Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in  Trichinella muscle larvae (ML) . To. To58]. eviously , 60, anderiments .Table 1TThe enzymatic activity of natural TsGSCP in crude soluble proteins of dsRNA-TsGSCP-transfected larvae was analysed and compared with that of untransfected larvae as previously described . BrieflyTo evaluate larval infectivity, survival and fecundity in the host intestine following dsRNA interference, 30 mice were assigned to 3 groups . Each mouse was inoculated orally with 500 ML treated with 40 ng/μL dsRNA-TsGSCP, dsRNA-GFP or PBS. The infected mice were euthanized 5 dpi, adult worms were removed from the intestine, the adult worm burden was assessed, and adult worm morphology and size were examined by microscopy. Thirty adult females from each group of infected mice were cultured, and female reproductive ability (fecundity) was ascertained on the basis of NBL production by each female within 72 h .P < 0.05 is considered to be statistically significant.All the data were analysis with SPSS 21.0 software. The results are shown as the means ± standard deviation (SD). TsGSCP expression level, worm burden and length, and NBL production among different groups were analysed with one-way ANOVA. Chi-square test was performed to analyse the larval invasion rate and TsGSCP expression after RNAi in the various groups. Trichinella species, the following results were obtained: the amino acid sequence of TsGSCP shared 98% identity with cysteine protease of T. nelsoni, Trichinella T8 and T. nativa, 97% identity with those of T. patagoniensis and Trichinella T6, 90% identity with those of T. papuae and T. zimbabwensis, and 89% with T. pseudospiralis , and the other was the nonencapsulated species clade .The CDS of TsGSCP is 991 bp, encoding 325 amino acids with a relative molecular weight of 36.9 kDa and isoelectric point (pI) of 6.88. TsGSCP does not contain a transmembrane region, but it has a signal peptide. The subcellular location is a secretory pathway, indicating that the TsGSCP protein may be a secretory protein. The tertiary structure of TsGSCP has 4 active enzymatic sites . Using BioEdit software to compare the TsGSCP amino acid sequences in different 5 following the final immunization, demonstrating that rTsGSCP induced immunogenicity. The results of the Western blot analysis showed that rTsGSCP was recognized by anti-rTsGSCP serum and infection serum but not by anti-MBP-tag serum and preimmune serum. Furthermore, natural TsGSCP of 35.2, 49.6, 56.5, 61.6 kDa in IIL soluble proteins was identified by anti-rTsGSCP serum but was not found among IIL ES proteins, suggesting that TsGSCP is a somatic worm protein, not a secretory protein.In the SDS-PAGE analysis, purified rTsGSCP was identified as a clearly visible individual band, and its molecular weight (74.2 kDa) was consistent with its predicted size     and showed an increasing trend with increasing IEC protein coating concentration   and showed a positive trend with increasing rTsGSCP dose   Figure B. The reThe IIF analysis results showed that after IECs were preincubated with rTsGSCP, green immunofluorescence staining was observed on the surface of the IECs probed with anti-rTsGSCP serum and infection serum but not with preimmune serum. The IECs preincubated with MBP alone did not show any immune staining upon treatment with anti-rTsGSCP serum or infection serum. No immune staining on the C2C12 surface was detected when C2C12 cells preincubated with rTsGSCP were probed with anti-rTsGSCP serum or infection serum Figure A. At higr = 0.950, P < 0.0001) and exhibited an increasing trend with increasing rTsGSCP dose , but MBP-tag did not facilitate intrusion . The suppression of IEC intrusion was dose-dependent for anti-rTsGSCP antibodies , and the suppressive effect was reduced with increasing serum dilution  , and the invasive ability of the larvae treated with dsRNA-TsGSCP was inhibited by 25.02% compared to that of the dsRNA-GFP group   Figure E. Furthe) Figure F. These 2 = 2.007, P > 0.05), indicating that electroporation has no evident effect on worm survival. When the ML were treated with 20, 40 and 60 ng/μL dsRNA and cultivated for 2 days, TsGSCP transcription levels were decreased by 14.82, 33.30 and 62.13%, respectively, compared to those in the PBS group (P < 0.05), and TsGSCP expression was reduced by 22.24, 45.28 and 60.51% (P < 0.05) , and TsGSCP expression was suppressed by 46.82, 1.73 and 2.26%, respectively (P < 0.05)   Figure F.F = 37.374 P < 0.001), and the control dsRNA-GFP group did not show any decrease in adult worm burden (P > 0.05)  , but there were no significant differences in female fecundity between the control dsRNA-GFP and PBS groups (P > 0.05)  Figure B, but ma) Figure C, demonsNecator americanus) are mainly localized in intestinal tissue [Angiostrongylus cantonensis; incubation of rat intestine with Ac-cathB-2 resulted in the detachment of epithelial cells, and antiserum reduced L3 larva penetration [T. spiralis cathepsin B (rTsCB) facilitated larva penetration into IECs, whereas anti-rTsCB antibodies and RNAi suppressed larva penetration into enterocytes, suggesting that TsCB participated in larva invasion into the gut epithelium and might be a molecular target against T. spiralis intrusion [Cysteine proteases usually consist of the enzyme precursor sequence and mature enzyme domain. They are usually nonenzymatic in the form of proenzymes but can self-hydrolyse as precursor peptides under acidic conditions and become catalytic enzymes . Cysteinl tissue . Previoul tissue . A catheetration . Our prentrusion .E. coli expression system. Following purification, rTsGSCP was used to immunize mice and produce anti-rTsGSCP antibodies. The anti-rTsGSCP IgG titre was 105 two weeks after the final immunization, which indicated that rTsGSCP has strong immunogenicity. In the Western blotting analysis, rTsGSCP was recognized by anti-rTsGSCP serum and infection serum. As shown in Figure T. spiralis cathepsin B family, TsGSCP has the same antigenic epitopes as cathepsin B [In the present study, bioinformatics analyses showed that TsGSCP has a functional cathepsin B domain; cathepsin B belongs to the C1A peptidase family. TsGSCP was cloned into the pMAL-c2x expression vector and expressed in an hepsin B , 49, 67.T. spiralis lifecycle. The results of the IIF analysis with intact worms showed native TsGSCP on the outer cuticle of intestinal IIL, AW and NBL but not on that of ML. As identified by immunohistological staining, native TsGSCP was principally located at the cuticle and intrauterine embryos of the nematode. The expression of TsGSCP in all lifecycles of the nematode suggested that this protease participates in T. spiralis growth, development and survival in the host [E. coli [T. spiralis larvae.The qPCR and Western blot analysis results showed that TsGSCP mRNA and protein expression at the IIL stage was higher than that at other developmental stages in the the host , 41. Morthe host , 26, 36.[E. coli . TherefoProtein–protein interactions between rTsGSCP and IECs were also investigated in this study. The far-Western blot and ELISA results showed that rTsGSCP specifically bound to IEC proteins in a dose-dependent manner. The IIF analysis results indicated that the binding sites of rTsGSCP and IECs were located in the cytoplasm. Previous studies have shown that when IIL were incubated with IECs, some proteases produced by IIL entered the IECs , 47. TheT. spiralis, RNAi is the primary technique used to investigate gene function [T. spiralis [Fasciola hepatica by RNAi reduced gut penetration [Schistosoma japonicum cathepsin B2 promoted skin invasion, while anti-rsjCB2 IgG clearly inhibited cercariae invasion into the skin [T. spiralis infection [In the parasitic nematode function . To verispiralis . Moreoveetration . In anotthe skin . These fnfection .T. spiralis lifecycle and primarily localized to the cuticle and intrauterine embryos of this parasite. The rTsGSCP showed good immunogenicity. rTsGSCP exhibited the ability to specifically bind with IECs, and the binding site is within the cytoplasm of the IECs. rTsGSCP accelerated larval intrusion into IECs, whereas anti-rTsGSCP antibodies impeded larval intrusion; the acceleration/inhibition effect was dose-dependent for rTsGSCP/anti-rTsGSCP antibodies. Silencing of the TsGSCP gene by specific dsRNA significantly decreased TsGSCP expression and TsGSCP ability to hydrolyse the cysteine protease substrate Z-FR-AMC; RNAi also impaired larval invasive ability, growth and fecundity. These findings demonstrated that TsGSCP plays a principal role in gut intrusion, worm development and fecundity during the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.In conclusion, TsGSCP was highly expressed at gut invasive IIL stages of the


E)-3- acrylate (Ph9), inhibited mushroom tyrosinase with an IC50 of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC50 of 2.1 nM compared to the positive control, kojic acid IC50 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (iK 0.093 nM) and non-competitive inhibition for Ph6 (iK 2.3 nM) revealed from Lineweaver–Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl ( This behavior of compounds Ph5 and Ph9 indicated that it inhibits tyrosinase by two different pathways; competitively forming enzyme inhibitor (EI) complex and interrupting enzyme–substrate-inhibitor (ESI) complex in a non-competitive manner. The secondary plots of slope versus concentration of compounds Ph5 and Ph9 showed EI dissociation constants (iK) iK′) were shown by secondary plots of intercept versus concentration of compounds Ph5 and Ph9 iK than iK′ pointed out stronger binding between enzyme and compounds Ph5 and Ph9 which suggested preferred competitive over non-competitive manners  complex. The secondary plot of slope against the concentration of Ph6 showed EI dissociation constant (iK) l-DOPA . Ph5 is a 3,5-dihydroxyl-substituted benzoic acid derivative and Ph9 is 2,4-dihydroxyl-substituted cinnamic acid derivative whereas, Ph6 is an unsubstituted cinnamic acid analogue. As the substrate l-DOPA bears 3,4-dihydroxy substitution, this could be the reason that Ph5 and Ph9 bearing hydroxyl substituent gave mixed-type inhibition, whereas Ph6 bearing no hydroxyl substituent was a non-competitive inhibitor.Among the synthesized Ph derivatives Ph5, Ph6 and Ph9 were selected to determine their inhibition type and inhibition constants on mushroom tyrosinase activity. The potential of these derivatives to inhibit the free enzyme and enzyme–substrate complex was determined in terms of enzyme inhibitor (EI) and enzyme–substrate-inhibitor (ESI) constants respectively. The kinetic studies of the enzyme by the Lineweaver–Burk plot of 1/ manners . Meanwhihttp://www.rcsb.org, accessed date 15 March 2021). To determine the amino acids of the protein involved in binding with the inhibitor, the selected poses of most active compounds Ph5, Ph6 and Ph9 were visualized.Computational docking studies were undertaken with Maestro Schrodinger (11.2) to predict the most favorable binding mode of the compounds (Ph1–Ph10) inside the binding pocket of mushroom tyrosinase (PDB ID:2Y9X). The crystal structure of mushroom tyrosinase (PDB ID:2Y9X) containing a tropolone ligand in the enzymatic pocket, was retrieved from RCSB Protein Data Bank (RCSB PDB)  with side-chain residues Asn260 and Asn81 with bond lengths of 2.93 Å and 3.04 Å, respectively. The para phenolic group is interacting by Cu400 (3.68 Å) and Cu401 (3.10 Å) and this ring is further stabilized by π–π stacking with His85, His263 and His259. The amide hydrogen of Ph9 is predicted to form a hydrogen bond (3.36 Å) with the nitrogen of the neighboring His244. For the most active compound Ph5 from benzoic acid derivatives (para methoxy moiety is picking two strong H-bond interactions with side-chain residues Asn81 (2.07 Å) and His85 (2.31 Å) respectively and this ring is additionally stabilized by π–π stacking with His85. The amide hydrogen of the inhibitor Ph5 interacts through