Min) and upper (CTMax) critical thermal limits of 17 species (12 genera) at two elevations (200 and 1500 m) in the Colombian Andes. In addition, we examined the influence of body size , hairiness (thoracic hair length) and coloration  on bees’ thermal tolerance. Because stingless beekeepers often relocate their colonies across the altitudinal gradient, as an initial attempt to explore potential social responses to climatic variability, we also tracked for several weeks brood temperature and humidity in nests of three species at both elevations. We found that CTMin decreased with elevation while CTMax was similar between elevations. CTMin and CTMax increased (low cold tolerance and high heat tolerance) with increasing ITD, hair length and lightness value, but these relationships were weak and explained at most 10% of the variance. Neither CTMin nor CTMax displayed significant phylogenetic signal. Brood nest temperature tracked ambient diel variations more closely in the low-elevation site, but it was constant and higher at the high-elevation site. In contrast, brood nest humidity was uniform throughout the day regardless of elevation. The stronger response in CTMin, and a similar CTMax between elevations, follows a pattern of variation documented across a wide range of taxa that is commonly known as the Brett’s heat-invariant hypothesis. Our results indicate differential thermal sensitivities and potential thermal adaptations to local climate, which support ongoing conservation policies to restrict the long-distance relocations of colonies. They also shed light on how malleable nest thermoregulation can be across elevations.Tropical pollinators are expected to experience substantial effects due to climate change, but aspects of their thermal biology remain largely unknown. We investigated the thermal tolerance of stingless honey-making bees, the most ecologically, economically and culturally important group of tropical pollinators. We assessed changes in the lower (CT Pollinators supply essential ecosystem services and bees (~20 000 spp.) are widely recognized as the most important pollinators of wild and cultivated plants . HoweverMin) and maximum (CTMax) temperatures at which an animal can maintain muscle control  than bees from low elevations. Small bees cool down and heat up more quickly than large bees because of their high surface area to volume ratio, which increases convective heat transfer (Min decreases while CTMax increases (higher cold and heat tolerance) with increasing body size. Because body hair may form an insulation layer that mitigates heat loss and increases retention of cool air  of stingless bees at two elevations (200 and 1500 m) in the Colombian Andes. Because temperature decreases with elevation, we predict that bees at high elevation will display lower CTFinally, plastic responses in thermal tolerance may result from the thermal environment in which the immature stages developed, and such responses are critical in the context of global warming because they can potentially compensate for the negative consequences of expected changes in environmental conditions . Given tStingless bees are social, pollen generalist, honey-making bees restricted to the tropics. They live in perennial colonies and pollinate a broad array of native and cultivated plants, including global commodities such as coffee e.g. . They haOxytrigona Cockerell and Scaptotrigona Moure, are highly defensive and may display suicidal biting  until we completed fieldwork. We tested bees within 1–2 h after captured in the field.We conducted field and experimental work during the dry season (January to April 2021) at two elevations on the western slope of the Oriental cordillera in Colombia (Department of Cundinamarca): Beltrán, a municipality within the tropical dry forest ecosystem along the Magdalena River  and San Antonio del Tequendama , a municipality situated within montane cloud forest . As it iAt each location, we measured ambient temperature and humidity using iButton data loggers , which we protected from solar radiation with aluminum foil and hung at 1 m above ground from tree branches (See 3) and plugged them with a moistened cotton ball  to ensure enough humidity during the assays. We used an initial temperature of 22°C and held bees for 10 minutes at this temperature before increasing it or decreasing it at a rate of 0.5°C min−1. We placed vials horizontally on the stage to avoid bees from climbing along the vial. To estimate the temperature inside the vials, we placed a K-type thermocouple inside two empty glass vials plugged with a cotton ball. We individually tracked these vial temperatures using a TC-08 thermocouple data logger . As an approximation of bees’ thermal limits, we used the temperature at which bees show signs of curling  protocol with the Elara 2.0 , a portable fully programmable heating/cooling anodized aluminum stage designed for precision temperature control of laboratory and field samples. The stage was modified with a Styrofoam cooler and clear acrylic lid to minimize the impact of airflow across the aluminum sample stage and maintain temperature stability across all vials. We placed bees individually inside glass vials  on the disc of mesoscutum, which is a component of the hue, saturation and lightness (HSL) digital color model. The lightness value represents the overall darkness or lightness of a color, and it ranges from 0 to 100%, with 0 being black and 100 white. We used digital images of the dorsum of five specimens per species randomly selected from each elevation and