranging from 0.4 to 0.6, they were induced with a final concentration of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 18°C for 18 h. The cells were harvested by centrifugation at 6,500 rpm at 4°C for 20 min. The subsequent purification process for TgFer was performed as described by 6-SUMO tag using SUMO protease (at a 1:500 ratio of protease to protein), the protein was further purified using a HiLoad 16/600 Superdex 200 pg gel-filtration chromatography column . Then, the purified protein was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit .TgFer cDNA has been reported to be 895 bp in length, consisting of a 163-bp 5′-untranslated region (UTR), a 213-bp 3′-UTR, and a 519-bp complete open reading frame that encodes a 172 amino acid polypeptide . The TgFE. coli BL21 (DE3) cells. The variant proteins were expressed and purified as described for the preparation of TgFer.The site-directed mutagenesis of TgFer was performed according to the instructions provided with the QuikChange Multi-site-directed mutagenesis kit . The primers used for site-directed mutagenesis were shown in −1 Tris–HCl, 150 mmol L−1 NaCl, pH 5.5). Twenty-milliliter portions of protein sample (approx. 0.5 mg mL−1) were dialyzed against 2 L of solution containing 2.5 mM CuCl2 at 4°C over 12 h as described by 2+ ions.The purified proteins were diluted using buffer solution (25 mmol L2∙dmol−1) was calculated as previously described (The protein samples (approx. 0.1 mg/ml) were prepared and transferred to a quartz cell with a 0.1 cm path length and 1.0 nm bandwidth. The Circular Dichroism (CD) spectra were recorded using a J-815 CD spectrometer . The secondary structure of ferritins was analyzed by Yang’s method by Jasco-Corp , and the molar ellipticity [θ] (deg∙cmescribed .−1) were prepared and digested using microwave digestion , as described previously  in 25 mM Tris-HCl, 150 mM NaCl, pH 8.0 was combined with 1 µL reservoir solution in the well of a 48-well plate and incubated at 18°C. TgFer crystals suitable for X-ray data collection were obtained in 4.0 M sodium formate. TgFer + Cu crystals were obtained in a condition containing 0.2 M ammonium acetate, 0.1 M Tris hydrochloride, pH 7.5 and 30% (v/v) 2-propanol. The sample of TgFer loaded with ferrous ions (TgFer + Fe) was prepared by the addition of [(NH4)2Fe(SO4)2]·6H2O  at a Fe2+ ion/TgFer molar ratio of 4,000:1 . TgFer + Fe crystals grew in solutions containing 0.1 M sodium cacodylate trihydrate, pH 6.5, 30% (v/v) (+/−)-2-Methyl-2,4-pentanediol. TgFer + CuFe crystals were obtained as reported previously by Preliminary screening for crystallization conditions was conducted using Crystal Screen I and II  through the sitting-drop vapor diffusion method. A volume of 1 µL protein solution (approx. 10 mgmLλ = 1.7389 Å) (X-ray data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) using a beamline BL17U1  at 100 K, combined with a Quantum 315r CCD detector  . The ano.7389 Å) . The dif.7389 Å) . The res.7389 Å) . The TgF.7389 Å) . Manual .7389 Å) , and aut.7389 Å) . The pos.7389 Å) . Water m.7389 Å) . The fin.7389 Å)  and PROC.7389 Å) . Data cohttp://web.expasy.org/peptide_mass/). The amino acid sequence searches for close homologs of TgFer were obtained from the GenBank database. A phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates using MEGA 5.0 software (http://superpose.wishartlab.com/) (https://pymol.org)  and predicted molecular weight (MW) were deduced by the ExPASy server (software . Structusoftware  and ESPrsoftware . The supab.com/) . All strmol.org) . The elemol.org) .−1) was dialyzed against sample buffer  containing 400, 800, 1,200, 1,600, 2000, or 2,400 µM CuCl2  at room temperature for 30 min. Ferritin was then purified by buffer exchange using a PD-10 desalting column  to remove unbound Cu2+ ions. The interactions of TgFer with the different concentrations of Cu2+ ions were determined by ultraviolet–visible (UV–vis) absorption spectroscopy, ranging from 190 to 500 nm. UV–vis absorption spectra were measured using a Nanodrop 2000 spectrophotometer . All analyses were performed in three replicates with three recordings each.The purified TgFer (approx. 1.0 mgmL2+ samples (50 mM) were prepared by dissolving Mohr’s salt in 0.1% (v/v) HCl under anaerobic conditions. The protein samples were diluted anaerobically to a final concentration of approx. 1.0 mgmL−1. Reactions examining iron oxidation kinetics by protein samples were performed in 0.2 mM HEPES, pH 6.5, containing 150 mM NaCl, with molar ratios of Fe2+ ions to ferritin of 1,000:1 at 25°C  at 25°C, as previously described . Oxygen- at 25°C . All kin at 25°C .2+ ion/ferritin molar ratio of 4,000:1 was added to TgFer and TgFer + Cu solutions  at 25°C for complete reactions. The samples were then dialyzed in 2 mM HEPES with stirring, and the solution was replaced every 4 h. The obtained TgFer + Fe and TgFer + CuFe samples were loaded onto HiLoad 16/600 Superdex 200 pg gel-filtration chromatography column . The iron content was determined by ICP-MS, as described above.To determine the iron storage ability of TgFer and TgFer + Cu, a freshly prepared solution of Mohr’s salt containing an Fep-value of less than 0.05 or 0.01 was considered significant.Statistical analyses were performed using SigmaPlot 12.0 , and significant differences were determined using a two-tailed Tukey’s honestly significant difference test, following an F-test. All data were plotted using Origin Pro 9.0 software . All data are presented as the mean ± standard error of the mean (SEM), and a Sinonovacula constricta ferritin (ScFer)  ferritins and is genetically closest to  (ScFer)   . The pre (ScFer) . CD meast 194 nm .S. constricta, with a sequence identity of 76.6%  is bound to the ferroxidase site with occupancy of 80%, where it is coordinated with the OE1 of Glu25 (helix A), the OE1 of Glu60 (helix B), the ND1 of His63 (helix B) and two waters (2+ ion (90% occupancy)   because rier map . After iFe2+ ion , and thiFe2+ ion .2+ ion entry channel to the ferritin cavity. One Fe2+ ion (Fe-2) with an occupancy of 60% is coordinated by the three OEs of Glu132 residues (distance of 2.63 ± 0.08 Å) from all three symmetrical subunits and three water molecules   are bound between the negative potential pore layer formed by Glu168 residues (2+ ions are found in the 3-fold channel (2+ ion (Cu-2) is located practically outside the channel bridged with three water molecules, and the intermetallic distance Cu-2–Cu-3 in the sites is 3.74 ± 0.23 Å (2+ ion (Cu-3) is located deep inside the channel, coordinated with the OD1 from Asp129 residue (distance of 2.71 ± 0.22 Å) and the OE2 from Glu132 residue (distance of 2.64 ± 0.36 Å) from the 3-fold symmetrical subunits  . In the  channel . The Fe- channel . The Fe-residues . Four irresidues . In the  channel . The Cu2± 0.23 Å . The othsubunits . The occsity map . Similarsity map . One copsity map . The othsity map .Rana catesbeiana bullfrog M ferritin (RcMf) variants (Chaetopterus ferritin (ChF)  atoms per cage, whereas the E168A mutant demonstrated only a slight decrease, with the copper content of 127 ± 0.72 (p > 0.05) atoms per cage. After the separate enrichment of iron for TgFer and TgFer + Cu, the target proteins were further purified by gel filtration, resulting in the elution of TgFer + Fe and TgFer + CuFe as symmetric peaks at approximately 15 ml on a calibrated gel-filtration chromatography column  . To assey column . As show2+ ion/TgFer molar ratio. The CD spectra of TgFer, TgFer + Cu, TgFer + Fe, and TgFer + CuFe showed that all proteins had positive and negative absorption peaks in the 190–198 and 198–254 nm wavelength ranges  and random coil (approx. 5.5%)   . In comp extents . Previou residue , possiblonserved , similaronserved . In the onserved , similar (MjFer) . By cont (MjFer) . Among t (MjFer) , which sitrogens , and Cu2ferritin . Thus, t2+ hexaaqua ion ([Fe(H2O)6]2+) that interacts with the side chains of Asp127 and Glu130 residues in human H ferritin (HuHF) (2+ ions with 6.4–6.9 Å in diameters to pass through the Glu130 constriction (approx. 