a hydrogen bond (2.40 Å) with the nitrogen of the neighboring His244. The large size and planner shape of Ph6 prevents it from entering the narrow binuclear copper-binding site and bounds to a shallow area at the surface of the enzyme pocket. However, a hydrogen bond (2.31 Å) is observed between the amide carbonyl and side-chain Asn81. Both para methoxy phenyl and cinnamic acid phenyl groups form π–π interaction with residues His85 and Phe192, respectively. The described binding mode would allow the substrate to enter the pocket, providing a useful non-competitive model. Our in vitro enzyme inhibitory assay showed a non-competitive inhibition for Ph6. The Ph6 is an unsubstituted cinnamic acid derivative and does not bear any hydroxyl substituent which is very important in ligand binding through hydrogen bonding, Coulomb and van der Waals interactions  ions and side-chain hydroxyl is picking up a hydrogen bond (1.73 Å) with amino acid residue Met280. The best ligand binding poses of Ph5, Ph6 and Ph9 were selected based on binding energy  , number ivatives b, the paractions c. His259−1). Elemental Analysis  was carried out on a Flash 2000 series elemental analyzer with thermal conductivity detector (TCD) system  and results are with ±0.3%. The 1H NMR and 13C NMR spectra (DMSO-d6) were recorded using a Bruker 400 MHz spectrometer . Chemical shifts (δ) are reported in ppm downfield from the internal standard tetra methyl silane (TMS). The purity of the compounds was checked by thin layer chromatography (TLC) on silica gel plate using n-hexane and ethyl acetate as mobile phase. Mushroom tyrosinase, l-DOPA and 2-(4-methoxyphenyl)ethan-1-amine were purchased from Sigma Aldrich . Stock solutions of the reducing substrates were prepared in phosphate buffer .All chemicals used for the synthesis of compounds (Ph1–Ph10) were purchased from Sigma Aldrich . Melting points were determined using a Digimelt MPA 160, USA apparatus and are reported uncorrected. The FTIR spectra were recorded with Shimadzu FTIR–8400S spectrometer  was synthesized by following the Sidhu et al., method with some modifications  mM−1. The EI-dissociation constant iK was determined by the secondary plot of 1/V versus inhibitor concentrations while ESI-dissociation constant iK′ was determined by intercept versus inhibitor concentrations.A series of experiments were performed to calculate the inhibitory kinetics of compounds Ph5, Ph6 and Ph9 by following the already reported method . The comAgaricus bisporus tyrosinase (PDB:2Y9X) from the RCSB Protein Data Bank (RCSB PDB). 2Y9X is a crystal structure of mushroom tyrosinase complex with tropolone. Tropolone was removed from the active site and all compounds (Ph1–Ph10) were docked in the active site. All ligands were prepared by LigPrep (Schrödinger) in their neutral form and their conformation optimized in the OPLS-3 force field. The protein structure was prepared by using one of the four monomers from the PDB entry and using the Protein Preparation (Schrödinger) for adding hydrogens and by removing all water molecules beyond 5.0 Å and setting protonation states appropriate for pH 7. The receptor grid box was defined as 20 Å. Docking was performed with Glide (Schrödinger) using extra precision (XP) with default settings and glide scoring function, reporting the 15 top ranked poses for each ligand. Visual inspection of the binding pose and generation of figure was done with Maestro (Schrödinger) [We studied all the synthesized compounds (Ph1–Ph10) using molecular docking simulations to determine possible binding orientations of the compounds in the crystal structure of the mushroom tyrosinase. For this purpose, we used the crystal structure of the ödinger) .In the present study novel tyrosinase inhibitors (Ph1–Ph10) containing amide and ester functionalities were synthesized and evaluated for their tyrosinase inhibitory potential against mushroom and melanoma cells human tyrosinase. Mushroom tyrosinase being commercially available has served almost in all studies conducted on tyrosinase inhibition so far. Phenolics are the largest groups in tyrosinase inhibitors and several polyphenols are accepted as substrates by tyrosinase, depending upon the presence and position of substituent . Cinnami50 0.00006 µM) much better than the standard kojic acid (IC50 16.7 µM). The enhanced inhibitory effect of Ph9 is potentially due to the presence of two hydroxyl groups at ortho and para positions of phenyl ring of cinnamic acid moiety. Whereas the Ph6 bearing an unsubstituted cinnamic acid moiety also displayed an excellent enzyme inhibitory activity (IC50 0.00204 µM). The inhibitor Ph5 with two hydroxyls at 3,5 positions of benzoic acid moiety also exhibited excellent tyrosinase inhibition (IC50 0.231 µM)  . The bioydroxyls . Here, w50) and human tyrosinase (% age) enzyme inhibitory activities showed that the compound Ph9  with 2,4-dihydroxycinnamic acid moiety, Ph6  bearing unsubstituted cinnamic acid and inhibitor Ph5  containing 3,5-dihydroxybenzoic acid unit showed excellent enzyme inhibitory potential, much better than the standard kojic acid . The presence of ortho and para hydroxyl substituents on phenyl ring and p-methoxy-substituted benzene ring on the other side of Ph9 played a vital role in enhanced tyrosinase inhibitory activity. Kinetic mechanism reflected the mixed type of inhibition for inhibitors Ph9 (iK 0.000093 µM), Ph5 (iK 0.219 µM) and non-competitive inhibition for Ph6 (iK 0.0023 µM) revealed from Lineweaver–Burk plots. In silico molecular docking studies showed that the predicted binding affinity of the synthesized compound Ph9 is excellent with a binding energy −7.93 Kcal/mol. It can be concluded from our results that the inhibitor Ph9 is highly potent and may be a model structure for the development of novel and potent melanogenic inhibitors.The mushroom and human tyrosinase inhibitors Ph1–Ph10 with excellent tyrosinase inhibitory potential compared to the reference kojic acid are presented in the current work. A series of compounds Ph1–Ph10 containing ester/amide functionalities were synthesized and evaluated to check their inhibitory potential against tyrosinase enzyme. Simple nucleophilic substitution synthetic routes are adopted to synthesize the desired compounds in good and excellent yield (71–84%). The mushroom (IC


Diminished inhibitory control of spinal nociception is one of the major culprits of chronic pain states. Restoring proper synaptic inhibition is a well-established rational therapeutic approach explored by several pharmaceutical companies. A particular challenge arises from the need for site-specific intervention to avoid deleterious side effects such as sedation, addiction, or impaired motor control, which would arise from wide-range facilitation of inhibition. Specific targeting of glycinergic inhibition, which dominates in the spinal cord and parts of the hindbrain, may help reduce these side effects. Selective targeting of the α3 subtype of glycine receptors (GlyRs), which is highly enriched in the superficial layers of the spinal dorsal horn, a key site of nociceptive processing, may help to further narrow down pharmacological intervention on the nociceptive system and increase tolerability. This review provides an update on the physiological properties and functions of α3 subtype GlyRs and on the present state of related drug discovery programs. Inhibitory neurotransmission plays a crucial role in the maintenance of a physiologically meaningful state of pain sensitivity and helps separating innocuous and noxious signal relay. Following injury and inflammation, reduced inhibition contributes to the phenomena of hyperalgesia (an increased sensitivity to input from nociceptive fibers) and allodynia, which describes a painful sensation elicited by input from non-nociceptive fibers. While both phenomena may help protect injured tissue from further damage and may foster its healing, under unfortunate conditions they outlast the healing process and may then severely compromise the quality of life of affected patients. The spinal dorsal horn is a key site for endogenous pain control and maladaptive plasticity which underlies many chronic pain conditions. Various processes triggered by peripheral inflammation or nerve damage compromise synaptic inhibition at this site and in corresponding brainstem areas. Among these processes are alterations in the excitatory drive to inhibitory dorsal horn neurons , a compr2) is a pivotal mediator of inflammation and inflammatory hyperalgesia. In response to peripheral inflammatory insults, PGE2 is not only produced in the periphery at the site of inflammation, but also in the CNS, especially in the spinal dorsal horn, where it contributes to the phenomenon of central sensitization. A series of reports published between 2002 and 2005 established the critical role of α3 GlyRs in inflammation and PGE2-mediated central sensitization. PGE2, but not other prostaglandins including PGD2, PGI2, or PGF2α, reduces glycinergic synaptic transmission in superficial dorsal horn neurons through a postsynaptic mechanism involving the activation of EP2 receptors, production of cAMP, and subsequent activation of protein kinase A (PKA) [substantia gelatinosa. This site constitutes the termination area of most nociceptive fibers arriving in the CNS from the periphery. A systematic in silico screen of the GlyR α3 protein sequence revealed a strong consensus site for PKA-dependent phosphorylation in the large intracellular loop between transmembrane segments 3 and 4. This consensus site comprises the amino acid sequence arginine-glutamate-serine-arginine (RESR), in which the serine at position 346 constitutes the actual site of phosphorylation. The critical role of this site for PGE2 triggered GlyR α3 phosphorylation was supported by results obtained in HEK293 cells. Heterologous expression of EP2 and α3 GlyRs reconstituted the inhibition of glycinergic membrane currents by PGE2. Replacing the serine residue at position 346 by an alanine, an amino acid of similar size but lacking the OH group required for phosphorylation, prevented inhibition by PGE2 [Cyclooxygenase-2 (COX-2) derived prostaglandin E2 (PGE A (PKA) . The expThe availability of mice lacking α3 GlyRs allowed assessing the contribution of α3 GlyRs and their regulation by PKA to baseline nociception and different forms of hyperalgesia. GlyRα3 deficient mice behaved normally in tests of baseline nociception . This lack of a pronociceptive phenotype may be linked to unaltered baseline glycinergic neurotransmission in GlyR α3 deficient mice and may hint at a compensatory up-regulation of other GlyR subunits. Pronounced alterations in the development of hyperalgesia became apparent when the mice were challenged with peripheral inflammation. In wild-type mice, subcutaneous injection of complete Freund’s adjuvant or with the yeast extract zymosan A induces thermal and mechanical hyperalgesia, which lasted for several days to weeks depending on the amount injected. GlyR α3 deficient mice showed strongly reduced thermal and mechanical hyperalgesia especially during the later phases of inflammation. A virtually identical phenotype was observed in a side-by-side comparison of GlyR α3 deficient mice and mice lacking prostaglandin EP2 receptors . substantia gelatinosa neurons in spinal cord slices of these mice demonstrated not only that the point-mutated α3GlyRs were resistant to inhibition by PGE2 but also confirmed the critical contribution of phosphorylation at this site to inflammatory hyperalgesia. Mice carrying the S346A point mutation were resistant to the hyperalgesic effects of intrathecally injected PGE2 and developed much less hyperalgesia after injection of zymosan A into one hindpaw.Although the reconstitution experiments in HEK293 cells supported the critical role of a direct PKA-dependent phosphorylation of α3 GlyRs, unequivocal proof for its relevance to in vivo hyperalgesia was still lacking. The recent generation of a genetically engineered