S-nitrosylation of cysteine residues in various proteins. Although PDI knockdown reduces α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)-mediated neuronal activity, the underlying mechanisms are largely unknown. In the present study, we found that under physiological condition PDI knockdown increased CaMKII activity (phosphorylation) in the mouse hippocampus. However, PDI siRNA inhibited protein phosphatase (PP) 2A-mediated GluA2 S880 dephosphorylation by increasing PP2A oxidation, independent of S-nitrosylation. PDI siRNA also enhanced glutamate ionotropic receptor AMPA type subunit 1 (GluA1) S831 and GluA2 S880, but not GluA1 S845 and GluA2 Y869/Y873/Y876 phosphorylations, concomitant with the enhanced protein interacting with C kinase 1 (PICK1)-mediated AMPAR internalization. Furthermore, PDI knockdown attenuated seizure activity and neuronal damage in response to kainic acid (a non-desensitizing agonist of AMPAR). Therefore, these findings suggest that PDI may regulate surface AMPAR expression through PP2A-GluA2-PICK1 signaling pathway, and that PDI may be one of the therapeutic targets for epilepsy via AMPAR internalization without altering basal neurotransmission.Protein disulfide isomerase (PDI) is a redox-active enzyme and also serves as a nitric oxide donor causing  N-methyl-D-aspartate receptor (NMDAR) is one of the major excitatory receptors, which contributes to regulation of neurotransmission, synaptic plasticity and neuronal damage. Although mechanistic regulations of NMDAR channel activity are basically relevant to the bindings of ligands (e.g. glutamate and NMDA) and modulators , redox modulation of reactive disulfide bonds on NMDAR also affects the conformational changes in the NMDAR functionality. For instance, reducing agents (dithiothreitol (DTT), H2S, glutathione (GSH)) inducing thiolation of disulfide bonds (S–S → HS– + –SH) reinforce NMDAR-mediated currents, while oxidizing agents (e.g. 5–5′-dithiobis-2-nitrobenzoic acid (DTNB) and oxidized glutathione (GSSG)) leading to disulfide formation (HS– + –SH → S–S) inhibit them. Furthermore, S-nitrosylation (–SH → –SNO) of cysteine (C) residues reduces NMDAR functionality3. Although the oxidation of NMDAR redox sites decreases NMDAR-mediated currents by inhibiting channel openings, it cannot block physiological NMDAR functions including synaptic plasticity, such as inductions of long-term potentiation (LTP) and long-term depression (LTD)5. However, NMDAR redox inhibits seizure activity induced by kainic acid (KA), bicuculline, pilocarpine, pentylenetetrazol, NMDA and low Mg2+ concentration6. In spite of the absence of reducing agents, furthermore, seizure activity causes the reduction of NMDAR redox sites leading to the potentiation of NMDAR function even after the cessation of epileptiform discharges5. Thus, it is likely that unknown endogenous reducing factors would be involved in these phenomena.The 7. Interestingly, PDI inhibition protects neurons from glutamate-induced oxidative cytotoxicity8. Similar to DTNB also known as PDI inhibitor9, PDI knockdown reduces the amounts of free thiols on NMDAR subunits (GluN1 and GluN2A) and attenuates acute seizure activity in response to pilocarpine and NMDA as well as spontaneous seizures in chronic epilepsy rats independent of S-nitrosylation, while it does not induce alterations in basal neurotransmission (paired-pulse response) and ER stress under physiological condition10. Thus, we have reported that PDI might be one of the reductive enzymes regulating the redox-mediated NMDAR activity.Protein disulfide isomerase (PDI) is a redox-active enzyme, which catalyzes disulfide bonds formation, reduction or isomerization of various proteins in the lumen of the endoplasmic reticulum (ER), cytoplasm and cell surfaceα-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) in response to NMDAR activity. Indeed, NMDAR-mediated Ca2+ entry, and the subsequent activations of Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) and protein kinase C (PKC) increase AMPAR channel conductance by increasing serine (S) 831 phosphorylation of glutamate ionotropic receptor AMPA type subunit 1 (GluA1). Furthermore, NMDAR activation enhances protein kinase A (PKA)-mediated GluA1 S845 phosphorylation, which regulates AMPAR peak response open probability and synaptic trafficking of AMPARs12. However, NMDAR activation also facilitates AMPAR internalization by enhancing PKC- and CaMKII-mediated GluA2 S880 phosphorylation14. On note, DTNB-induced NMDAR oxidation cannot affect AMPAR- and γ-aminobutyric acid receptor (GABAR)-mediated synaptic responses, although it attenuates seizure activity16. Therefore, it is likely that NMDAR redox would not involve the activity-dependent regulation of AMPAR functionality. However, we have found that PDI knockdown reduces the amplitude and frequency of neuronal discharges in response to AMPA as well as NMDA without altering GABAergic inhibitions10. These discrepancies would be consequences from properties of DTNB and PDI siRNA: DTNB oxidizes C residues on proteins exposed to the extracellular milieu, because of poor membrane permeability17. Unlike DTNB, PDI siRNA regulates the thiol modification of proteins in ER, cytosol and cell surface19. Furthermore, PDI serves as a nitric oxide (NO) donor causing S-nitrosylation of C residues that is a posttranslational protein modification21. Therefore, it is noteworthy exploring the underlying mechanisms of the blockade of AMPAR-mediated neuronal activity induced by PDI knockdown, which has been largely unknown.On the other hand, activity-dependent alterations in synaptic strength are mediated by S-nitrosylation. Furthermore, PDI knockdown attenuated seizure activity and neuronal damage in response to KA, a non-desensitizing agonist of AMPAR23, by rapidly increasing protein interacting with C kinase 1 (PICK1)-mediated AMPAR internalization. Therefore, these findings indicate that PDI may regulate surface AMPAR expression via PP2A-mediated GluA2 S880 dephosphorylation, and suggest that PDI may be one of the therapeutic targets for epilepsy via AMPAR internalization without altering basal neurotransmission.Here, we found that under physiological condition PDI knockdown inhibited PKC, but increased CaMKII activity (phosphorylation) in the mouse hippocampus without affecting PKA activity. However, PDI siRNA enhanced GluA1 S831 and GluA2 S880, but not GluA1 S845 and GluA2 Y869/Y873/Y876 phosphorylations, and diminished surface AMPAR expression. These phenomena were relevant to the inhibition of protein phosphatase (PP) 2A by enhancing disulfide bond formation (oxidation), independent of 25. PDI siRNA decreased PDI protein level to 0.64-fold of control siRNA level in the mouse hippocampus  and GluA1 S831 phosphorylation ratio (p-GluA1 S831 ratio) to 1.41-fold of control siRNA level  and GluA1 S845 phosphorylation ratio  and GluA2 Y869/Y873/Y876 phosphorylation ratio . Thus, PDI knockdown elevated GluA2 S880 phosphorylation ratio (p-GluA2 S880 ratio) to 1.41-fold of control siRNA level  and GluA2 surface expressions , as compared to control siRNA . Although PDI siRNA did not change PKC total protein level  and PKC T497 phosphorylation ratio (p-PKC T497 ratio) to 0.57-fold of control siRNA level  without affecting CaMKII protein level . Thus, CaMKII T286 phosphorylation ratio (p-CaMKII T286 ratio) was also increased to 1.37-fold of control level following PDI knockdown  was unaltered by PDI siRNA . PKA T197 phosphorylation  and PKA T197 phosphorylation ratio  residues regulates phosphatase activity34. Given that PDI is a redox-active enzyme and a NO supplier21, it is likely that PDI siRNA may enhance CaMKII-mediated GluA1 S831 and GluA2 S880 phosphorylation by regulating redox and/or S-nitrosylation of PP1 and PP2A. In the present study, co-immunoprecipitation data showed PDI bound to PP2A, but not PP1, under physiological condition  = 12.532, p < 0.001, n = 7, respectively, Friedman test; Fig. p = 0.007, n = 7, respectively, Mann–Whitney test; Fig. To confirm whether PDI siRNA-induced GluA2 S880 phosphorylation decreases the neuronal activity by enhancing AMPAR internalization, we applied KA  to animals. This is because AMPAR responses are not desensitized during exposure to KA2(3) = 20.502, p < 0.001, n = 7, respectively, Kruskal–Wallis test; Fig. 2(3) = 21.259, p < 0.001, n = 7, respectively, Kruskal–Wallis test; Fig. 2(3) = 1.577, p = 0.665, n = 7, respectively, Kruskal–Wallis test; Fig. 2(3) = 22.84, p < 0.001, p = 0.004, Tukey post-hoc test, n = 7, respectively, Kruskal–Wallis test; Fig. 2(3) = 22.84, p < 0.001, p = 0.032, Tukey post-hoc test, n = 7, respectively, Kruskal–Wallis test; Fig. p = 0.995, Tukey post-hoc test; Fig. Finally, we evaluated the effect of PDI knockdown on AMPAR internalization in response to KA. KA did not affect surface expressions of GluA1 and GluA2 in control siRNA-treated animals Fig. a–c. As c37. Therefore, we validated whether PDI knockdown also accelerates GluA2:PICK1 interaction. PDI siRNA did not influence PICK1 level under physiological- and post-KA condition (χ2(3) = 0.785, p = 0.853, n = 7, respectively, Kruskal–Wallis test; Fig. p < 0.001, Tukey post-hoc test). KA did not alter GluA2:PICK1 binding in control siRNA-infused animals , but further enhanced it in PDI siRNA-infused animals (χ2(3) = 21.857, p < 0.001, p = 0.028, Tukey post-hoc test, n = 7, respectively, Kruskal–Wallis test; Fig. GluA2 S880 phosphorylation reinforces its internalization by facilitating GluA2:PICK1 binding, which is regulated by PP2A38. Among them, GluA2 regulates the preference of ion-permeability of AMPAR. GluA2-containing AMPAR activation leads to Na+ and K+, but not Ca2+, influx, while GluA2-lacking AMPAR activation makes Ca2+ influx39. GluA2-containing AMPAR also shows low channel conductance and a linear rectification pattern, whereas GluA2-lacking AMPAR exhibits high channel conductance and an inward rectification pattern23. Thus, GluA2 subunit dominantly inhibits AMPAR-dependent Ca2+ permeability as well as channel conductance. Furthermore, GluA2 homotetramer generates steady-state currents when the receptors are desensitized40. In the present study, PDI knockdown increased GluA2 S880 phosphorylation concomitant with the reduced surface GluA2 expression. In addition, PDI knockdown enhances GluA1 S831 phosphorylation that increases channel conductance25. Thus, it is presumable that PDI knockdown would increase the surface expression of Ca2+-permeable GluA2-lacing AMPAR or the preponderance of homomeric GluA1 tetramers that would result in much larger single-channel conductance than heteromeric GluA1-GluA2 tetramers through GluA1 S831 phosphorylation27. However, the present study also shows that PDI siRNA decreased surface GluA1 expression and neuronal activity in response to KA without altering the GluA1/GluA2 ratio. Furthermore, PDI knockdown did not affect GluA1 S845 phosphorylation that drives surface trafficking of GluA1-containing AMPAR and facilitates AMPAR insertion into synaptic membrane to potentiate the peak AMPAR current42. In contrast to S845 phosphorylation, GluA1 S831 phosphorylation is not required for AMPAR trafficking, although it directly enhances the function of AMPAR27. Given that the GluA1/2 heterotetramer is the most dominant AMPAR subtype in the hippocampal pyramidal cells and > 95% of AMPARs contain the GluA2 subunit under physiological condition43, our findings indicate that PDI knockdown may diminish neuronal activity in response to KA by reducing surface heteromeric GluA1-GluA2 tetramers through GluA2 S880 phosphorylation without increasing the proportion of homomeric GluR1 tetramers, and that the increased GluA1 S831 phosphorylation may be an adaptive response to the decrease in the total number of AMPAR