2.3 Å diameter) in the 3-fold channel of RcMf (2O)6]2+ must occur for ion passage through 3-fold channel, resulting in a direct coordination between the metal ions and Asp/Glu residues in order for the Fe2+ ions to reach the ferroxidase center in ferritin  (Phascolosoma esculenta ferritin (PeFer and Fer147) (Dendrorhynchus zhejiangensis ferritin (DzFer)   and RcMfn (HuHF) . However of RcMf . Therefoferritin . Moreoveous ions . Besides (ScFer) , Phascol Fer147)  and Dend (DzFer) .2+ ion located at the deepest part of the channel pore can travel toward the ferroxidase center with the help of amino acid residue  over the iron ion (For TgFer and TgFer + Fe, the free iron ions outside of ferritin can be attracted by the His116 and Asp120 residues located at the entrance of the 3-fold channel, then guided into the 3-fold pore, where they interact with the side chains of Asp129 and Glu132 residues, as illustrated in  residue . In TgFeectively . The obsectively . The resiron ion .via 3-fold channel can be monitored by determining the kinetics of the catalytic oxidation reaction occurring at the ferroxidase centers in ferritin (2+ ion oxidation kinetics, while E168A mutant inside 4-fold channel has little effect on the oxidation rates. Similar results were observed with the mutations around the 3-fold channel of HuHF (2+ transport into the cavity, and thus to greatly affect the ferroxidase activity (2+ ions were captured by Glu130 residue first and then directed to active sites by Asp127 residue in frog ferritin (2+ ion binding at 3-fold channel may affect the function of iron uptake in the structure of TgFer.The iron transit efficiency ferritin . To elucferritin  (Supplemferritin . We foun of HuHF  and RcMf of HuHF . It is c of HuHF . Previouactivity . Moreoveferritin . CollectThe structural analyses indicate that the 4-fold channel of TgFer presents the special electrostatic arrangement . Althoug2+ ions based on its biochemical characterization such as UV-vis and CD analyses. Nevertheless, there are still several other limitations to our study. On the one side, since the crystals of TgFer + Cu suffered severe cracking after long exposure times to the salt, we set the soaking time to 5 min. Although the crystals are performed with a short soak in mother liquor containing iron atom reagent, it seems highly likely that the soaking process can cause rough crystal surfaces and thus occur some low-resolution X-ray diffraction data and limited structure information. In this work, the crystal structures of TgFer + Cu and TgFer + CuFe had been solved by X-ray diffraction analysis at 2.3 and 3.9 Å resolution, respectively. Obviously, a low-resolution diffraction data not only present a challenge to structure determination, but also hamper interpretation of mechanistic details (In this study, we also find that there are obviously distinct differences among the TgFer structures before and after adding Cu details . Moreove details . Therefo2+ ion oxidation. However, the structural and biochemical analyses indicated that the 4-fold channel of TgFer could be serviced as potential binding sites of metal ions. Structural comparisons revealed that although Cu2+ ion can compete with Fe2+ ion to bind to the structure of TgFer, Cu2+ binding to the 3-fold channel as well as ferroxidase site can potentially block Fe2+ binding, inhibiting the ferroxidase activity of TgFer. Further study will shed light on the role played by Cu2+ ion binding in strengthening the integrity and stability of the TgFer cage and for catalytic promotion by competing iron ions.This study was performed to determine the crystal structural characteristics of TgFer, TgFer + Cu, TgFer + Fe, and TgFer + CuFe at resolutions of 1.78, 2.30, 1.85, and 3.90 Å, respectively. These structures exhibited a typically cage-like spherical shell composed of 24 identical subunits, in which the highly conserved organization of both the ferroxidase center and the 3-fold channel suggested the common function of directing iron into the cavity for Fe


The median intensity of the Raman peaks at 670 cm-1, 731 cm-1, 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 show a significant difference between the CMA3≤41 and CMA3>41groups. The Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to label-free detect sperm with chromatin abnormalities, such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level.Chromatin condensation is one of the main factors essential for sperm function. Evaluation of chromatin condensation by current methods render the assessed sperm unsuitable for assisted reproduction. We examined the Raman spectra of normal morphology sperm to determine whether a non-invasive confocal Raman spectroscopy can detect spectral differences between groups having different levels of chromatin condensation. Semen samples from 85 donors who underwent ICSI were obtained. Chromomycin A3, aniline blue and acridine orange staining were performed to evaluate the protamine deficiency, histone retention and DNA fragmentation respectively. Raman spectra were obtained from 50 normal morphology sperm for each donor. Spectral analysis was performed using home written programs in LabVIEW software and samples were grouped based on chromomycin A3 staining. Raman peaks intensities at 670 cm According to the World Health Organization (WHO)’s criteria, infertility is the inability of couples of a reproductive age to achieve pregnancy within one year in spite of unprotected intercourse –3. RoughSeveral factors lie behind that phenomenon such as chromatin condensation which is necessary for sperm function and later embryonic development. Abnormal sperm chromatin condensation is associated with natural reproductive failure such as spontaneous abortion and failure of assisted reproduction procedures –12. One However, the selection of sperm during assisted reproductive technology (ART) methods like intracytoplasmic sperm injection (ICSI), depends solely on sperm shape and its motility. But there is evidence and developing consideration that sperm of normal shape are not necessarily completely functional and may feature abnormal chromatin condensation –20. FertRaman spectroscopy is a technique based on the Raman effect which studies the inelastic scattering of light by the vibrating atomic bonds. The scattered photons can either lose some of their energy (Stokes scattering) or acquire energy (anti-Stokes scattering) . Generalet al. [et al. [et al. [et al. [Few studies have been done on sperm using Raman spectroscopy. Interestingly, the first living cell that has been studied using Raman spectroscopy was salmon sperm. They assessed the extracted sperm DNA and found that it had a B-type conformation . Huser eet al.  studied  [et al.  used con [et al.  used Ram [et al.  employedIn the present study we examined the Raman spectra that were obtained from normal morphology sperm in order to determine whether confocal Raman spectroscopy can detect spectral differences between the studied groups having different levels of chromatin condensation.Semen samples were collected from donors who underwent ICSI for infertility treatment at the Prince Rashid Ben Al-Hasan Military Hospital, Irbid, Jordan. Patients gave written consent after they were approved to use these samples for research purposes based on the approval of the Royal Medical Services Human Research Ethics Committee number 8/2018). Samples were provided by masturbation after 3-6 days of sexual abstinence and processed immediately after complete liquefaction at 37°C for 30 min. Semen parameters were tested according to WHO guidelines and are summarized in Table 8. SampleFractions of the fresh ejaculate samples were frozen at -18°C in Jordan, transferred under cooled conditions to Germany and stored again at -18°C within 10h. This study was carried out in the Laboratory of Biochemistry and Molecular Biology of Reproductive Medicine, Department of Obstetrics and Gynecology at the University Hospital of Saarland, Homburg, Germany, and at the Department of Informatics and Microsystems Technology, University of Applied Sciences Kaiserslautern, Campus Zweibrücken, Germany.A fraction from the raw semen samples was washed and centrifuged at 250g for 10 min in two steps: first samples were washed twice with phosphate-buffered saline (PBS). Then, the obtained sperm pellet was washed twice with deionized water. Finally, the pellet was resuspended in deionized water and stored at -20 °C until the use in the Raman measurement.et al. [2+ -Mg2+ free PBS (1 volume semen: 2 volumes PBS) followed by centrifugation at 250g for 10 min. Then, the washed sperm were smeared and fixed using methanol/acetic acid, 3:1 (Carnoy’s solution) at 4°C for 5 min. Then, each slide was stained in dark for 20 min with 100 μl of CMA3 stain solution. (CMA3 stain prepared in McIlvaine buffer (pH=7.0) supplemented with 10 mM MgCl2 to a final concentration of 250 μg/ml). Then, each slide was rinsed in PBS buffer, dried and mounted with buffered glycerol (1:1). The evaluation of chromatin condensation each slide was examined using a fluorescence microscope (Olympus) at 100X oil immersion magnification. 500 normal morphology sperm were evaluated for each donor, by differentiating between the CMA3 positive sperm (bright yellow-stained) and CMA3 negative sperm (dull yellow-stained). The percentages of CMA3 positivity were calculated by dividing the number of sperm with positive CMA3 (protamine deficient sperm) by the total number of the evaluated sperm.As described by Bianchi et al.  chromatiet al. [As described by Hammadeh et al.  