S346A point mutated mouse line allowed filling this gap . This mo2 to neuropathic pain has been proposed [Besides inflammation, neuropathy is another major source of chronic pain and hyperalgesia. A contribution of COX-2 or PGEproposed  but is sproposed . Both wiproposed . It shouproposed  and 5-deproposed , reduce 2 led to a decrease in glycinergic membrane currents with no change in their inactivation kinetics or plasma membrane expression. In single channel recording experiments, activation of PKA progressively reduced single current amplitudes to about 66%, which is close to the inhibition of glycinergic synaptic currents observed in mouse spinal cord slices [50) of GlyR currents. Substituting S346 with phospho-deficient alanine left the single channel amplitude and open probability almost unaffected. Additional insights have also been gained into the molecular mechanisms that link phosphorylation at S346 to decreased glycinergic currents . In HEK2d slices ,8,14. Ind slices , the intTo fully understand of the role of α3 GlyRs in spinal control of nociception, precise knowledge of the neural circuits controlled by these receptors is essential. On a very gross scale, spinal hyperalgesia may be viewed as an imbalance of excitatory nociceptive input and local inhibitory control. In reality, the situation is likely much more complex. The simplest mechanism by which GlyRs may contribute to spinal nociceptive control might be the direct inhibition of nociceptive output from the spinal cord. This output occurs mainly via projection neurons located in lamina I of the dorsal horn, which relay nociceptive signals to the brainstem. These neurons receive direct excitatory drive from nociceptors and are controlled by GABAergic and glycinergic input . The glyA receptors and GlyRs, these neurons become excitable also by input from Aβ fibers through a polysynaptic pathway [However, compared to neurons of the deeper dorsal horn, projection neurons of lamina I receive relatively little spontaneous inhibitory input , potenti pathway . A receptors) on their surface has been directly demonstrated [The first neuron type identified in this pathway were protein kinase Cγ (PKCγ) expressing excitatory interneurons , which anstrated ,27,28. Tnstrated . It is bnstrated . Consistnstrated . Similarnstrated ,32. DespA receptors but no GlyRs. Accordingly, glycine does not contribute to so-called primary afferent depolarization or presynaptic inhibition of primary afferent input. However, GlyRs reside on presynaptic terminals of central neurons, where they increase transmitter release. Such presynaptic GlyRs have first been found in neurons of the auditory brainstem, where their activation increases glycine release [Inhibitory dorsal horn neurons do not only provide classical postsynaptic inhibition to intrinsic dorsal horn neurons, but also target axon terminals of primary sensory afferent nerve fibers. These terminals express GABA release . A simil release . The sub release .Direct evidence to support the presence of glycinergic pain control in humans is difficult to obtain given the present lack of compounds suitable for clinical testing in humans. A recent study in human patients suffering from inherited hyperekplexia provides however supporting evidence . Most hyGLRA1 and GLRB as well as in SLC6A5 (encoding for GlyT2) are well-established causes of hyperekplexia, GLRA3 has not been linked to any human disease yet. This may suggest that pharmacological targeting of α3 GlyRs may be relatively safe, but it also means that human genetic evidence supporting a specific role of α3 GlyRs is lacking. On a positive note, the PKA consensus site in the large intracellular loop of α3 GlyRs is conserved in humans. It is also noteworthy that the phenotype described in α3 GlyR deficient mice (reduced inflammatory pain) might not be easily detectable in humans. Furthermore, a screening of the human genetic variation database (www.ncbi.nlm.nih.gov/variation/) for loss of function (non-sense) variants revealed few hits. All of which occurred with frequencies too low to allow systematic clinical trials in such persons .A receptors, GlyRs have remained therapeutic orphans. Nevertheless, several drug companies  and aca., AMGEN ,41,42 ha., AMGEN . Figure Recent drug screening efforts at AMGEN have led to the discovery of novel glycine receptor potentiators ,37,38. O9-tetrahydrocannabinol (THC) modulates GlyR function, in addition to activating G protein coupled CB1 and CB2 receptors. Chemical modification of THC has led to the derivatives 5-desoxy-THC , 1-desoxy-THC, and di-desoxy-THC. Further, 5-desoxy-THC/DH-CBD and di-desoxy-THC are devoid of activity at CB1 and CB2 receptors but still bind to GlyRs. In addition, 5-desoxy-THC/DH-CBD acts as a positive allosteric modulator and di-desoxy-THC as its competitive antagonist of GlyRs [1 and CB2 deficient mice [Other evidence supporting potential therapeutic benefit of GlyR potentiators comes from cannabinoid derivatives. Δof GlyRs . In mousent mice . A receptors. At higher concentrations, it also acts as a GlyR modulator. The propofol derivative 2,6-di-tert-butylphenol  is devoid of activity at the major GABAA receptor subtypes [2 or when it was tested in spinal cord slices prepared from animals with inflamed paws [Propofol  is an intravenous anesthetic that targets primarily GABAsubtypes  but retasubtypes  and posssubtypes . Criticasubtypes . Interesmed paws . Molecules with positive allosteric activity at GlyRs have also been isolated from a natural compound library generated from Australian and Antarctic marine invertebrates and algae ,49,50. TGlyRs are in addition modulated by a number of endogenous molecules or synthetic compounds that either lack drug-like properties or activate primarily targets different GlyRs. Although such compounds are unlikely to be used therapeutically, they have led to the discovery of sites for allosteric modulation of different GlyRs. As such, they may provide starting points for drug discovery programs. Below, we provide a relatively short summary of this work. A more comprehensive coverage of this topic can be found elsewhere ,52,53,541 and CB2 receptors. Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) are pivotal endogenous activators of these receptors. Related arachidonoyl conjugates potentiate GlyR function with partially different effects on the different GlyR subtypes [Endocannabinoids are lipid signaling molecules that primarily activate the G protein coupled cannabinoid CBsubtypes ,56. Whilsubtypes . Buildinsubtypes . Tropeines are antiemetic drugs that act as antagonists at ionotropic serotonin (5-HT3) receptors. Two tropeines, MDL-72222 and tropisetron, also potentiate GlyR activity at nanomolar concentrations ,59,60. T+ channels and T-type Ca2+ channels and it is unclear which target is responsible for its antiepileptic effect. Some evidence suggests that zonisamide possesses antihyperalgesic activity in different preclinical models [Zonisamide is an antiepileptic drug, which has recently been reported to facilitate activation of recombinant and native α1, α2, and α3 GlyRs at therapeutic concentrations . Zonisaml models . This isl models . Lastly, glycine receptor activity can also be modulated in a bidirectional fashion by zinc, where sub-micromolar concentrations (20 nM–1 µM) lead to potentiation and micromolar concentration (20–50 µM) to inhibition , revieweThis question cannot be answered yet . GlyRs d


C1 lateral mass and C2 pedicular screws insertion are used for C1-C2 posterior fusion. Fluoroscopy Guided technique is routinely used for screw placement but it is associated with risk of injury to spinal cord and vertebral artery. 3D printing has developed rapidly in the fields of medicine. It is helpful in improving precise treatment and used for instrumentation in spine. We want to evaluate the accuracy of C1 lateral mass screws and C2 pedicle screws insertion by Pre-Fabricated Template made by three-dimensional (3D) printing.Five cervical samples were obtained from cadavers. Based on fine-cut CT scan 3D-images reconstructed and the path of the screws designed by special software. A template produced by 3D-printer from 3D images. After printing the templates, they were fixed on the relevant vertebra in the operation room and based on the template path, C1 lateral mass screw and C2 pedicular screws were inserted. Placement of the screws was evaluated using CT scans post-operatively.A total of 14 screws were inserted by above-mentioned method. After evaluation with CT scans none of the screws were entered in the spinal canal. Two screws had vertebral artery canal perforation with less than 50% breach. Violation was judged as noncritical and would probably not have resulted in injury to vertebral artery.The accuracy of C1 lateral mass screw and C2 pedicle screw insertion is acceptable with pre-fabricated template and can provide a useful aid for screw placement. Similar to the other parts of the human body, this structure is prone to a number of diseases and injuries. A wide range of diseases, including inflammatory diseases, e.g., rheumatoid arthritis, congenital conditions, and trauma, can damage this structure leading to atlantoaxial instability4. The surgical treatment of atlantoaxial instability is performed by fixing the first and second vertebrae using a number of different methods. Among these methods, fixing with screws has been considered in recent years. The surgical treatment of this area is associated with high risk, even if it is performed by the most experienced surgeons, due to the complexity of the anatomy and the sensitivity of the elements in this area. Also, there is several anatomic variations in the general population.The first and second cervical vertebrae are among the most important and complex components of the vertebral column. The anatomy of these two vertebrae and their joints is one of the most complex and sensitive parts of the human body as they are connected by multiple ligaments and joints5.The 3D printing has been employed in many surgical and medical fields. Such may include surgical planning, printing of models to aid with intra-operative guidance and anatomy definition, prosthesis development, medical education, patient-specific implant creation, and numerous other purposesDue to the sensitivity of the atlantoaxial area and the complexity of the anatomy, it seems that pre-fabricated templates, which can guide the correct screw fixation path according to each person’s anatomy, can reduce the risk of surgery and its adverse consequences. The main objective of this study is to investigate the screw fixation technique for a C1 lateral mass screw and a pedicle screw in the second vertebra through pre-fabricated templates on cadaver samples.At first, five cervical samples were obtained from the Anatomy Department of the University of Tehran. After numbering the samples, CT scan was performed with a delicate cut of 2mm  at Sina Hospital Imaging Centre. The CT scan data were transferred to the three-dimensioning mimics software [Materialize Mimic software version 19] in the form of DICOM files for preparing Three-Dimensioning CT Scans. In this software, the CT scans taken from the samples are reconstructed in three-dimensional form. The software environment is shown in .Then, in the same environment, the correct direction of the screws was determined using cylinders in the software environment. The path for each cylinder was followed in all cuts of axial, coronal and sagittal planes of the CT scan, where each path of the cylinder was in its correct path. None of the cylinders had entered the canal or towards the holes of the vertebral artery .With regards to the first and second cervical vertebra, the path of the screw was determined for each sample and examined in various cuts in terms of correctness of the path. Finally, the path of the screws in the three-dimensional view was examined ,4,5.After designing the path of the screws, the information was stored as an STL