used the average value. Using a standardized gray background, we generated images with the Macropod Pro 3D (www.macroscopicsolutions.com). Then, we used the eyedropper tool with a 31 by 31 average pixel radius in Adobe Photoshop  to sample the RGB color model  and then transformed them to a HSL color model (We estimated body size by measuring the minimum intertegular distance (ITD)  of each Voucher specimens are in the Laboratorio de Abejas of the Universidad Nacional de Colombia, Santa Fé de Bogotá and in the Division of Entomology, University of Kansas Natural History Museum (Biodiversity Institute), Lawrence, Kansas.Frieseomelitta paupera (Provancher) at the low-elevation site , Melipona eburnea Friese at the high-elevation site  and Tetragonisca angustula (Illiger) at both elevations . In Colombia, the first species is restricted to dry forests, the second species typically occurs at mid elevations and the third species is widely distributed across the elevation gradient, from sea level up to 1800 m , we implemented a similar LMM for each species individually. To evaluate the relationship between CTMin and CTMax, as well as between each morphological trait and CTMin and CTMax, we implemented a linear regression analysis using the lm function. To compare the slope of regression between elevations, we specified a model that included the interaction between CTMax and elevation (low and high) and between each morphological trait and elevation. We used a mixed-model ANCOVA to compare CTMin and CTMax between elevations while controlling for the effects of each morphological trait. We implemented a LMM with elevation and species as fixed factors, a morphological trait as covariate and nest identity as a random nested variable within species. To assess the relative importance of morphological traits on critical thermal limits, we first implemented a linear model with either CTMin or CTMax as the response variable and all morphological traits as predictors. Then, we used the function stepAIC from the MASS package  whereas that of the high-elevation site (Tequendama) was 18.8°C , and such a difference was significant . However, the magnitude of the daily variations in temperature were similar between the two elevations, ranging from 10 to 13°C of difference between the maximum and minimum values at each location (25–35°C at the low-elevation site and 15–28°C at the high-elevation site). Mean hourly air relative humidity was lower at the low-elevation site  in comparison to that of the high-elevation site , and that difference was also significant . However, daily changes in humidity were greater in the low-elevation site than in the high-elevation site. The difference between the maximum and minimum humidity values were always higher than 40% at the low-elevation site, reaching the lowest value at 14 h when temperature was highest. In contrast, ambient humidity was relatively constant throughout the day and night at the high-elevation site, with the difference between the maximum and minimum values always less than 20%.Temperature and relative humidity differed significantly between elevations. The mean hourly air temperature at the low-elevation (Beltrán) site was 27.7°C , hair length from 0.04 mm in Paratrigona eutaeniata Camargo and Moure to 0.92 mm in M. compressipes and lightness from 22% in Oxytrigona daemoniaca Camargo to 53% in Tetragona ziegleri (Friese). While ITD was similar between elevations, and the interaction between species and elevation was not statistically significant, hair length and lightness differed between elevations and the interaction between species and elevation was significant (L = 27.7%) than bees from the low-elevation site . At each elevation, and across all species, both CTMin and CTMax increased significantly with increasing values of each morphological trait, except for the relationship between lightness and each thermal limit at the low-elevation site, which was not significant , which is 2.2°C lower than the CTMin of bees from the low-elevation site . CTMin varied significantly across species  and the difference in CTMin between elevations was significant after accounting for body size  while those from the high-elevation site showed a mean CTMax of 43.34°C . CTMax also varied significantly across species  but the difference in CTMax between elevations was not significant after accounting for body size  were the least cold tolerant species among the bees tested in the low-elevation site, with an average CTMin of 12.96°C and 13.59°C. These two species, as well as Cephalotrigona femorata (Smith), displayed a CTMax that was on average between 1.05°C and 2.76°C higher than the average CTMax estimated for the bee community. Among the species from the high-elevation site, Parapartamona zonata (Smith) displayed the lowest CTMin and a low CTMax .There is a tradeoff between cold and heat tolerance among stingless bees. At each elevation, some species appeared to be more warm or cold adapted than others, as judging by their thermal limits . For exaow CTMax . While aion site . An ANCOevations , and a rS. magdalenae, T. angustula and T. fulviventris, were tested at both elevations. For these species, ITD was similar between elevations for S. magdalenae (P = 0.086) and T. fulviventris (P = 0.331). However, bees of T. angustula from the high-elevation site were significantly larger . However, after accounting for body size, CTMin was similar between elevations for S. magdalenae (P = 0.610) and T. fulviventris (P = 0.096), but it was significantly lower for T. angustula at the