A. bisporus). Our results revealed that scytonemin monomer (ScyM), a pure compound from our compound library and also the monomeric last-step precursor in the biosynthesis of the well-known cyanobacterial sunscreen pigment “scytonemin,” consistently showed the highest tyrosinase inhibitory score. Determination of the half maximal inhibitory concentration (IC50) further indicated that ScyM is more potent than the commonly used commercial inhibitor standard “kojic acid” . After a scaled-up chemical synthesis of ScyM as well as its O-methyl analog (ScyM-OMe), we conducted a series of follow-up studies on their structures, inhibitory properties, and mode of inhibition. Our results supported ScyM as the second case ever of a novel tyrosinase inhibitory compound based on a marine cyanobacterial natural product. The excellent in vitro performance of ScyM makes it a promising candidate for applications such as a skin-whitening agent or an adjuvant therapy for melanoma cancer treatment.Tyrosinase, an important oxidase involved in the primary immune response in humans, can sometimes become problematic as it can catalyze undesirable oxidation reactions. Therefore, for decades there has been a strong pharmaceutical interest in the discovery of novel inhibitors of this enzyme. Recent studies have also indicated that tyrosinase inhibitors can potentially be used in the treatment of melanoma cancer. Over the years, many new tyrosinase inhibitors have been discovered from various natural sources; however, marine natural products (MNPs) have contributed only a small number of promising candidates. Therefore, in this study we focused on the discovery of new MNP tyrosinase inhibitors of marine cyanobacterial and algal origins. A colorimetric tyrosinase inhibitory assay was used to screen over 4,500 marine extracts against mushroom tyrosinase ( The market for skin-whitening products, especially in Asian countries, such as China, India, and Japan, is experiencing unprecedented growth . The funl-tyrosine (or l-DOPA) to dopaquinone by tyrosinase , using a well-validated classic colorimetric fungal-tyrosinase inhibitory assay. After multiple validations through dose-dependent testing, our results revealed that scytonemin monomer  using a Thermo Fisher Scientific HPLC system comprising a Thermo Dionex UltiMate 3000 pump, RS autosampler, RS diode array detector, and automated fraction collector. 1D NMR and 2D NMR spectra were acquired using a Bruker Avance III DRX-600 NMR and a JEOL ECZ 500 NMR spectrometer. Deuterated solvents for NMR were purchased from Cambridge Isotope Laboratories, Inc. Samples were passed through C18 SPE  before LC injections. All other general solvents were HPLC grade as purchased from Thermo Fisher Scientific.All starting materials were purchased from Sigma-Aldrich. Other synthetic reagents and solvents were purchased from Fischer Scientific. All solvents employed in synthesis were dried by distilling from CaHl-alanine (l-DOPA) was obtained from Tokyo Chemical Industry Corp. Sodium Phosphate buffer  was purchased from bioWORLD Corp. The positive control compound, Kojic Acid (KA), was purchased from Thermo Fisher Scientific Corporation. DMSO from Sigma-Aldrich was used in small amounts to dissolve samples as well as in controls for consistency within the assay procedure. A Millipore Milli-Q system  was used to purify water for use in dilutions.Most of the compounds, fractions and extracts used in this Medium Throughput Anti-tyrosinase Screening campaign were purified from a variety of tropical marine cyanobacterial and algal collections produced in our laboratory during past expeditions. A few of the compounds from the collection of Pure Compounds had been synthesized by former researchers in our laboratory. All samples were stored in DMSO at −80°C before dilution and testing. Mushroom tyrosinase (polyphenol oxidase) was obtained from Sigma-Aldrich and Worthington Biochemical Corp. The substrate 3--5  in 12 ml of 4:1 CH2CH2/ (CH2Cl)2 in a round bottom flask under N2 at RT were added 4-hydroxybenzaldehyde  and TsO·H2O . The flask was then equipped with a Hickman still holding 3 Å molecular sieves and a condenser on top of the Hickman still. The reaction mixture was then refluxed under vigorous stirring overnight. Upon cooling to RT, the reaction mixture was filtered through a plug of silica gel and rinsed with EtOAc. The volatiles were removed under vacuum from the combined crude product solution. The crude solid was dissolved in 1:9 MeOH/CH2Cl2 and subjected to silica gel flash column chromatography employing a gradient mobile phase from 100% hexanes to 1:1:18 EtOAc/CH2Cl2/hexanes, then to 1:10:10:80 MeOH/EtOAc/CH2Cl2/hexanes, and then finally to 1:3:3:6 MeOH/EtOAc/CH2Cl2/hexanes. A yellow-brownish solid  was obtained. This material contained a trace amount of starting material 6 and was therefore further purified by reverse-phased HPLC with an isocratic mobile phase consisting of 1:1 acetonitrile/H2O containing 0.1% formic acid. Pure synthetic compound 1 showed the following: TLC Rf = 0.54 (1:1 EtOAc/hexanes); Mp > 210°C; IR  νmax 3,375 (br), 2,963, 2,921, 1712, 1,599, 1,483, 1,441, 1,274, 1,231, 1,165, 1,123, 745 cm−1; 1H and 13C NMR data, see To a solution of the indole 5  in CH2Cl2 (10 ml) under N2 in a round bottom flask at RT were added anisaldehyde  and TsO·H2O . This flask was then equipped with a Hickman still holding 3 Å molecular sieves and a condenser on top of the Hickman still. The reaction mixture was refluxed overnight. Most of the solvent had evaporated at this point, leaving behind a dark brown solid. The crude material was dissolved in CH2Cl2 and subjected to silica gel flash column chromatography with a gradient mobile phase ranging from 1:49 acetone/hexanes to 1:4 acetone/hexanes. A yellow solid  was obtained that showed the following: TLC Rf = 0.40 (1:3 acetone/hexanes); 1H NMR  δ 8.21 , 7.60 , 7.55 , 7.35 , 7.25 , 7.17 , 7.16 , 7.04 , 3.90 , 3.55 ; 13C NMR  δ 204.6, 160.3, 140.2, 138.8, 129.6, 128.7, 127.8, 124.3, 124.3, 120.9, 120.3, 119.8, 114.8, 111.8, 55.5, 36.5; HRMS (EI) calcd for C19H15NO2Na [M + Na]+ 312.0995, found 312.0998 (Δ1.0 ppm).To a solution of the indole l-DOPA as substrate. Experiments were constructed in 96-well microplates format with slight modifications made from previously described methods  for absorbance measurement of each well at 475 nm  and 490 nm (as a wavelength duplicate) in the end-point mode. Forty-five seconds of shaking was performed by the instrument before the wells were read.The contents of each well were first mixed and pre-incubated at RT for 20 min after which 40 μl of 3 mM The percentage inhibition of the tyrosinase activity was calculated by the equation: % Inhibition = {[(A − B) − (C − D)]/(A − B)} × 100, where A, B, C and D are all OD values from the corresponding wells at the same time point. It reflects the inhibitory strength of the testing material by comparing to the blank condition where no inhibitor is added.l-DOPA as substrate. Experiments were carried in triplicate in 96-well microplates using the same 200 μl reagent mixture system as described in the broad screening section, with the difference being that the concentration of the sample  was varied within the 2 μl of sample that was added to each well. Respectively, the final concentration gradient of the test samples was set to be 200, 150, 75, 25, 5, and 1 μM. For OD measurements, the plate was read in the dynamic mode for 60 min with measurements recorded every minute, using the SpectraMax M3 plate reader at 475 nm and 490 nm. Forty-five seconds of shaking was also performed before first reading to retain method consistency. The IC50 values and curves of average percent inhibition (triplicates) versus sample concentration were generated using data obtained at either 5 min or 20 min with GraphPad Prism 9.2.0.Similar to the end-point assays, the dynamic tyrosinase inhibitory activity of materials was also evaluated using Km) and maximal velocity (Vmax)] of tyrosinase were determined by Lineweaver–Burk plot analysis using various concentrations of l-DOPA  as substrate. To accommodate for solubility issues, the inhibitor concentrations for ScyM were 10, 5, 2.5, 1.25 and 0 μM; for KA the concentrations were 25, 10, 5, 2.5 and 0 μM. The Lineweaver–Burk plot was generated using Graphpad Prism 9.2.0. Experiments were performed in triplicate.The mode of inhibition and inhibition parameters   program in which the docking results were manipulated using the GBVI/WSA dG scoring function that estimates the free binding energy of the ligand from a given orientation. Among the various tyrosinase enzyme crystal structures available in the Protein Data Bank, the  1.02 Å] .4 cells/ml of Roswell Park Memorial Institute (RPMI-1640) medium with standard fetal bovine serum (FBS), leading to 180 μl/well. The cells were then pre-incubated for 24 h at 37°C in 96-well plates before drug was added. Thereafter, with addition of diluted compounds of interest dissolved in DMSO, plates were incubated for an additional 48 h and then stained with MTT  for 25 min, after which the optical densities (OD) were recorded at 630 and 570 nm for each well on a SpectraMax M3 plate reader . Doxorubicin and 1% DMSO in RPMI-1640 without fetal bovine serum were used as positive and negative controls, respectively. Polyethylene glycol (PEG) was used to facilitate dissolution of compounds with limited solubility.The cytotoxicity assay was performed using a protocol previously published . Brieflyp < 0.05 were considered to be statistically significant.Tyrosinase inhibitory scores of the best candidates from the broad screening were analyzed using ordinary one-way analysis of variance (ANOVA) on Graphpad Prism 9.2.0. The Tukey–Kramer multiple comparison post-hoc test was used to determine the significant difference among groups. Scytonemin, the pigment material found in the sheaths of many species of cyanobacteria, is a potent UV-absorbing metabolite that is used as a defense against UV radiation, and is often referred to as a “cyanobacterial sunscreen pigment” due to this property . Additioebo genes in a recent study, in which the ebo genes were proven to be responsible for the export of the final precursor from the cytoplasm to the periplasm, the location where the final steps of scytonemin biosynthesis takes place  as seen in O-methyl analog 4 in 50 value for comparison with other inhibitors, (2) using Lineweaver-Burk plots for kinetic analysis on its mode of inhibition, and (3) deducing its potential positioning inside the tyrosinase binding site through in silico molecular docking.The biological activities of scytonemin have been reported in a number of reports since its discovery and characterization. It has been shown to possess anti-inflammatory, anti-tumor and calcium antagonistic properties by different research laboratories , in addi50 = 5.4 μg/ml = 37.9 μM). These experiments established that the protocol was working properly, and thus formal screening of natural product samples could proceed.Before the formal screening of our samples, several test trials were performed using the positive control KA so as to validate the experimental design and evaluate the performance of the reagents. The validating experiments used the same experimental setup as for the dose-dependent assay experiments, with KA being tested at various concentrations . After raw data processing, the inhibitory activity of KA at various concentrations see  was compFor the broad screening campaign, over 4,500 extracts and fractions and all 169 pure compounds in the UCSD Marine Natural Product library were tested in the assay. Among all of these samples, only six passed our pre-set criterion of 20% inhibition, with scytonemin monomer (ScyM) being the only pure compound. By comparison, the KA control showed >60% inhibition