file and transferred to the Geomatics Studio program. Then, based on the outer cortex and the posterior cortex of the first and second vertebrae, special templates were designed. These templates are perfectly fixed on the posterior section of each vertebra so that the path of the screws passes through the pre-fabricated templates . Once thAfter printing the templates, they were fixed on the relevant vertebra in the operation room at Sina Hospital. Then, based on the template path, a hole was created using a 2.7 drill. Next, a 3.5mm screw  was fixed in the drilled position .et al classification6. The grades are as follows: Grade 1: None of the canals were damaged. Grade 2: Less than 50% of the screw is inside the canal. Grade 3: More than 50% of the screw is inside the canal.After fixing the screws, the sites of the screws were checked by CT scan. In the performed CT scans, samples were examined in terms of extending into the spinal canal and the vertebral artery canal by Kaneyama et al classification for screw6.Four cervical samples were obtained from the anatomy department. Then, four screws were fixed in C1 and C2 of each sample according to the method described. Due to damage to the posterior arch of the cervical vertebra in one of the samples, it was impossible to place the screw in the first vertebra of this sample. Finally, 14 screws were fixed using the abovementioned method. Based on the post-operative CT scan . the scrIn this study, none of the screws entered the spinal canal. Two screws were entered in vertebral artery canal that were grade 2, while none of the screws were in grade 3.11. Patient-specific drill guide templates have been developed as an inexpensive, accurate method to guide screw insertion in the spine based on advances in 3D computing and printing technologies. Ryken et al fixed screws on four cadaver samples for the first time using pre-fabricated templates12.The fixation of a screw in the lateral masses of the first vertebra and the fixation of a screw inside the second vertebra’s pedicles require high skill and knowledge about the anatomy of this area. Surgery around theses area is difficult and dangerous due to the relatively small vertebral pedicle and the key anatomical structures around them. Therefore, comprehensive evaluation and precise navigation are very important for the surgery. Even with high levels of experience, there is a risk of damage to the spinal cord and the vertebral artery. The normal navigation methods face the problem of intra-operative drift; thus, the screw may easily break through the bone cortex when the pedicle is smallet al showed that the specific template for each vertebra had high accuracy in screw fixation13. In addition, Van Cleynenbreugel et al used two types of templates for screw fixation using the Magerl method14. Hu et al used a pre-fabricated template for screw fixation using the Magerl method15. Sugawara et al used template for posterior C1-C2 fusion. they inserted a total of 48 screws with accuracy of 100%16. Guo et al compared C1 and C2 screw fixation in patients guided by the template and the patients which screws were fixed under guidance of fluoroscopy. In the template guided group, the acceptability rate was 94.6% compared to 70.27% in control group. The acceptability rate in test group was higher than the rate in control group. The operation time and fluoroscopic frequency for each screw were decreased, compared with the control group17.Moreover, D’urso 18. The navigation template used in the current study was built by several reverse engineering software packages; thus, it required manual adjustments. These factors affect the accuracy of the method, and different studies report different navigation accuracy rates 22.In another study comparing a total of 68 screws placed at the pedicle in the 19 patients who underwent atlantoaxial pedicle screw assisted by a 3D-printed guide plate, while a total of 76 screws were placed at the pedicle in 24 patients using the traditional method. The accuracy of screw placement in the 3D-printed plate group (94.1%) was far higher than that in the traditional fixation group (76.3%). atlantoaxial pedicle screw fixation assisted by a 3D-printed plate significantly improved the accuracy rate of screw placementPre-fabricated samples based on the patient’s CT scans try to reduce the error during the surgery and minimise adverse complications of these operations. The screw fixation technique based on pre-fabricated templates is a modern technique, which reduces the risk of such operations. The results of the current study are comparable to the findings of the previous studies. However, the screws damaged the cortex of the vertebral canal in two cases, which may have been due to the very small diameter of the samples’ pedicle. Therefore, the screw fixation method is not recommended if the diameter of the pedicle is small.To our knowledge, this is the first study in Iran for assessment of feasibility and accuracy of a 3D-printed template for spinal instrumentation. The first limitation of this study is the low number of cadaver samples. Unfortunately, access to cadaver samples has many problems in our country and samples often do not have good quality. The soft tissue of these cadaver samples does not have good consolidation. In addition, samples undergo changes in bone density due to long-term storage, which are evident in the CT scans and differ from living samples. Another limitation of this study is that common screws were used, due to the high cost of cervical screws, and these common screws cause artifacts in the post-operative CT scans.As such, we expect to summarise the advantages and disadvantages of the 3D printing technology in a follow-up work in vivo and promote the relevant clinical applications.Cervical pedicle screw placement using a rapid prototyping drill template based on a 3D model is technically feasible, and it can enable accurate pedicle screw placement without the need for extra radiation. This study indicates the ability to perform such techniques in Iran; however, further studies are needed in this area.


Cinnamomum verum), ginger , and white pepper (Piper nigrum) are the spices used in the preparation of the ice cream as they are freely available and used as complementary spices in Sri Lanka. Physicochemical characteristics and sensory attributes of coconut milk-based spicy ice cream were compared with the existing normal coconut ice cream. In preparation of the ice cream, the same ice cream manufacturing process was followed with some modifications. Three different formulas  were developed by changing the percentage of spices added. The 0.018% spice-added sample was selected as the most acceptable ice cream with desired sensory attributes. pH (6.33 ± 0.01), titratable acidity (0.33 ± 0.05%), moisture (61.86 ± 0.33%), ash (0.41 ± 0.25%), total solids (38.02 ± 0.14%), overrun (66.76 ± 1.44%), protein (4.18 ± 0.16%), and fat content (11.66 ± 0.60%) were evaluated as physicochemical properties. Total phenolic content of the ice cream was expressed as 0.093 ± 0.002 mg gallic acid equivalents (GAE) per gram of sample in dry weight (mg/g). DPPH radical scavenging activity was 60.39 ± 0.02 mg ascorbic acid equivalents per gram of sample in dry weight (mg/g), and total antioxidant capacity was expressed as 0.36 ± 0.04 mmol ascorbic acid equivalent (AAE)/g of dry weight. Physicochemical properties of spicy coconut ice cream were more or less similar to that of normal coconut ice cream and in compliance with the Sri Lankan standards. Coconut milk-based spicy ice cream could be introduced to the market as a potential marketable nondairy product with spicy flavor, aroma, and smooth texture.Spices have been a major influence on Sri Lankan cuisine since times immemorial. Spices are identified as one of the most distinctive ingredients for their indigenous flavor, aroma, and medicinal properties. In this study, coconut milk-based spicy ice cream was developed in compliance with the Sri Lankan standards to introduce a new perception of flavor using spices to the ice cream industry. Although coconut ice cream is commercially available in the local market, spicy flavored coconut ice cream is not yet available. Cinnamon ( Cinnamomum verum) is a spice obtained from the inner bark of several trees from the genus Cinnamomum that is used in both sweet and savory foods. Moreover, cinnamon is a powerful spice that has been used medicinally around the world for thousands of years [Zingiber officinale) is an underground stem or rhizome used as a flavoring and medicine in Asian and Arabic herbal traditions. Adding ginger as a flavoring makes dishes more delicious. Ginger is used as an ingredient for preparation of tea and production of some sweets and beverages [Piper nigrum) is also one of the most popular culinary spices in the world. White pepper spice has had the black outer shell of the peppercorn removed, giving it a smooth mellow flavor. White pepper itself also contains immunity-boosting properties and anticancer, energy-boosting, anti-inflammatory, and antioxidant properties due to the compounds of capsaicin and piperine [Cocos nucifera L) kernel with or without addition of water. As a coconut-producing country, coconut milk plays a vital role in the Sri Lankan diet [Ice cream is a delicious, wholesome, and nutritious frozen dairy dessert, which is widely consumed globally, and it is very popular among people of all ages because of its taste and cool sensation property . The maiof years . Cinnamoof years . Ginger everages . It impaeverages . White ppiperine . Though kan diet . It is vkan diet . Developkan diet . Moreovekan diet . HoweverCinnamon quills (7 g) were purchased from a supermarket and washed using hot water (45°C) and dried at room temperature. Then, dried cinnamon quills were finely ground to obtain a powder with the help of a grinder .Fresh white peppercorns (4 g) were purchased from a supermarket and cleaned using hot water (45°C) and dried at room temperature. Dried peppercorns were finely ground using a grinder  to obtain the white pepper powder.Fresh ginger rhizomes (15 g) were purchased from a supermarket. A mixture of 200 g of fresh, peeled, and cleaned ginger roots and 100 mL of filtered hot water (45°C) was blended using a medium speed level for 2 minutes using a blender (Wipro ANGEL WAM-L-55). Then, the blended mixture was filtered using a muslin cloth.Mature fresh coconuts were split and scraped with the use of a stainless steel electric coconut scraper . Milk was extracted by blending the scraped coconut with water (coconut : water = 3 : 1). 