high-elevation site than in the low-elevation site .Forager bees of three species, Min  nor CTMax  displayed significant phylogenetic signal (T. angustula from the high-elevation site displayed a significantly lower estimate of CTMin than foragers of this species at the low-elevation site (see above). Thus, we ran another test using the estimate of CTMin for foragers of T. angustula from the high-elevation site and found non-significant results .Neither CTc signal . ForagerAt both elevations, ambient and internal temperature of unoccupied control hives (empty wooden boxes) were either similar (low-elevation site) or significantly different but very close (high-elevation site) . HoweverT. angustula (28.8°C ± 0.06) and F. paupera (27.6°C ± 0.07 and 28.5°C ± 0.07) were similar or significantly different but very close to each other (≤ 1°C higher) , unoccupied control hive (27.9°C ± 0.07) and brood temperature of  higher) . Brood t higher) . In cont the day  and 7.6– the day . Mean ho the day . In the humidity . In the humidity .Min decreased with elevation while CTMax was similar between elevations. Thus, these results are partially in agreement with our expectations because CTMax did not decrease with elevation. However, although unanticipated, these results are consistent with studies in other organisms , the CTMax of bees from the low-elevation site is only between 3.5°C and 8°C higher than the highest daily ambient temperature recorded (39°C). Thus, our data suggests that stingless bees from the low-elevation site are living closer to their maximum thermal limit and that mountain habitats might represent important refuge for them under global warming. Indeed, studies under climate change scenarios using niche modeling approaches predict shifts in elevation for some stingless bees to compensate for the increase in temperature  with increasing values of the morphological traits examined, except for the relationship between lightness and thermal limits at the low-elevation site, which was not significant , and both CTMin and CTMax were not related with lightness at the low-elevation site. However, although most relationships were statistically significant, these are weak and the morphological traits explained no more than 10% of the variance . To date, CTMin has only been assessed in bumble bees  than estimates for either honey or stingless bees. This is not surprising given that bumble bees are known for being largely cold-adapted species. In addition, information on bumble bees’ thermal limits is from species occurring in temperate areas where they experience much lower temperatures than in the tropics. Furthermore, other morphological traits might be more informative for stingless bees’ thermal limits. For example, water content, cuticle thickness and sculpturing, hair density and color, type of hair (simple and erect vs. branched and decumbent) and color of metasoma. Further studies should explore them, as some of these traits influence ants’ thermal limits  or cold (P. zonata) adapted based on their high CTMax or low CTMin. Our analyses indicated that performance at low and high temperatures might be inversely correlated, as bees tended to be either cold or heat tolerant but not both (F. paupera) to large (M. favosa) in body size, they all nest inside empty cavities, and they build an involucrum surrounding the brood area, except for F. paupera. The cold-adapted species, P. zonata, is relatively small, nests in the ground and builds an involucrum. Thus, the high heat or cold tolerance displayed by these bees might be driven by genetic mechanisms and tied to aspects of local climate, as documented for bumble bees , have already resulted in total colony loss or low establishment of nests. Tetragonisca angustula is the most common species of stingless bees used in meliponiculture in Colombia and an informal market of hives from low elevation areas, where the species is abundant, to mid elevation areas where agricultural production is high, is increasingly common. Local adaptations to other environmental conditions besides temperature, such as humidity, might be also important for stingless bees. Unfortunately, the desiccation tolerance of stingless bees, or bees in general, remains to be explored.Our results have significant implications for the conservation and sustainable use of stingless bees. We showed that bees have differential thermal sensitivities, as judging by their critical thermal limits, and thus not all species are going to be affected the same way to heat or cold stress. We showed that species or populations might be climatically adapted, so that bees from high elevations can handle lower temperatures than those from low elevations. In addition, our work suggests that bees from low elevations are living closer to their maximum thermal limit than those from high elevations. This means that some species might be more vulnerable to global warming than others, which matches the differential responses predicted under climate change scenarios for stingless bees . From a This work was supported by a Fulbright Colombia-National University of Colombia Distinguished Chair in Biodiversity and Sustainable Development award, Fulbright U.S. Scholar Program, National University of Colombia’s Office of Research (Hermes #51171), as well as the University of Kansas’ Center of Latin American and the Caribbean Studies, and National Science Foundation .The data underlying this article are available in the article and in its online supplementary material.Web_Material_coac073Click here for additional data file.