at 10 μg/ml see . The fiv1) and KA (3) suggested that additional experiments were required to evaluate their relative potencies, and this required the chemical synthesis of additional ScyM (1) as detailed below.Although multiple re-tests of ScyM all showed that it retained >20% inhibition, because ScyM is a colored material, there was concern that its pigment nature could interfere with the colorimetric assay. Therefore, the OD shifts for six different conditions over 20 min were monitored to prove ScyM’s promising inhibitory activity was a real inhibition and not the result of pigment absorption see ,D,F, conm/z 275, while the MS2 spectrum showed a detailed pattern and suggested a potential [M-28]+ fragmentation pattern (2 pattern matched perfectly with previously recorded high resolution MS data when ScyM (1) was first produced, which was also performed in the positive ion ESI MS mode  showed a parent peak at  pattern S7. This MS mode . We hypoble ScyM .m/z 123 peak eluting at RT = 2.78 min was recognized to be remaining 4-hydroxybenzaldehyde (5) that was used as starting material in ScyM’s synthesis. As a result, a further HPLC purification was performed using ~10 mg of crude ScyM dissolved in MeOH, and yielded 4.4 mg of pure material. This purified ScyM was assayed by LCMS and showed a much better purity profile with a clean m/z 275 peak plus fewer and less intense impurity peaks  provided additional validation of its structure  were used as internal chemical shift references. Additionally, 2D NMR spectra including HSQC and HMBC were performed to unequivocally assign carbon and proton atoms (1), and thus allowed for further biochemical and bioassay studies.The NMR data for synthetic ScyM  met our expectation in that the inhibition was more complete upon increasing concentrations (l-DOPA substrate. This suggested that ScyM might reach its saturation point in the assay system at a concentration less than 75 μM. ScyM’s poor solubility in water or buffer is in agreement with previous reports (1).The inhibition of tyrosinase by KA , HPLC purified ScyM material was used at concentrations from 0 to 10 μM. As a result, ScyM was calculated to have a lower molar IC50 value compared to KA , which is the only other reported tyrosinase inhibitor from a cyanobacterial source . Next, different concentrations of ScyM and KA with a 1:2.5 ratio were mixed and tested, and the IC50 of the mixture was determined as 2.05 μM ScyM +5.12 μM KA. With these IC50 values, the isobologram for ScyM and KA was constructed  and KA (3). An interaction index (γ) has been used to help better understand and mathematically describe the strength of the interacting effect of drugs  and maximal velocity (Vmax), are also visualized in this plot. The x-intercept represents −1/mK and the y-intercept represents 1/Vmax . Interestingly, a mixed-mode of inhibition has been frequently observed in kinetic studies of mushroom tyrosinase inhibitors , its O-methoxylated analog ScyM-OMe (4), the positive control KA (3) and the natural substrate of tyrosinase l-DOPA (see Molecular docking simulations can be used to predict the favored orientation of ligands toward target protein(s) to form stable complexes . In this studies . Using MDOPA see , as descDOPA see . The outl-DOPA. The structure of l-DOPA was extracted directly from the Protein Data Bank (PDB: 4P6S). According to the best predicted conformation of the L-DOPA substrate inside the binding pocket .After validation of docking accuracy using tropolone as described in the Methods section, the docking experiments started with the natural substrate g pocket , the depg pocket . Other i3), was found to form a metal contact with the copper ion and to form a hydrogen bond with Met280  in mushroom tyrosinase was modeled, and the binding site was found to be close to that of the natural substrate l-DOPA. ScyM was also predicted to form a metal contact with the copper atom at the catalytic site  and diphenolase  activities. Considering the similarity of the phenol head of ScyM which is shared by l-tyrosine . Therefore, a dose-dependent assay was also performed for scytonemin. Curiously, the results revealed that at all concentrations tested, from 1 up to 200 μM, there was almost no inhibition by scytonemin (2) molecule is too large to enter the catalytic site of tyrosinase and thus can no longer participate in ligand interactions with surrounding residues as does ScyM (1).The tyrosinase inhibitory properties that we found for ScyM led us to wonder if similar properties would be present for its dimeric structure, the cyanobacterial sunscreen pigment scytonemin  is predicted to interact with Phe264 and Val248 residues of tyrosinase, both of which are quite distant from the catalytic copper-binding pocket and the conserved histidine residues  and ScyM (1) show similar activities in the cytotoxicity assay. Our cytotoxicity assay revealed ScyM to be non-cytotoxic, as no significant effect was observed with H460 human lung cancer cells at concentration as high as 254 μM. Scytonemin was also reported to be non-cytotoxic in several research reports  to possess excellent in vitro mushroom tyrosinase inhibitory activity. Its inhibitory activity is greater than that of the commercially available tyrosinase inhibitor standard, kojic acid , as well as many other tyrosinase inhibitors reported in the literature. We also revealed the ScyM retains slowly reversible mixed-type inhibition against mushroom tyrosinase. It was also shown that there is a modest level of synergistic effect between ScyM and KA regarding mushroom tyrosinase inhibition. On the other hand, the O-methoxylated analog of ScyM (4) and its dimeric form the cyanobacterial sunscreen pigment scytonemin (2), both showed complete loss of inhibitory activity in vitro. Overall, we successfully identified ScyM as the second case ever of a novel tyrosinase inhibitory compound based on a marine cyanobacterial natural product. Together with the fact that it is non-cytotoxic, ScyM can be a promising candidate for applications such as a skin-whitening agent or an adjuvant therapy for melanoma treatment. We hope this work opens the possibility that future research on other cyanobacterial-derived natural products, as well as some mycosporine-like amino acids (MAAs), will reveal additional anti-tyrosinase compounds with promising pharmaceutical and cosmeceutical value.From a screening campaign of marine algal and cyanobacterial extracts, fractions and pure compounds using a colorimetric assay, we discovered scytonemin monomer  and NSF-CHE-2102225 (TS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A‐ergic inhibition, within the immobilized hemisphere only in the immobilization group. Previous research has demonstrated that decreases in GABAA‐ergic inhibition are necessary for the induction of LTP‐like plasticity in M1; therefore, decreased intracortical inhibition after short‐term arm immobilization may provide a novel mechanism to enhance the capacity for LTP‐like plasticity within M1 and may be a potential target for strategies to augment plasticity capacity to enhance motor learning in health and disease.Previous research has suggested that short‐term immobilization of the arm may be a low‐cost, non‐invasive strategy to enhance the capacity for long‐term potentiation (LTP)‐like plasticity in primary motor cortex (M1). Short‐term immobilization reduces corticospinal excitability (CSE) in the contralateral M1, and interhemispheric inhibition (IHI) from ipsi‐ onto contralateral M1 is increased. However, it is unclear whether reduced CSE and increased IHI are associated with changes in intracortical inhibition, which has been shown to be important for regulating neuroplasticity in M1. The current study used transcranial magnetic stimulation to evaluate the effects of short‐term (6 h) arm immobilization on CSE, IHI, and intracortical inhibition measured bilaterally in 43 neurotypical young adults (23 immobilized). We replicated previous findings demonstrating that immobilization decreased CSE in, and increased IHI onto, the immobilized hemisphere, but a significant change in intracortical inhibition was not observed at the group level. Across individuals, decreased CSE was associated with a decreased short‐interval intracortical inhibition, an index of GABA Previous research has suggested that short‐term immobilization of the arm may be a low‐cost, non‐invasive strategy to enhance the capacity for long‐term potentiation (LTP)‐like plasticity in primary motor cortex (M1). The current study used transcranial magnetic stimulation (TMS) to evaluate the effects of short‐term (6 h) arm immobilization on M1 excitability and cortical inhibition, in 43 neurotypical young adults. We replicated previous findings demonstrating that immobilization decreased M1 excitability and increased IHI onto, the immobilized hemisphere suggesting that decreased intracortical inhibition after short‐term arm immobilization may provide a novel mechanism to enhance the capacity for LTP‐like plasticity within M1. AvanzinA‐ergic processes and assessed with paired pulse TMS , has been found to be decreased  arm immobilization on intra‐ and inter‐cortical circuit excitability compared to an equivalent period without immobilization. We used a battery of TMS22.1Forty‐four healthy participants  aged 18–35 years old (mean = 24.5 ± 4.7 years) participated in this study. Inclusion criteria included (1) no history of movement impairment or neurodegenerative disease, (2) right‐handedness according to the Edinburgh Handedness Scale  electrodes were placed in a belly‐tendon montage to measure TMS‐motor evoked potentials (MEPs) of the FDI muscles during all TMS assessments. Additionally, a 30 mm plate ground electrode was affixed over the dorsum of the hand. The skin under each electrode was prepared using sandpaper and alcohol wipes to ensure optimal contact between the skin and the electrodes. EMG data were sampled using a 16‐channel bipolar system  at a rate of 5000 Hz, across a range of ±16.384 mV, and band‐pass filtered at 10–1000 Hz.2; MagStim) were delivered in the posterior–anterior direction using a 70 mm figure‐of‐eight coil. Once an MEP was elicited at a given stimulation intensity, the coil was systematically moved to determine the scalp location resulting in the largest MEP at that intensity. Then, the resting motor threshold (RMT) was determined for the M1 hotspot in each hemisphere using the ML‐PEST method  was used to register surface head landmarks to an MNI template image. The hand representation in the posterior bank of the precentral gyrus M1) was identified manually and used as the starting point for motor hotspot identification for the target FDI muscle. Monophasic TMS pulses . Paired pulse (PP) paradigms involved a conditioning stimulus followed by a test stimulus at a given interstimulus interval  out of the 43 included participants were randomly assigned to undergo arm immobilization. These individuals were instructed to wear a finger control mitt on the left (non‐dominant) hand that secured positioning of the fingers in a padded mitt to restrict finger movement and sensory input. In addition to the hand mitt, the arm was placed in a sling to reduce movement of the wrist, elbow, and shoulder joints. Participants were instructed to move their left arm as little as possible during the immobilization period but to use the non‐immobilized arm as they would normally. The other 20  participants were randomly assigned to be in the No Immobilization control group. These individuals did not wear a mitt or a sling and were told to use their arms normally throughout the day. Activity monitors  were worn on both wrists by all participants to measure bilateral arm movements between sessions and determine compliance with the immobilization procedure.2.4p‐values