Chromatiet al. [2HPO4·7H2O: pH=2.5). Then, slides were gently washed with deionized water and covered before drying. The evaluation of chromatin DNA integrity was performed using fluorescence microscope (Olympus) at 100X oil immersion magnification. 200 normal morphology sperm were evaluated for each donor by distinguishing the green-stained sperm (sperm with intact double-stranded DNA), from yellow- or orange- or red-stained sperm (sperm with single-stranded DNA). The percentages of the DNA fragmentation for each donor were obtained by dividing the number of yellow- or orange- or red-stained sperm (sperm with single-stranded DNA) by the total number of the evaluated sperm.As described by Tejada et al.  DNA fragSamples were classified based on the result of sperm chromatin condensation evaluation by Chromomycin A3 (CMA3) , 34. Usi-1 (LabSpec 6 Software). No further processing of the spectra beyond accumulation was performed on the Instrument’s software. Spectra also were saved as comma separated values text files for further processing. A total of 50 normal morphology sperm per sample were chosen from different microscopic fields of view. To acquire each spectrum, the cell was centered and the laser directed at the postacrosomal region of the sperm head.Aliquots of 20 μl of the pre-prepared sperm suspensions were smeared onto stainless steel slides and allowed to air dry. The Raman spectra of sperm were measured using a confocal Raman spectrometer , equipped with an Olympus BX41 microscope, 660 nm diode laser (100 mW), motorized notch filter selector, adjustable confocal pinhole, two switchable gratings, and CCD detector. The acquisition of all spectra were performed by 8 accumulations of 5 seconds each with slit pinhole apertures of 250 μm at 600 grooves/mm diffraction grating using the Olympus X100 objective and a wavenumber range from 600 to 1850 cmThe acquired raw Raman spectra (n=4250) were automatically batch processed using home written custom programs in LabVIEW software .A baseline correction of the original spectra was performed to remove the spectral background caused by autofluorescence of cell components. All acquired Raman spectra were filtered by applying a 5 points baseline model using home-written program in LabVIEW software. This program automatically subtracts the background from the raw spectra without providing a significant distortion of the Raman peaks of the measured samples resulting in virtually background-free Raman spectra. Then, another program normalizes the spectra by setting the lowest intensity of the spectra to zero followed by dividing the whole spectrum by the average intensity of the whole spectrum. Then, multiplying the resulting spectra by 3 to set the maximum intensity to around 1.The normalized spectra files were analyzed by home-written LabVIEW program. To determine the spectral differences among the studied groups and the variations within each group, this program was used to extract the average spectrum and standard deviation over wavenumber for each selected group. To determine the differences among the studied groups in each Raman peak, this program was used to extract the average and standard deviation of any selected Raman peak intensities.Data analysis was performed using origin program . Data were expressed as mean ± SD, median and range. Data were tested for normality using the Anderson-Darling test. The relationship between chromomycin A3, aniline blue, acridine orange, Raman peak intensities and their standard deviation were analyzed using nonparametric correlation (Spearman's test). The Mann-Whitney U test was used to compare the two groups (CMA3≤41 versus CMA3>41). The results were considered statistically significant when the p-value was smaller than 0.05. The effect size for the Mann-Whitney U test, r, was calculated by dividing z by the square root of n (r = z / √n).Three sperm functional parameters for all studied samples (n=85) were evaluated. These parameters include chromomycin A3 staining (protamine deficiency), aniline blue (histones retention) staining and acridine orange staining (DNA fragmentation). A notable variation in the percentages of the positively stained sperm in all sperm parameters was observed as shown in Table All three examined functional sperm parameters were tested for correlations in all studied samples (n=85). Chromomycin A3 staining was significantly positively correlated with acridine orange staining , but it has not been significantly correlated with aniline blue staining . Aniline blue staining was not significantly correlated with acridine orange staining .-1, 731 cm-1, 785 cm-1, 858 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1, the assignment of these Raman peaks for DNA or protein are shown in Table Several Raman peaks show a high variation in their intensities among the studied samples. These peaks were 670 cm-1 , 731 cm-1 , 785 cm-1 , 1062 cm-1 , 1098 cm-1 , 1185 cm-1  and 1372 cm-1  as shown for 1098 cm-1 in Fig. -1 , 1424 cm-1 , 1450 cm-1 , 1532 cm-1 , 1618 cm-1  and 1673 cm-1 . Aniline blue staining was significantly negatively correlated with the DNA related Raman peak intensity around 1098 cm-1  and 1372 cm-1 . Acridine orange staining was significantly negatively correlated with the DNA related Raman peak intensities around 731 cm-1 , 1098 cm-1 , 1372 cm-1  and 1424 cm-1 , while it was significantly positively correlated with the protein related Raman peak intensity around 858 cm-1  and 1450 cm-1 . Finally, the Raman peak intensities ratio (1050 cm-1/1098 cm-1) was significantly positively correlated with the percentage of the chromomycin A3 positive , aniline blue staining  and acridine orange .Chromomycin A3 staining was significantly negatively correlated with the DNA related Raman peak intensities around 670 cmDifferent thresholds values were tested to determine a cut-off value of CMA3 for donor group differentiation regarding fertilization rates, and 41% (positive) was used. Samples were classified into two groups: CMA3≤41% and CMA3>41%. The ROC result for this threshold yields an area under the curve of 0.8589 with 87.8% specificity and 81.8% sensitivity. This means that CMA3 is an excellent diagnostic test in predicting fertilization rates. The median fertilization rates of the CMA3≤41% and CMA3>41% differed significantly at 82 % ± 15.5 for the CMA3≤41% group vs 57 % ± 19.7 for the CMA3>41% group -1, 731 cm-1, 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1) show a significant difference between the CMA3≤41 and CMA3>41 groups in their median peaks intensities as illustrated in Fig. -1 (CMA3≤41 (0.223±0.013), CMA3>41 (0.201±0.015), p=0.001), 731 cm-1 (CMA3≤41 (0.479±0.029), CMA3>41 (0.462±0.028), p=0.002), 785 cm-1 (CMA3≤41 (0.763±0.05), CMA3>41 (0.691±0.064), p=0.015), 1062 cm-1 (CMA3≤41 (0.327±0.018), CMA3>41 (0.314±0.024), p=0.002), 1098 cm-1 (CMA3≤41 (0.594±0.031), CMA3>41 (0.553±0.033), p<0.001), 1185 cm-1 (CMA3≤41 (0.251±0.015), CMA3>41 (0.238±0.015), p=0.007) and 1372 cm-1 (CMA3≤41 (0.763±0.0123), CMA3>41 (0.748±0.0171), p<0.001) showed higher medians intensities in the CMA3≤41 group, while the Raman peaks at 1424 cm-1 (CMA3≤41 (0.444±0.019), CMA3>41 (0.462±0.017), p=0.001), 1450 cm-1 (CMA3≤41 (0.715±0.0137), CMA3>41 (0.719±0.0213), p=0.028), 1532 cm-1 (CMA3≤41 (0.0864±0.0153), CMA3>41 (0.0998±0.0139), p=0.003), 1618 cm-1 (CMA3≤41 (0.295±0.025), CMA3>41 (0.332±0.031), p<0.001) and 1673 cm-1 (CMA3≤41 (0.643±0.027), CMA3>41 (0.694±0.041), p<0.001) showed higher medians intensities in the CMA3>41 group. Generally, the CMA3>41 group shows a higher variation than the CMA3≤41 group as indicated by the higher standard deviation values throughout the spectra. This is most pronounced at the Raman peaks intensities at 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 showing significant differences in their standard deviations as illustrated in Fig. Figure The evaluation of male infertility has become more important and revealing with the availability of new diagnostic and treatment methods . Sperm cTo establish a new diagnostic technique based on the Raman spectroscopy that is able to distinguish between sperm with normal and abnormal chromatin condensation it is essential to understand the Raman spectra of the sperm components and Raman spectral differences between normal sperm and sperm with chromatin condensation abnormalities.