Of the six derivatives examined, 1–3 showed inhibitory activity with IC50 values of 3.7 ± 0.1, 10.3 ± 1.0, and 41.3 ± 3.2 μM, respectively. Based on enzyme kinetics, 1–3 were confirmed to be competitive inhibitors with Ki values of 2.8, 9.0, and 51.9 μM, respectively. Molecular docking analysis revealed the formation of a binary encounter complex between 1–3 and the tyrosinase catalytic site. Luteolinidin (1) and petunidin 3-O-glucoside (2) may serve as tyrosinase inhibitors to block melanin production.The enzyme tyrosinase plays a key role in the early stages of melanin biosynthesis. This study evaluated the inhibitory activity of anthocyanidin ( Tyrosinase EC 1.14.18.1) is a multifunctional copper-containing enzyme that plays a role in melanin biosynthesis in mammals, plants, insects, and microorganisms .14.18.1 . MelaninO-glucoside, delphinidin 3-O-glucoside, and pelargonidin 3-O-glucoside are the most abundant naturally occurring anthocyanins, which are widely found in Morus nigra, Berberis vulgaris, and Ipomea batatas  × 100, where C and S are the intensity of control and inhibitor after 20 min, respectively.To assess the inhibitory effect of compounds on tyrosinase, 130 μL tyrosinase in 0.05 mM phosphate buffer (pH 6.8) was aliquoted into 96-well plates , and 20 The three-dimensional structure of the protein encoded by 2Y9X was determined from the Research Collaboratory for Structural Bioinformatics homepage. Hydrogen atoms were added to this and assigned Gasteiger charges using AutoDockTools. Ligands were built and their energy minimized by MM2 using Chem3D Pro. A grid containing the active site was established . A DPS file was constructed to set up a Lamarckian genetic algorithm for ligand docking with the receptor . The results were presented using Chimera  and LIGPLOT .All inhibitory concentration data were obtained from independent experiments carried out in triplicate. Results are shown as the mean ± standard error of the mean (SEM). The results were subjected to analysis using Sigma plot 10.0 . O-glucoside and cyanidin 3-O-glucoside) were confirmed to decrease MMP-2 and u-PA secretion in SCC-, Huh-7, and HeLa cells [Arctium lappa L. leaves, which contained trans-5-caffeoylquic acid, rutin, kaempferol 3-O-rutinoside, 3,5-di-O-caffeoylquic acid, and 4,5-di-O-caffeoylqunic acid, was found to decrease tyrosinase activity and melanin levels [The anthocyanin-rich fractions of blueberry extract decreased the proliferation of B16-F10 melanoma murine cells by inducing apoptosis . The majLa cells . Recentln levels . This don levels .1) from Sorghum bicolor [O-glucoside (2) from Vitis vinifera L. [O-galactoside [3) showed moderate inhibitory activity. Many studies have examined the inhibitory effects of anthocyanin-rich fractions on tyrosinase [1) and anthocyanins (2–3) showed the inhibitory activity within the concentration range of micromole on tyrosinase. Some flavonoids have recently been reported to interact directly with the tyrosinase catalytic site [1–3, as competitive inhibitors, were confirmed to be bound into a common active site in an in vitro assay. The molecular simulation calculated that they were anchored into the cavity hole in tyrosinase. In particular, two inhibitors, 1 and 3, formed cluster 1 stably in the left position next to the active site. Based on enzyme kinetic theory, it can be confirmed that this cluster, formed by the two inhibitors, had a logical error. Therefore, cluster 2, formed by the two inhibitors on the catalytic site, was determined to be the predicted binding calculated by the Autodock program. According to the above results, it was confirmed that glycosides 2 and 3 form a similar Autodock score and binding poses. Inhibitors 2 and 3, which are similar to the backbone of inhibitor 1, were bound as different poses in the catalytic site by their sugars. Luteolinidin ( bicolor  and petuifera L.  were theactoside  (3) showrosinase ,24, whertic site . In addi1), petunidin 3-O-glucoside (2), and delphinidin 3-O-galactoside (3) acted as tyrosinase inhibitors at micromolar concentrations. These compounds bound competitively, with Ki values of 2.8, 9.0, and 51.9 μM, respectively. A molecular simulation of the binding of the compounds to the catalytic site of tyrosinase confirmed that inhibitors 1 and 2 interact with tyrosinase with an excellent inhibitory effect and low Autodock binding energy. Therefore, we suggest that luteolinidin (1) and petunidin 3-O-glucoside (2) should be the lead compounds for developing new tyrosinase inhibitors.Many studies have examined the tyrosinase inhibitory activity of anthocyanin extracts from plants . We eval