between 0.05 and 0.10 were considered non‐significant trends in the data. Data are presented in figures as change for visualization purposes. All data met the assumptions of normality and homogeneity of variance.A custom Matlab (2016a) script was used to automatically identify the minimum and maximum peak amplitudes of the TMS‐evoked response in the FDI to calculate the peak‐to‐peak amplitude for all MEPs. Each MEP was also confirmed visually. Mean MEP amplitude for each TMS assessment was analyzed before and after immobilization, and between‐group analyses were performed to examine changes in excitability between those who were immobilized compared to those who were not immobilized. Mixed‐effects analyses, within‐subject factors of time (AM [pre‐immobilization] or PM [post‐immobilization]) and hemisphere (target or nontarget) were performed for each group (control or immobilization group) to evaluate within‐group effects of immobilization. To assess associations between immobilization effects on different outcome measures within individuals, we performed bivariate correlation analyses with Pearson's correlation coefficients between changes in CSE with changes in interhemispheric and intracortical inhibition, adjusted for multiple comparisons. A mixed‐effects analysis was performed to determine the effects of arm (target or non‐target) and group (control or immobilized) on activity counts and assess compliance with the immobilization protocol. Prism GraphPad 8 statistical packages were used for all statistical analyses. Significance level was set as α ≤ 0.05. Non‐significant 33.1F = 163.7, p < 0.0001) and group , and there was a significant Group × Arm interaction  on wrist activity counts .A mixed‐effects analysis showed significant effects of arm  on RMT, where RMT was higher in the target hemisphere in both groups across testing sessions. Additionally, there was a non‐significant trend for an effect of time , where RMT increased from the AM to the PM session. There was no effect of group  on RMT.A mixed‐effects analysis showed a non‐significant trend for an effect of hemisphere , and there was a significant effect of immobilization on the change in CSE in the immobilized group , and SIHI was greater onto the nontarget hemisphere . In the immobilized group, there was a significant effect of hemisphere with higher IHI onto the non‐target hemisphere across test sessions .In both IHI conditions, control participants showed a bilateral release of interhemispheric inhibition over the course of the day, with less IHI observed at the PM session compared to the AM session. However, immobilization led to an increase in inhibition from the nontarget hemisphere onto the target hemisphere. Additionally, the decrease in inhibition from target to nontarget hemisphere observed in controls was blocked for both short interhemispheric inhibition (SIHI) and long interhemispheric inhibition (LIHI) Figure . AnalyseF = 25.2, p < 0.0001). Additionally, immobilization significantly modulated the change in LIHI over the course of the day   and was greater onto the nontarget hemisphere  in control participants. In the immobilized participants, LIHI was also greater onto the nontarget hemisphere .For LIHI, a mixed‐effects analysis demonstrated greater IHI onto the nontarget hemisphere (Hemisphere: ) Figure . Analyse3.5F = 3.7, p = 0.06) and an interaction between time and hemisphere  on SICI  and did not change throughout the day  in non‐immobilized control participants. In immobilized participants, SICI was not significantly different between hemispheres , but there was a non‐significant trend for lower SICI in the target hemisphere at both time points .A mixed‐effects analysis showed non‐significant trends for differences in SICI between hemispheres , and there was a non‐significant trend for an interaction between time and hemisphere  on LICI  , IHI10 , or IHI50  in the target hemisphere in immobilized individuals. In the control group, change in CSE was not significantly correlated with change in any measure of inhibition .Our results demonstrated that change in SICI was negatively correlated with change in CSE  to assess whether the downscaling of excitability within M1 is specific to inhibitory circuits or if it is indeed a more global phenomenon.Changes in the activity of other regions that are interconnected with M1 may also contribute to the decrease in CSE following short‐term immobilization. Given the observation of profoundly reduced movement in the immobilized arm, which necessarily reduces both motor output and sensory input, one potential cortical region of interest is the primary somatosensory cortex (S1). Previous research has demonstrated that M1 and S1 are highly interconnected, and these connections strongly influence motor behavior  and independent samples t‐tests demonstrated that there was not a significant difference between males and females in the outcome measures at baseline . Although, it remains possible that these or other factors may have contributed to inter‐individual variability of the neuromodulatory effects of immobilization.Measures of spinal excitability were not collected in the current study which may also have influenced TMS‐based assessments of CSE. It has been demonstrated that longer durations of immobilization  led to an increase in spinal excitability (Lundbye‐Jensen & Nielsen, 5A‐ergic intracortical inhibition, suggesting that short‐term arm immobilization may lead to a global downscaling of synaptic strength in M1. Reduced intracortical inhibition may provide a novel mechanism by which short‐term immobilization could enhance the capacity for LTP‐like plasticity within M1 and may be a potential target for strategies to augment plasticity capacity to enhance motor learning in health and disease.Overall, our results demonstrate that immobilization reduces CSE and increases IHI, consistent with our hypotheses and previous findings. Interestingly, decreased CSE was significantly associated with a decrease in an index of GABAThe authors have no conflicts of interest to disclose.


Compared with traditional pedicle screw trajectory, cortical bone trajectory (CBT) increases the contact surface between the screw and cortical bone where the screw is surrounded by dense cortical bone, which does not deform remarkably due to degeneration. We aimed to provide detailed information about the improvement of three-dimensional (3D)-printed navigation templates for modified CBT screw placement in the lumbar spine and evaluate the safety and accuracy thereof.Four human cadaveric lumbar spine specimens were selected. After CT scanning data were reconstructed to 3D models, either the left or right side of each specimen was randomly selected to establish a 3D-navigation template, mutually complemented with the surface anatomical structure of the lateral margin of the lumbar isthmus, vertebral plate, and spinous process. The corresponding 3D centrum was printed according to the CT scanning data, and a navigation template of supporting design was made according to modified cortical bone technique. The same template was used to insert CBT screws into 3D printed and cadaveric specimens. After the screws were inserted, the screw path of the 3D printed specimens was directly observed, and that of the anatomical specimens was scanned by CT, to determine the position and direction of the screws to analyze the success rate of screw placement.Twenty cortical bone screws were placed in each of the 3D printed and anatomical specimens, with excellent rates of screw placement of 100% and 95%, respectively.We report the easy, safe, accurate, and reliable use of a 3D-printed navigation template to carry out screw placement by modified cortical bone technique in the lumbar spine. Cortical bone trajectory (CBT) was a new lumbar screw trajectory proposed by Santoni in 2009 . CompareThe current traditional CBT technique still has imperfections reported in previous clinical, imaging, and anatomical studies , 12. To Recently, 3D printing techniques have been applied in orthopedic clinics , 15, shoThe experiments were completed in the Anatomy Teaching-Research Office and the Department of Spine Surgery at the Orthopedic Center of Xinjiang Medical University from April 2019 to June 2022.Four intact human wet cadaver lumbar specimens , aged 61–77 years (average: 71 years), were provided by the Anatomy Teaching-Research Office of Xinjiang Medical University , 14. TheHigh-resolution computed tomography (CT) data  was performed on the four lumbar specimens. In Mimics 19 , the CT scanned original data (DICOM format) underwent a reverse reconstruction operation. First, the coronary section at the midpoint of the long axis of each lumbar pedicle was taken as a hypothetical dial, to select the position of screw placement . In our The lumbar segment was split layer by layer, and single vertebra data (STL format) were imported into Geomagic Studio  for model repair and then into Z Brush for model reconstruction and optimization with the DynaMesh tool. After exporting the OBJ document, the data were imported into MAYA software  using the polygons module for the design. The designed pedicle implanted screw trajectory and Kirschner wire fixation hole were produced and performed, the split model set was exported as an OBJ document, and imported into Geomagic Studio. The final navigation template pattern was obtained and finally imported into MIMICS 19 to perform a Boolean calculation with the vertebra, under a tolerance of 0.3 mm, to complete the design of the guide hole of the navigation template screw.In the Matic-3 STL 10 software , the Wave Brush Mark tool was used to extract the anatomy data required for the margin of the spinous process basilar part, lateral margin of the lumbar isthmus, and vertebral plate superficial structure. This was skewed by 2.0 mm, and the model was exported from STL to Geomagic Studio. Simultaneously, the screw trajectory model, navigation template grip, and Kirschner wire fixation hole manufactured by MAYA were imported to perform the Boolean calculation. Finally, the screw trajectory of the navigation template and the trimmed boundary were connected, and the design and manufacture of the navigation template was complete.A CT scan was performed on the four anatomy specimens. After 3D reconstruction of data in Mimics 19, each intact lumbar vertebra model was inputted into a 3D printer, MBot Grid2 , to print in 1:1 proportion with anatomical specimens. This was then attached to the 3D-printed navigation template, so as to facilitate follow-up improvement and the experimental operation of screw placement.All screw placements in specimens were performed by spine surgeons without any experience of lumbar cortical bone screw placement. One side of each specimen was randomly selected to place each screw. To avoid wasting the specimens, a preliminary experiment was performed on the 3D printed vertebra. The navigation template was optimized and updated and, once confirmed to be accurate, safe, and reliable, the final test was performed on the anatomical specimens.The navigation template screw placement processes on the 3D printed vertebra and anatomical specimens were as follows. First, the navigation template, with the isthmus lateral margin, vertebral plate, and spinous process as anatomic landmarks, was attached to the corresponding vertebra. The fit between the navigation template and the above-mentioned skeletal anatomical structure of the related vertebra was confirmed. We initially found that, when using a drill or Kirschner wire to drill through the guide hole of the navigation template, the template shook significantly during the rotation, whether held by the surgeon alone or with an assistant. This may have been caused by the small volume and weight of the navigation template itself and the small area held by the operator and may affect the accuracy of cortical bone screw placement. To solve this, a Kirschner wire was used to temporarily fix the navigation template, the attachment carefully checked, and a 2.7-mm drill used to trepan prior to screw placement. We drilled with an electric drill along the guide hole, to a depth of 35.0/40.0 mm, depending on the screw length. A 4.5-mm screw tap was used to extend the screw trajectory, and a probe was used to gently confirm the screw trajectory. Once the walls of the hole were confirmed to be smooth and continuous, a 4.5 × 35.0/40.0 mm titanium alloy lumbar back cortical bone screw was inserted.In total, 40 specific cortical bone screws were placed in the corresponding specimen, with 20 placed in the 3D printed vertebra and the remaining 20 placed in human anatomical specimens.Due to the absence of current criteria for evaluation of CBT screw placement, a less than ideal alternative was adopted. The evaluation criterion was scored as previously described : grade IAfter the completion of screw placement, a CT scan was repeated under the same conditions. The scan was imported into Mimics 15.01 to evaluate the success rate of lumbar posterior cortical bone screw placement.