360 g sugar, 10 g ice cream stabilizer Cremodan (Cuisine tech), and 14 g gelatin (Motha Confectionery) as a thickening agent were mixed together and added to the coconut milk (1.4 mL) . The mixHeating was continued to 90°C, and salt (8 g) was added. The mixture was pasteurized at 90°C for 20 minutes and cooled to room temperature. The mixture was filtered and homogenized. Then, it was kept for around 4 hours for aging. The mixture was fed into the instant ice cream-making machine . Ice cream was filled into a clean container (3 L) and stored in a freezer at -18°C prior to analysis.The pH of the melted ice cream sample was measured by using a pH meter (Starter 3000) .Titratable acidity of the melted ice cream sample was calculated as the percentage of lauric acid was determined by titration with 0.1 N sodium hydroxide .Moisture content of the spicy coconut ice cream and normal coconut ice cream (control) was measured according to the oven-dried method 925.10 as described in AOAC .The empty moisture cans and lids were dried in the oven  at 105°C for 3 hours and transferred to the desiccator to cool. The weight of cans with lids was taken. About 5 g each of spicy ice cream and control sample was weighed to the dishes, and samples were spread uniformly. The cans with samples were placed in the oven overnight.After drying, the dishes with partially covered lids were transferred to the desiccator to cool.Finally, the cans and samples were reweighed.W1 is the weight (g) of the sample before drying and W2 is the weight (g) of the sample after drying.Calculation :(1)MoisAsh content of spicy coconut ice cream and control samples was determined using the dry ash method 925.10 as described in AOAC .The crucibles and lids were placed in the muffle furnace  at 550°C for 5 hours to ensure that impurities on the surface of the crucible were burned off. The crucibles were cooled in a desiccator. Then, the clean and dry crucibles and lids were weighed.5 g each of dried spicy ice cream and control samples was weighed into crucibles and placed in the furnace without covering with the lids. The sample was incinerated at 550°C for 5 hours. The lids were placed after completed heating to prevent loss of fluffy ash. After cooling down in the desiccator, ash with crucibles and lids was weighed.W1 is the weight (g) of ash and W2 is the weight (g) of the dried sample.Calculation :(2)Ash Both spicy ice cream and control samples were subjected to the following testing. Samples were transferred to a beaker and gradually warmed in a water bath (35-40°C). Then, samples were cooled to room temperature. Dishes and lids were heated in an oven at 102°C for 1 hour. The lids were placed on the dishes and immediately transferred to the desiccator to cool. Then, weights were taken. About 5 mL of spicy ice cream and control samples was poured into the dishes separately. Lids were placed, and weights were taken. The dish was placed in boiling water bath without a lid . Heating was continued till most of the water is removed. The dish was removed from the water bath and placed in the oven at 102°C for 2 hours alongside lids. Thereafter, lids were placed and kept in a desiccator to cool.The weights were taken, and dishes were heated again with lids alongside in an oven for 1 hour. Then, lids were placed and transferred immediately to the desiccator, and weights were recorded .M1 is the mass in g of dish + lid + sample, and M2 is the mass in g of dish + lid + dried sample.Calculation :(3)TotaMelt down of spicy coconut ice cream and control samples was determined according to method 941.08 as described in AOAC .Melt down of ice cream samples was evaluated using a 50 g ice cream block  which was placed on a metric test sieve that was supported by a previously weighed beaker. The mass of melted ice cream collected in the beaker was recorded at 5-minute time intervals for a 60-minute duration. The mass of melted ice cream (g) was plotted against the time (min) .Overrun of the ice cream was determined by using about 20 mL of ice cream mix and frozen ice cream.Calculation :(4)OverCrude protein content of the spicy coconut ice cream and control was determined by the Kjeldahl method: 920.87 as described in AOAC .N∗6.25). 0.25 N HCl was standardized by titrating 0.25 N sodium carbonate solution.Approximately 0.5-1.0 g of dried ice cream sample was placed in a dry and cleaned digestion flask. 25 mL of concentrated sulfuric acid and Kjeldahl tablet were added to the digestion flask. Digestion was run for 3 hours using a mini Kjeldahl unit (Block Digestion Unit Model: K-424). After, the digestion flask was allowed to cool and conducted distillation using an automated distillation unit . Finally, the sample was titrated with 0.25 N HCl solution, and crude protein content was determined (Fat content of the spicy coconut ice cream and control was determined by the Soxtherm method as described in AOAC .W1) was weighed accurately by using analytical laboratory balance in a weighed extraction thimble (W2). The rubber ring was selected, and thimbles were placed in a fat analyzer. 80 mL of petroleum ether was filled into each tube. Then, the fat analyzer was programmed for 5 minutes in immersion, 20 minutes in washing, and 30 minutes in recovery.Oven-dried samples were used to measure crude fat using a fat analyzer . 1 g of sample Fat v/v) and vortexed at high speed for 30 minutes. Then, the mixture was centrifuged  for 10 min at 792 rpm. After the centrifugation, the extracts were subsequently filtered through a filter paper . Finally, the prepared extracts were evaporated in a rotary evaporator  at 40°C under vacuum and stored at -18°C until assayed within 1 week [Ice cream samples were dried by lyophilization  for 24 h. An ice cream sample (13 g) was mixed with a mixture of 70 mL methanol/water  was added. The samples were vortexed and kept in the dark for 2 hours. The absorbance was measured at 760 nm using a spectrophotometer . The calibration curve of gallic acid was plotted to evaluate the activity capacity of the samples. The result was expressed as milligram of gallic acid equivalents (GAE) per gram of ice cream sample (mg GAE/100 g of dry weight) [About 1 mL of the melted ice cream sample was added to a 1.5 mL diluted Folin-Ciocalteu reagent. Samples with the reagent were left for 5 minutes, and then, 1 mL 7.5% sodium carbonate ( weight) .About 3 mL of prepared DPPH solution was added to 1 mL of melted ice cream sample. Then, solutions in the test tubes were shaken well. Samples were incubated in the dark for 15 minutes at room temperature. Finally, the absorbance was measured at 517 nm using a spectrophotometer  .Ac is the absorbance of the control and As is the absorbance of the sample [The DPPH radical scavenging activity (%) was calculated ase sample .About 0.1 mL of the ice cream sample was added into a test tube and mixed with 1 mL of reagent solution .The tube was capped with an aluminum foil and incubated at 95°C for 90 minutes. Then, the tube was cooled down to room temperature, and the absorbance was measured at 695 nm using a spectrophotometer  .The acceptability of the coconut milk-based spicy ice cream was evaluated by conducting a sensory evaluation with 30 untrained, both male and female panelists, in the age between 22 and 30 years. The sensory attributes tested were color and appearance, body and texture, aroma and flavor, melting quality, and overall acceptability. A 9-point hedonic scale was used. The values were scored on least preference (1) to most preference (9).p < 0.05 for the results of all analyses with both control and coconut spicy ice cream. The final results obtained were expressed as the mean values ± standard deviation of three replicates. Sensory evaluation data were analyzed using the Kruskal-Wallis test.MINITAB version 14 was used to statistically analyze all the results obtained from physicochemical and sensory evaluation. A one-way analysis of variance (ANOVA) was performed, and the significant difference was defined at w/w of spices, is the most acceptable formula. Color and appearance, body and texture, spicy flavor and aroma, and overall acceptability of sample 02 were higher than that of 0.025% spice-incorporated sample 01 and 0.010% spice-incorporated sample 03. Melting quality of sample 02 is lower than that of samples 01 and 03  between moisture content, titratable acidity, and overrun of the spicy coconut ice cream when compared to the normal coconut ice cream.pH of the spicy coconut ice cream is lower than the pH of the normal coconut ice cream. There is no any significant difference  between fat content, protein content, total solids, and ash content of the spicy coconut ice cream when compared to the normal coconut ice cream (There is no any significant difference (ce cream .p < 0.05) in total phenolic content and DPPH radical scavenging activity of spicy coconut ice cream when compared with normal coconut ice cream. There was no any significant difference (p > 0.05) in the total antioxidant capacity of spicy ice cream when compared to the normal coconut ice cream. Total phenolic content and DPPH radical scavenging activity of spicy coconut ice cream are higher than that of normal coconut ice cream  in both spicy coconut ice cream and normal coconut ice cream was increased with the storage time. There were no any yeast and mold counts .This study revealed that the spicy coconut ice cream which consolidated 0.018% spices (sample 02) demonstrated a higher mean rank for the five attributes, in particular, color and appearance, body and texture, spicy flavor, aroma, and overall acceptability from sensory evaluation . CompareMoisture content of both ice creams was similar. Moisture basically comes to the ice cream by milk and maintained through the freezing process. Since the same quantity of coconut milk was used to make both ice creams and the same freezing conditions (continuous freezing) were used, moisture content of both ice creams was similar. The recommended moisture content for ice cream is 61.7% , and spiAsh content of spicy coconut ice cream (0.41 ± 0.25%) and normal coconut ice cream (0.38 ± 0.05%) was not considerably different because the addition of spices in minor concentrations did not alter the ash content of spicy ice cream. Moreover, the result lies within the recommended range for ash content (0.3-0.6%).Titratable acidity of dairy ice cream expressed as lactic acid is ranged from 0.19 to 0.22%  that comAccording to the results, the percentage of total solids of spicy coconut ice cream was similar to the total solid percentage of normal coconut ice cream because the same fat source was used in the ice cream mix. Ice cream with fat content of 12-15% usually consisted of 32-40% of total solid content . Based oThe overrun of spicy coconut ice cream was indicated as 66.76 ± 1.44%, and it falls within the recommended range (60-90%) . The oveFrom the melt down curves , it can Spicy coconut ice cream and normal coconut ice cream contain fat content of 11.66 ± 0.66% and 11.06 ± 0.38%, respectively. This property indicates that both ice creams have similar fat contents as coconut milk plays a role as the major fat source. Recommended fat content of an ice cream is 8-12% , and spiAccording to the results, protein content of both ice creams was not significantly different. Results indicate that the protein content of spicy coconut ice cream (4.18 ± 0.16%) was higher than the recommended protein content of ice cream (4%) . The KjeAn antioxidant is a major bioactive compound present in spices. In this study, total phenolic content, DPPH radical scavenging activity, and total antioxidant capacity were analyzed to examine the antioxidant activity of ice cream. The developed spicy coconut ice cream contained higher values for all three antioxidant properties compared to normal coconut ice cream as 0.093 ± 0.002 , 60.39 ± 0.02 , and 0.36 ± 0.04 , respectively, due to addition of spices. Cinnamon contains cinnamaldehyde and cinnamic acid, ginger contains gingerol, and white pepper contains piperine as the phenolic compounds. Polyphenols have the ability to quench DPPH radicals by providing hydrogen atoms or by electron donation. The higher the percentage of inhibition of free radical activity, the more potent the antioxidant activity of the extract in terms of hydrogen atom donating capacity.Total plate count reflects the viable microbial population. It reveals the microbiological quality of the product examined and also reflected on the quality of raw materials, degree of hygiene, and the cleanliness maintained during manufacture, handling, and storage of the product. In the four weeks of study, all the total plate counts were less than 500 per 1 mL, which is given in the SLS 223 . As showAn ice cream was developed using coconut milk as an alternative for cow milk with the incorporation of locally available spices  as a novel product. Among the developed three different formulas, 0.018% spice-incorporated sample 02 achieved higher overall sensory acceptability. This spicy coconut ice cream contained considerably similar values for physicochemical properties compared to normal coconut ice cream but potentially higher antioxidant properties when compared with the normal coconut ice cream. Accordingly, coconut milk-based spicy ice cream can be introduced to the market as a nondairy product with desired sensory attributes and functional properties.