Dreissena mussels in the late 1980s, the nearshore waters of the Laurentian Great Lakes were home to diverse assemblages of native aquatic insects, including the net-spinning caddisfly Hydropsyche alternans, which occurred in rocky surf-exposed habitat throughout the system from Lake Superior downstream to Lake Ontario. These surf zone caddisflies are still abundant in the largely Dreissena-free waters of Lake Superior where the present study was conducted. They have not been reported in the lakes below Lake Superior for decades, and are presumed to have been extirpated from Dreissena-infested habitats. The ecological profile presented here documents the life history of H. alternans in Lake Superior, and reveals details of its feeding biology that shed light on the roles that surf zone net-spinners play in the native nearshore food webs of the Great Lakes. The H. alternans life history in Lake Superior begins in mid-summer shortly after oviposition, which features females swimming to surf-exposed lake bottom substrates. Larval development takes approximately 10 months, but most of it occurs during their first 100 days. Adult emergence is broadly synchronous, with large numbers present from the summer solstice through mid-July. Gut content analyses showed that larvae opportunistically feed on algal, animal, and detrital material of aquatic and terrestrial origin. δ13C and δ15N stable isotope data indicate that they function as omnivores that link coastal, nearshore, and pelagic food webs. These energetic links, and the very existence of surf zone insect assemblages in the Great Lakes, depend on the Dreissena populations in Lake Superior remaining relatively small and isolated.Prior to the invasion and spread of Hydropsyche alternans in four rocky sites located along the south-central coast of Lake Superior. The H. alternans life history and broad trophic niche space were similar to those of its riverine relatives. Quantitative sampling over the course of one ice-free season revealed that most individuals lived univoltine life histories that featured early to mid-summer mating, and oviposition and rapid growth and development through summer into fall. Most individuals overwintered as ultimate or penultimate larval instars. Pupation followed ice-out in the spring. Gut content sampling and δ13C and δ15N stable isotope analyses indicated that the typical larval diet is a mix of benthic, pelagic, and terrestrial food resources, including diatoms, small arthropods, sloughed periphyton, and in one site, fugal hyphae apparently of foredune origin. As a suspension-feeding omnivore that relies on waves and currents to deliver food to its nets, H. alternans larvae form energetic links between coastal, nearshore, and offshore food webs. These connections have been lost throughout the lower Laurentian Great Lakes as a consequence of the invasion and spread of Dreissena mussels.We studied the life history, diet, and trophic ecology of  Antocha crane flies (Diptera: Limoniidae) [Net-spinning caddisflies (Trichoptera: Hydropsychidae) are among the most well-known aquatic insects. Larval hydropsychids filter food particles from flowing water using silken mesh nets rigged to retreats made of silk and bits of mineral and plant material. These little “fishing shacks” are common features of stream bottoms worldwide. Hydropsychids also inhabit the rocky, wave-swept shorelines of large lakes alongside other familiar stream insects such as flathead mayflies (Ephemeroptera: Heptageniidae), perlid stoneflies (Plecoptera: Perlidae), riffle beetles (Coleoptera: Elmidae), and oniidae) ,2,3.2 in surface area [The flows that impart a riverine nature to the nearshore waters of large lakes are produced by wave energy surging up beaches and running back out, forming longshore currents during prolonged wind events . In veryace area , wind enace area ,6,7,8. Race area .Waves and currents deliver suspended food particles to surf zone hydropsychid nets. By limiting the accumulation of fine sediments ,2, wave Like many aquatic insects, larval Hydropsychidae are omnivorous feeders . Gut conStable isotope studies have shown that larval Hydropsychidae tend to fall in the middle of available energy sources within a system, and that their trophic position ranges from primary consumer (trophic level 2) to predators of small invertebrates (trophic level 3) ,22,23,24Dreissena mussels, hydropsychids were common in the nearshore waters of all five Laurentian Great Lakes [Dreissena polymorpha) and quagga mussels (Dreissena bugensis) expanded throughout Lakes Michigan, Huron, Erie, and Ontario over the past three decades [Dreissena populations are thriving [Hydropsyche alternans , still flourishes