α = 0.05 on both sides, and P < 0.05 indicated a statistical significance.We choose the random matching design, because the statistical efficiency of this design will be higher, hoping to make up for the small sample size of this study. The specific measures are as follows: the order of anatomical specimens is determined by simple random sampling one by one without putting back by drawing lots . After sorting, the anatomical specimens are selected in order, and two numbers are simply and randomly selected from 0 to 100.If the first number is greater than the second number, we use the modified CBT method to place the nail on the left side of the corpse. If the first number is less than the second, the right side of each corpse is fixed with the modified CBT method. Follow this method until the completion of all four specimens of one side screw implantation. The data were collected and analyzed with SPSS 17.0 . Statistical analyses (chi-square test) were used to evaluate success rates of screw placement among groups. The test criterion was Approval was obtained from the ethics committee of the Anatomy Teaching-Research Office of Xinjiang Medical University. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.P > 0.05).When the screws were inserted into the printed and anatomical specimens with the aid of the 3D printed navigation template, we observed good process and stability of the navigation template. By visual inspection, all 20 screws placed in the 3D printed template were grade I, with a favorable rate of 100%. Of the 20 screws placed in the human anatomical specimens, the CT scan results showed that all except one screw placed on the right of L2 of one specimen were completely placed inside vertebral canal. This screw was evaluated as grade III; all other screws were evaluated as grade I, with an excellent rate of 95% . The ratTwenty cortical bone screws were placed in each of the 3D printed and anatomical specimens, with excellent rates of placement of 100% and 95%, respectively. Our 3D-printed navigation template successfully aided easy, safe, accurate, and reliable screw placement.At present, the clinical application of 3D-printing techniques mainly involve the manufacture of a physical model, printing of surgical auxiliary materials, or printing of implants , 19–21. Compared to traditional pedicle screw techniques, the insertion point of the cortical bone screw technique is closer to the spinal canal, so it requires higher screw placement skill of the clinicians, and surgical staff and patients both receive larger doses of x-ray radiation . performA series of improvement measures were also taken in the design of the navigation template, which differ from other traditional cortical bone screw 3D placement navigation templates. First, to increase 3D navigation template stability in screw placement, we referred to other template designs, and added the contact area between the navigation template and vertebral plate bone surface, to enable improved attachment. Meanwhile, the lateral arms on both sides of the navigation template were connected through the top beam structure in the middle, which is also a method to increase the contact area between the navigation template and bone, increase the stability of the navigation template when drilling, and prevent offset. We also designed a wider holding platform on top of both lateral arms, allowing the surgeon to easily hold the template to place the screw. By applying downward force with the hands, the navigation template attached more closely and tightly with the vertebral plate surface, which is good for follow-up temporary fixation with a Kirschner wire. In all our screw placements, only one placed into the L2 spinal canal of an anatomical specimen was evaluated as grade III. A follow-up improvement will be to increase the guide hole integrity to cover at least 3/4 of the diameter; at least 270° around the sleeve and drill. In cases where the spinous process hinders the side wall of the guide hole, the length of the guide hole will need to be reduced as much as possible. Even if the guide hold is 5-mm short but still complete, with a stable work sleeve and follow-up screw placement, the screw can be inserted in the appropriate direction, avoiding placement error. If the spinous process is blocked, we consider thinning the bone on both sides of the spinous process to make the guide plate adhere to the bone surface to the greatest extent. If it is still ineffective, the bone of the lower 1/3 spinous process can be removed and the proximal bone can be retained as much as possible, so as to protect the attachment of the supraspinal ligament as much as possible and increase the local stability. Modern medicine is becoming more individualized, precise and digital. We can choose CBT or MCBT according to the the exact level or position of the lumbar spine and preoperative imaging data of patients, and even combine the two .Second, due to the supraspinous ligament, we designed the left and right arms of the 3D navigation template and middle top beam in the form of an arch bridge. This may help the surgeon and assistant to observe when the Kirschner wire is inserted from both sides, preventing inserting the screw too deep or even penetration into the vertebra to cause a neurological function lesion. The arch shape design is also good for mechanical stability of the navigation template. Furthermore, we increased the thickness of the side arms of the navigation template, so that it remains stable when the Kirschner wire penetrates the spinous process from one side. In addition, the height of the arch can be designed according to the thickness of the supraspinous ligament, enabling the 3D navigation template to straddle the spinous process and the supraspinous ligament above it. This helps to maintain distance with the supraspinous ligament; thus, the bottom of the side arms of the template can be well attached to the surface of the vertebral plate, rather than suspended , 25.Third, it was found that, due to the small size of the 3D navigation template, during intraoperative guiding, it was not convenient for the surgeon or assistant to hold the template for a long time, and the rotation of the drill may change its direction at any time. To solve this problem, we preserved two fine holes in the navigation template use a 2-mm Kirschner wire for temporary fixation. The original design, to use one Kirschner wire to fix the template on the spinous process, cannot provide a solid attachment between the template and the vertebral plate surface, and the navigation template still shakes during drill rotation. As improved with time, the final Kirschner wire fixation scheme adopted a cross placement method at two different planes  to temporarily fix the 3D navigation template solidly at multiple planes. As we are operating through a small incision, the angle of the two crossed Kirschner wires in the coronal plane should be small as possible; if this angle is too large, it hinders placement of the wire. The ends of the Kirschner wire should cross properly in the sagittal plane, so that it may smoothly enter the middle of the side arms on both sides of the navigation template, increasing navigation template stability. We eventually improved the navigation template material and used transparent macromolecule polyethylene, so that the surgeon could observe the wire in detail, to prevent inserting it too deep.Fourth, during practical guiding using the navigation template, we discovered that, because the navigation template is made from macromolecule polyethylene, the direction of drilling may deviate when the inner wall of the guide hole makes contact with the drill. This may also be caused by the hardness of the navigation template material, reducing navigation template accuracy. The only way to prevent this is to further increase the strength of the inner wall of the guide hole, making it strong enough to prevent drill deviation. We improved the inner wall structure of the guide hole in the navigation template by placing a stainless steel cannula used for limb fracture fixation inside it, and the problem of deviation was solved. This also solved the problem of the numerous chippings generated during drilling in the original inner wall of the navigation template. These chippings are likely to enter the vertebra of a patient during drilling, causing immunological reactions. In addition to the metal cannula, we found that a series of puncture tools used in the treatment of balloon dilation for spinal compression fractures can combine with the design of this navigation template, to make screw placement safer and more reliable .However, the metal sleeve used in limb fractures and bone cement forming surgery has complicated surgical steps: the drill bit and wire are used to approach the opening in the vertebral body, the Kirschner wire and 3D navigation template are removed, and the screw inserted. A relatively simple solution is to use a hollow screw to implant the drill bit along the previous opening. In a previous study, a disposable 3D navigation template made of stainless steel or alloy was also considered, but was not appropriate because of its high production cost and long production time. Combined with the knowledge from previous studies, and the operation experience of screw placement, we designed a detachable screw placement tool that can provide the inner wall strength of metal and is suitable for minimally invasive implantation. By seamless connection with the guide hole of the fifth generation 3D navigation template, this ensures that the screw is inserted accurately in the direction of the guiding hole, with a metal-to-metal interface and without any debris . With thIn summary, the cortical bone screw trajectory technique is a new lumbar posterior internal fixation technique, with improvements and a screw trajectory that differs from traditional pedicle screws where the failure rate is always high and depends upon the manual screw placement experience of the surgeon. We designed a safe insertion angle, screw diameter, and other indexes through 3D reconstruction and reverse engineering techniques, and utilized a 3D printing technique, to verify the anatomical vertebra and navigation template. We proved that our technique makes it easier and safer for spine surgeons without any experience to place screws using a navigation template. In clinical practice, our 3D printed navigation template and special tools can further improve the accuracy and safety of modified cortical bone screw placement.