-1 represents the ring breathing mode of guanine, indicates that the DNA backbone was in B-form conformation [-1 and 785 cm-1, which are the ring breathing modes of the nitrogen bases in the DNA nucleotides, are correlated to the DNA content and can be used to differentiate between sperm with X- or Y-chromosome [Previous studies analyzed Raman spectra and correlated spectral features to various properties , 45–52. ormation . Raman promosome , 50.-1 and 785 cm-1 that were significantly higher in the low chromomycin A3 (CMA3≤41) group compared to the high chromomycin A3 (CMA3>41) group (731 cm-1 (CMA3≤41 (0.479±0.029), CMA3>41 (0.462±0.028), p=0.002), 785 cm-1 (CMA3≤41 (0.763±0.05), CMA3>41 (0.691±0.064), p=0.015)) with medium effect size of r=0.34 for 731 cm-1 and small effect size of r=0.26 for 785 cm-1. This indicates that chromatin condensation quality could affect the sperm’s relative DNA content. The highly condensed chromatin in the low chromomycin A3 (CMA3≤41) group produces more intense DNA Raman signals due to the presence of highly compacted DNA in the laser beam path compared to the high chromomycin A3 (CMA3>41) group in which the loosely compacted DNA produces less intense DNA Raman signals. The highly compacted chromatin protects the sperm DNA against DNA fragmentation factors such as nucleases and polymerases [4) peak around 1098 cm-1 showed the same trend and confirmed the previous finding. It was significantly higher in the low chromomycin A3 (CMA3≤41) group compared to the high chromomycin A3 (CMA3>41) group (CMA3≤41 (0.594±0.031), CMA3>41 (0.553±0.033), p<0.001) with a large effect size r=0.6. It was reported that Raman peaks intensity and position are affected by nucleotide composition and sequence [4) peak around 1098 cm-1 is the least affect one [-1, it was significantly higher in the low chromomycin A3 positive group compared to the high chromomycin A3 positive group. A similar finding was reported by Hud et al. [The results of the current study showed a significant difference in the Raman peaks median intensities of 731 cmymerases , 55 or oymerases  and thissequence , and thefect one . TherefoMA3≤41 0.4±0.031, d et al.  in which-1 and 1372 cm-1 in which the median intensities of these Raman peaks were significantly higher in the low chromomycin A3 (CMA3≤41) group compared to the high chromomycin A3 (CMA3>41) group (1185 cm-1 (CMA3≤41 (0.251±0.015), CMA3>41 (0.238±0.015), p=0.007) and 1372 cm-1 (CMA3≤41 (0.763±0.0123), CMA3>41 (0.748±0.0171), p<0.001)) with medium effect size r=0.3 for 1185 cm-1 and r=0.42 for 1372 cm-1. Several studies identify these Raman peaks and assigned them to ring breathing modes of DNA nitrogen bases [The same trend was observed in the Raman peaks around 1185 cm A3 CMA3≤ group coen bases –49, 51. en bases .et al. [-1 with protamines packaging efficiency. With low intensity indicating highly packaged chromatin. The result of the current study showed a significant negative correlation  between this peak and the chromomycin A3 positivity indicating that the intensity of this peak is significantly decreased in poorly condensed chromatin (785 cm-1 (CMA3≤41 (0.763±0.05), CMA3>41 (0.691±0.064), p=0.015)). This result is in the same line as Hud et al. [-1 Raman peak being higher in the salmine-DNA complex compared to the native B-form DNA. This peak results from the contribution of cytosine and sugar-phosphate backbone vibrational modes. The increased intensity could be explained by the disturbance of the vibrational modes that occur as a result of binding to arginine residues in the protamine [et al. [On the other hand, Huser et al.  linked td et al.  in whichrotamine . Amaral  [et al.  found th [et al. .-1 was significantly higher in the high chromomycin A3 (CMA3>41) group compared to the low chromomycin A3 (CMA3≤41) group (CMA3≤41 (0.715±0.0137), CMA3>41 (0.719±0.0213), p=0.028) with medium effect size r=0.34. This peak is associated with the protein and lipids content in the cell [et al. [-1 . Two possible explanations could be offered here. The first one is based on the lipids contribution in this peak. Unsaturated fatty acids are the main component of the biological membranes and are very vulnerable to oxidative attack. Therefore, alteration in the lipids content can be associated or may be caused by lipid peroxidation, which is a well-recognized indicator of oxidative DNA fragmentation [The Raman peak of the methylene deformation mode around 1450 cmthe cell , 29, 52.the cell . A simil [et al. , 64. Alsentation . The secentation . They ar-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 in which the median intensities of these Raman peaks were significantly higher in the high chromomycin A3 (CMA3>41) group compared to the low chromomycin A3 (CMA3≤41) group (1424 cm-1 (CMA3≤41 (0.444±0.019), CMA3>41 (0.462±0.017), p=0.001), 1532 cm-1 (CMA3≤41 (0.0864±0.0153), CMA3>41 (0.0998±0.0139), p=0.003), 1618 cm-1 (CMA3≤41 (0.295±0.025), CMA3>41 (0.332±0.031), p<0.001) and 1673 cm-1 (CMA3≤41 (0.643±0.027), CMA3>41 (0.694±0.041), p<0.001)) with medium effect sizes r=0.36 for 1424 cm-1, r=0.32 for 1532 cm-1, r=0.4 for 1618 cm-1 and r=0.42 for 1673 cm-1. Several studies identify these Raman peaks and assigned them to protein [The same trend was observed in the Raman peaks around 1424 cm protein , 49, 51.et al. [-1/1098 cm-1 and 1445 cm-1/1098 cm-1 in order to discriminate between normal morphology sperm from abnormal sperm. The ratio distribution was not accurate and most abnormal morphology sperm were found in the normal morphology range. Mallidis et al., Huang et al. [et al. [Huser et al.  plotted g et al. , 52 and  [et al. , in whic-1/1098 cm-1) was significantly positively correlated with the percentage of the chromomycin A3 positivity  and this ratio was significantly higher in high chromomycin A3 (CMA3>41) group compared to the low chromomycin A3 (CMA3≤41) group (CMA3≤41 (0.544±0.036), CMA3>41 (0.561±0.05), p=0.039). A similar trend was found for DNA fragmentation evaluated by acridine orange. DNA fragmentation was significantly correlated with Raman peak intensities ratio (1050 cm-1/1098 cm-1) . This result is in agreement with that of Sanchez et al. [-1/1098 cm-1) being correlated with induced oxidative DNA fragmentation evaluated by flow cytometry also based on acridine orange. The same trend was observed by Mallidis et al. [-1/1098 cm-1) was increased with UV-induced DNA fragmentation. As mentioned before, the phosphate (PO4) Raman peak around 1098 cm-1 is the least variable DNA peak and is assumed not to be affected by nucleotide composition or sequence [-1 could be due to the changes in the groups and their force-bearing environment resulting from covalent bonds breakages between the deoxyribose and phosphate groups [et al. and Mallidis et al. [-1 and it appears as a separated peak. In the current study and other several studies [-1 they observed could be caused by the extensive DNA fragmentation caused by their sample preparation methods or could alternatively partially be explained by the substrate used during the spectral acquisition. They used quartz that contributes with a significant Raman signal in the vicinity of this Raman peak at 1050 cm-1 . This spectral background contribution might not have been fully corrected by the authors.The results of the current study showed that the Raman peak intensities ratio  group compared to the low chromomycin A3 (CMA3≤41) group. The magnitude of Raman peak intensity is directly related to the concentration of corresponding molecules of that peak. Therefore, these variations reflect variations in the biochemical content of the assessed sperm. It is known that sperm either possess a X-chromosome or a Y-chromosome that contain different amounts of DNA. Although this difference is small, it is reported that Raman spectroscopy can differentiate between X or Y bearing chromosome sperm , 50. TheIn summary, the spectral analysis of Raman peaks indicates that the Raman DNA related peaks intensities and with it the DNA densities decreased when chromatin condensation quality decreased, while the Raman proteins related peaks intensities and with-it protein densities increased when the chromatin condensation quality decreased.The result of this study indicate that Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to detect sperm with chromatin abnormalities such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level. Unlike the currently used chromatin integrity tests that destroy the sample under analysis, Raman spectroscopy should have the ability to detect chromatin integrity noninvasively in living sperm. Therefore, Raman spectroscopy represents a promising technique that could be accompanied with an ICSI procedure and used to select sperm with proper chromatin condensation and intact DNA. But, this ultimate goal still needs further evaluation, e.g. establishing laser power and exposure time conditions that are actually noninvasive for the living sperm.


Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.  Mycobacterium abscessus is a rapid-growing nontuberculous Mycobacterium (NTM) and is often present in soil and water [M. abscessus infection is increasing in hospitals in Taiwan [M. abscessus is highly resistant to multiple antibiotics, treatment of M. abscessus infections is often difficult [M. abscessus is resistant to first-line anti-tuberculosis agents such as rifampin and ethambutol [M. abscessus infection consists of intravenous amikacin combined with oral macrolide such as clarithromycin, as suggested by the American Thoracic Society and the Infectious Diseases Society of America [M. abscessus pulmonary disease is only 30–50%, much lower than other NTM diseases [M. abscessus infections have also been reported, especially after lung transplantation [M. abscessus can infect both immunodeficient and immunocompetent patients [nd water ,2. It hand water ,4,5,6 annd water ,8. The pn Taiwan ,10. Becaifficult ,12. It hhambutol ,14. Currdiseases . Fatal Mantation ,17. Sevepatients . M. abscessus exhibits two different colony morphotypes, smooth and rough [M. abscessus, M. smegmatis, M. chelonae, and M. avium complex [M. abscessus pairs [nd rough , with dind rough ,21,22. Tnd rough ,24. Stud complex ,26. Singus pairs . Cells oM. abscessus. In our previous study [M. abscessus cs1c-R strain. A mutant with a smooth morphotype designated mab_3083c::Tn was isolated. Results of bioinformatic analyses suggest that mab_3083c is a homologue of ribonuclease J. RNase J is an enzyme present in many bacteria and plays an important role in all aspects of mRNA metabolism including ribosomal RNA maturation, mRNA decay and stability [mab_3083c gene, homologue of RNase J, in colony morphology switching and surface-associated properties of M. abscessus.In order to investigate the mechanism of colony morphotype switch, we attempted to identify genes involved in colony morphology and virulence of us study , a transtability . In thisM. smegmatis mc2155 was purchased from the American Type Culture Collection (ATCC), and the clinical strain M. abscessus cs1c was obtained from the Veterans General Hospital, Taichung, Taiwan. M. abscessus cs1c-R was a spontaneous mutant derived from strain cs1c [M. abscessus and M. smegmatis were grown at 37 °C on Middlebrook 7H11 agar  containing 10% OADC   or in 7H9 broth  with 10% OADC and appropriate concentration of Tween 80. When required, selective media were prepared with 7H11 agar supplemented with 10% OADC and apramycin (50 μg/mL) or kanamycin (50 μg/mL).ain cs1c . Cells oTM<KAN-2>Tnp TransposomeTM Kit was carried out according to the manufacturer’s instructions , and the Tn5 insertion site in the target mutant was identified by inverted PCR with primers KAN-2 FP-1 (5′-ACCTACAACAAAGCTCTCATCAACC-3′) and KAN-2 RP-1 (5’-GCAATGTAACATCAGAGATTTTGAG-3′).The mutant library made by EZ-Tn5mab_3083c was amplified by PCR using primers mab_3083c-F-BglII (5′-TAAGATCTGTGGCAGTTACCCCACCC-3′) and mab_3083c-R-NheI (5′-ATGCTAGCCTAGTCGACGGTGAGCAC-3′), and that of msmeg_2685 was amplified with primers msmeg_2685-F-Acc65I (5′-TGGGTACCATGAGCGCCGAACTCGCG-3′) and msmeg_2685-R-NheI (5′-ATGCTAGCTCAGATCTCTATGACGGTC-3′) containing restriction enzyme sites (bold-faced). The amplified products were then cloned into the vector pYUB412A [mab_3083c. The D89K,H90Amab_3083c mutations were generated by site-directed mutagenesis with a three-step PCR as described previously [mab_3083c-mut-F (5′-ATAGGATCGCATCACCATCACCATC-3′), mab_3083c-mut-R (5′-ATAAGACCAGACGCTCGCCAAC-3′), mab_3083c-mut-Fm2 (5′-GCATGAGAAAGCCATCGGGGCCATTCCGTTCCTGCTGAAG-3′), and mab_3083c-mut-Rm2 (5′-CTTCAGCAGGAACGGAATGGCCCCGATGGCTTTCTCATGC-3′) containing mutated sequence (bold faced). The constructed plasmid was introduced into M. abscessus strains by electroporation  at 2500 V, 1000 Ω, and 25 μF. The pYUB412A , generateviously . This thM. abscessus cells were inoculated on 7H9 with 0.3% agar. After an incubation at 37 °C for 3 days, the sliding distance was measured.Sliding motility was examined as previously described . M. abscM. abscessus cells with an optical density of OD600 = 0.1 were cultured in a tube containing 5 mL of 7H9 medium with 10% OADC. After an incubation at 37 °C with continuous rotation for 2 days, the cultures were allowed to stand still at room temperature for 10 min. The upper portion of each culture containing dispersed cells was then removed to determine the OD600 value. The aggregated cells at the bottom of each tube were completely suspended by vortexing with glass beads of 4.5 mm in diameter , and the cell suspension was measured for its OD600 value. The aggregative index of each culture was calculated as the ratio of OD600 value of aggregated cells to that of dispersed cells [The aggregation assay was performed as previously described with modifications . M. absced cells .M. abscessus lipid was extracted as previously described [M. abscessus cells were cultured for 4–5 days on agar plates, harvested, and incubated in 1 mL of chloroform/methanol  at 56 °C for 2 h in a water bath sonicator (Branson 5200). After centrifugation , the supernatant (500 μL) was purified by extracting with 500 μL chloroform/methanol (2:1) and then with 1000 μL distilled water. The organic extracts were dried, weighted, and resuspended in chloroform/methanol (2:1). For saponification, 1:1 volume of 0.2 N NaOH and resuspended total lipid were mixed, incubated at 37 °C for 45 min, and then neutralized with glacial acetic acid . The saponified lipid was dissolved in chloroform/methanol, spotted on an aluminum-backed silica gel60 TLC plate , and resolved with 10 mL of chloroform/methanol/H2O (100:14:0.8). The TLC plate was soaked briefly in 10% H2SO4 in methanol and then heated to visualize lipids.escribed ,28,31. M2 incubator overnight in 24 well plates (SPL Lifesciences) with a total amount of 500 μL, 2 × 105 cells per well. The culture medium was refreshed, and the culture was incubated for 2 more days. The cells were then infected with M. abscessus at a multiplicity of infection (MOI) of 1 for 2, 24, 48, or 72 h. The infected cells were washed to remove extracellular bacteria. The washed cells were then lysed by incubation with 1% Triton X-100 for 30 min, and the cell lysate was plated on 7H11 agar plates to determine the number of intracellular mycobacteria [This assay was performed as previously described . The humbacteria ,33.M. abscessus cells were first cultured in 7H9 broth supplemented with 10% OADC and 0.5% Tween 80 at 37 °C for 3–4 days. Approximately 3 × 107 of these cells were inoculated into 3 mL of 7H9 broth without OADC and with various concentrations of lysozyme  for 24 h or with H2O2  for 2 h at 37 °C. The cultures were then serial diluted and plated (20 μL) on 7H11 agar plates for determination of bacterial CFU.http://www.genome.jp/kegg/ (accessed on 23 March 2021)) analysis, the functional role of Mab_3083c was recommended as RNase J. The following amino acid sequence alignments were performed by clustalW2 and NCBI program.According to the result of Kyoto Encyclopedia of Genes and Genomes . A mutant named mab_3083c::Tn with the colony morphotype switched from rough to smooth was obtained  was found to revert back to rough  between nucleotides 365 and 366 downstream from the initiation codon (To determine the insertion site of Tns of Tn5 B. The PCon codon B.mab_3083c in the genome of the complemented strain (mab_3083c::Tn/mab_3083c), PCR was performed with primers , suggesting that some copies of the introduced mab_3083c on pYUB-mab_3083c were integrated into the chromosome of mab_3083c::Tn/mab_3083c.To