tetramers.The AMPAR is composed of the homomeric or heteromeric tetramer of four subunits  encoded by different genes45. GluA2 Y869/Y873/Y876 phosphorylation destabilizes GluA2:GRIP1 interaction and promote its internalization47. Phosphorylation of GluA2 S880 site also causes dissociation of GRIP1 and subsequently binds to PICK1, then leads to the endocytosis of AMPAR48. When GluA2 S880 site is dephosphorylated by PP2A, PICK1 dissociates from GluA2 and GRIP1 binds to GluA249. Thus, the phosphorylation of S880 on GluA2 is an essential step for AMPAR internalization, which is inhibited by Y876 phosphorylation50. Compatible with these reports, PDI siRNA increased GluA2 S880 phosphorylation and GluA2:PICK1 binding without affecting GluA2 Y869/Y873/Y876 phosphorylation, which was caused by oxidation-mediated PP2A inhibition under physiological condition, and facilitated GluA2:PICK1 interaction following KA treatment. Regarding that tyrosine phosphorylations of GluA2 is are required for AMPAR internalization46 and GluA2-PICK1 interaction is not influenced by tyrosine phosphorylation of GluA247, these findings indicate that PDI knockdown may facilitate GluA2:PICK1 binding and AMPAR internalization independent of tyrosine GluA2 phosphorylation.Surface GluA2 expression is reversely regulated by interactions with glutamate receptor interacting protein 1 (GRIP1) and PICK1. GRIP1 increases surface GluA2 expression, while PICK1 diminishes it14. NMDAR activation initially increases Ca2+-dependent PKC activity followed by persistent CaMKII activation51. Furthermore, NMDAR-stimulated Ca2+ entry activates CaMKII, and subsequent phosphorylates GluA1 at S831 site to increase channel conductance52. Since PDI enhances NMDAR-mediated neuronal activity through the sulfhydration (reduction) of cysteine residues on the NMDAR redox site10, it is plausible that PDI siRNA would result in the decrease in GluA1 S831 and GluA2 S880 phosphorylation, accompanied by the reduced PKC T497 and CaMKII T286 phosphorylations  between C266 and C269 in PP2A is an inhibitory redox switch in its phosphatase activity33. Furthermore, PDI has the reductive activity at the cell surface, although it acts predominantly as an oxidase in the ER57. In the present study, we found that PDI bound to PP2A, but not PP1, and PDI siRNA decreased PDI:PP2A interaction and the amount of free thiols (oxidation) without altering that of SNO-thiols under physiological condition. These findings indicate that PDI downregulation induced by siRNA may decrease the PDI:PP2A binding, which would subsequently diminish the amount of total thiol on PP2A, and suggest that PDI may act as a reductase rather than NO transporter of PP2A. The present data also show that KA increased PDI:PP2A and GluA2:PP2A bindings without altering surface GluA1/2 expression and GluA2:PICK1 interaction in control siRNA-infused animals. Although we could not provide the underlying mechanisms in the present study, it is likely that these phenomena may be maladaptive responses to KA-induced seizures: KA may augment PDI-mediated sulfhydration (reduction) of PP2A and the subsequent PP2A-mediated GluA2 S880 dephosphorylation, which would prolong seizure activity in response to KA. Since GluA2 S880 phosphorylation facilitates AMPAR internalization, in turn, PP2A-mediated GluA2 S880 dephosphorylation may result in the unchanged GluA2 surface expression upon KA treatment by abrogating PICK1-mediated AMPAR internalization. These are compatible with previous studies demonstrating that KA is a non-desensitizing agonist of AMPAR23. In contrast, PDI siRNA attenuated seizure activity in response to KA and further decreased surface GluA1/2 expression concomitant with augmented GluA2:PICK1 interaction following KA injection as compared to saline treatment, although it did not change GluA2:PP2A binding. Regarding that KA activates PP2A and decreases CaMKII activity and its autophosphorylation in the mouse hippocampus at 2.5–6 h after injection59, these findings also indicate that PDI may abolish AMPAR internalization by activating PP2A. Given that the clear loss of PP2A activity correlates with the cysteine oxidation60, our findings suggest that PDI siRNA may increase GluA1 S831, GluA2A S880 and CaMKII T286 phosphorylations by inducing oxidation-mediated PP2A inhibition under physiological condition, which would facilitate PICK1-mediated AMPAR internalization following KA treatment.PP1 and PP2A, but not PP2B, dephosphorylates GluA2 S880 site61. In the amygdala, KA receptor (KAR)-activation induces a depression of NMDA and AMPA-mediated evoked excitatory postsynaptic current (eEPSC) amplitude, which is prevented by PKA, but not PKC, inhibitors62. In the hippocampus, KAR activation also negatively modulates glutamate release by convergence with group II metabotropic glutamate receptors  through PKA64. In contrast, KAR activation facilitates glutamate release through Ca2+-CAM-mediated PKA activation in the hippocampus and thalamocortical synapses66. Thus, it is presumable that PDI would regulate seizure susceptibility to KA by affecting presynaptic KAR. However, the present study shows that PDI knockdown inhibited PKC, but increased CaMKII activity without affecting PKA activity under physiological condition. Considering that activation of PKA, but not PKC, is required for KAR-mediated regulation of presynaptic glutamate release66, our findings suggest that PDI may not influence presynaptic glutamate release in response to KA.On the other hand, KA shows a biphasic effect on presynaptic transmission in the hippocampus and other brain regions. Low KA concentrations (50–100 nM) increase glutamate release, while higher concentrations decrease itIn conclusion, the present data indicate that PDI played a crucial role in PP2A activation by reducing disulfide bonds, which abolished AMPAR internalization by dephosphorylating GluA2 S880 and CaMKII T286 sites and abrogating GluA2:PICK1 interaction . This study was also carried out in compliance with the ARRIVE guidelines.Male C57BL/6J mice (8 weeks old) were used in the present study. Animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions . All reagents were obtained from Sigma-Aldrich (USA), except as noted.2O:O2). A brain infusion kit 3  was implanted into the right lateral ventricle  and connected to an osmotic pump  containing a non-silencing RNA (control siRNA) or PDI siRNA (CCUUUGCUAGCGAAUCUCAGAGCC), which were continuously infused over 6-day period. Some animals were also implanted monopolar electrode  into the left dorsal hippocampus 21.Mice were anesthetized with Isoflurane  or an equal volume of normal saline instead of KA. Saline-treated mice were used as controls. Diazepam  was administered 2 h after KA injection. Electrode-implanted animals were also given KA  after baseline recording for at least 30 min. Electroencephalographic (EEG) signals were recorded with a DAM 80 differential amplifier  and digitized (1000 Hz) using LabChart Pro v7 software . EEG total power was obtained from integration of EEG amplitude in the frequency bands from 0 to 80 Hz. Frequency-power spectral temporal maps were generated with filtrations of the frequency domain (0–80 Hz) and the amplitude domain (0–50 mV). Total power was measured during the 2 h recording session and spectrograms were automatically calculated using a Hanning sliding window with 50% overlap by LabChart Pro v7 software . Total EEG power was normalized by the baseline power obtained from each animal. Latency of seizure on-set was defined as the time point showing more than 3 s and consisting of a rhythmic discharge between 4 and 10 Hz with amplitude of at least two times higher than the baseline EEG. After recording, animals were used for biochemical experiments21.Animals were sacrificed by decapitation under urethane anesthesia . The hippocampus was dissected out and homogenized in lysis buffer (50 mM Tris containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), ethylene glycol tetraacetic acid (pH 8.0), 0.2% Tergitol type NP-40, 10 mM ethylenediaminetetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Total protein content was measured by BCA protein assay kit. Western blot was performed according to standard procedures (Table 21.The tissues were lysed in radioimmune precipitation buffer (RIPA) with protease and phosphatase inhibitor cocktails (Roche Applied Sciences) and 1 mM sodium orthovanadate. After quantification of total protein concentration, equal amounts of protein (amount in 150 μg) were precipitated with the PDI, PP1, PP2A or PICK1 antibody and subsequently incubated with protein G sepharose at 4 °C overnight. Beads were collected, eluted in sample buffer and boiled at 95 °C for 5 min. The elution step was performed under the same conditions and in parallel. To analyze membrane expressions of GluA1 and GluA2, subcellular Protein Fractionation Kit for Tissues  was used according to the manufacturer’s instructions. Next, Western blot was performed according to standard proceduresS-nitrosylation Western Blot Kit  according to the manufacturer's protocol. Briefly, lysates were reacted with ascorbate in HENS buffer for specific labeling with iodoTMTzero reagents with MMT pretreatment (for SNO-thiol), or not . Protein labeling can be confirmed by Western blot using TMT antibody. Thereafter, TMT-labeled proteins were purified by Anti-TMT Resin, eluted by TMT elusion buffer, and identified by Western blot according to standard procedures. For technical controls, we omitted ascorbate for each sample. The ratio of SNO-protein or total thiol-protein to total protein was described as S-nitrosylation or total thiol levels21.Modified biotin switch assay was performed with the 4 μm2) were captured, and were selected. Thereafter, cells count was performed using AxioVision Rel. 4.8 software21.Animals were anesthetized with urethane anesthesia  and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer . Brains were post-fixed in the same fixative overnight and then cryoprotected and sectioned at 30 μm with a cryostat. Free-floating coronal sections were incubated in PDI antibody in PBS containing 0.3% Triton X-100 overnight at room temperature. Tissue sections were incubated with a mixture of guinea pig anti-NeuN and rabbit p-GluA2 S880 antibodies in PBS containing 0.3% Triton X-100 overnight at room temperature. Thereafter, sections were visualized with Cy2- and Cy3-conjugated secondary antibodies. Immunoreaction was observed and analyzed using an AxioImage M2 microscope . To establish the specificity of the immunostaining, a negative control test was carried out with preimmune serum instead of the primary antibody. No immunoreactivity was observed for the negative control in any structures. All experimental procedures in this study were performed under the same conditions and in parallel. To analyze the neuronal damage, we also applied FJB staining. Three days after KA treatment, sections were prepared as the same methods aforementioned. Sections were placed on slides, dried, and immersed in 80% ethanol containing 1% sodium hydroxide. Next, slides were immersed in 70% ethanol solution for 2 min and in purified water for 2 min. After immersion in 0.06% potassium permanganate solution for another 10 min, slides were rinsed with purified water for 2 min. Then, slides were were incubated for 30 min in 0.001% FJB , freshly prepared by adding 20 ml of a 0.01% stock FJB solution to 180 ml of 0.1% acetic acid, with gentle shaking in the dark. After mounting, areas of interest (the CA3 pyramidal cell layer, 10post-hoc test. A p < 0.05 is considered to be statistically different.Comparisons of data among groups were analyzed by Mann–Whitney test, Friedman test or Kruskal–Wallis test followed by Tukey Supplementary Information.