Occipitocervical fusion is an effective surgical method for treating various upper cervical disorders. However, complications such as implant failure due to rod breakage have been reported. Therefore, we devised a surgical technique for occipitocervical fusion with a triple rod connection to prevent implant failure. Occipitocervical fusion with triple rod connection was performed in two cases with a high risk of instability such as athetoid cerebral palsy and rheumatoid arthritis. A multiaxial screw (diameter: 4.5 mm) was inserted into the screw hole in the middle of the occipital plate, and subsequently, an additional rod was attached. It was connected to the main rod using an offset connector at the caudal side. The connection of the additional rod was simple and did not interfere with the fusion bed for bone graft between the occipital bone and axis. The head of the screw was crimped to the occipital plate, and the plate was firmly fixed. Moreover, since the head of the screw did not protrude to the dorsal side, the tension of the soft tissue and skin did not increase. No complications occurred after surgery in both cases. In addition, no special instruments were required to connect the additional rod to the main rod in this procedure. Therefore, our technique may be useful as an option to prevent implant failure due to rod breakage at the craniocervical junction. Occipitocervical fusion is an effective surgical method for treating various upper cervical pathologies such as degenerative, traumatic, neoplastic, congenital, and infective lesions ,2. PreviSurgical technique of occipitocervical fusion with triple rod connectionThe instrument used was VuePoint Ⅱ . Depending on the case, pedicle, laminar, lateral mass, and paravertebral foremen screws were used . An occiOccipitocervical fusion with triple rod connection was performed in two patients with severe instability of the upper cervical spine after confirming that the placement of the additional rod could be easily performed using model bone Table . The patCase 1A 61-year-old man with athetoid CP visited our hospital with complaints of clumsiness of both hands and gait disturbance that worsened over several months. The Japanese Orthopaedic Association (JOA) score was 4 points. Posterior cervicothoracic fusion (C1-T1) and decompression (C3/4) were performed for myelopathy with the instability of C1/2 and C3/4. Three months after the operation, finger movements improved, and gait supported with a walker became possible (JOA score: 10.5 points). However, six months after the operation, neck pain, clumsiness of both hands, and gait disturbance worsened again (JOA score: 8.5 points). Bilateral C1 screw breakages were observed, and the recurrence of myelopathy due to atlantoaxial subluxation (AAS) was diagnosed Figure , 3B. AftCase 2A 68-year-old woman with a history of RA for 25 years and osteoporosis (young adult mean: 62%) was referred to our hospital for limb weakness and gait disturbance that had worsened over several months (JOA score: −0.5 points). Myelopathy with not only AAS but also spinal instability and cord compression at C3/4 was diagnosed, and posterior decompression (C1 and C3/4) and occipitocervical fusion (Oc-C7) were performed Figure . After tIn both cases, the additional rod was connected in the midline; therefore, the paraspinal muscle detachment could be limited to the extent necessary to place the occipital plate and cervical screws. The head of the MA screw was crimped to the occipital plate; therefore, the occipital plate was firmly fixed. The additional rod was easy to connect and did not interfere with the fusion bed of the bone graft between the occipital bone and axis Figure . FurtherWe devised a novel triple rod connection technique for implant reinforcement during occipitocervical fusion. In this study, the procedure was performed as revision surgery. However, it may be also useful as a primary surgery for upper cervical lesions with a high risk of instability.In cervical posterior instrumentation surgery, thick titanium rods with 4.0 mm diameter or cobalt-chromium rods were used to increase fixation strength. However, the devices with 4.0 mm diameter titanium rods are limited. Furthermore, it has been reported that junctional kyphosis may occur more frequently with cobalt-chromium systems than with titanium alloy systems after multiple intervertebral fusions . TherefoIn recent years, a procedure using multi-rod constructs has been reported to prevent rod breakage in long spinal fusion for adult spinal deformity . BecauseIf an additional rod is to be connected to the exterior of the main rod during occipitocervical fusion, the invasion of soft tissue may be increased because of the widening of the range of detachment of paraspinal muscles. In our procedure, of the three screws in the middle of the occipital plate, the longest screw could have been inserted into the most cranial side with a thick bone cortex. However, the head of the screw might protrude to the dorsal side, causing skin and soft tissue problems. Therefore, the MA screw was inserted into the central screw hole. In both cases, a screw with a length of 10 mm or more could be inserted, and the occipital plate was firmly fixed. Since the additional rod was placed in the middle and had a low profile, this procedure was considered useful for older patients or patients with RA who had fragile soft tissue and skin.This study has several limitations. First, there are no biomechanical data to demonstrate the superiority of the mechanical strength of the construct in this technique compared to that in the conventional method; therefore, it will be investigated in the future study. Second, the instruments used in this study had good compatibility with the MA screw and occipital plate. However, it is necessary to investigate whether this procedure can be performed using other instruments. Third, the length of the MA screw inserted into the occipital bone was the shortest at 10 mm. However, there may be patients in whom this procedure cannot be performed because of the risk of protrusion of the screw into the skull depending on the shape of the occipital bone. Therefore, it is important to create an appropriate preoperative plan using computed tomography.Occipitocervical fusion with triple rod connection was performed for upper cervical lesions with a high risk of instability. In this procedure, no special instruments were required to connect the additional rod to the main rod without affecting the fusion bed of the bone graft, and no adverse events such as skin or soft tissue problems occurred. Therefore, this technique may be useful as an option for both primary and revision surgery to prevent implant failure due to rod breakage at the craniocervical junction.


Garcinia mangostana. In previous studies, xanthones were reported to be highly active inhibitors of FAS. In the present study, the detailed inhibitory mechanism of garcinone E on FAS was investigated. We found that garcinone E inhibited the activity of FAS in a concentration-dependent manner with a half-inhibitory concentration value of 3.3 μM. The inhibition kinetic results showed that the inhibition of FAS by garcinone E was competitive with respect to acetyl-CoA, mixed competitive and noncompetitive with respect to malonyl-CoA, and noncompetitive to NADPH. In addition, garcinone E showed irreversible inhibition on FAS, which was different from all other xanthones. Since FAS is believed to be a therapeutic target for obesity and cancer treatment, these findings suggest the clinical potential of garcinone E in the prevention and treatment of both obesity and cancer.Fatty acid synthase (FAS) is highly expressed in human adipocytes and cancer cells and is considered as a dual therapeutic target for obesity and cancer treatment. Garcinone E is a natural xanthone and exists in the pericarp of  Garcinone E exhibits both fast-binding reversible and time-dependent irreversible inhibition on the activity of fatty acid synthase. However, human adipocytes and cancer cells express high levels of FAS and undergo significant endogenous fatty acid synthesis.5,6 In adipocytes and cancer cells, the functional connection between FAS and cell proliferation, lipid deposition, progression, aggressiveness, and metastasis has been provided by the discovery and design of small molecule drugs that inhibit the catalytic activity of FAS.7–10 Previous studies have shown that FAS inhibitors such as C75, cerulenin and epigallocatechin gallate (EGCG) lead to weight loss, preadipocyte proliferation inhibition, and cancer cell apoptosis.11–13 Therefore, FAS deemed as a potential bi-target for anti-obesity and anti-cancer drugs.14–17Fatty acid synthase  catalyzes the synthesis of saturated long-chain fatty acids, predominately palmitate, from acetyl-CoA (Ac-CoA) and malonyl-CoA  precursors, in the presence of NADPH.H-xanthen-9-one) is a xanthone which exists in the pericarp of Garcinia mangostana.18 Experimental results have demonstrated that the xanthones isolated from G. mangostana exhibit antioxidant, antitumoral, antiallergic, anti-inflammatory, antibacterial, and antiviral activities.19–21 However, the literature related to the application of garcinone E in prevention and treatment of obesity or cancer is limited. It was reported that garcinone E induced significant cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cell lines, as well induced apoptosis and inhibited migration and invasion in ovarian cancer cells.22,23 Nevertheless, the precise mechanisms involved are still not demonstrated clearly.Garcinone E -9G. mangostana, has been found strong inhibitory activity on FAS overall reaction. Compared with the wellknown FAS inhibitors such as C75 and EGCG, garcinone E showed stronger inhibitory activity.18 In the present study, we further investigated the inhibitory kinetics of garcinone E on FAS activity.In previous studies, garcinone E, as well as other xanthones isolated from the pericarp of 2.2.1Garcinone E (98% HPLC), Ac-CoA, Mal-CoA, NADPH, ethyl acetoacetate, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Other reagents were of analytical grade.2.224 The amino acid sequence of chicken FAS has 63% identity with the sequence of human enzyme.25 All animal operations followed the Guidelines for the Care and Use of Laboratory Animals established by the National Research Council (US) Institute for Laboratory Animal Research. The purified FAS was homogenized by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, respectively. The concentrations of FAS and its substrates were determined by UV-vis spectrophotometer  using the following experimental parameters: FAS, 4.83 × 105 M−1 cm−1 at 279 nm; Ac-CoA, 1.54 × 104 M−1 cm−1 at 259 nm, pH 7.0; Mal-CoA, 1.46 × 104 M−1 cm−1 at 260 nm, pH 6.0; acetoacetyl-CoA, 1.59 × 104 M−1 cm−1 at 259 nm, pH 7.0; NADPH, 6.02 × 103 M−1 cm−1 at 340 nm, and 1.59 × 104 M−1 cm−1 at 259 nm, pH 9.0 26.The preparation of FAS from chicken liver  was performed as described previously.2.32PO4–K2HPO4 buffer, 1 mM EDTA, 1 mM dithiothreitol, 3 μM Ac-CoA, 10 μM Mal-CoA, 35 μM NADPH, and 10 μg FAS in a total volume of 2 ml as previously described.26,27 The assay for β-ketoacyl reduction activity of FAS was catalyzed by KR containing 100 mM KH2PO4–K2HPO4 buffer, 200 mM ethyl acetoacetate, 35 μM NADPH, and 20 μg FAS in a total volume of 2 ml (pH 7.0).The FAS activity was measured at 37 °C by spectrophotometer at 340 nm of NADPH absorption. The overall reaction system contained 100 mM KH2.4Ai and A0 respectively. The value of Ai/A0 × 100% was defined as the relative activity (R.A.) of FAS. The half-inhibitory concentration value (IC50) was obtained from a plot of R.A. versus garcinone E concentration. In the experiments the largest volume added to the reaction was far less than 0.5% (v/v); thus the DMSO existing in the reaction system had no influence on the results of the activity assay.Reversible inhibition was determined by adding the inhibitor to the reaction system before the reaction was initiated. The activities of FAS in the presence and absence of garcinone E (dissolved in DMSO) were designated as 28The activity of KR was determined at 37 °C by measuring the change of absorption at 340 