confirm the presence of ab_3083c B,C. As tfragment C was detab_3083c B. Both tab_3083c C and 862ab_3083c C were gemab_3083c::Tn, and complemented (mab_3083c::Tn/mab_3083c) strains, lipids of these strains were extracted and analyzed by TLC. The lipid profiles of three strains were no significant difference  and the mab_3083c::Tn mutant at an MOI of 1, and the CFU of intracellular bacteria was counted at 2, 24, 48, and 72 h post infection. As shown in 3) was similar to that of the wild type ((3.7 ± 0.6) × 103). No significant difference was observed at 24, 48, and 72 h post infection between the wild type ((2.8 ± 0.5) × 105 at 24 h; (1.9 ± 0.5) × 107 at 48 h; (5.6 ± 1.0) × 107 at 72 h) and the mutant ((3.6 ± 0.2) × 105 at 24 h; (1.5 ± 0.3) × 107 at 48 h; (5.2 ± 1.5) × 107 at 72 h).It has been shown that cells of the rough strain of  cleared . To invemab_3083c::Tn mutant to oxidative stress and lysozyme, the bacteria were incubated with 10–30 mM hydrogen peroxide for 2 h or 0.5–2.5 mg/mL lysozyme for 24 h. Bacteria incubated with broth without hydrogen peroxide or lysozyme were used as controls. The susceptibility was expressed as the percentage of CFU of bacteria with treatment divided by that without treatment. The results showed that the percentages of susceptibility of the wild type and the mutant were 52.3 ± 8.7 and 46.8 ± 9.4 to 10 mM H2O2, 7.4 ± 0.8 and 5.6 ± 0.7 to 20 mM H2O2, and 1.4 ± 0.4, and 0.9 ± 0.3 to 30 mM H2O2, respectively . The mab_3083c complemented strain (mab_3083c::Tn/mab_3083c) was similar to the wild type with a low sliding phenotype (1.44 ± 0.29 mm).According to previous studies, smooth strains of pability ,30. To t plotted A,B. The mab_3083c affects the aggregation capability of M. abscessus, the variants were grown in 7H9 media with 10% OADC and 0.05% Tween 80 for two days. Cultures of the mab_3083c::Tn mutant were found to be turbid, whereas those of the wild type and the complemented (mab_3083c::Tn/mab_3083c) strains had bacterial aggregates at the bottom and clear supernatants. To quantify the aggregation ability, the aggregation index of each culture was calculated and plotted. As shown in mab_3083c::Tn mutant (1.70 ± 0.03) displayed a lower aggregation ability than the wild type (4.49 ± 0.62), and the mab_3083c complemented strain (9.49 ± 0.69) regained the aggregative phenotype. These results suggest that mab_3083c is associated with a low sliding and a high aggregation ability of M. abscessus.To test whether M. smegmatis mc2155 (msmeg_2685) indicated that they share a 75.4% identity  failed to complement the mab_3083c::Tn mutation. Taken together, these results provided evidence that Mab_3083c functions as an RNase J.It has been shown that D85K and H86A mutations generated at the active site of activity . To provrphotype B and higrphotype C, the saMycobacterium promotes bacterial aggregation and loses the ability to produce biofilm and sliding ability [mab_3083c::Tn mutant) was selected. Interestingly, no significant differences were found between wild type and mutant in GPL profiles, intracellular survivability, susceptibility to hydrogen peroxide and lysozyme. According to bioinformatics analysis, the role of mab_3083C was recommended as RNase J. Phenotype alterations regulated by RNase J have been reported in Bacillus subtilis [Streptomyces venezuelae [Bacillus subtilis affected its spore formation and maturation as well as alteration in cell appearance with disordered peptidoglycan layer and long spiral filaments [Streptomyces venezuelae also has defects in cell development, sporulation, and ribosome assembly. In our study, the RNase J mutant of M. abscessus (mab_3083c::Tn) was viable, suggesting that RNase J is not essential for its growth, unlike Streptococcus pyogenes in which both RNase J1 and RNase J2 are essential [GPLs are a member of glycolipids produced by many mycobacteria genus involved in morphotype switching, biofilm formation, sliding motility, pathogenicity, and immunomodulation ,35,36,37 ability . In thissubtilis  and Strenezuelae . RNase Jilaments . The RNassential .Thermus thermophilus RNase J [Msmeg are located at the active site of the β-lactamase domain, and D85K and H86A mutations of M. smegmatis RNase J result in the loss of its exonuclease activity [As some ribonucleases, RNase J regulates mRNA stability or ribosomal RNA maturation ,41,42,43 RNase J , RNase Jactivity .Bacillus subtilis was found to have increased levels of approximately 300 mRNA transcripts and decreased levels of equal numbers of mRNA transcripts [mab_3083c) of M. abscessus affects the stability of some mRNAs related to its colony morphology, sliding motility, and aggregation ability. Further studies are warranted to identify the genes encoding these mRNAs.Some studies have described the role of RNase J in mRNA turnover. For example, the RNase J1/J2 double mutant of nscripts . Studiesnscripts ,46. It iM. abscessus were associated with GPL locus. Our study demonstrated that the non-GPL locus gene mab_3083c also plays a role in this switch. The disruption of mab_3083c led to colony morphology switching from rough to smooth, increased sliding motility, and reduced aggregation ability of M. abscessus. Complementation of the mab_3083c::Tn mutant with RNase JMsmeg (msmeg_2685) switched its colony morphology from smooth back to rough. These results suggest that mab_3083c is the RNase J gene of M. abscessus.Most reports on the morphotypic switch of


Moreover, it is unclear whether NG2 glia express auxiliary transmembrane AMPA receptor related proteins (TARPs), which modify AMPA receptor properties, including their Ca2+ permeability. Through combined molecular and functional analyses, here we show that hippocampal NG2 glia abundantly express TARPs γ4, γ7, and γ8 as well as cornichon (CNIH)-2. TARP γ8 undergoes profound downregulation during development. Receptors of adult NG2 glia showed an increased sensitivity to blockers of Ca2+ permeable AMPA receptors, but this increase mainly concerned receptors located close to the soma. Evoked synaptic currents of NG2 glia were also sensitive to blockers of Ca2+ permeable AMPA receptors. The presence of AMPA receptors with varying Ca2+ permeability during postnatal maturation may be important for the cells’ ability to sense and respond to local glutamatergic activity and for regulating process motility, differentiation, and proliferation.Synaptic and axonal glutamatergic signaling to NG2 glia in white matter is critical for the cells’ differentiation and activity dependent myelination. However, in gray matter the impact of neuron-to-NG2 glia signaling is still elusive, because most of these cells keep their non-myelinating phenotype throughout live. Early in postnatal development, hippocampal NG2 glia express AMPA receptors with a significant Ca The subunit TARP γ2, also called stargazin, is necessary for surface translocation and synaptic expression of AMPA receptors as demonstrated in cerebellar granule cells . Meanwhieceptors . Specifi TARP γ7 . In gransynapses . TARP γ8synapses . Geneticsynapses . The presynapses . Anothersynapses . CNIHs isynapses . CNIH-2 synapses .2+ permeable AMPA receptors, while activation of P2Y receptors led to a decrease  from mouse forebrain revealed abundant expression of TARP γ4, γ8 and, to a lower extent, γ5 . For acute cell isolation, slices were stored in HEPES-buffered solution lacking nominal Ca2+ .Transgenic mice [NG2ki-EYFP ] of post+ channels, Na+ channels and GABAA receptors, Ba2+ (100 μM), quinine (200 μM), TTX (0.5 μM) and picrotoxin (100 μM) was added to the bath solution. AMPA receptor agonists, modulators and blockers