Considerably higher isotopic niche overlap and generally shorter isotopic distance were found between pelagic and benthic food web components in 2005 than in 2016, suggesting weaker coupling in the latter, low-ice year. δ15N values indicated more refractory food consumed by benthos in 2016 and fresher food reaching the seafloor in 2005. Higher δ13C values of zooplankton indirectly suggested a higher contribution of ice algae in 2005 than 2016. The difference in pelagic–benthic coupling between these years is consistent with higher energy retention within the pelagic system, perhaps due to strong stratification in the Amerasian Basin in the recent decade. Weaker coupling to the benthos can be expected to continue with ice loss in the study area, perhaps reducing benthic biomass and remineralization capacity; monitoring of the area is needed to confirm this prediction.Pelagic–benthic coupling describes the connection between surface-water production and seafloor habitats via energy, nutrient and mass exchange. Massive ice loss and warming in the poorly studied Arctic Chukchi Borderland are hypothesized to affect this coupling. The strength of pelagic–benthic coupling was compared between 2 years varying in climate settings, 2005 and 2016, based on δ Important insights into the global carbon cycle can, therefore, be gained from understanding the processes connecting ocean surface and seafloor through dynamics of organic matter, nutrients and energy cycling, i.e. pelagic–benthic coupling, in deep-sea ecosystems3. Pelagic–benthic coupling is considered to be tight when organic matter from surface production sinks to the seafloor with little reworking in the water column. Conversely, in weakly coupled systems, most of the energy is retained in the pelagic realm with low inputs to the benthos. Organic fluxes to the seafloor and strength of pelagic–benthic coupling vary in different ocean regions and largely depend on biological and physical processes in the water column7. One of the least studied regions in terms of trophic structure and carbon flux is the Arctic Ocean Basin with its complex morphological features along the perimeter8.The deep sea is considered the world's largest sink for biogenic carbon10. In addition, nutrient concentrations in the surface water are often low, mostly due to strong stratification11. Therefore, the amount of pelagic primary production in the Arctic Basin is among the lowest recorded in the world, with average estimates of 1 to 25 g C m−2 y−113. In addition to phytoplankton, sea-ice algae can contribute significantly to total primary production in this region. For example, ice algae have been previously reported to contribute more than 50% to the total primary production in the Arctic Basin14, a fraction that is much higher than their contribution of 4–30% on seasonally ice-covered Arctic shelves16. Other nutrient inputs, such as from advection of terrestrial matter and shelf production18 or from large food falls20, may be of local importance but are overall minor contributions for the Arctic Basin as a whole11.The Arctic Basin region is characterized by strong seasonality, with seasonal, and regionally multiyear, ice cover and the polar night lasting for up to 6 months, jointly constraining light availability needed for primary production22. Specifically, high pelagic grazing efficiency leads to a decrease in sedimentation of fresh phytoplankton, while little grazing facilitates higher sedimentation of intact phytoplankton cells and aggregates to the deeper water layers. The particulate organic matter (POM) can settle out in the form of intact cells, phytodetritus, fecal pellets, zooplankton carcasses, and marine snow8. During the descent, the POM undergoes additional biodegradation by bacteria and heterotrophs23. The amount and quality of material reaching the seafloor also depend on the water depth, as stronger vertical flux attenuation is expected in deeper areas of the Arctic Ocean24. Indeed, only a very small portion of carbon produced at the surface is estimated to reach the bottom of the Arctic deep sea (1–10%)27. Thus, typically very little and largely reworked organic particles reach the benthic fauna in the central Arctic, although export of fresh ice algal production has occasionally been observed28. Therefore, benthic trophic pathways in deeper areas of the Arctic Ocean have generally been described as longer than in shallower regions, with up to five trophic levels recorded for benthic species in the very few published studies from Arctic and sub-Arctic deep-sea environments29.The combined primary production sources serve directly as food for ice-associated and pelagic fauna. Their grazing intensity, in turn, has a strong impact on the amount and composition of organic matter reaching the seafloor30, although extremely high particle flux to the seafloor has recently also been observed during low sea ice cover on the Chukchi Sea shelf31. Ice algal production is mostly represented by large-sized diatoms that contribute significantly to a relatively fast transport of undisturbed organic matter to the seafloor33. In areas where open-water conditions dominate, pelagic phytoplankton is often characterized by a higher proportion of dinoflagellates than present in the sea ice community that might be retained more efficiently in the upper water column33. Similar connections between sea ice presence and stronger pelagic–benthic coupling have been observed28 or modeled for the Arctic deep sea34. Knowledge of food webs and pelagic–benthic coupling in the Arctic deep sea is, however, very scarce (but see36) due to logistical challenges related to sampling , leading to few observations mostly scattered over different Arctic deep-sea areas with the majority of studies being a snapshot in time.The presence or absence of sea ice may alter the strength of pelagic–benthic coupling in the Arctic marine ecosystems. Based on work on Arctic shelf systems, pelagic–benthic coupling is traditionally considered tighter in areas where sea ice is present39. This decline is due to the Arctic currently experiencing strong warming of about four times the global average air temperature40. Thinning of sea ice allows higher light penetration42 and increases in primary production in several areas, primarily on shelves, of the Arctic Ocean44. However, small-sized primary producers  are expected to dominate in warmer, fresher, and nutrient-poor water46, like the Beaufort Gyre47. Smaller phytoplankton cells are more resistant to sinking48. In addition, pelagic grazing pressure can increase in response to increased primary production49, as well as due to increased advection of zooplankton with Pacific and Atlantic water into the Arctic Ocean50, leading to higher retention of organic matter in the water column. Thereby, physical and biological alterations related to climate change can lead to a weakening of pelagic–benthic coupling and carbon sequestration in deep-sea sediments, and, therefore, decrease in benthic food supply. However, it has not yet been evaluated whether the strength of the coupling in the central Arctic has been modified as a consequence of climate change since this is difficult or impossible to determine because few or no baseline data are available from former years (but see36).The Arctic sea ice cover, however, is undergoing significant thinning and decrease in extent53. While time series have been established on benthic biomass and food supplies, and coupling have been modeled on the adjacent Chukchi Sea shelf55, temporal comparisons in adjacent deep waters are lacking. We here consider 2 years characterized by different sea-ice settings—2005 and 2016  compared to later, lower ice years (represented by 2016). Following earlier studies on pelagic–benthic coupling in Arctic regions60, we used stable nitrogen and carbon isotope analysis of POM endmembers and pelagic and benthic consumers to investigate pelagic-benthic coupling, specifically by comparing food source use and trophic niche space between the 2 years. This approach is based on the well-established concept that nitrogen stable isotope ratios indicate trophic position of organisms as tissues are progressively enriched in the heavier isotope with increasing trophic level in a reasonably predictable manner61. Thus, lower δ15N values of benthic taxa can be expected in a food web where pelagic-benthic coupling is tight. Carbon stable isotope ratios in consumers are indicative of carbon endmember utilization based on different isotopic ratios of different primary producers or habitats62. For example, sea-ice algae are often enriched in 13C compared to phytoplankton 64. Thus, higher consumer carbon isotope values can be found in areas where ice algae are a main food source. Both trophic markers (15N and 13C) combined describe trophic niches in isotope biplot space65. A high overlap of isotopic niches of pelagic and benthic members in a given food web can indicate tight coupling between these two realms. Therefore, we hypothesized a decrease in pelagic-benthic coupling strength would be reflected in a lower overlap of pelagic and benthic isotopic niches, higher δ15N values of benthic organisms, as well as lower δ13C values in benthic consumers from reduced ice algal uptake associated with lower ice extent. In this study, we aim to assess potential changes in pelagic–benthic coupling in the Arctic Chukchi Borderland within the Canada Basin, where only few benthic studies on the topic have been conducted before15N values of sPOM (sediment POM) and benthos  collected in 2005 were significantly lower than those collected in 2016  in 2005 and over 2.5‰ (6.3–8.8‰) in 2016 (Tables 15N of benthic consumers was 9.1‰ (10.4–19.5 ‰) in 2005 and 8.1‰ (12.4–20.5‰) in 2016 , however, did not differ significantly between the 2 years, although mean δ15N of pPOM in 2005 was slightly lower than in 2016. The range of δ15N of pPOM was 3.7‰ (from 1.4 to 5.1‰) in 2005 and 5.7‰ (from 1.9 to 7.6‰) in 2016  in 2005 and 6.4‰ (between 8.3 and 14.7‰) in 2016  in 2005 and 2.9‰ (from − 28.9 to − 26.0‰) in 2016. The δ13C range of zooplankton comprised 8.1‰ (from − 27.8 to − 19.7‰) in 2005 and 6.9‰ (from − 28.6 to − 21.7‰) in 2016  and over 8.4‰ (− 24.6 to − 16.2‰) in 2005 and 2016, respectively  for the benthos components between years. Compared to the benthos, the isotopic niche size of zooplankton was more similar between years, with an 86% probability of difference; as this probability was below the threshold of 95%, zooplankton niche sizes were not considered statistically different , of the benthic component differed between 2005 and 2016 with a wider niche in 2005 Fig. . This dient Fig. .Figure 4SEAc overlap between consumer groups and between endmembers was also different for the 2 years. Specifically, overlap between benthos and zooplankton was considerably higher in 2005 (57.9%) than in 2016 (5.5%). The SEAc overlap between sPOM and pPOM was generally low, but also higher in 2005 4.8%) than in 2016, when the two SEAc did not overlap , was too patchy to be captured by our sampling, or in fact was not isotopically enriched enough to be visible in benthic taxa and sPOM. In summary, some evidence points to the possibility of ice algae playing a role in the apparent difference in pelagic-benthic coupling between the study years, but unequivocal conclusions are difficult based on a single sampling period in each year.The