nm. The KR reaction mixture (2 ml) contained 40 mM ethyl acetoacetate, 35 μM NADPH, 1 mM EDTA, 1 mM DTT and 10 μg of FAS in 100 mM phosphate buffer, pH 7.0.13 The FAS solution was mixed with garcinone E and incubated at 25 °C, and then aliquots were taken to measure the remaining activity at the indicated time intervals to obtain the time course. This time-dependent inhibition is usually caused by a chemical combination of the inhibitor with the enzyme, and is irreversible. The rate constant of FAS inactivation can be calculated from a semi-log plot of the time course, which is based upon the formula ln At/A0 = −kobst. The At/A0 expresses the remaining activity at t time, and kobs is the observed rate constant. Based on earlier studies, the inhibition of FAS activity is due to both fast-binding and time-dependent inhibitions, although sometimes the fast-binding reversible inhibition is not potent enough to affect the enzyme.Assay of time-dependent inhibition activity was carried out as previous described.2.5Possible interference by the inhibitor at each substrate binding site was examined by holding the concentration of the inhibitor at several fixed levels respectively, and increasing one substrate concentration while keeping the concentrations of the other substrates constant. Double reciprocal plots for every concentration of the inhibitor were yielded to estimate the competitive relationship between the variable substrate and inhibitor concentrations. This study is based on fast-binding inhibition.3.3.1in vitro. Garcinone E exhibited strong fast-binding inhibition of chicken FAS. The data obtained from The inhibitory activity of garcinone E on overall reaction and KR of FAS was assayed 3.2kobs values were 0.009, and 0.0004 min−1 for overall reaction and KR, respectively. The more than 20-fold difference in the inactivation rate between overall reaction and KR suggested that the irreversible inhibition of garcinone E should be rarely related to KR activity.Garcinone E exhibited time-dependent inhibition of FAS overall reaction. 3.3Y axis, indicating that garcinone E inhibited FAS competitively with Ac-CoA. The dissociation constant for inhibitor binding, Kis of 1.77 μM, was obtained from the secondary plot of the slopes of these lines versus garcinone E concentration. The lines for Mal-CoA , Kii of 5.99 μM, was obtained from the secondary plot of the intercepts versus garcinone E concentrations. However, the lines for NADPH  or from the secondary plot of intercepts versus garcinone E concentrations (Kii of 2.67 μM). The inhibition types and inhibition constants of FAS for every substrate by garcinone E were summarized in Inhibition kinetics study was carried out to elucidate the mechanism of the reversible inhibition of FAS by garcinone E. Possible interference by garcinone E at each substrate-binding site was examined by holding the concentration of garcinone E at a constant value, and measuring the effect of increasing one substrate concentration (the concentrations of the other two substrates were fixed) on the initial reaction rate. Double-reciprocal plots of the results  yielded  Mal-CoA  had a poor NADPH  intersecFurthermore, substrate protection for time-dependent inhibition was examined in the measurement of FAS inactivation by garcinone E with 10 μM Ac-CoA, 10 μM Mal-CoA, or 10 μM NADPH, respectively. Inactivation in the same condition but without any substrate was the control. The result of substrate protection on the inactivation of FAS by 10.0 μM garcinone E was given in 4.in vivo, FAS has been indicated as a feasible target for the treatment of both obesity and cancer. However, no anti-obesity and anti-cancer drugs have been developed from FAS inhibitors so far. It is necessary to discover more safe and effective FAS inhibitors that may be applied practically in treatment of obesity and cancer. The amino acid sequences of human and other animal FASs are very similar,29 however, the human FAS preparations showed lower activity than the FASs of other animals.25 So the chicken and duck FAS were the most commonly used materials for the study of FAS inhibitors. In this study, chicken FAS was used to measure the inhibitory activity as well as the inhibitory mechanism of garcinone E. In the present work, we found that garcinone E was a very potent FAS inhibitor with an IC50 value of 3.3 μM on the overall reaction of FAS, which was much lower than those of cerulenin (IC50 = 89 μM)30 and EGCG (IC50 = 52 μM).13 The strong inhibitory activity of garcinone E towards FAS, plus its edible plants source, opens up excellent prospects for its application as an anti-obesity and anti-cancer agent.In recent years, the morbidity of obesity or cancer is increasing at an epidemic rate. As a key enzyme which can regulate energy metabolism 50 value of β-ketoacyl reduction was 4.57 times higher than overall reaction. Garcinone E showed strong reversible inhibition on the overall reaction of FAS but showed weak inhibition on KR reaction. These results indicated that KR was not the main active site in FAS that attacked by garcinone E, which also was confirmed by kinetic results since garcinone E was a noncompetitive inhibitor of FAS with respect to NADPH.In order to demonstrate the inhibition mechanism of garcinone E on FAS, we further determined the kinetic parameters of FAS. We found that garcinone E could inhibit both FAS and KR domain. However, the ICAs a wellknown FAS inhibitor, EGCG inhibits mainly the KR domain of FAS, and it binds to the same site competitively with NADPH. As for C75 and cerulenin, two other known FAS inhibitors, it has been reported that they inhibit FAS by irreversibly binding to the KS domain of FAS, and that their inhibition of FAS is related to the binding site of the substrate Mal-CoA. However, we found that garcinone E inhibited FAS in a mixed competitive and noncompetitive type with Mal-CoA. Therefore, as regards inhibition mechanism, garcinone E was totally different from the other FAS inhibitors previously reported. Garcinone E possibly affected the KS or the MAT domain of FAS because it inhibited FAS competitively with respect to Ac-CoA, Although the inhibitory activity of xanthones and other polyphenols on FAS was reported before, no slow-binding irreversible inhibition on FAS was found in these compounds. In the present work, we tested the time-dependent inhibitory effects of α-mangostin, β-mangostin, γ-mangostin, whose structures are similar to garcinone E, on FAS. However, none of them showed slow-binding inactivation towards FAS (data not shown). Since garcinone E comprises one more isoprene group compared with the structures of α-mangostin, β-mangostin, and γ-mangostin, the calculated lipid/water partition coefficient of garcinone E (7.9) was much bigger that those of α-mangostin (6.3), β-mangostin (6.6), and γ-mangostin (5.9). Maybe this could explain why garcinone E showed irreversible inhibition on FAS since it was easier to enter the interior of the FAS and combined with the active site of the enzyme because of the hydrophobic effect.11 Cerulenin was found to inhibit adipocyte differentiation and lipid accumulation.31 α-Mangostin was reported to prevent human preadipocytes 3T3-L1 cells differentiation and reduce lipid accumulation as a potent FAS inhibitor. A number of studies have revealed the anti-obesity and anti-cancer effects of FAS inhibitors in human cancer cells and adipocytes.32–34It has been reported that inhibition of FAS could selectively inhibit the growth and proliferation of human adipocytes and cancer cells because FAS activity is vital for the growth and survival of these cells. C75 could obviously reduce body weight and food intake in fat mouse.The potent anti-cancer effect of garcinone E was reported. However, the detailed mechanisms of apoptosis induction and invasion inhibitory effects of garcinone E on human cancer cells were not fully understood. We now speculate that garcinone E possibly performed anti-cancer activity, at least in part, by inhibiting FAS activity. Our present work not only demonstrated that garcinone E was a potent FAS inhibitor, but also revealed the possible mechanism of its inhibitory activity. Since garcinone E exists in edible plants, it might have great security for the human body and have promising application potential. This study may supply some useful idea and new clues in developing drugs in treatment of obesity and cancer.Yan Liang, Hao Wu as the principal investigators, were responsible for the concept and design of the study. Xuan Gao, Yan Liang, Di Luo did the whole experiments of the study, conducted the research and wrote the manuscript. All authors participated in the preparation of, and had approved the final version of the manuscript.The authors confirm that this article content has no conflict of interest.Ac-CoAAcetyl CoAACPAcyl carrier protein;DHDehydraseDMSODimethyl sulfoxideEREnoyl reductaseFASFatty acid synthaseFBSFetal bovine serum50ICHalf-inhibitory concentrationKRKetoacyl reductaseKSKetoacyl synthaseMal-CoAMalonyl-CoAMATMalonyl/acetyltransferaseTEThioesterase


Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease. Protein phosphorylation is the most common and widely studied post-translational modification (PTM) in eukaryotes. Phosphorylation is involved in numerous signal transduction processes including proliferation, differentiation, apoptosis, and metabolic processes that maintain cellular homeostasis and direct physiological responses to stimuli . PhosphoAlthough substantial progress has been made in elucidating signaling events and uncovering therapeutic strategies, many challenges remain in understanding the relationship between signaling dynamics and disease progression. Specifically, the functions of signaling networks are based on the spatiotemporal dynamics that emerge from extensive protein groups and interactions collectively, rather than the simple presence or absence of individual proteins or interactions ,26,27. TComputational modeling is also becoming increasingly important in understanding the collective behaviors of signaling proteins which are difficult to comprehend intuitively. Many different modeling paradigms and inference algorithms have been proposed to facilitate modeling, simulation, and inference of different types of biological systems . In part32P-labeled ATP to monitor kinase activity towards specific substrates [Current research in protein phosphorylation is focused on identifying phosphorylation sites, quantifying their abundance, and characterizing functional impact. From an analytical standpoint, there are inherent challenges in phosphorylation analyses due to the labile nature of the modification and sub-stoichiometric concentrations . While dbstrates . More rebstrates ,39. Altebstrates . Unfortubstrates . ConsequThe development of Mass Spectrometry (MS)-based proteomics has revolutionized the study of protein phosphorylation. Conceptual and technical breakthroughs in MS-based phosphoproteomics have enabled the detection and quantification of tens of thousands of phosphosites per sample ,41,42. GThe general workflow for targeted phosphoproteomics is outlined in The careful selection of target peptides is a crucial step in the identification and quantification of target proteins. The primary criteria for selection of surrogate peptides are that they must uniquely represent the protein of interest and be experimentally detectable within a proteome. They are often referred to as ‘proteotypic peptides’ ,56. ProtOngoing analytical challenges for targeted analyses include high peptide lability, sub-stoichiometric concentrations, heterogenous iso-forms, and low mass spectrometric response to protein phosphorylation . It is e3+, Ga3+, Ti4+, and Zr4+ can chelate and capture negatively charged phosphopeptides [The most common phosphoenrichment technique introduced over three decades ago is IMAC. It is based on the principle that positively charged metal ions such as Fepeptides ,63,64,65peptides . Howeverpeptides  and its peptides . Though peptides ,70. An apeptides . It is apeptides .4+ and Ti4+ with phosphates and carboxylic acid groups, and this has been implemented in approaches like the Ti4+-IMAC technology [4+-IMAC has been shown to be able to enrich