were applied via the bath solution. The artificial cerebrospinal fluid (aCSF) contained (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 10 glucose, 26 NaHCO3 . The pipette solution was composed of (in mM): 130 KCl, 0.5 CaCl2, 2 MgCl2, 5 BAPTA, 10 HEPES, and 3 Na2-ATP (pH 7.28). Recordings were obtained at room temperature.Membrane currents were measured with the patch-clamp technique in the whole-cell configuration. Current signals were amplified , filtered at 3 and 10 kHz and sampled at 10 and 100 kHz by an interface connected to a computer system, which also served as a stimulus generator. The resistance of the patch pipettes was 2–4 MΩ . Capacitance compensation was used to improve voltage clamp control. The input resistance was determined by stepping the membrane from −70 to −60 mV. To block K2, 0.5 CaCl2, 5 BAPTA, 10 Hepes, 3 Na2-ATP, 10 TEA. Stimulation of Schaffer collaterals was performed through a chlorinated silver electrode inserted in a low resistance (<1 MΩ), aCSF-filled glass capillary which served as a monopolar stimulation electrode. Pulse sequences were generated with an AM-Systems isolation pulse stimulator . The stimulation electrode was positioned close to the patch electrode . Responses were analyzed with custom-written macros in Igor Pro 8 software (Wavemetrics). Stimulus artifacts were offline compensated by subtracting averaged failures. Recordings were obtained at 35°C.For analysis of postsynaptic currents, a CsCl-based pipette solution was used (in mM): 120 CsCl, 2 MgCl+ channel blockers  as well as AMPA receptor agonists, blockers and modulators were applied by transferring the cells with a tube electrode to the different solutions . The pipette solution was the same as for slice recordings. For recordings in high Ca2+ solution, 150 mM NaCl was replaced by 50 mM CaCl2 and adjusted with N-methyl-D-glucamine (NMDG) to an osmolarity of 320 mOsm (pH 7.4). As for recording synaptic currents (cf. above), CsCl-based pipette solution was used. Recordings were obtained at room temperature. Salts and buffers were purchased from AppliChem , kainate and CTZ were received from Abcam , Naspm from Alomone Labs (Israel), and IEM-1460, JNJ 55511118 and GYKI53655 from Tocris .Freshly isolated cells were obtained from slices after protease treatment as described . K+ chanolutions . MembranTM , random hexamer primers , and reverse transcriptase . The reaction mix was added to the harvested cell content, final volume was 10 μl, and the reaction mix was incubated at 37°C for 1 h. A multiplex two-round single-cell PCR was performed with primers for TARPs, CNIH-2 and the housekeeping gene PDGFRα. The first PCR was performed after adding PCR buffer, MgCl2 (2.5 mM), dNTP (4 μM × 50 μM), primer (200 nM each), and 5 U Taq polymerase (Invitrogen) to the RT product . Thirty-five cycles were performed . An aliquot (2 μl) of the PCR product was used as a template for the second PCR  using nested primers  was added. Products were identified with gel electrophoresis. As a positive control, total RNA from mouse brain was run in parallel. Negative controls were performed using distilled water or bath solution for RT-PCR.Single-cell transcript analysis was performed as previously reported . Briefly primers . The con2+- and Mg2+-free). After centrifugation  the supernatant was discarded and the cells were suspended in 200 μl lysis/binding buffer , frozen in liquid nitrogen and stored at −80°C. Messenger RNA was isolated from isolated cells by cell lysis in the lysis/binding buffer and by using oligo(dT)25-linked Dynabeads (Invitrogen). The beads with adherent mRNA were suspended in DEPC-treated water (20 μl). For first strand synthesis, RT was performed using oligo-dT24-primer . The reaction mix was incubated for 1 h at 50°C .NG2ki-EYFP mice  were sacrificed and their brains were dissected and the hippocampus was isolated under microscopic control . A cell suspension was prepared by mincing the tissue, digesting in papain  and incubating with DNAseI . Fluorescent NG2 glial cells were identified by their EYFP fluorescence (emission at 527 nm) and sorted by a FACSAriaIII flow cytometer  into tubes containing Hanks’ balanced salt solution  and Taqman primer/probe mix . One μl of the RT product was added, the reaction volume was 12.5 μl. PCRs for the respective target genes and β-actin, as a housekeeping gene, were run in parallel wells for each sample (triplicates for each sample). Water served as a negative control in each run. After denaturation , 50 cycles were performed . Fluorescence intensity was read out during each annealing/extension step. Target gene/β-actin expression ratios were determined by comparing CT the cycle number at threshold. The amplification efficiency for TARP γ2 was 1.89, for γ4 1.90, for γ7 1.92, for γ8 1.96, for CNIH-2 1.98, and for β-actin 1.94. For the AMPA receptor subunits the amplification efficiencies were 1.95  and 1.96 .yielding a gene ratio with X being the input copy number and CThe rectification index (RI) of rector currents was determined by comparing the chord conductance of agonist/blocker-sensitive currents at –70 and +70 mV or +40 mV according to:rev its extrapolated reversal potential.where I is the amplitude of the currents at +70, +40, and −70 mV, respectively, and Et-test or Mann–Whitney-U-test with or without Welch correction for equal or diverse variance. In case of paired data, a paired t-test or Wilcoxon signed rank test was performed. For group analyses, we used two-way ANOVA followed by Tukey or Holm’s post hoc test, in case of non-parametric data, aligned rank transformed ANOVA followed by Mann–Whitney-U-Test or Wilcoxon signed rank test with Holm’s correction for multiple comparisons. Tests were performed with the software R (R Development Core Team1). Non-Gaussian distributed data are displayed in box plots showing median , quartiles  and whiskers (±1.5 times the interquartile range). Statistical significance of gene expression by single-cell RT-PCR was analyzed by χ2 test. Significance level was set to P < 0.05. Except box plots, data are given as mean ± SD. n and N refer to the number of cells and mice, respectively.Data were tested for Gaussian distribution by the Shapiro–Wilk tests and for homogeneity of variance with Levene’s test, followed by a two sample + cells located next to blood vessels were excluded from this study to avoid mix up with NG2+ pericytes  and Ba2+ (100 μM) to the bath shifted the resting potential of the NG2 glial cells from −85 mV  to −50 mV (−55 to −44 mV) and increased the input resistance from 225 MΩ (165–320 MΩ) to 1,000 MΩ . Subsequent application of kainate (250 μM) induced inward currents of −5.85 pA/pF (−5.14 to −7.93 pA/pF)   . Next we co-applied kainate (250 μM) with 1-naphtyl-acetyl-spermine  to detect putative currents through Ca2+ permeable AMPA receptors , RI did not change   . Althoug60–1.94) . The rev60–1.94) . Increaseceptors . While t, N = 5) . Naspm r+ channel blockers induced a depolarization from −87 mV (−84 to −90 mV) to −65 mV (−70 to −52 mV) while the input resistance increased from 80 MΩ (66–104 MΩ) to 714 MΩ . The lower input resistance in these older mice reflected the cells’ increased Kir conductance  und were completely blocked by NBQX (10 μM) (p < 0.001) might have indicated a more efficient spermine block and hence an elevated Ca2+ permeability in NG2 glia from the older mice. In the presence of Naspm, RI was 1.36 , i.e., lower than at juvenile stage (V = −100 mV) did not change during maturation.At p60, the Kductance . Applica (10 μM) . This stle stage , but the2+ permeable AMPA receptors and that this block, in