Nikko Bay is a highly sheltered system with organisms showing low connectivity with surrounding environments, making this bay a unique site for evaluating adaptation and acclimatization responses of organisms to warmed and acidified environments. Seawater pCO2/Ωarag showed strong gradation ranging from 380 to 982 µatm (Ωarag: 1.79–3.66), and benthic coverage, including soft corals and turf algae, changed along with Ωarag while hard coral coverage did not change. In contrast to previous studies, net calcification was maintained in Nikko Bay even under very low mean Ωarag (2.44). Reciprocal transplantation of the dominant coral Porites cylindrica showed that the calcification rate of corals from Nikko Bay did not change when transplanted to a reference site, while calcification of reference site corals decreased when transplanted to Nikko Bay. Corals transplanted out of their origin sites also showed the highest interactive respiration (R) and lower gross photosynthesis (Pg) to respiration (Pg:R), indicating higher energy acquirement of corals at their origin site. The results of this study give important insights about the potential local acclimatization and adaptation capacity of corals to different environmental conditions including pCO2 and temperature.Ocean warming and acidification caused by increases of atmospheric carbon dioxide are now thought to be major threats to coral reefs on a global scale. Here we evaluated the environmental conditions and benthic community structures in semi-closed Nikko Bay at the inner reef area in Palau, which has high  Coral mass bleaching events are now occurring more frequently compared to the past, and ocean warming is predicted to further increase the susceptibility of corals to bleaching in the coming decades4. Some studies have indicated that zooxanthellate corals may have the ability to adapt to high temperature environments, such as by shuffling to Symbiodiniaceae types that have higher tolerances to warmer temperature7. In addition to global warming, increases of seawater partial pressure of CO2 (pCO2) and decreases of aragonite calcium carbonate saturation (Ωarag) have been reported to decrease calcification rates of most reef calcifiers including corals and crustose coralline algae (CCA)9. Meanwhile, it has been shown that the tolerance of organisms to high pCO2 can differ among species and even within species10. Hence, there is now wide interest in understanding how reef organisms will respond to ocean warming and acidification at the community level, and to examine if organisms are able to acclimatize or adapt to these environmental changes.Increases in atmospheric carbon dioxide  in the inner reef area in Palau Fig.  with hig2 vent sites20 and naturally acidified sites23 have been found on coral reefs and utilized as essential models for evaluating the effects of ocean acidification at the ecosystem level. However, most of these systems are open or semi-closed lagoon ecosystems with significantly shorter residence times compared with that of Nikko Bay, and thus they may continuously receive recruitment from the surrounding ocean; this may limit local adaptation of organisms to high pCO2 conditions. On the other hand, Nikko Bay is of particular interest because it is a highly sheltered system with a long seawater residence time of 71 days, and with organisms showing low connectivity with surrounding populations24. Additionally, the seawater conditions found in this bay have been suggested to have been maintained at least for the past 150–500 years12 and hence, long-term selection of coral larvae may have allowed local adaptation or acclimatization responses to environmental conditions found within this bay. Finally, this unique bay provides opportunities to evaluate the effects of the co-stressors of ocean warming, ocean acidification, low oxygen and high chlorophyll-a (Chl-a) levels, at the community scale. Recent studies in mangrove habitats25, tidal inshore reefs26, and upwelling systems27 have demonstrated the need and importance of evaluating these multiple co-varying factors in understanding the mechanisms sustaining complex ecosystems and in predicting future coral reef scenarios28.COarag gradient found within the bay. We also conducted reciprocal transplantation experiments of the most dominant coral species, Porites cylindrica, to evaluate the potential acclimatization and adaptive responses of corals to warm and acidified conditions.Here we evaluated the environmental conditions and benthic community structures along with the Ωarag and temperatures (SST) showed a strong gradient at the entrance into the bay  and had lower pH (7.83 ± 0.06), lower Ωarag, (2.44 ± 0.34) and higher pCO2 (619 ± 104 μatm) compared to parameters outside the bay , Ωbay Fig. a, b, e ap < 0.01, Fig. pCO2 within Nikko Bay were mainly caused by low seawater TA due to calcification and by high DIC due to respiration , mean net calcification (Gn) and net primary production (Pn) rates within the bay were calculated to be 22.7 mmol CaCO3 m−2 d−1 and − 6.9 mmol C m−2 d−1. These values were lower than the net calcification rates found at most reefs31, however the positive net calcification at seawater Ωarag of 2.44 within the bay contradicts with previous studies suggesting that coral reef formation is restricted to seawater Ωarag higher than ca. 2.8 (pCO2 lower than 560 μatm)32, and also with CO2 seep studies indicating that reef development ceases where pH is lower than 7.7 (Ωarag 2.1) in Papua New Guinea18, and lower than 7.9 in the Mariana Islands20. When CO2 gas exchange is considered, the heterotrophy of Nikko Bay becomes even higher. Although there are no wind speed data measured directly inside Nikko Bay, wind speed measured at PICRC station was 0.51 ± 0.80 m s−1 (N = 4320) during this period, and wind can be expected to be weaker in Nikko Bay, which is surrounded by islands. Using the average pCO2 of seawater in the bay , and gas exchange coefficient33 utilized; gives the largest gas flux under this condition), the pCO2 flux was calculated to be about 5.6 mmol m−2 d−1. Using this value, the Pn in Nikko Bay would be approximately -12.5 mmol m−2 d−1.Daytime average total alkalinity (TA) and dissolved inorganic carbon (DIC) were significantly lower within the bay compared to outside the bay  inside the bay and one site outside of Nikko Bay (N1) were selected for benthic community observation , CCA , macroalgae, or seagrass coverage , which follows previous results at a CO2 vent at Iwotorishima in southern Japan showing high coverage of soft coral at a high pCO2 site19. Turf algae coverage increased with Ωarag , contradicting previous observations at a CO2 vent in the Mariana Islands20 , Chl-a, nitrate plus nitrite (NO3−  + NO2−) concentration, pCO2 and temperature , low pCO2 (395 μatm), low temperature (29. 3 °C), low Chl-a (0.55 μg/L), high DO (6.09 mg/L), and was dominated by Acropora spp. (coverage 16.5 ± 4.1%), while site N5 was characterized by low Ωarag (1.28), high pCO2 , high temperature (30.5 °C), high Chl-a (1.68 μg/L), low DO (4.52 mg/L), and was dominated by Merulinidae spp.  showed the highest coverage, accounting for 22 to 79% of hard coral cover inside the bay  seawater.To evaluate the correlation among seawater carbonate chemistry and benthic community structure, six sites (N2-N7) along with the Ωgradient .28–3.51 ion Fig. . Even thion Fig. . There w20 Table , Fig. S1ure Fig. . Site N1bay Fig. , and theP. cylindrica were reciprocally transplanted between two inner reef bays; a reference site at Malakal Bay (site M1) and a site in Nikko Bay  rate of P. cylindrica transplanted to site N5 had a significantly higher value (p = 0.04) regardless of their origin  and lower Pg:R compared to corals outside of the lagoon25. Acclimatization of corals at the New Caledonia lagoon was suggested to be caused by high respiration through potentially high heterotrophy of corals within the lagoon, which has high organic carbon sedimentation25. A comparatively high heterotrophy of the corals in Nikko Bay is also suggested as zooplankton abundance (particularly copepod abundance) was observed to be higher at site N5 compared to reference site M1 , as well as Durusdinium43 (former Symbiodinium ‘Clade D’), which are known to be tolerant to high temperatures44, while P. cylindrica from the other sites only hosted Cladocopium subclade C1 , and water quality parameters including chlorophyll-a (Chl-a), turbidity (FTU), dissolved oxygen (DO) and inorganic nutrients  were measured once each during daytime and nighttime from 40 sites around Nikko Bay , dissolved inorganic carbon (DIC), aragonite saturation (Ωa (fluorescent chloride sensor), dissolved oxygen  and turbidity  were measured by vertical casting  using a multi-parameter sensor . Salinity data was calibrated by a salinometer , and Chl-a (3 replicates) by a fluorometer . Surface water samples for TA, DIC (2 replicates) and nutrient  measurements (4 replicates) were collected from the same sites. TA and DIC were measured within 24 h after seawater collection using an auto burette titrator , which has an accuracy of ± 2 µmol Kg−1 for TA and ± 3 µmol Kg−1 for DIC and standardized by certified reference materials obtained from A. Dickson Laboratory . When samples were not measurable within 24 h, they were poisoned with saturated mercury chloride and kept within air-tight glass vial bottles, and these samples were measured within a few days after collection. Nutrient samples were stored in a freezer (-20 °C) until measurement by nutrient autoanalyzer . Carbonate chemistry was calculated from measured TA, DIC and surface salinity and temperature measured at 30 cm below the seawater surface by a casting sensor using CO2SYS45 with the constants of Mehrbach et al.46 as refit by Dickson and Millero47, and aragonite solubility of Mucci48. The accuracy of calculated pCO2 is estimated as approximately 2–3% of the measurement range of Nikko Bay .Water quality measurements in Nikko Bay were conducted during daytime before sunset (15:00–18:00) and during nighttime around sunrise (5:00–8:00) between 17 to 19 November, 2014 at 40 sites , by simply assuming these TA and DIC differences were accumulated in the bay during this residence time; mean net calcification rate (Gn) was calculated from the TA differences, τ, d, and seawater density (ρ), and primary productivity (Pn) rate was calculated from DIC and TA differences, τ, d, and ρ . For the calculation TA and DIC were normalized at S = 33.02 which was the mean salinity of Nikko Bay.Mean net calcification and primary production in Nikko Bay were calculated from the differences between the average of inside Nikko Bay TA and DIC , nutrients (4 replicates), and suspended