multiple basic amino acid residues in addition to acidic residues [3+-Fe3+-IMAC where Ga3+ is initially applied to enrich multi-phosphorylated peptides followed by Fe3+ to capture monophosphorylated peptides [To increase specificity and improve robustness of phosphoenrichment, several modifications have been applied to IMAC methodologies. Elements like Nickel have been linked to NTA-based resins to improve specificity . Metal 3 have been tested as matrixes for enrichment of phosphopeptides [2-based precolumn. These were subsequently eluted in alkaline buffers for LC-MS/MS analysis [2 and thus, increased the binding affinity for phosphopeptides [2 microcolumn can be first eluted with acidic methylphosphonate buffer followed by successive basic elution of multi-phosphopeptides with disodium hydrogen phosphate, ammonium hydroxide, and pyrrolidine [MOAC enrichment is based on the Lewis acid-base interaction principle where metal oxides and hydroxides interact with oxygen atoms of phosphate groups to form complexes. Compounds like TiOpeptides . The firanalysis . The bufpeptides ,78. Howerolidine .3+-IMAC and multi-phosphopeptides are eluted in alkaline conditions, followed by TiO2 enrichment of the unbound acidic fraction for mono-phosphopeptides [2 resin followed by Fe3+-IMAC [Since IMAC and MOAC possess different affinities for phosphopeptides, it can be beneficial to combine both enrichment strategies. IMAC and MOAC typically favor multi-phosphorylated peptides and mono-phosphorylated peptides, respectively ,80. One peptides ,82. Likee3+-IMAC . Combini2 methods [PolyMAC is another adaptation of IMAC but using soluble nanopolymers instead of solely metal ions. The nanopolymers are dentrimers multifunctionalized with metal ions that bind to phosphopeptides in solution. The PolyMAC-phosphopeptide complexes are subsequently recovered on a solid phase by coupling to hydrazine-agarose gels, followed by washing and phosphopeptide elution. The PolyMAC-Ti formulation has been well studied and appears to display high recovery, reproducibility, and enrichment compared to IMAC and TiO methods ,85,86.Many traditional biochemical separation approaches are based on antibodies binding to specific epitopes. This has been applied to phosphoproteomics using antibodies raised against phosphorylated serine, threonine, and tyrosine residues. The metal-based methods discussed above are efficient for pSer and pThr residues, but not pTyr due to their significantly low relative abundance . FortunaFractionation is a step employed to reduce sample complexity either prior to phosphoenrichment or MS analysis. These strategies separate complex peptide samples into fractions that are more suitable for further enrichment or to enhance coverage depth of the phosphoproteome .Strong cation exchange (SCX) and strong anion exchange (SAX) are chromatography techniques that fractionate peptides according to their charge. In SCX, positively charged cations interact with the negatively charged SCX matrix. The pH of the loading and elution buffers determine the binding and elution of different peptides. Acid buffers (pH 2.7) are used to protonate the C-terminal arginine and lysine residues and N-terminal amino acid groups, separating phosphorylated peptides from non-phosphorylated peptides by net charge states . Bound pHigh pH reversed-phase liquid chromatography (HpH RPLC) is another fractionation strategy based on hydrophobic interactions . It is cOther fractionation strategies like hydrophilic interaction chromatography (HILIC) and electrostatic repulsion-hydrophilic interaction (ERLIC) are based on hydrophilic interaction ,110. HILCurrent targeted methods of phosphoproteomics are based on selected reaction monitoring )  to provi3−)-modified fragments are detected in Q3 by the addition of 79 Da, which is only ideal for highly stable phosphopeptides. Alternatively, neutral loss scan mode detects the loss of a neutral group (H3PO4) from a phosphorylated peptide after low-energy CID. The loss of H3PO4 results in a mass shift of 98 Da, 49 Da and 32.7 Da for singly, doubly, and triply charged phosphopeptides, respectively. However, tyrosine-specific phosphopeptides are more stable and show no neutral loss, thus Q3 scans for an immonium ion (216.042 Da) marker [m/z) values of precursor-fragment ion pairs monitored by Q1 and Q3 are called ‘transitions.’Selected Reaction Monitoring (SRM), also known as Multiple Reaction Monitoring (MRM), is an approach for reliably measuring phosphoproteins in a highly reproducible manner within and across laboratories and instrument platforms ,113. Imp) marker . The acqm/z fragment ions due to low specificity and ensure that chosen fragments have m/z values above the precursor for high selectivity and spectral noise reduction [In establishing SRM assays, selecting optimal transitions for each peptide is important. First, it is best practice to select the most intense fragments because the intensities of fragment ions derived from one precursor differ substantially. Low-energy CID induces the generation of b/y series fragment ions. In this fragmentation environment, y-ion signals are more common, robust, and stable compared to b-ion signals . Even theduction . In geneeduction ,118.The first utilization of SRM for large-scale targeted phosphoproteomics was reported by White’s group in 2007 . They weFor absolute quantification workflows, addition of chemically synthesized heavy isotope-labeled peptides containing the phosphorylated sites of interest is the gold standard for analyzing phosphopeptides and their stoichiometries ,112,120.In general, standard-based quantification for targeted phosphoproteomics is gaining relevance with increasing experimental demonstrations by different groups . The larIn the ongoing effort to avoid limiting the number of targets, technical advancements in high-resolution mass-measuring instruments like the hybrid quadrupole-orbitrap mass spectrometer has enabled the development of Parallel Reaction Monitoring (PRM) . Like SRPRM data acquisition takes advantage of certain Quadrupole-Orbitrap instrumentation parameters that can be optimized to improve analysis of complex samples. The quadrupole isolation width can be narrowed to directly control the selectivity of precursor ions and exclude interferences, and the trapping fill time can be longer to enhance sensitivity and widen dynamic detection range . PRM hasm/z range of a selected precursor, followed by fragmentation in sequential small m/z windows. These acquisitions are continuously repeated to collect information on all detectable precursor and fragment ions within the specified windows [Sequential window acquisition of all theoretical mass spectra (SWATH-MS) operating in a data-independent acquisition (DIA) mode is an approach designed to combine the advantages of both shotgun and targeted proteomics to achieve reproducibility and consistency in a high-throughput manner ,138. Thi windows . Assay d windows . A recen windows . Althoug windows ,141,142.Altered phosphorylation drives processes underlying numerous diseases. Disease-associated phosphorylation cascades can be restricted to specific pathways or, in many cases, involves crosstalk with other pathways. Hence, altered phosphorylation-mediated signaling can include tens to hundreds of proteins. These can potentially be followed and analyzed by SRM-based phosphoproteomics . One priMS-based instrumentations have revealed extensive phosphorylation modifications in the proteome ,148,149.The complexity of phosphorylation-mediated signaling and the subsequent heterogeneity make systems-level models most useful. To highlight some relevant sources of cellular complexities: (1) the presence of potential phosphorylation sites on receptors does not mean all sites can be phosphorylated at the same time, or ever at all . SometimPredictive modeling in cellular signaling aims to develop mathematical tools that are capable of computing expected outputs from given inputs under defined conditions . With a A few studies have undertaken modeling approaches using different approaches of generating phosphorylation data . Our groIn general, mathematical models are generally useful for: (1) aiding experimental designs to test both simple and complex hypotheses; (2) reconciling discrepancies in experimental data; and (3) consolidating knowledge about a system. Simple models like the partial least squares regression (PLSR) and multiple linear regression (MLR) are based on linear regressions . The datIn complex biological systems, reducing the biochemistry (reactions) of the system to construct a network of ordinary differential equations (ODE) or partial differential equations (PDE) is a common approach . BeginniRule-based models offer an approach which factors in the combinatorial complexities of cellular signaling mechanisms . These mRule-based and ODE/PDE models rely heavily, and in some cases entirely, on generated experimental knowledge of well-studied systems , which iPrevious studies have shown the utility of experimental data for constraining models and estimating model parameters. In considering the power of different experimental methods we argue that absolute quantification methods, such as targeted phosphoproteomics, provide much greater advantage in parameter estimation than relative quantification of phosphorylation levels. Absolute quantification can greatly reduce parameter space by directly providing values for protein abundance (concentration) and phosphorylation levels. Most kinetic models use “lumped” rate constants that combine both protein abundance and activity. Since protein abundance varies across cell types , knowingTargeted MS-based quantitative proteomics is becoming increasing popular across scientific and clinical research areas due to significant advancements in assay sensitivity, specificity, and reproducibility. These exceptional characteristics make them suitable for multiple applications such as biomarker discovery in basic and translational research. Classic SRM/MRM-based proteomics has been applied to verify protein biomarkers for different types of diseases such as melanoma, prostate cancer, lung cancer, Parkinson’s disease, cardiovascular diseases, and type 1 diabetes using different specimens like plasma, serum, urine, cerebrospinal fluid, and tissue . ClinicaHowever, the broad utility of phosphoproteomics is not without existing challenges. Despite great improvements in proteomics workflows, sample throughput in MS-based proteomics is still relatively low compared to antibody-based techniques, such as Reverse Phase Protein Arrays . PhosphoAnother challenge is that due to the low abundance levels of phosphorylation sites, a large amount of input material is usually required for enrichment and subsequent analysis. This could pose a limitation for applications that generate only a small amount of sample/specimen. With the introduction of single-cell proteomics, there is the possibility for pushing the sensitivity of targeted phosphoproteomics to measure target analytes in small amounts of samples, such as those generated by flow cytometry. In terms of specificity, mass spectrometers cannot always differentiate all target phosphopeptides, especially if multiple phosphorylations result in similar mass or structural changes to the peptides. Consequently, utilizing recent tools like structure for lossless ion manipulation (SLIM) in resolution-type analysis, which can distinguish targets by size and shape in addition to mass, may help to resolve this issue . FurtherAs biomedical research gravitates towards a systems approach to answer important and increasingly complex questions, the integration of experimental and computational tools will continue to increase. To support this work, targeted phosphoproteomics has provided a platform to obtain high quality data with scalable throughput. It also provides important information for building predictive models that can assist in experimental designs and clinical decision making. Experimental and computational integration hold promise to better decipher phosphorylation mechanisms underlying disease pathogenesis and identify potential biomarker/therapeutic candidates for use in traditional and precision medicine.