contrast to intracellular spermine block, does not depend on TARP expression . At p60, the blocking effect of intracellular Naspm was also stronger than at p10 (p < 0.01)  .2+ permeable, GluA2-lacking AMPA receptors . We found heterogeneous expression of auxiliary subunits in NG2 glia. At p10, a majority of cells expressed TARP γ4 , TARP γ7 , TARP γ8 , and CNIH-2  while TARP γ2 and TARP γ5 were less frequent  (n = 21), TARP γ7  and CNIH-2  were most abundant while TARPs γ2 and γ5 were less frequent . However, the expression of TARP γ8 differed, declining to 23% at p60 , and CNIH-2 was also less abundant than at p10  (Since TARPs and CNIHs affect the intracellular polyamine block and may accompany Cactively) . At p60, = 0.02) .n = 8), followed by TARP γ7  and CNIH-2 . Expression of the hippocampus-specific subunit γ8 was low in NG2 glia . TARP γ2 and γ5, which are abundantly present in the cerebellum, were expressed at a low level while TARP γ3 was absent   . Expressn = 10 for all)  , followed by GluA3 (0.050 ± 0.023), GluA4 (0.030 ± 0.014) and GluA1  . A similfor all) .2+ permeable AMPA receptors, Naspm and IEM-1460  to the bath depolarized the cells from −28.2 ± 15.8 mV to −16.2 ± 8.0 mV and increased their input resistance from 1,285 ± 847 MΩ to 2,152 ± 2,044 MΩ   (rev) of −1.5 ± 8.9 mV and RI = 1.26 ± 0.27 , confirming previous findings  (Vrev = −2.8 ± 10.2 mV).After fresh isolation, NG2 glia was identified by their intrinsic fluorescence. At p10, addition of quinine and Ba N = 13) . With sp −70 mV) . The I/Vfindings . GYKI536, N = 9) , with th2+ permeable AMPA receptors and putative involvement of TARP γ8 in kainate-induced responses of NG2 glia, JNJ was applied subsequent to Naspm or IEM-1460. Naspm (50 μM) decreased the inward currents  while the subsequent application of JNJ (10 μM) led to a further block of the kainate-induced currents   while addition of Naspm led to a further reduction by 30.2 ± 11.8%  , which was less compared to block by 10 μ JNJ      . With th; N = 4) . To avoi; N = 4) . These d N = 11) , indicat, N = 6) . Notably, N = 4) .2+ permeable AMPA receptors was used, IEM-1460. IEM-1460 (100 μM) blocked 38.8 ± 10.8% of the kainate responses . Compared to Naspm, this blocking efficacy was lower (p = 0.03). Subsequent application of JNJ further reduced the receptor currents   and additionally by 28.2 ± 10% through IEM-1460 . Again, 40.5 ± 9.4%  of the responses were insensitive to both inhibitors    . Vice vehibitors . IEM-sen, N = 4) . In cont, N = 4) .2+ permeable receptors. Thus, TARP γ8 is mainly associated with Ca2+- impermeable AMPA receptors.JNJ still blocked part of the Naspm- or IEM-1460-insensitive currents. Vice versa, after initial application of JNJ, Naspm and IEM-1460 still blocked Ca2+ depolarized the cells (from −23.6 ± 11.5 mV to −15.9 ± 8.1 mV) and increased the input resistance  . With spermine (50 μM) in the pipette solution, the kainate-induced currents reversed at −0.5 ± 6.0 mV. RI was 0.98 ± 0.26 (n = 20) indicating linear I/V relationships, which differed from the outward rectifying responses seen in juvenile NG2 glia (p = 0.0005). GYKI53655 almost completely blocked the responses . RI of GYKI-sensitive currents was 1.00 ± 0.13 (Vrev = −2.8 ± 8.3 mV)  . Co-appl 8.3 mV) .n = 10, N = 6, p = 0.002). Subsequent co-application of JNJ (10 μM) led to further reduction of the responses   while subsequent co-application of Naspm further reduced the currents by 44.0 ± 24.0%   was weaker (p < 0.05) (p < 0.05). Subsequent block by Naspm (50 μM) was as efficient as after application of JNJ at the higher concentration , but stronger than at p10 (p = 0.01). The reversal potential of Naspm-sensitive currents was −1.3 ± 9.0 mV, with RI = 0.74 ± 0.39 mV , indicating that the current were partially mediated by Ca2+ permeable receptors. JNJ-blocked currents reversed at −1.4 ± 7.1 mV and showed outward rectification    exerted a much stronger inhibition than at p10  . With th, N = 3) . At a co < 0.05) , but mor; N = 3) . The pro 0.0001) . In 6/9 V = −70 mV, n = 6, N = 3) of the receptor currents, thus being more efficient than at p10 (p < 0.001) but less efficient than Naspm at p60 (p = 0.028). IEM-sensitive currents showed strong inward rectification   . Inhibition by JNJ was more effective than at cells at p10 (p = 0.00007), and the proportion of currents insensitive to the co-applied blockers was lower than at the early stage  (IEM-1460 (100 μM) inhibited 63.1 ± 6.4%  . Subsequ n = 12) . When JN< 0.001) . In cont< 0.001) .2+ impermeable receptors. In line with the observations at p10, the data suggested that JNJ blocked Ca2+ impermeable receptors. Although molecular analysis revealed developmental downregulation of TARP γ8, the modulatory effect of JNJ was stronger in adult NG2 glia.Together, the data demonstrate that extracellularly applied spermine analogs inhibit AMPA receptors in hippocampal NG2 glia. The efficiency of inhibition by Naspm and IEM-1460 was higher in NG2 glia from adult mice. Naspm and JNJ showed a partly overlapping block. Since there was almost no overlap of IEM-1460- and JNJ-mediated inhibition, it seems that Naspm also blocked some Ca2+ permeability of NG2 glia AMPA receptors, Na+-free extracellular solution with Ca2+ (50 mM) as the sole charge carrier was used. Kainate/CTZ induced currents in high Ca2+ solution, indicating Ca2+ permeability of the glial AMPA receptors. The permeability coefficient PCa/PCs was calculated according to the constant field equation   .Ca/INa = 5.5 ± 1.6%, a reversal potential in high Ca2+ solution at −35.1 ± 10.7 mV, and PCa/PCs = 0.24 ± 0.13   . None of; N = 5) . Thus, tn = 6, N = 4 and 41.1 ± 16.8%, n = 5, N = 3; p60, by 31.5 ± 10.8%, n = 7, N = 4 and 32.8 ± 4.8%, n = 6, N = 5)  . The eff2+ permeable AMPA receptors , we observed inward rectifying receptor currents in the presence of intracellular spermine, indicating expression of Cad by p10 . In rat,d by p10 . Naspm ad by p10  while in2+ permeability of AMPA receptors in juvenile NG2 glia has already been demonstrated with Ca2+ imaging (2+ as the sole charge carrier confirmed functional expression of Ca2+ permeable receptors. Masking of intracellular spermine block of outward currents through Ca2+ permeable AMPA receptors, as observed here, was previously demonstrated in heterologous expression systems and cultured cortical glial cells (2+ permeable AMPA receptors in mature NG2 glia. A recent publication demonstrated that intracellular Naspm confers complete and TARP-independent block to Ca2+ permeable, GluA2-lacking AMPA receptors (2+ permeable AMPA receptors were located on postsynaptic glial membranes (A Ca imaging . In the al cells , and migeceptors . In our embranes .Some anti-epileptic drugs, for example perampanel , target 2+ permeable AMPA receptors to endogenous polyamines. Ca2+ influx through somatic AMPA receptors may regulate proliferation and differentiation (NG2 cells in hippocampus primarily express the TARP subunits γ4, γ7, and γ8 as well as CNIH-2. These auxiliary subunits may slow receptor desensitization, increase kainate efficacy and reduce the sensitivity of Cantiation , and delntiation . SystemaThe original contributions presented in the study are included in the article/Ethical review and approval was not required for the animal study because we only performed organ harvesting after anesthesia.CS and GS designed and supervised the experiments. SH, DT, SP, AT, RJ, and GS performed and/or analyzed the experiments. SH, GS, and CS wrote the manuscript. All authors contributed to the article and approved the submitted version.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.