source of primary production can also influence pelagic-benthic coupling. Based on the higher sea ice cover in 2005, we might assume that the abundance of ice algae was also higher in that year, though ice-algal biomass was not measured in the present study. In the adjacent northeastern Chukchi Sea, however, the ice algal signal at the seafloor, assessed by the isoprenoid trophic marker IP23. Mean δ15N values of sPOM and benthos were significantly lower in 2005 compared to 2016, which indicates that organic matter available to the benthos was generally less reworked in 2005 than in 2016. In addition, the isotopic niche of benthos was significantly wider in 2005 than in 2016, even though fewer benthic samples were available in 2005  and increased contribution of small cells (such as flagellates) in 2016 related to sea ice loss83, freshening of the area70, and strengthened stratification in recent years11. As a result, vertical organic matter export flux would have been dominated by faster sinking and, thus, fresher food sources85 for benthic consumers in 2005 than 2016. While we lack direct evidence for this hypothesis from our region, a study from deep Arctic Fram Strait indeed supplies indirect evidence in that the authors documented higher organic matter export efficiency in regions with than without seasonal sea ice86.Besides the amount and sources of primary production, freshness and, hence, quality of food has an effect on benthic trophic structure5 Tables , S2. The34 which might be expected if zooplankton densities increased with stronger advection of Pacific species into the basin or perhaps by locally increased reproductive output50. The few available inter-annual zooplankton studies from the Canada Basin region88, however, do not suggest a trend for increasing zooplankton populations between 2007 and 2017, nor do Abe et al.’s88 model results imply zooplankton increases in the region in the study period.Further, the strength of pelagic-benthic coupling is affected by grazing efficiency of zooplankton, which largely depends on the density, species composition, and developmental stages of herbivorous zooplankton present at the time of primary production. At high zooplankton densities and grazing rates, downward carbon flux can be reduced36. In the present study, we compared pelagic-benthic coupling in 2005, at the end of a decade with only early signs of warming56, and 2016, when years of intense climate warming had been documented and impacts on system drivers were observed. Our results suggest stronger coupling of benthic and pelagic realms in 2005 compared to 2016 and may indicate that ice-algal contribution was potentially higher in zooplankton diets in 2005 compared to 2016. This inference is consistent with observations from the nearby NE Chukchi Sea shelf and comparisons of vertical carbon export in ice-covered versus open water areas in deep Fram Strait, yet seasonal sampling in our study area would have been needed to provide firm evidence. Benthic communities received fresher organic material in 2005 than in 2016, as evidenced by δ15N values of benthic consumers and sPOM. The inferred decoupling in 2016 is consistent with physical and biological changes that were observed in the region in recent years. Specifically, a shift from perennial to seasonal sea ice39 may have resulted in an overall shift in primary producer composition and vertical carbon export within this system. Strengthening of the halocline within this region89 has resulted in a decrease in primary production in the area69 and a shift to small-celled phytoplankton70. We propose that these changes likely lead to a longer residence time of organic matter in the water column, a higher level of organic matter biodegradation before it reaches the seafloor, and, thus, a decrease in overall organic matter flux to the seafloor. This situation would reduce carbon storage in deep-sea Arctic benthos. Since ecosystem responses to climate change varies depending on local environmental and biological settings, it is recommended that time-series observations, similar to those on the adjacent Chukchi Sea shelf54, be extended into the deep Arctic Ocean basin.Evaluation of climate change effects on pelagic-benthic coupling in the deep Arctic Ocean is difficult due to limited availability of long-term data sets90 for both study years. The concentration is defined as the fraction of the area of the grid cell covered by sea ice and is given in percentage from 0 (no ice) to 100 (fully covered by ice) percent ice (https://nsidc.org/cryosphere/seaice/data/terminology.html). Average sea ice concentration for July and September (minimum ice month) was downloaded from the National Snow and Ice Data Centre (https://nsidc.org/data/NSIDC-0051/versions/1). The data were then imported into ArcGIS 10.591 software and projected spatially.To illustrate the difference in sea-ice cover between the sampling years, we plotted average sea-ice concentration data derived from satellite Nimbus‐7 SMMR and DMSP SSM/I‐SSMIS Passive Microwave at a grid cell size of 25 × 25 kmHEALY between 28 June and 25 July 2005 (expedition HLY05-02) and 2 July and 10 August 2016 (expedition HLY16-01) (Table 91). In 2005, five stations were sampled at depths ranging from 621 to 2090 m  Table . Samplin m Table , Fig. 5. m Table , Fig. 5.Pelagic POM (pPOM) was collected at each station in both years from the chlorophyll maximum layer from Niskin bottles attached to a SBE9/11 + CTD rosette equipped with an in-situ fluorometer. The chlorophyll maximum layer varied from 30 to 60 m depth in 2005 and from 50 to 70 m in 2016. Two to three water samples per station were collected from different bottles of the same CTD cast, totaling 13 pPOM samples in 2005 and 21 pPOM samples in 2016 Table . The col2 box core samples at 3 stations in 2005 and from 0.25 m2 box core samples at each of the 7 stations in 2016 (Table Sediment POM (sPOM) was collected into a sterile plastic bag from the top ~ 1 cm sediment from 0.06 m16 Table .Calanus glacialis (grazer), Calanus hyperboreus (grazer), Paraeuchaeta glacialis (predator), the amphipod Themisto abyssorum (predator/omnivore), and the chaetognath Eukrohnia hamata . Often, mass of individual zooplankton organisms was insufficient for isotopic analysis; thus, several individuals of the same species were pooled by station. A total of 71 zooplankton samples were collected in 2005 and 66 in 2016. Replication varied from 1 to 3 samples of each zooplankton species per station  in 2005 and at 6 stations with the same Multinet in 2016 Table . Five zoon Table .Global Explorer, Deep-Sea Systems Inc. in 2005, and Oceaneering International in 2016) in both years. The ROVs were equipped with a suction hose and a manipulator arm enabling targeted sample collection. Infaunal benthic consumers were collected with a 0.25 m2 box core in both years. All benthic samples were washed to remove sediments  and fauna were identified to the lowest taxonomic level possible. Vouchers of invertebrate taxa were collected when identification was uncertain and identified later by experts (see acknowledgments). Taxon names were verified with WoRMS . Benthic consumers were then subsampled for muscle tissue, where possible, to represent a tissue with slow turnover rate93. Where muscle tissue was not distinguishable or unavailable, tissue was sampled from body walls , tube feet , and entire organisms were collected when body mass was small . A total of 29 and 85 benthic organism samples were collected in 2005 and 2016, respectively, with replication varying from 1 to 3 per species per station  beam trawl and a Remotely Operated Vehicle  vapor for 48 h and dried before analysis. sPOM samples were thawed and each sample was homogenized by mixing. Approximately 1 ml of the sediment was treated with 1 N HCl until bubbling stopped and then rinsed with distilled water until pH of the sediments was close to neutral, after which the samples were freeze-dried before analysis2 as standard for nitrogen. Instrument error was < 0.2 ‰ for δ 13C and < 0.4 ‰ for δ 15N in 2005, and < 0.2 ‰ for both δ 13C and δ 15N in 2016. Sample isotopic ratios were expressed in the conventional δ notation as parts per thousand (‰) according to the following equation:13C or 15N of the sample, and R is the corresponding ratio of 13C/12C or 15N/14N.All carbon and nitrogen stable isotope analyses were performed at the Alaska Stable Isotope Facility at the University of Alaska Fairbanks on a Thermo Finnigan Delta Isotope Ratio Mass-Spectrometer with Vienna PDB as standard for carbon and atmospheric N13C versus δ15N were generated based on station-averaged values of each of the two carbon end-members  and each of the consumer groups (zooplankton and benthos). The isotopic niche widths of these four food web components  were then calculated as Standard Ellipse Areas corrected for small size (SEAc)97. To compare the isotopic niches of food web components between years statistically, we used a Bayesian approach to calculate 100 000 posterior iterations of SEA97 that produced a range of probable SEAs (Bayesian SEA = SEAB) for each of the food web component from each year. This enabled robust statistical comparison of SEAB between the sampling years by calculating the probability of difference between them97. Following97 and98, we considered a probability higher than 95% a meaningful difference. In addition, the overlap of SEAc of different food web components was calculated as the percentage of ellipse area shared by two components in order to test the hypothesis that benthic–pelagic coupling (expressed here as isotopic niche proximity) was tighter (= stronger overlap in SEAc) in 2005 than 2016. These analyses were conducted using the SIBER package  in R 4.0.3. statistical software99.For the analysis of potential differences in benthic–pelagic coupling between sampling years, we included only station pairs that were geographically close to each other and located in similar bathymetric features  Fig. , Table 315N and δ13C between different food web components as a measure of pelagic-benthic coupling were calculated by subtracting the mean δ15N (δ13C) of one food web component from the mean δ15N (δ13C) of another food web component. This metric was used to test the hypothesis that distance between food web components was lower in 2005 than in 2016.Isotopic distances of δ15N was overall lower in benthos (reflecting fresher food reaching the seafloor through tighter pelagic-benthic coupling) and δ13C was higher  in 2005 than in 2016, the means of δ15N and δ13C of each food web component were compared between the 2 years. The following tests were used for the comparison: a two-sample t-test , a Welch’s two sample t-test , and a Wilcoxon rank sum test . The Shapiro–Wilk test was applied to test for normality, followed by the Bartlett-test to verify the equality of variances. Values are presented as mean ± standard error (SE) in the text and tables. The analysis was conducted in R99.To test the hypothesis that δSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Mycobacterium tuberculosis complex (MTBC) variants are clonal, variant tuberculosis is a human-adapted pathogen, and variant bovis infects many hosts. Despite nucleotide identity between MTBC variants exceeding 99.95%, it remains unclear what drives these differences. Markers of adaptation into variants were sought by bacterial genome-wide