Pseudomonas syringae from −1 °C to −2 °C. The addition of an antifreeze protein inhibited the action of the tested ice-initiating agents.The classical nucleation theory shows that bulk water freezing does not occur at temperatures above ≈ −30 °C, and that at higher temperatures ice nucleation requires the presence of some ice-binding surfaces. The temperature and rate of ice nucleation depend on the size and level of complementarity between the atomic structure of these surfaces and various H-bond-rich/depleted crystal planes. In our experiments, the ice nucleation temperature was within a range from −8 °C to −15 °C for buffer and water in plastic test tubes. Upon the addition of ice-initiating substances , ice appeared in a range from −3 °C to −7 °C, and in the presence of the ice-nucleating bacterium  On our planet, liquid water is an extremely widespread compound. It is crucially important for all organisms living here. Thus, a transformation of liquid water into ice has serious consequences for the affected organisms, and since conditions that favor water-to-ice transition exist (at least sometimes) on a large fraction of the Earth’s surface, many organisms living here should (and do) have special adaptations for that case, including actions of many biomolecules of very different chemical natures.Given this great importance, it is natural that the processes of water freezing and ice melting have been investigated for a very long time. There are many excellent papers considering different aspects of these processes and the participation of biomolecules in them, but many important details of the emergence of ice—especially in living bodies—are still unclear.In this article, we focus on the nucleation of ice rather than on its growth . Hence, the “freezing temperature” refers to the “ice nucleation temperature” everywhere in this paper. We evaluate the temperature dependence of the characteristic time of nucleation of the “new” crystalline phase (i) in the volume and (ii) on surfaces adjacent to the “old” liquid phase (being interested in the vicinity of the point of thermodynamic equilibrium of these phases), and then discuss some experimentally investigated biological and non-biological agents that can drastically reduce this typically long (when temperature approaches 0 °C) time.It is common knowledge that water freezes at 0 °C. More precisely, 0 °C is the temperature at which ice coexists with water, but this does not mean that water begins to freeze at 0 °C when cooled. Freezing is a first-order phase transition ,2, whichThe formation of ordered phases from melts is described in ,2,3,4. FAccording to the classical nucleation theory, the basic estimate of the nucleation time for phases arising from a first-order phase transition can be achieved using the transition state theory ,7 when td-dimensional (d > 1) new phase of n >> 1 particles  or perimeter (for d = 2) of the “new” phase; and n particles. Given This change for a piece of the articles  is knownonal d >  new phase theory ,8, we maThe nucleation of a new phase is a multistep reaction . It begiT is just a little lower than We are interested in the case when the “new” phase is stable (d > 1) system, n, passes through its maximum—which occurs in the transition state corresponding to the Gibbs’ “critical nucleus” of the new phase—and then decreases P. syringExperiments on freezing and melting were carried out using a Julabo F-25 thermostat (Germany). The thermostat and measuring thermometers (thermocouples) were checked using an LT-300-N TERMEX laboratory thermometer (Russia), at a resolution of 0.01 °C, and with an accuracy of ±0.05 °C.®). The liquid volume was 0.5 and 1 mL.Freezing of the solutions was carried out in standard plastic (polypropylene) microcentrifuge test tubes  oxide (CuO) was from Reachem . It was added to the test tubes in amounts of 2 mg. This non-soluble CuO powder was at the bottom of the test tubes.P. syringae cells (Pseudomonas syringae pv. syringae) were grown on medium L  at a temperature of +26 °C. Cells were grown in a liquid medium up to the cell density of 1.0 optical units (by absorption at 600 nm), and then precipitated on a centrifuge at 6000 g, and washed twice with a solution of 20 mM Tris-HCl (pH 7.5). The initial cell solution was diluted with a buffer of the same composition to the desired cell density . The concentration of P. syringae cells in the experiments was controlled by absorption at 600 nm.The experiments were repeated at least 10 times, which made it possible to determine the ice-nucleating temperature range for the used solutions.We report that the kinetics of ice nucleation (i) prevent ice formation in bulk water at temperatures above −35 °C, (ii) prevent ice emergence on smooth ice-binding but not ice-nucleating surfaces at temperatures above −4 °C, and (iii) allow ice nucleation and growth on ice-binding surfaces with a grooved (corrugated or fluted) atomic structure at nearly 0 °C.In the absence of in-water shock waves or water overcooling by evaporation from an open vessel, the presence of an ice-binding surface with a specific (fluted or corrugated) atomic structure that disrupts interactions between molecules within the ice layers arising on this surface seems to be the only possibility to achieve ice nucleation at or very close to 0 °C in any closed vessel, including animal or plant fluids, tissues, and cells.Pseudomonas syringae the ice nucleation temperature is about −1 to −2 °C. The addition of an antifreeze protein inhibits the action of both of these ice-initiating agents.Our experiments show that the ice nucleation temperature is about −10 °C for a buffer in plastic test tubes, while with CuO added, it ranges from −5 to −7 °C , and in the presence of the ice-nucleating bacterium


Importantly, INT-777 reversed STZ-induced downregulation of TGR5 and glucose usage deficits. Our results further showed that INT-777 suppressed neuronal apoptosis and improved neurogenesis which were involved in tau phosphorylation and CREB-BDNF signaling. Moreover, INT-777 increased action potential firing of excitatory pyramidal neurons in the hippocampal CA3 and medial prefrontal cortex of ICV-STZ groups. Taken together, these findings reveal that activation of TGR5 has a neuroprotective effect against STZ-induced cognitive impairment by modulating apoptosis, neurogenesis, and neuronal firing in the brain and TGR5 might be a novel and potential target for Alzheimer's disease.Takeda G protein-coupled receptor 5 (TGR5) is the first known G protein-coupled receptor specific for bile acids and is recognized as a new and critical target for type 2 diabetes and metabolic syndrome. It is expressed in many brain regions associated with memory such as the hippocampus and frontal cortex. Here, we hypothesize that activation of TGR5 may ameliorate streptozotocin- (STZ-) induced cognitive impairment. The mouse model of cognitive impairment was established by a single intracerebroventricular (ICV) injection of STZ (3.0 mg/kg), and we found that TGR5 activation by its agonist INT-777 (1.5 or 3.0  One-way ANOVA revealed that INT-777 treatments did not significantly alter the total distance  for the hippocampus and frontal cortex, Figures P < 0.05 (INT-777 1.5 μg) and P < 0.01 (INT-777 3.0 μg) for the hippocampus and frontal cortex, Figures The CREB-BDNF pathway is critical for hippocampal neurogenesis and apoptosis which are closely involved in the pathogenesis of AD . To evalα-mCherry vector before STZ and INT-777 injections  were used to block presynaptic roles of other glutamatergic and GABAergic neurons in whole-cell patch-clamp recording. One-way ANOVA showed that bath application of TGR5 agonist INT-777 (10 μM) significantly increased frequency of action potential firing of hippocampal CA3 excitatory pyramidal neurons in control and ICV-STZ mice  significantly increased the frequency of action potential firing of mPFC excitatory pyramidal neurons in control and ICV-STZ mice  and the GABAAD can be divided into SAD and FAD. SAD, caused by multiple etiologic factors, including environmental, genetic, and metabolic factors, is the majority of AD cases in the clinic . STZ cytβ-cell destruction and is widely used experimentally to produce a model of diabetes mellitus [β-cell number [Glucose hypometabolism and energy deficit are hallmarks of AD, and previous studies have showed that reduced glucose utilization in brain regions affects patients with AD . The monmellitus , 7, we smellitus . In thismellitus . In the l number . Here, wβ pathway.Cellular longevity is associated with apoptosis and neurogenesis, which are associated with energy metabolism. Decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells . ApoptosStructural plasticity and network maintenance of the hippocampus rely on neurogenesis, and increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells. Dysfunctional neurogenesis resulting from early subtle disease manifestations may exacerbate neuronal vulnerability to AD and contribute to memory impairment . Recent β1–42-induced or lipopolysaccharide-induced AD models [κB signaling and production of proinflammatory cytokines TNF-α and IL-1β while suppressing microglia activation in ICV-STZ mice  is at a central converging point of pathways and mechanisms activated during the processes of synaptic strengthening and memory formation . CREB siD models , 86. In Neuronal firing homeostasis is a master regulator of the integrative homeostatic network that maintains the stability of neural circuits and safeguards from neurodegeneration . ExtraceOur results suggest that activation of TGR5 by INT-777 ameliorates STZ-induced cognitive impairment by modulating apoptosis, neurogenesis, and neuronal firing, which might provide new insight into the mechanism of AD and highlight TGR5 as a novel and promising target for the prevention or treatment of AD.