in the last stronghold of the Great Lakes surf zone insects.Prior to the invasion of at Lakes . As popu decades ,26, with decades ,28, surfthriving ,30. Alththriving , it presHydropsyche recurvata  of the Fox River, the largest tributary of Lake Michigan, persisted in shallow water lake margins despite massive cyanobacterial blooms during summer months and the dramatic water quality changes that followed. This apparent tolerance of widely fluctuating lacustrine water quality was aptly deemed “remarkable” by the authors.While describing a southern Lake Michigan population of the species he knew as nd Hynes  suggestend Hynes  indicateH. alternans suggests a strong influence of Pleistocene glacial–interglacial cycles in shaping its broad niche space [Hydropsyche alternans dominated the insect assemblage of the large lake that formed there during the final interglacial (warm) period before the advance of the Laurentide Ice Sheet that ultimately carved out the modern Great Lakes. Eleven interglacials, which are separated by long periods of cold temperatures and advancing glaciers, have occurred over the past 800,000 years [Hydropsyche alternans range expanded from unglaciated refugia streams into large glacial scour lakes formed during interglacial periods, as it has during the present (Holocene) interglacial, which began approximately 11,700 years ago.The present distribution of he space . Williamhe space  surveyed00 years . AlthougH. alternans may also have occurred in glacial meltwater lakes, including the glacial Lake Agassiz, the immense proglacial lake that extended up to 500 km outward from the Laurentide Ice Sheet  are located along a 7 km stretch of northeast facing coast. Granitic headlands to the north (Thoney Point) and south (Little Presque Isle) exert considerable influence on the surf and current exposure. All of these sites periodically receive strong surf from long-duration gale-force (≥40 km per hour) north, northwest, northeast, and easterly winds. The larval retreat sites in Site 4 occur along the west side of a granitic headland that forms an embayment which captures wind swells arriving from the northwest, north, and northeast.2 configuration [Larvae used for life history analyses were collected with multiple-plate samplers  in an 8-guration . Samplerguration ; adult iguration ,56.H. alternans larvae over the course of a full growing season and by accurately measuring the widths of their heads, we were able to reliably differentiate among larval instars. Additionally, identification of 5th instar Hydropsychidae by head capsule width can be verified by the presence of bud-like pupal gills on the sides of the abdomen [Larval Hydropsychidae pass through five instars prior to pupation. As they grow, the head capsule width of successive instars increases in a consistent geometric progression , typical abdomen .H. alternans adults from each sample were identified using male genitalia characteristics, which allowed us to establish adult presence. Peak abundance was determined when the sampling jars containing 95% ethanol were overflowing with thousands of specimens after approximately 6 h of sampling.The approximate beginning, peak, and end of the adult flight period were determined with evening collections made with a portable light trap equipped with a 395 nm LED flashlight  six timeH. alternans, we calculated the length:width ratio of the first mesothoracic tarsal segments of 10 females and 10 males with an ACU-RITE linear encoder measuring system. Although not definitive evidence of swimming behavior, significant sexual dimorphism in this character has been linked to swimming and deep oviposition in hydropsychid species [Most female Hydropsychidae oviposit underwater and some species are known to swim to substrates using flattened mesothoracic legs, a behavior that would presumably be adaptive in Lake Superior where suitable habitat extends from shore for hundreds of meters in many locations. To assess the potential for female swimming and deep oviposition in  species ,61.Gut content analyses were performed on 39 larvae from Site 4 (9 fourth instar and 13 fifth instars) collected on 6 October 2017 and 17 larvae from Site 3  collected on 16 October 2017. Gut content samples were processed and prepared using Cummins’ standard protocols . Foregut13C and δ15N stable isotope analyses.After removing the foregut and midgut for gut content analyses, specimens were processed for stable isotope analysis by removing the hindgut and the head, which was retained as a voucher specimen. The rest of the specimen was then dried at 55 °C and sent to the Alaska Isotope Facility for sample preparation and δH. alternans