association study of single nucleotide polymorphisms extracted from 6,362 MTBC members from varied hosts and countries.While The search identified 120 genetic loci associated with MTBC variant classification and certain hosts. In many cases, these changes are uniformly fixed in certain variants while absent in others in this dataset, providing good discriminatory power in distinguishing variants by polymorphisms. Multiple changes were seen in genes for cholesterol and fatty acid metabolism, pathways previously proposed to be important for host adaptation, including Mce4F , 4 FadD and FadE genes (playing roles in cholesterol and fatty acid utilization), and other targets like Rv3548c and PTPB, genes shown essential for growth on cholesterol by transposon studies.bovis and variant tuberculosis, and suggest that adaptation to new hosts could involve adjustments in uptake and catabolism of cholesterol and fatty acids, like the proposed specialization to different populations in MTB lineages by alterations to host lipid composition. Future studies are required to elucidate how the associations between cholesterol profiles and pathogen utilization differences between hosts and MTBC variants, as well as the investigation of uncharacterized genes discovered in this study. This information will likely provide an understanding on the diversification of MBO away from humans and specialization towards a broad host range.These findings provide a robust set of genetic loci associated with the split of variant The online version contains supplementary material available at 10.1186/s12920-023-01695-5. Mycobacterium tuberculosis complex (MTBC) has afflicted human and animal health since the dawn of civilization. This ancient pathogen, typified by M. tuberculosis variant tuberculosis (MTB), infects humans primarily and is considered specialized for this niche [M. tuberculosis variant bovis (MBO) is a generalist pathogen – its host range includes foxes, seals, cattle, cervids, lions, dogs, mustelids, badgers, and others [The is niche , 2. Its is niche , 4. MTB is niche , 6. On td others , 7–10. Td others , 11, 12,d others , 14, andd others . From thd others . Within d others , and metd others , 19, amoTo help address this gap in knowledge, WGS datasets were collected for MTBC variants from diverse hosts and countries. Paired-end read SRA datasets with metadata including country and host of isolation, and MTBC variant  were used to create a set of 9,755 SNPs for a bacterial genome-wide association study (bGWAS). This sought to detect loci associated with classification as MTB or MBO, as well as any detectable host-specific markers . Using RAxML-NG , prewas caprae, and 8 variant orygis . All sequence data used in this project are publicly available through NCBI and ENA. Accession IDs for all data are recorded in the table Additional file Core SNP extraction was successful for 6,362 isolates. Isolates were from 27 countries Fig. A; includgis Fig. B; and acgis Fig. C. We addroup n = . All seqBovidae also produced the same 120 significant loci by the same subsequent test but did not produce loci for the simultaneous test  yielded no significant hits  showed an unusual pattern by subsequent statistical test where nearly all SNPs clustered just around the significance cutoff, yielding hundreds of loci technically exceeding the cutoff yet are tightly clustered with those under it .TreeWAS runs three tests for statistical significance – the terminal, simultaneous, and subsequent tests. The terminal test identifies broad associations between genotype and phenotype looking only at terminal nodes in the tree; the simultaneous test more stringently identifies deterministic relationships of genotype and phenotype, without necessitating the relationship occur at all branches; the subsequent test utilizes the terminal test but adds ancestral state reconstruction to analyze all nodes of the tree . A thoroest Fig. . Anotherest Fig. . A phenoThe 32 loci identified by the simultaneous test for MBO are listed in Table Bovidae, MBO, and Homo sapiens by the subsequent test are listed in Table As mentioned for the spurious hit for UK samples, the simultaneous test can report loci as associated even if a SNP is only present in a few isolates. These false positives are included in the data table but are marked by bolding to indicate they are not meaningful. Subtracting these spurious hits, the simultaneous test identifies 22 loci, all of which are also identified by the subsequent test. The 120 loci concordantly identified as associated by n = 22) are presented in Table The subset of loci called by both tests in MBO  or not (0), and Homo sapiens (1) or not (0).These 10 isolates’ VCF files were checked against the SNP barcode , with liDespite remarkable similarity between MTB and MBO, evidence of clear divides was present in SNPs highlighted as associated by treeWAS analysis.The Fad family of proteins are important in MTBC, with MTB known to carry 36 FadD and FadE loci , 27. GWAmce4f SNP separated MTB/MOR and MBO/MCP.Cholesterol intake in MTBC requires a functional Mce4 system . A misseppsD, and synonymous changes in ppsB and pks15, all polyketide synthase genes. Genes annotated in roles of cholesterol and fatty acid metabolism or in pathways downstream of these processes are among all loci identified by the MBO subsequent tests are shown in Table Catabolized cholesterol products fuel core acyl-CoA metabolism pathway, as well as polyketide synthesis, a pathway already known to differ between MBO and MTB . GWAS idksgA and ispE, genes involved in ribosome maturation and cell wall synthesis, respectively [Other identified loci with functions separate from cholesterol and lipid metabolism include AccD1, involved in leucine degradation , which bectively . After aMTBC physiology and function remain uncertain, and 48/120 loci identified are in genes annotated only by locus identifier and generically, like “conserved protein” or “possible oxidoreductase” even after PE/PPE gene filtering, removing a largely uncharacterized family comprising ~ 10% of MTBC genes. Even among genes with fuller annotations, nearly all include “probable” in their descriptions. The genes presented in Table –10 mutations per cell division) [. reporting cholesterol utilization in MTB is key to host adaptation [Any associated loci may signify adaptive roles in differentiation from a specialist infection by MTB and a pathogen with a much broader host range, like MBO. It is well-reported that members of the MTBC are clonal, and not only is horizontal gene transfer vanishingly rare, mutation rates in members of this complex are low (~ 2 × 10ivision) . Given 9ivision) , 37. Whiaptation . MTB driaptation –41. The aptation , as is taptation , a modelaptation  vs. the aptation , 43, 44,aptation , 46. Theaptation . Others aptation . MTB is aptation –40. Foamaptation , indicataptation , 45, 48.aptation , 50. BioM. canettii, performing an MBO-specific bGWAS to try to identify host adaptation within the variant, or even by constructing an MTBC pangenome to call SNPs against for the maximum possible number of informative protein-coding changes. We hope our research serves as a launching point for these studies, for more routine sequencing and better metadata classification of isolates across the MTBC, and for better understanding of MTBC divergence, including through generation of time-measured phylogenies to estimate SNP occurrences through evolutionary history.Adaptation to specific hosts was not detectable with this approach, which could be improved through routine sequencing of isolates from non-standard host types, which are currently rare and geographically biased. While efforts were taken to select from countries worldwide, the dataset itself is necessarily a biased sampling as well – very few isolates of the vast number of infections worldwide are ever sequenced, and of those that are, even fewer meet the inclusion criteria for metadata and sequencing coverage. Furthermore, in many clinical MTB genomes utilized in this work, evolutionary pressures imparted by antibiotic use are a powerful influence on SNP distribution, and drug-resistance associated SNPs were not filtered from the input dataset. This adds an additional layer of bias in that antibiotic selection pressures vary wildly across MTBC members. For future studies and adaptations of these findings, it is critical to note that our use of MTB H37Rv as a reference to study the entire MTBC and its evolutionary history is necessarily missing data. While the use of this reference as a standard is common practice and the H37Rv coordinates are typically how MTB SNPs are defined, the H37Rv lineage is a modern one, and any regions of difference absent in H37Rv but present in other MTBC members will not be SNP-called by our method, excluding potentially tens of thousands of base pairs. Future studies should consider using more “ancestral” variants like Mycobacterium is a major outstanding knowledge gap. While tremendous work has been performed by groups worldwide, the mechanisms of how exactly MTBC members present different and infect different hosts is unresolved. Detail transcriptomic profiling has been performed in cattle infections by MTB and MBO, which demonstrate that macrophages have very distinct response profiles to each variant, as well as what appears to be a fine-tuned engagement with the bovine innate immune system for MBO [Analysis of the genetic differences between MTBC isolates and their association with classification as certain variants or isolation from certain hosts by GWAS was performed. The 120 SNPs identified through this analysis provide a trove of genes and pathways implicated in adaptation towards a generalist lifecycle, including loci across cholesterol and fatty acid uptake, catabolism, and downstream processing pathways, important for central metabolism in MTBC organisms and critical for pathogenesis , 39, 51. for MBO . It is k for MBO , and tha for MBO . As disc for MBO , and pri for MBO . Multipl for MBO . While tIn summary, these SNPs reliably differentiate MTBC variants, and importantly, they may inform research into genes that differ between variants, narrowing the pool of uncharacterized proteins to study in the MTBC.. in designing the MTBC SNP barcode are a validated set of primarily human MTB isolates [Mycobacterium. Remaining paired-end read sets were selected  to build the final “snippy-core” core SNP alignment, along with the H37Rv reference and M. canettii (GCF_000253375.1) as an outgroup, while masking PE/PPE genes using the H37Rv-specific.bed file provided by default in Snippy. Core SNPs were used for phylogenetic tree generation in RAxML-NG [. SNP barcode and SNP-IT tool were used [Existing datasets were collected, including those where bGWAS was performed to answer other questions. Dong et al. 2022) genome-sequenced 74 Chinese cattle MBO isolates, and performed bGWAS analysis on 3,227 total MBO isolates from around the world . Additioisolates . Both daisolates , formattisolates , 55 and isolates  run withisolates  revealed22 genomeRAxML-NG ; bootstrRAxML-NG , R (v4.0d n = 6,3 to buildRAxML-NG  were useRAxML-NG . In prewRAxML-NG  environmRAxML-NG , OpenMPIRAxML-NG , and R (RAxML-NG  was instRAxML-NG  and treeRAxML-NG  from Gitere used , 25. Snpere used . TreeWASAdditional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.Additional file 8.Additional file 9.