solids (3 replicates). Seawater carbonate chemistry (TA and DIC) was measured by collecting two replicate water samples three times  during transplantation. pH and temperature loggers  were also deployed at the three transplantation sites for ca. 5 days. The pH data for the loggers were calibrated using TRIS-AMP buffers and are reported in the total scale.In addition to seawater multi-station measurements across the entire bay, several measurements were conducted during the subsequent benthic survey and coral transplantation survey. Seawater qualities (3 m depth) for the seven sites where benthic surveys were conducted were measured using the AAQ-Rinko in the same manner as above. Additionally, seawater samples were taken at 3 m depths using Van Dorn samplers to measure TA, DIC, and nutrients. During coral transplantation, bottom seawater was sampled using Van Dorn samplers on October 17, 2015 for measurement of Chl-2 of benthos per depth and site. We measured coral community composition and water quality of one site outside of Nikko Bay (N1) and six sites within Nikko Bay (N2-N7) at similar depths (~ 5 m) showing different Ωarag conditions (1.39–3.45) for the analyses.Benthic coverage including coral community cover (at the genus level) was determined by taking 0.5 × 0.5 m photo-quadrats every meter along five 50 m transects at 3 m depth. A total of 50 photographs were taken per transect and per depth at each site; representing an area of 12.5 m51. The benthic substrate directly below five random points per photograph was classified into benthic categories as described in several previous studies . The benthic categories included live corals (to the genus level), fleshy macroalgae (identified to the genus level), turf algae, other invertebrates , crustose coralline algae (CCA), and non-living substrates . The percentage cover of benthic categories at each site and depth was the result of the average of percentage cover among the five transects.Sites N1 to N5 were surveyed in November 2015 while sites N6 and N7 were surveyed in August 2014. No major disturbances within Nikko Bay (e.g. bleaching events) were observed between the two benthic survey dates. Benthic photographs were analyzed using CPCe softwarePorites cylindrica colonies in September 2015 from Nikko Bay (sites N5) and a reference site at Malakal Bay (site M1) at 2–3 m depths, respectively. The M1 site was selected as reference site because the coral community structure was similar to Nikko Bay and dominated by P. cylindrica, while seawater carbonate chemistry and seawater temperatures were close to the conditions found outside the bay such as at site N1. Sampled corals were put within a container with seawater bubbled with an air-pump, and transported by boat after sampling to the Palau International Coral Reef Center, arriving within 1 h  according to Davis53, and calcification rates were calculated by the change of skeletal dry weights during transplantation and normalized by the surface areas measured using the aluminum foil technique54. Additionally, photosynthesis and respiration rates were measured for 9 out of 12 nubbins in each condition. Coral nubbins  were first cleaned to remove any attached algae on the coral surface, and each nubbin was then placed individually in an airtight glass container (volume = 400 ml) filled with seawater collected from the same site where the corals were transplanted, placed under LED light (250 μmol photon m−2 s−1) and photosynthesis was measured during daytime (11:00–14:00). One extra incubation container without a nubbin was also added as a control. Seawater within the container was continuously stirred with a magnetic stirrer (450 rpm) during incubation and oxygen concentrations were measured at 0, 20 and 40 min using an oxygen sensor  calibrated with 100% saturated and 0% oxygen water prepared by Na2SO3. The same procedure was also conducted at night (20:00–22:00) under dark conditions to measure the respiration rate. Net photosynthesis (Pn) and respiration (R) rates were calculated by the following equation, where V is volume (ml), T is time in hours, and SA is the surface area of the coral (cm−2). Gross photosynthesis (Pg) was calculated by Pn minus R.Eighteen days after transplantation, all nubbins were recovered and the buoyant wet weights of all nubbins were measured. Skeletal dry weights were calculated using aragonite density  from three sites  using a 100 m Nansen plankton net (30 cm diameter). Horizontal tows were conducted 5 times at 1–3 m depth per site, and the filtered volume was recorded with a flowmeter. Zooplankton samples were split and one-half of the sample was preserved in 5% borated-buffered formalin. Individual numbers of all zooplankton and copepods of the formalin fixed samples were counted under a microscope.55. In the present case, the Hellinger distance was used. Generalized linear models  were used to evaluate the relation between Ωarag and coral and other benthic coverage. The calcification rates of the transplantation experiment were evaluated with a Generalized Linear Mixed Effects model (family = Gamma) with origin site and transplanted site and its interaction as fixed effects and colony as random effect. Net photosynthesis, respiration (log transformed), and Pg:R (log transformed) of the transplantation experiment were evaluated with a linear mixed-effect model (REML) with origin site and transplanted site and its interaction as fixed effects and colony as random effect. Tukey’s HSD test was conducted when there was a significant interaction. Differences of zooplankton and copepod abundances among sites were tested with student t-test. All statistical analyses were conducted using R (version 3.6.3)56.Differences in seawater qualities inside and outside Nikko Bay were analyzed using Wilcoxon signed-rank test. Redundancy analysis (RDA) was conducted as a constrained ordination technique to relate the coral communities to seawater environmental variables. Input for the RDA consisted of coral coverage data that were first transformed using the decostand function in the R package veganSupplementary Information 1.Supplementary Information 2.


This paper presents an experimental study on surface icing on leaves in six plant species having different surface micromorphology and wettability properties. Contrary to previous studies on ice crystallization, which have been mainly performed by using infrared video thermography, we applied a Cryo-SEM approach allowing not only characterization of plant surfaces in their native conditions but also visualization of ice crystal formation on the native plant surfaces at the micro- and nanoscales. The Cryo-SEM was also used as an experimental device to freeze water vapor, thaw ice crystals, and freeze fluid water on the plant surface again. The experiments clearly demonstrate that trichome coverage  and 3D wax projections can be recognized as anti-icing strategies of plants. Trichomes can prevent and delay ice formation by being nucleation points for the formation of ice from vapor and protect the plant surface from overcooling, when fluid water freezes in contact with the leaf surface. The study shows for the first time two important effects that might reduce plant cell freezing rate: the presence of air pockets between wax projections that protect from direct contact between ice crystals and the plant cuticle and elimination of fluid water after thawing and preventing further re-freezing on the surface. The detailed knowledge obtained here is not only important for plant ecology, evolution, and plant protection but also for looking for potential biomimetic strategies that reduce/avoid icing of cultural plants and artificial technical surfaces. Vegetative and generative organs of many herbaceous plant species of temperate zones are often exposed to temperatures below zero and can be easily iced on their surfaces Fig. . In ordeHowever, ice can also be easily formed on the outer surfaces of plants and this ice can form quickly and in large amounts. The epicuticular wax layer, cuticle, and cell walls of epidermal cells can prevent the ice crystal’s nucleation from moving inside cells and damaging them, but some plant cuticles are rather thin and vulnerable toward potential mechanical damage from ice crystals. Moreover, plant surfaces are rather different in their physico-chemical properties , which certainly might have an effect on ice nucleation on the plant surface. Especially less hydrophobic surfaces, having strong contact with water when plants are initially wet, may generate heavy crusts of ice when environmental temperatures go below zero presenting a potential damage source for the plant surface. It has also been previously reported that plant surfaces, made hydrophobic by artificial particles, limited the spreading of ice crystal formation and delayed plant freezing , Fragaria ananassa (Rosaceae), Lupinus polyphyllus (Fabaceae), Campanula portenschlagiana (Campanulaceae), Ficaria verna (Ranunculaceae), Bellis perennis (Asteraceae), Cerastium brachypetalum (Caryophyllaceae), Narcissus pseudonarcissus (Amaryllidaceae), and Tulipa gesneriana (Liliaceae). Leaves of the following species were collected and their upper  surfaces were used in the Cryo-SEM experiments , B. perennis and C. brachypetalum (surfaces covered with trichomes), and N. pseudonarcissus and T. gesneriana (surfaces covered with 3D epicuticular waxes).Natural occurrence of ice on leaf surfaces of plants was studied in early Spring in the Botanical Garden of the University of Kiel . The following species were observed, and icing on their leaves was documented by taking images in nature and at various magnifications under the binocular microscope Figs. , and 3: nts Fig. : P. laurn = 10 leaves of 10 individual plants for each species.Native surfaces of plant leaves were examined using cryo scanning electron microscopy (SEM). Small samples (1 cm × 1 cm) from the middle part of the leaves were cut out from living plants, immediately attached with their lower  side to the metal holder using polyvinyl alcohol Tissue-Tek® OCTTM compound , and frozen in a cryo-stage preparation chamber at − 140 °C . Frozen samples were sputter-coated with gold–palladium (thickness 6 nm) and studied in the frozen condition in a Cryo-SEM Hitachi S-4800  at 3 kV accelerating voltage and − 120 °C temperature. Description of the trichomes was performed mainly according to Voigt et al. . Types o−3, surface tension = 72.1 mN m−1, dispersion component = 19.9 mN m−1, polar component = 52.2 mN m−1; Busscher et al. n = 20 leaves of 10 individual plants for each species.Contact angles of double-distilled water  from the middle part of the leaves were cut out from living