The direct chromogenic assay is easy to perform, fast, reproducible, and suitable for high-throughput screening of pigmented α-amylase inhibitors.The inhibition of carbohydrate digestion by plant bioactive compounds is a potential dietary strategy to counteract type 2 diabetes. Indeed, inhibition of α-amylase, a key enzyme that carries out the bulk of starch digestion, has been demonstrated for a range of bioactive compounds including anthocyanins; however, sample pigmentation often interferes with measurements, affecting colorimetric assay outcomes. Therefore, the present study compared the performance of a direct chromogenic assay, using 2-chloro-4 nitrophenyl α-D-maltotrioside (CNPG3) as a substrate, with the commonly used 3,5-dinitrosalicylic acid (DNS) assay. The direct chromogenic assay demonstrated a 5–10-fold higher sensitivity to determine α-amylase inhibition in various samples, including acarbose as a reference, pure anthocyanins, and anthocyanin-rich samples. The IC Anthocyanins, a sub-class of flavonoids, are water-soluble naturally occurring bioactive compounds responsible for the red to dark blue colour of most flowers, fruits, and vegetables ,2. Over Dietary carbohydrates are hydrolysed by salivary and pancreatic α-amylases in the mouth and small intestine, respectively, to smaller fragments such as maltose and maltodextrins. The resultant products of α-amylase action are further hydrolysed by intestinal brush border α-glucosidases into absorbable glucose units . AlthougAntidiabetic drugs, such as acarbose, miglitol, and voglibose, have been approved for their use in diabetes management. They reduce postprandial hyperglycaemia by inhibiting the activity of digestive enzymes ,10. HoweThere are several methods reported in the literature to determine the α-amylase inhibitory properties of polyphenolic compounds. These include methods to detect reducing sugars, such as the 3,5-dinitrosalicylic acid (DNS) assay and the Nelson–Somogyi reagent, as well as the iodine–starch test, turbidity test, and chromogenic methods . ChromogThere is a rationale to explore the use of alternative assays using synthetic substrates in order to determine the inhibition of α-amylase activity by pigmented polyphenols, i.e., anthocyanins. Recent research  highlighOn the basis of the above-mentioned shortcomings in measuring pigmented polyphenol inhibitory activity, the aim of the current study was to test the performance of a direct chromogenic assay that utilizes CNPG3 as the substrate, in comparison with the DNS assay, to determine the α-amylase inhibitory properties of anthocyanin-rich extracts and purified anthocyanins.50) at 37.6 μg/mL  using a similar substrate.Acarbose, a well-known α-amylase and α-glucosidase inhibitor, is a commonly used antidiabetic drug  and is typically used as a positive control when assessing the enzyme inhibitory properties of plant extracts in vitro ,26. Inde.6 μg/mL A. These .7 µg/mL  and 77.5.7 µg/mL  for α-am72 μg/mL B. This op-nitrophenol was recorded at 1 min intervals over the 10 min reaction period and plotted against time. For acarbose  were tested for α-amylase inhibitory activity using both assays. The blackcurrant sample, which consisted of anthocyanins only (>95% purity) , inhibitectively A,B. Simiectively C and wavectively D of blac50 24.4 μM)  (0–5 mM) in the presence or absence of inhibitors. The Lineweaver–Burk plots, shown in While ICIn contrast, with blackcurrant anthocyanins, the Km remained unaffected, whereas Vmax decreased, showing mixed-type inhibition of α-amylase C. Previop-nitrophenyl-linked maltose derivatives such as CNPG3 exhibit a lower affinity for amylase ), and Km and Vmax were calculated.To detail the inhibitory mechanism, a series of experiments were conducted by maintaining PPA at 1 U/mL and varying the concentration of inhibitors  and substrate CNPG3 (0–5 mM). A Lineweaver–Burk plot was constructed for every inhibitor, and Km and Vmax were calculated.50 values (concentration at 50% enzyme inhibition) of pure compounds and purified and non-purified extracts were calculated using a non-linear regression model. The calculated inhibition rates at every concentration of anthocyanins/anthocyanin-rich extracts and acarbose were fitted with a nonlinear curve fit in GraphPad Prism. The IC50 values were then obtained from the respective curve. The mode of enzyme inhibition was graphically determined using the Lineweaver–Burk plot through nonlinear regression of all data sets at once using GraphPad Prism software.All experiments were conducted at least in triplicates and analysed using Prism software program v. 9 . The IC50 compared to the DNS assay. Due to this higher sensitivity, as well as a lack of interference from pigmented and sugar compounds, this method should be favoured for enzyme inhibition measurements in pigmented samples. The microplate format makes the assay efficient for high-throughput screening purposes, with application in academic and industrial research to screen for potential antidiabetic components and assess food formulations for functional properties. However, it should be noted that α-amylase has lower affinity for CNPG3 compared to natural starch substrates. The inhibitory activities of the various anthocyanin samples varied from sample to sample, with the highest potential for blackcurrant and blueberry. Variations in outcomes linked to anthocyanins may be associated with anthocyanin structure, in particular hydroxylation, glycosylation, and acylation pattern. Enzyme inhibitory properties may also be affected by the presence of other inhibitors in the extracts, such as other polyphenols. Further investigations elucidating the inhibitory potential of differently structured anthocyanins on α-amylase, their mode of inhibition, and validation against in vivo inhibition will need to be conducted for the most effective use of anthocyanins in the prevention and management of diabetes.This study tested the performance of a direct chromogenic assay that utilizes CNPG3 as the substrate, in comparison with the DNS assay, to determine the α-amylase inhibitory properties of anthocyanin-rich extracts and purified anthocyanins. The chromogenic assay consistently showed lower IC


The inhibition of tyrosinase (TYR) activity is an effective measure to inhibit melanin synthesis. At present, there are many methods with discrepant details that study the TYR inhibitory activity of samples. Under the same experimental conditions, this paper systematically studies whether enzyme species and sample addition methods are the key factors that determine the TYR inhibitory activity of samples. TYRs extracted from B16F10 cells, apple and mushroom, called BTYR, ATYR and MTYR, respectively, were selected to implement this study. Results showed that TYR inhibitory activities of samples were obviously affected by the above two factors. It was necessary to select the appropriate enzyme according to the problems to be explained. It was speculated that indirectly inhibitory activity reflected the comprehensive effects of samples on TYR catalytic activity and intracellular TYR synthesis pathway, while directly inhibitory activity reflected the effects of samples on TYR catalytic activity. Additionally, kojic acid could be used as a positive control for both B16F10 cells and MTYR models. The TYR inhibitory activity of β-arbutin was complicated and fickle, while that of epigallocatechin gallate (EGCG) was universal and stable, which is to say, EGCG always inhibited TYR activity in a dose-dependent manner. In conclusion, the TYR inhibitory activities of samples were affected by enzyme species and sample addition methods. Compared with the unstable β-arbutin, EGCG was more valuable for clinical research. The key rate-limiting enzyme in melanin biosynthesis is tyrosinase (TYR) . TYR is TYR is widely found in bacteria, fungi, plants and animals. TYRs have similar physiological functions in vivo, but their physical and chemical properties are different. For example, their molecular weight (about 53 kD to 75 kD of different species) and isoelectric point have a wide range of changes . In addiIn addition, there are differences in the methods used to detect the effects of samples on TYR activity in animal cells. Some researchers firstly extracted TYR from mammalian cells and then reacted with the substrate (L-DOPA) in the presence of the sample. For example, Funayama et al.  reportedBased on these, we hope to systematically study the effects of species and sample addition methods on the TYR inhibitory activities of samples under the same experimental conditions , because these two factors would directly affect the reliability and application range of results. B16F10 is a cell with strong ability to synthesize and secrete melanin, and it is a commonly used cell model to study the anti-melanogenic activity of samples . The TYRIn conclusion, TYRs from three sources, BTYR, ATYR and MTYR, were used to study the influence of species on the TYR inhibitory activity of samples. In terms of sample addition methods, (a) TYR extraction being followed by sample treatment and (b) sample treatment being followed by TYR extraction were designed. This study could prove the factors that might affect the TYR inhibitory activity of samples in experimental system, and provide reference for researchers who are just starting out with the anti-melanogenic experiments to select appropriate enzyme sources and experimental methods.Two sample adding times were designed for different sample addition methods. (a) TYR extraction was followed by sample treatment. In detail, TYR was extracted from B16F10 cells or apples without sample treatment, and then mixed with samples and substrates to start the enzymatic oxidation reaction. In this process, sample only affects the catalytic activity of TYR and is in direct contact with TYR, so the effect of sample on TYR was called directly inhibitory activity; (b) Sample treatment was followed by TYR extraction. Specifically, TYR was extracted from B16F10 cells or apples treated with samples, and then mixed with substrates. Because the sample does not exist directly in the enzymatic reaction system, the effect of sample on TYR was called indirectly inhibitory activity.50 was 0.54 ± 0.02 mM). The ability of β-arbutin to directly inhibit BTYR activity (IC50 was 59 ± 11 mM) was only 1/100 of that of kojic acid. Although 0.025–2.4 mM EGCG directly inhibited the activity of BTYR, the inhibition rate did not reach 50%. In fact, we also studied the directly inhibitory effects of 3.2 mM EGCG and 4 mM EGCG on BTYR. However, when EGCG at above two molarities were mixed with BTYR, the reaction system becomes opaque, affecting the determination of absorbance  on BTYR was significantly stronger than β-arbutin (IC50 of 541 ± 20 µM), and both were stronger than kojic acid (IC50 was not reached at 1200 μM).The effect of samples on cell viability was detected before the BTYR indirectly inhibitory experiment. Cell viability results  showed t50 = 1.77 ± 0.02 mM). Low concentration (12.5–400 µM) of kojic acid promoted ATYR activity with EC50 of 0.096 ± 0.004 mM, and the lower the concentration, the stronger the promoting effect. High concentration (800–4000 µM) of kojic acid inhibited the activity of ATYR in a dose-dependent manner (IC50 = 2.11 ± 0.02 mM). 0.025–320 mM β-arbutin did not directly inhibit ATYR activity, but weakly promoted ATYR activity (max to 21%).As a fruit prone to browning, apple was also used to implement related experiments in order to enhance the accuracy, reliability and commonality of results about sample addition methods. When the sample was in direct contact with ATYR in enzymatic oxidation reaction , EGCG inp < 0.05) except for apple slices soaked by water at 4.17 h. Therefore, soaking contributed to reduce a* increasing. Compared with water, apple slices soaked by compounds could significantly weaken a* (apart for β-arbutin at 0.17 and 24.17 h), so all the three compounds could inhibit the browning of apple slices. The a* of apple slices soaked by EGCG was significantly lower than apple slices soaked by β-arbutin at 0.17 to 8.17 h, and lower than β-arbutin at 24.17 h without significant difference. There was no significant difference between EGCG and kojic acid at 0.17 and 8.17 h, while EGCG significantly lower than kojic acid at 4.17 h. These results showed that EGCG effectively reduced the increase in a* value. The b* values of each treatment had little difference (p < 0.05). Referring to ΔE, without soaking kept the largest ΔE at all times  and kojic acid (704 μM) were used as samples, and BTYR was selected as the target enzyme. The effects of two samples on intracellular BTYR activity, catalytic activity of BTYR in non-cellular system and intracellular BTYR mRNA expression level were analyzed. As shown in Although kojic acid significantly inhibited the catalytic activity of BTYR compared with EGCG and no difference was found between EGCG and kojic acid on TYR mRNA expression, the ability of kojic acid to inhibit intracellular BTYR activity was significantly lower than EGCG. This phenomenon indicated that the influence of samples on intracellular physiological metabolism is very complex, including not only TYR synthesis, but also possibly TYR degradation and other processes. This provides a path for researchers to carry out more in-depth research, that is study extending from effective combination of two sample addition methods.In addition to sample addition methods, the species of enzyme may be another factor affecting the TYR inhibitory activity of the sample. Firstly, whether TYR inhibitory activity of samples being influenced by TYR species was investigated when samples were in direct contact with TYR in enzymatic oxidation reaction. As shown in 50 of 1421 ± 61 μM) was only 1/15 of that of kojic acid (IC50 of 94 ± 1 μM), which was consistent with the result that EGCG was weaker than kojic acid in the directly inhibitory activity of BTYR. While in ATYR directly inhibitory activity, EGCG was stronger than kojic acid.Except for the compounds showed either promoting or inhibiting activity in different species of enzymes, there were also species differences in sample activity comparison. As shown in Secondly, there were also species differences in the TYR indirectly inhibitory activities of samples b. The thIn summary, the TYR inhibitory activity of the samples was affected by the enzyme species.We were not able to obtain purified TYR extracted from apple or B16F10 cells so as to further study the inhibition types of these enzymes treated by samples. Therefore, this part was mainly carried out for commercial MTYR. The effect of β-arbutin on the directly inhibitory activity of MTYR was fickle a. β-arbumK increased with increasing concentration of EGCG, but maxV remained unchanged. The intersection of lines was on the Y-axis. Based on these results, it was speculated that EGCG was a competitive inhibitor of MTYR  inhibitor of MTYR  were set up to obtain the initial rate of enzymatic reaction of each treatment. 1/V against 1/[S] was plotted to get a series of lines with different slopes. The results showed that  of MTYR a,e. With of MTYR a,e 20]..Km increKi, was calculated by replot of slopes (Lineweaver–Burk plot) vs various molarities of inhibitor [Ki is relevant to inhibitory activity of inhibitor, being the smaller the value of Ki the stronger is the inhibitory activity [Ki values of EGCG and kojic acid were 170.4 μM  determines the mechanism, and its value decides the degree to which the binding of inhibitor alters the affinity of the enzyme to the substrate. α of kojic acid was 2.579 d,e, whicIn addition, slope or intercept was replotted versus sample concentrations b–d, whicTYR is a key rate-limiting enzyme in melanin biosynthesis. Inhibition of TYR activity is an effective measure to reduce the formation of melanin, and is also a common indicator to screen compounds with anti-melanogenic activity. This study mainly explored some interesting and contradictory phenomena about enzyme activity founded in previous studies, that is, whether the species of enzyme and the sample addition methods were the influencing factors for the TYR inhibitory activity of sample.The TYR inhibitory activity of sample was affected by sample addition methods. In the directly inhibitory activity of BTYR, all of EGCG, kojic acid and β-arbutin, inhibited the activity of BTYR in a dose-dependent manner, however, the activity of kojic acid was stronger than that of EGCG and β-arbutin. In the indirectly inhibitory activity of BTYR, although EGCG, kojic acid and β-arbutin indirectly inhibited BTYR activity in a dose-dependent manner, the activities of EGCG and β-arbutin were stronger than that of kojic acid. When using ATYR as an enzyme source, 0.025–320 mM β-arbutin directly promoted the activity of ATYR, while 1 mM β-arbutin significantly and indirectly inhibited the activity of ATYR, showing a completely different action of mode. This was a thought-provoking and interesting result. Lim et al. , in orde50 was 8.4 mM), but as a TYR diphenolase inhibitor in B16 mouse melanoma cells, α-arbutin was 10 times more potent than β-arbutin. In other words, α-arbutin and β-arbutin showed different inhibitory activities in TYR of different species. Mann et al. [50 = 4400 mM) was 1/4000 of that on MTYR (IC50 = 1.1 mM) [It was found that the TYR inhibitory activity of samples was also affected by enzyme species. For example, (1) in direct inhibition, kojic acid inhibited BTYR and MTYR activities in a dose-dependent manner. With the decrease in kojic acid concentration, its inhibitory activity on BTYR and MTYR was close to zero. Kojic acid has a double effect on ATYR activity. Low concentration of kojic acid promoted ATYR activity, while high concentration of kojic acid inhibited ATYR activity. (2) In direct inhibition, the effects of β-arbutin at lower concentration 0.025–0.8 mM) on BTYR activity were unstable, while higher concentration (0.8–320 mM) of β-arbutin inhibited BTYR activity in a dose-dependent way. β-arbutin slightly increased ATYR activity in a dose-dependent manner (the maximum increase was only 21%). 0.48–40 mM of β-arbutin weakly promoted MTYR activity (4%–13%), whereas 80–320 mM of β-arbutin dose-dependently inhibited MTYR activity 5%–27%). (3) In the directly inhibitory activity of MTYR and BTYR, EGCG was weaker than kojic acid. In the directly inhibitory activity of ATYR, EGCG was stronger than kojic acid. (4) In indirect inhibition, kojic acid inhibited the activity of BTYR in a dose-dependent manner, but 1 mM kojic acid significantly increased the activity of ATYR. In addition, which activity of EGCG or arbutin was stronger was affected by species. Similar contradictory results have been reported in the previous literatures. Funayama et al.  found thn et al.  thought n et al. . For exa 1.1 mM) ,24. In f–0.8 mM o 1.1 mM) . Liu Sha%. (3) In®-8). The orange-yellow formazan dye obtained by the oxidative reduction of WST®-8 triggered by intracellular dehydrogenase is able to dissolve in the medium. The amount of formazan dye generated is proportional to the number of living cells [In this study, CCK-8 method was used to determine the changes of cell viability under each treatment, and cell viability was used to represent cell survival rate, thus indicating the toxicity of samples to cells. In our previous study , 43.8–70ng cells . Therefong cells . The difIn this experiment, kojic acid had a double effect on ATYR activity. Low concentrations 12.5–400 µM) of kojic acid promoted ATYR activity, while high concentrations (800–4000 µM) of kojic acid inhibited it. Similar results have been found in the previous literature. Pyrogallol is an organic gallic acid conversion compound with three hydroxyl groups and belongs to the phenol family . When py0 µM of kKi results showed that kojic acid had stronger inhibitory activity against MTYR than EGCG when the compound was in direct contact with MTYR, further confirming the results of IC50.In addition, the results of inhibition types showed that EGCG was a competitive inhibitor of MTYR diphenolase activity, while kojic acid was a mixed inhibitor of MTYR diphenolase activity. In this study, two conceivable and potential factors, enzyme species and sample addition methods, that might affect TYR inhibitory activity of samples were systematically studied under unified experimental conditions, which can help the researchers who are new to anti-melanogenic activity to avoid traps and carry out more valuable research faster. At the same time, it was found that EGCG had universal and stable TYR inhibitory activity, which might have a better clinical study value compared with the unstable β-arbutin.Dulbecco’s modified Eagle’s medium (DMEM), α-MSH (A1025), kojic acid (≥99%), 3,4-dihydroxyphenylalanine (L-DOPA), and MTYR were obtained by the same way as our previous literature ,19.Fetal bovine serum (FBS) was purchased from Gibco . D-PBS, cell counting kit-8 (CCK-8), dimethyl sulfoxide (DMSO) and P0013J lysis buffer were purchased from Beyotime . NaOH was purchased from Sinopharm chemical reagent Co., Ltd. . EGCG (98%) was a generous gift from the Tea Research Institute of Chinese Academy of Agricultural Sciences. β-arbutin (≥99%) and polyvinylpolypyrrolidone (PVPP) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. .2 cell culture dish were washed with D-PBS two times and incubated for 30 min at −20 °C in 1.16 mL of lysis buffer. The lysates, called BTYR, were centrifuged at 12,000 rpm for 10 min at 4 °C.B16F10 cells at the exponential phase in 10 cmv0) of each reaction was represented by the slope of the initial linear range of the progress curve, and was used to calculate the relative enzyme activity of each treatment [The reaction system in 96-well plate was 100 μL. 20 μL of BTYR was mixed with 20 μL compounds and incubated at 25 °C for 10 min, then 60 μL of 8 mM L-DOPA was added to start the reaction. A Synergy H1 microplate reader  was used to dynamically monitor the optical densities at 475 nm. Absorbance was measured every minute and sustained 30 min. The initial velocity ; Sample control group ; Negative control group ; Blank control group (there are TYR only).2 at 37 °C.Referring to our previous methods . In shor4 cells/well and incubated for 24 h. Then, the culture medium was replaced with complete medium containing compound and further incubated at 37 °C for 48 h. After rinsing once with D-PBS, 100 µL DMEM containing 10% CCK-8 was added to each well. Followed by another 1 h incubation, the absorbance at 450 nm was measured.Measurement of cell viability was determined according to our previous procedure . In brieThe molarities of kojic acid, β-arbutin and EGCG were 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 μM. The cell viability was expressed as a percentage of the control.Measurement of intracellular melanin content was determined according to our previous procedure . BrieflyThe BTYR indirectly inhibitory activity was assayed according to our previous procedure  with sliIn a reaction system of 100 μL, 20 μL of BTYR was mixed with 60 μL of 8 mM L-DOPA (the remaining volume was supplemented with 20 mM pH 6.8 PBS), and immediately placed at 475 nm for dynamic detection for 30 min. The absorbance was measured once per minute. The control group containing only BTYR (blank control and sample control) was set for synchronous detection to exclude background interference.With negative control as the reference, the indirectly inhibitory activity of BTYR was calculated in the same way as in Colorimetric measurements were carried out using Shanxi Fuji apples, following the procedure of  with sliThe preparation of fresh-cut apple slices was the same as in 20 g of pitted apple slices (about two apple slices) were weighed into a juicer  containing insoluble PVPP (2 g) and pre-cooling phosphate buffer , followed by homogenized for 1 min and centrifuged at 12,000 rpm for 10 min at 4 °C. After the supernatant was divided, it was quick-frozen with liquid nitrogen, and then stored in the refrigerator at −80 °C. The supernatant was used for ATYR activity analysis.In a reaction system of 200 μL, 40 µL ATYR was mixed with 40 µL corresponding compound in 96-well plates. After incubated at 25 °C for 10 min, L-DOPA  was added to each well as a substrate. Once the substrate was added in reaction system, the formation of dopachrome was immediately measured at 475 nm for 30 min at 1 min intervals. The molarities of kojic acid were 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2 and 4 mM. The molarities of β-arbutin were 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4, 5, 10, 20, 40, 80, 160 and 320 mM. The molarities of EGCG were 0.00004, 0.0002, 0.001, 0.005, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2 and 4 mM. The group setting and the calculation of ATYR directly inhibitory activity were the same as in Apple slices, immersing in various test solutions for 10 min, were used to extract crude ATYR solution. The test solutions were water (negative control groups), kojic acid (1 mM), β-arbutin (1 mM), and EGCG (1 mM).In a reaction system of 200 µL, 40 µL of ATYR was mixed with 120 µL of 8 mM L-DOPA (the deficient part of the system was supplemented with 20 mM pH 6.8 PBS), and the absorbance was measured once per minute at 475 nm for 30 min. The control group containing only ATYR (blank control and sample control) was set for synchronous detection to exclude background interference. With negative control as the reference, the indirectly inhibitory activity of ATYR was calculated in the same way as in The MTYR directly inhibitory activity was assayed according to our previous procedure . BrieflyThe molarities of kojic acid were 0.00004, 0.0002, 0.001, 0.005, 0.025, 0.05, 0.1, 0.2 and 0.4 mM. The molarities of β-arbutin were 0.03, 0.06, 0.12, 0.24, 0.48, 0.96, 2, 4, 5, 8, 10, 20, 40, 80, 160 and 320 mM. The molarities of EGCG were 0.00004, 0.0002, 0.001, 0.005, 0.025, 0.1, 0.2, 0.4, 0.8 and 1.6 mM.5 cells/mL). Total RNAs prepared were followed and qPCR analysis of TYR were carried on with β-actin as an internal control.Measurement of mRNA expression of TYR was determined according to our previous procedure . BrieflyThe experimental method was the same as mK and maxV of MTYR were determined by the Michaelis–Menten equation. The Lineweaver–Burk plot was used to display the data. Secondary replot of Lineweaver–Burk plot or intercept was used to display inhibition constant (Ki) and α.The molarities of L-DOPA were 0.0625, 0.25, 1, 4 and 8 mM. The molarities of kojic acid were 25, 50 and 100 µM, while that of EGCG were 200, 400 and 800 µM. The p < 0.05). Michaelis–Menten equation and Lineweaver–Burk plot were analyzed and drawn using GraphPad Prism .All experiments were carried out at least in three independent trials. The experimental data were expressed as the mean ± standard deviation. One-way analysis of variance was carried out by SPSS 21.0  and was used to compare the value differences (In summary, this study found that the TYR inhibitory activity of samples was affected by the species of enzyme and the sample addition methods. In view of the important effect of enzyme species on the TYR inhibitory activity of samples, it is necessary to select the appropriate enzyme according to the problems to be explained. In addition, the results of indirectly inhibitory activity reflect the comprehensive effects of samples on TYR catalytic activity and intracellular TYR synthesis pathway, while the results of directly inhibitory activity mainly reflect the effects of samples on TYR catalytic activity. Kojic acid directly inhibited the activity of BTYR and MTYR in a dose-dependent manner, and kojic acid also indirectly inhibited the activity of BTYR in a dose-dependent manner. Therefore, kojic acid could be used as a positive control for both B16F10 cells and MTYR models. In terms of directly inhibitory effect, β-arbutin could inhibit or promote TYR activity according to different species of TYR, or even promote TYR activity first and then inhibit TYR activity in a dose-dependent manner. Therefore, the directly inhibitory activity of β-arbutin on TYR was unstable. β-arbutin could indirectly inhibit the activities of BTYR and ATYR. In directly inhibitory activity, EGCG maintained a stable inhibitory activity on TYR of different species. EGCG also indirectly inhibited BTYR activity in a dose-dependent manner. It could be speculated that the TYR inhibitory activity of EGCG is universal. Compared with the unstable β-arbutin, EGCG might be more valuable for clinical research.