plants, immediately attached with their lower side to the metal holder using polyvinyl alcohol Tissue-Tek® OCTTM compound, and frozen in the vicinity of liquid nitrogen. Water vapor from the air generated icing, the intensity of which on the plant surface was dependent on the time of exposure to the liquid nitrogen. Usually, we stopped icing formation after 1–2 min and quickly transferred the sample in a vacuum to the cryo stage preparation chamber at − 140 °C. Frozen samples were not sputter-coated but directly studied in the frozen condition in the Cryo-SEM Hitachi S-4800 at 3 kV accelerating voltage and − 120 °C temperature.After examination, samples were taken out of the microscope and held under room conditions for 3 – 5 min until the ice crystals started to thaw and then quickly frozen by submersion into the liquid nitrogen (1–2 min) and transferred in vacuum to the cryo stage preparation chamber at − 140 °C. Frozen samples were directly studied in the frozen condition in the Cryo-SEM . After examining for ice damage on the plant surfaces, samples were transferred back to the cryo stage preparation chamber at − 140 °C. Ice was sublimated (freezing-drying) by heating the samples up to − 90 °C and further studied in their frozen condition in the Cryo-SEM .lightly iced condition, when single scattered ice crystals up to a monolayer of ice crystals are situated on the surface, whereas heavily iced condition contains many layers of ice crystals.It is important to note that the Cryo-SEM experimental conditions are different from environmental conditions in many respects. The freezing temperature in the vicinity of the liquid nitrogen is difficult to control, and at some point of the experiment, we had to go to − 140 °C in order to keep the formed surface ice under the vacuum condition of SEM. Plants under natural conditions are never exposed to such low temperatures. In spite of these differences, this experiment clearly shows the interaction between the ice, formed from the water vapor, and cold plant surfaces. In the description of the results, we use the term P. laurocerasus and F. verna have rather smooth upper leaf surfaces. P. laurocerasus bears a relatively thick and mechanically stable cuticle (together with the smooth epicuticular wax), and epidermal cell shapes are only hardly visible in SEM, whereas F. verna , whereas those of N. pseudonarcissus are nanoscopical irregular platelets .rna Fig. , B with tip Fig. , B. Tricase Fig. , B. Tricxes Fig. , B. The P. laurocerasus, 77 ± 13° on F. verna, 88 ± 22° on B. perennis, 93 ± 9° on C. brachypetalum, 152 ± 7° on N. pseudonarcissus, and 157 ± 8° on T. gesneriana.Contact angles of water were 97 ± 5° on Ice on smooth plant surfaces started with regularly distributed nucleation sites Figs. , B, whicF. verna; that is why the contact area between fluid water and epidermal cell surfaces became very large.After thawing, fluid water filled the depressions between elevated middle parts of epidermal cells Fig. , F. WateCampanula portenschlagiana and Fragaria ananassa , water can penetrate the boundary layer and form ice crusts directly on the surface of epidermal cells. In this case, trichomes usually become sites of ice nucleation and the ice crystal orientation is clearly dependent on the arrangement of trichomes on the surface  with Fig. T. gesneriana, 3D wax), or Cryo-SEM images Fig. F. verna, smooth) with Fig. T. gesneriana, 3D wax), it is hard to recognize any difference. It seems that ice formation on plant surfaces covered with wax projections started at more or less regularly distributed nucleation sites, which, with an increased amount of frozen water vapor, built larger hexagonal ice crystals . If one compares binocular microscope images Fig. ls Figs. , J. Latels Figs. , F. The ls Figs. . That isls Figs. .Fig. 8TuAfter thawing, water drops rolled off the surface, even at very small angles (2–3°). This behavior, due to the surface superhydrophobicity, immediately cleaned the surface of water that could be potentially re-frozen and in frozen conditions represents potential danger of further damage to the plant surface Fig. , L. Due Due to the fact that ice crystals were often deposited between single wax projections, we expected strong damage in the 3D wax layer on iced surfaces. However, the wax layer remained absolutely intact after ice thawing and water escaping from the surface Fig. , F.It is well known that ice can be formed on surfaces in general and on outer surfaces of plants in particular. Depending on environmental conditions, this ice can be formed quickly and in large amounts. The extracellular surface structures as well as the outermost cell walls of epidermis can stop the nucleation of ice crystals from moving inside cells, but depending on the contact area between ice and plant surface, the freezing rate within the cell’s interior might be potentially different. Additionally, some smooth plant cuticles are rather thin and vulnerable to potential mechanical damages by ice. That is why it was plausible to assume that the structure and chemistry of plant surfaces may influence plant resistance against damages caused by freezing.Plant surfaces vary greatly in their texture , orientation, bases, cellular arrangement, branching, shape, etc. (see review Gorb and Gorb ). TrichoSalvinia effect), but no evidence for their role in reduction of the freezing damage of plant surfaces was demonstrated. Our study shows two important effects that might reduce plant cell freezing rate: (1) the presence of air pockets between wax projections that protect from direct contact between ice crystals and cuticle . ResearT. gesneriana), and therefore, their low adhesion/friction on the surface  and 3D wax coverage can be recognized as anti-icing strategies of plants.


Decapterus maruadsi) hydrolysates (RSHs). In this study, RSHs were obtained by using three proteases . Among them, the RSHs of 6-h hydrolysis by neutrase displayed the strongest XO inhibitory activity and had an abundance of small peptides (<500 Da). Four novel peptides were purified by immobilized metal affinity chromatography and identified by nano-high-performance liquid chromatography mass/mass spectrometry. Their amino acid sequences were KGFP (447.53 Da), FPSV (448.51 Da), FPFP (506.59 Da) and WPDGR (629.66 Da), respectively. Then the peptides were synthesized to evaluate their XO inhibitory activity. The results indicated that the peptides of both FPSV (5 mM) and FPFP (5 mM) exhibited higher XO inhibitory activity (22.61 ± 1.81% and 20.09 ± 2.41% respectively). Fluorescence spectra assay demonstrated that the fluorescence quenching mechanism of XO by these inhibitors (FPSV and FPFP) was a static quenching procedure. The study of inhibition kinetics suggested that the inhibition of both FPSV and FPFP was reversible, and the type of their inhibition was a mixed one. Molecular docking revealed the importance of π-π stacking between Phe residue (contained in peptides) and Phe914 (contained in the XO) in the XO inhibitory activity of the peptides.The objective of the present study was to investigate the xanthine oxidase (XO) inhibitory effects of peptides purified and identified from round scad ( Hyperuricemia is currently recognized as the fourth highest-risk chronic disease after hyperglycemia, hypertension, and hyperlipidemia, which could cause complications such as gout, hypertension and diabetes disease . It is aDecapterus maruadsi) is a kind of marine fish belonging to the family of mackerel, which is widely found in distributed warm, nearshore waters of China  is the quencher concentration. Kq is the quenching rate constant of biomolecule. Kq = Ksv/τ0, τ0 is the average lifetime of biomolecule in the absence of quencher and its value is approximately 10−8 s. Ksv used to be estimated by linear regression plot slope of F0/F versus [Fluorescence spectra were measured on an F-7000 Spectrofluorimeter  with a 1.0 cm quartz cell and a thermostatic bath. Briefly, 2 mL XO (0.05 U/mL) was titrated by continuous addition of peptides solution  versus the XO (U/mL) at different concentrations of the inhibitors (peptides) were constructed to determine the reversibility of inhibitory behavior on XO [The plots of ν ΔOD0nm/min vKm and Vmax values, as corresponding kinetic parameters, were obtained from the Lineweaver and Burk double reciprocal plots [The type of the inhibition of XO activity by the inhibitors (peptides) was investigated using Lineweaver and Burk kinetic analysis. It was performed via evaluating the initial rate of reaction in the presence and absence of inhibitors at different xanthine concentration. Briefly, the peptide (0 and 5 mM) and XO (0.05 U/mL) were mixed and incubated in the buffer  for 30 min at 37 °C. Subsequently, different xanthine concentrations  were successively added into the solutions. Then, the dynamic changes of the absorbance at 290 nm were recorded, and ν was calculated using the same method above. al plots .http://www.pdb.org/pdb/home/home.do, accessed on 30 September 2020). The process was performed according to Li et al. (2019) [In order to explore the inhibitory effect of peptides on XO, molecular docking studies were used to explore the potential binding sites and binding affinities between XO and peptides. The molecular docking studies were operated by Discovery studio 2016. The 3D structures of peptides were generated using Chem 3D. The X-ray crystal structure of bovine XO complex with inhibitor TEI-6720 (PDB ID: 1N5X) was downloaded from the Protein Data Bank website (. (2019) . In dockt-tests were performed using one-way analysis of variance (ANOVA) on the IBM SPSS statistics 25.0 software. Differences were considered statistically significant at p < 0.05.Duncan’s (Decapterus maruadsi) protein hydrolysates (6-h hydrolysis by neutrase). The XO inhibitory peptides, especially for FPSV and FPFP, displayed great potential for XO inhibitory activity. Fluorescence spectra assay demonstrated that the fluorescence quenching mechanism of XO by the inhibitors of both FPSV and FPFP was a static quenching procedure. According to the inhibition kinetics study, the inhibition of both FPSV and FPFP was reversible, and the type of inhibition by both FPSV and FPFP was a mixed one. Molecular docking results revealed that the π-π stacking between Phe residue (contained in peptides) and Phe914 (contained in the XO structure) played an important role in the XO inhibitory activity of peptides. These results suggested that peptides obtained from marine fish (Decapterus maruadsi) exhibited XO inhibitory effect and could be used as potential natural XO inhibitors.In the present study, four novel XO inhibitory peptides  were purified and identified from round scad