The “In–Out–In” (IOI) posterosuperior screw was common in screw fixations of femoral neck fractures. The impacts of the IOI screw on the blood supply of the femoral head have not yet been clarified. The nutrient foramen was damaged when the screw was present in their corresponding cortex surface. This study aimed to evaluate the damage degrees of the nutrient foramina in the femoral neck as the IOI posterosuperior screw was placed in different posterosuperior locations.t-tests were used for comparative analyses before and after damage.One hundred and eight unpaired dry human cadaveric proximal femurs were scanned by a three-dimensional scanner. Digital data obtained from the proximal femur surface were employed for subsequent analysis. All nutrient foramina in the femoral neck were identified and marked in each subject. A simulation of the anteroposterior, lateral, and axial views was then performed, and regions of interest (ROIs) for IOI posterosuperior screws, with 6.5 mm diameter, were determined in the posterosuperior femoral neck on the axial graphs. Nutrient foramina were counted and analyzed in ROIs and femoral neck, and its damage from the IOI posterosuperior screw was also calculated in different conditions of screw placement. Paired P < 0.01). The risk zone determined by these locations was located in a posterosuperior square of ROIs with an edge length of 9.75 mm.Most nutrient foramina were located in the subcapital region and the least in the basicervical region in the femoral neck, while the most were located in the transcervical and the least in the subcapital in the ROIs. In addition, most nutrient foramina in ROIs were located in the superior–posterior area of the femoral neck. There were four main locations of IOI posterosuperior screws where the decrease in the nutrient foramina was statistically significant (To minimize iatrogenic damage to the blood supply of the femoral head, screw positions could be assessed in anteroposterior and lateral radiographs using a risk zone. The IOI posterosuperior screw in ROIs can be applied to fix femoral neck fractures when feasible in clinical practice. This study could provide surgeons with more alternatives for screw placement in the posterosuperior femoral neck. Femoral neck fractures are common hip trauma, and their incidence increases with population aging and traffic accidents. Multiple cannulated screws may be used to fix femoral neck fractures. The “In–Out–In” posterosuperior screw may penetrate the cortex anatomically, but not fluoroscopically, because of the specific morphology of the femoral neck, with a reported incidence of 70% , 2. TherAn ideal design of fracture fixation can provide sufficient biomechanics, with no interruption of blood supply. A superior biomechanical property of fixation of femoral neck fractures can be attributed to the “In–Out–In” posterosuperior screw in inverted triangular screws . To dateThis study aimed to evaluate the damage degrees of the nutrient foramina in the femoral neck when the “In–Out–In” posterosuperior screw was placed in the different positions of the posterosuperior regions. We hypothesized that there was a risk zone of damaging nutrient foramina for the “In–Out–In” posterosuperior screws.We obtained 108 unpaired dry human cadaveric proximal femurs from the Department of Anatomy. General information regarding these dry specimens of adult femurs, such as age and gender, was unknown. Using a 3D scanner , the surfaces of the proximal femur were scanned. An image resolution of 1,310,000 pixels was obtained by a scanning distance of 200 mm, precision of 0.01 mm, and measuring point of 0.04 mm. The digital 3D model was recorded from the proximal femur surface. Data were then processed using 3-matic 12.0  software.Based on the center of the nutrient foramen, the positions of nutrient foramen were defined in 3D models . The poiThere were four synchronized windows available in the 3-matic software. One window was set as the front view, while the other was set as the lateral view. When the image opacity was set to 50%, the front and the lateral views, respectively, appeared as an anteroposterior (AP) and a lateral radiograph of the femoral neck.Based on two points in three-dimensional space, the femoral neck axis was determined in the AP view. One point was at the center of the femoral neck's smallest cross section, and the other was at the center of the femoral head. To correct the femoral neck anteversion angle after the axis was created, the femoral neck was rotated to ensure that the above two points were on the axis in the lateral view. The screw placement based on the guide wire was parallel to the femoral neck axis Fig. .Fig. 2SiBps) and two intersection points  between the femoral neck curve and two lines, between three dots, were determined  line was parallel to the femoral neck axis in AP and lateral radiographs, and the Bps was also tangential to the posterior and superior curves of the femoral neck in the lateral and AP radiograph, respectively. The boundary posterior–inferior (Bpi) and anterior–superior (Bas) lines were set in a similar principle. Finally, the simulated view was rotated until the boundary lines were observed as dots in order to create a projective graph along the femoral neck axis. So, the point of the Bps    for “In–Out–In” posterosuperior screws. In the projective graph, the ROIs were determined by three points (PPp) Fig. B. Thus, Bps Fig. C.y, z). The start and end positions of the “In–Out–In” posterosuperior screw were defined as the positions intersecting the critical points of ROIs, including PBps, Ps, and Pp. The travel distance of each screw movement was set to a radius of the screw, 3.25 mm  and the center of the screw was less than the radius of the screw, the nutrient foramina were damaged. The initial position of the center of the screw was located in the anterior–inferior site of the PBps , and it was 3.25 mm far away from the PBps . The formula was as follows:Nutrient foramina damaged by 6.5-mm-diameter screws were sought. The standardized coordinate of the screw center position was . The statistical significance of paired data was tested using paired In the femoral neck, the distribution ranking of nutrient foramina was in the subcapital, the transcervical, and the basicervical, with a mean percentage of the local number over the total number of 35.56%, 34.94%, and 29.51%, respectively. In the subcapital region, the mean percentage was 15.75% in the superior–posterior area and 19.81% in the non-superior–posterior area. In the transcervical region, a mean of 15.33% of nutrient foramina was found in the superior–posterior area and 19.61% in the non-superior–posterior area. In the basicervical region, the 12.00% of the nutrient foramina were in the superior–posterior area and the 17.51% were in the non-superior–posterior area.In the ROIs, most nutrient foramina were located in the transcervical region and the least in the subcapital region Table . In the There were six placements of “In–Out–In” posterosuperior screws that can damage nutrient foramina. Their movement directions and distances from the initial screw center along the z-axis and the y-axis were as follows: , , , ,, and . In the location of , nutrient foramina of only three of 108 subjects were damaged, with the number ranging from one to three. In the location of , one nutrient foramina of only one of 108 subjects were damaged. Thus, these last two conditions were the most extremely rare, and the former four conditions were used for the subsequent analysis.P < 0.0001). In the condition of , the decrease in the nutrient foramina was statistically significant in the superior–posterior area of the femoral neck (P < 0.0001). In the condition of , the nutrient foramina reduction was statistically significant in the superior–posterior area of subcapital, transcervical, and basicervical regions, with a P-value of < 0.0001, 0.0026, and < 0.0001, respectively. Therefore, the “In–Out–In” posterosuperior screw mostly damaged the nutrient foramina in the superior–posterior regions. When the screw center was in the surrounding site of PBps, a risk zone, there was a risk of damaging nutrient foramina. This zone was located in a square of ROIs with an edge length of 9.75 mm, and PBps was its posterosuperior vertex  >  >  >  (Table Bps based on traditional AP and lateral radiographs. The impacts of the “In–Out–In” posterosuperior screws, in different placements, on nutrient foramina were illustrated in this study, and our results confirmed that not all “In–Out–In” posterosuperior screws can damage the blood supply of the femoral head. Therefore, the risk zone in the posterosuperior femoral neck should be avoided when intraoperatively placing the posterosuperior screw for femoral neck fracture fixation.This study demonstrated that there was a risk zone of damaging nutrient foramina when the posterosuperior screws were “In–Out–In” in the management of femoral neck fractures. And, this zone was located around the site of PMost nutrient foramina of ROIs were located in the transcervical region, and the damage of nutrient foramina from “In–Out–In” posterosuperior screws was likely to be mild. Consistent with previous reports, most nutrient foramina were located in the subcapital region of the femoral neck , 12. In The risk zone in this study could provide a reference for ideal screw positions in the clinical treatment of femoral neck fractures. With the insufficient precision of CT or MRI, the nutrient foramina could not be directly assessed in the clinic. In this study, there were four main locations of “In–Out–In” posterosuperior screws, which caused a significant reduction in nutrient foramina. The distribution of the terminal branch of the nutrient vessels matched that of the nutrient foramina . TherefoOur findings could provide a theoretical basis for updating the treatment principle, while the unacceptable concept of all “In–Out–In” was still used in clinical practice and research. Previous studies reported a variety of attempts to avoid the presence of the “In–Out–In” status of the posterosuperior screw, but there was still a lack of consensus on preventive measures –8. KumarThe “In–Out–In” posterosuperior screws were traditionally inevitable, but this screw may result in better biomechanical properties. Due to the complex osseous anatomy of the femoral neck, a high incidence of “In–Out–In” posterosuperior screws was observed based on conventional AP and lateral radiograph views , 3. MostThis study has several limitations. Firstly, intact dry human proximal femurs were used to generate the digital data. Therefore, before considering the posterosuperior screw placement, the anatomic reduction is essential in a clinical situation. Secondly, the 6.5-mm-diameter screw was only chosen for analysis in this study, although it was the most commonly used one. When considering other screws with a greater diameter in some special cases, screw locations that were away from the risk zone could be recommended.Not all “In–Out–In” posterosuperior screws can damage the nutrient foramina of the femoral neck. To minimize iatrogenic damage to the blood supply of the femoral head, screw positions could be assessed in AP and lateral radiographs using a risk zone. With a biomechanical advantage to the fracture site, the “In–Out–In” posterosuperior screw can be applied to fix femoral neck fracture when feasible in clinical practice. Therefore, this study provided surgeons with more alternatives for screw placement in the posterosuperior femoral neck.