larvae included in the body tissue analysis. The other sample contained silk glands from two 4th instar H. alternans and one similarly sized Cheumatopsyche larva. Data from both samples, which were isotopically similar and thus of general value in assessing the relationship between 4th instar H. alternans body and silk gland isotopic compositions, are included in the results. Specimens from Sites 1 and 2 were processed using a modified protocol. While removing the midgut and hindgut, which is standard practice, particularly for predators or omnivores , we notiAlthough the focus of the present study was on larval trophic ecology, three adult males and three adult females from Sites 1 and 2 were analyzed in order to characterize differences in resource allocation that occur as male and female reproductive tissues differentiate. These samples will also serve as the baseline data for future investigations of energy flow from the surf zone to the swash zone, and nearshore terrestrial food webs in the Lake Superior basin.p ≤ 0.05 were used to evaluate the observed differences in adult vs. larval body δ13C and δ15N, adult male vs. female body δ13C and δ15N, and sexual dimorphism in adult legs. Single factor ANOVA models were used to test the effects of larval instar and site on isotopic ratios, and the effects of instar and site on larval silk gland and body isotopic ratios. Post hoc Tukey–Kramer tests were used to test for pairwise differences among treatment means. All data analyzed met the assumptions of independence and normality (Kolmogorov–Smirnov tests). All isotope data met the assumption of homogeneity of variance (Levine’s tests). Adult leg data were log transformed to correct for heteroskedasticity.Unpaired (independent samples), two-tailed t-tests, with an a priori significance level of UV light sampling detected a small number of adults on 18 June, establishing the approximate beginning of the 2020 flight period. Adults were abundant from late June through mid-July, tapering off dramatically by early August. During the daylight hours, adults nervously rested on lakeside vegetation and other shaded habitats. Small, loose swarms, approximately 2 to 5 m above ground along the forest–beach edge were generally present throughout the day. At dusk, adults appeared en masse along the shoreline. Pre-mating behavior featured a dizzying blend of low swarming flights, frenetic scrambling over, under, and around lakeshore rocks, and excited nuzzling by males alongside of females emitting pheromonal signals from paired abdominal glands .H. alternans eggs were observed on larval samplers in late July, although adult collections showing oviposition were likely occurring at least a month earlier. This is further supported by the presence of large numbers of 2nd and 3rd instar larvae in the late July samples. Larval development appeared to be largely completed by fall, with most individuals overwintering as 5th instars. However, the presence of some 4th instar larvae in early winter samples indicates a degree of developmental flexibility at the end of a larva’s first open-water season.Head width data, instar determination cutoff points, and the relative sizes of egg and larval life stages are shown in p < 0.00001)  = 3.54 ± 0.08 for females, 6.99 ± 0.22 for males, t(18) = 18.32, 0.00001) . Our sam0.00001) B.H. alternans collected in October from Sites 3 and 4 indicates that a wide variety of food items were consumed  had broadly similar diets and were clearly omnivorous. Approximately half of the larvae examined had recently consumed arthropod prey, supplementing common diets comprising diatoms, green algae, and detritus . There w13C and δ15N isotope analyses were conducted on the specimens used in the 2017 gut content analysis from Sites 3 and 4, and also on some of the larvae and adults collected from Sites 1 and 2 as part of the 2020 life history analysis. The data from these analyses, which are treated in this section as four separate studies, are presented together in δ13C data detected significant effects of instar on the principal energy pathway . Specifically, 3rd instars were, on average, depleted in 13C relative to the 4th and 5th instars, but some overlap with both older instars was apparent as well . A post-hoc analysis showed that this pattern was driven by apparent broad trophic separation between 3rd instars and the more 15N-enriched 5th instars  . Post-hoc analysis showed this effect resulted from apparent trophic separation between the most 15N-enriched group, 5th instars from Site 4, and the most depleted group, 4th instars from Site 3 (This study compared δ 0.0003) . Post-hognatures . The corm Site 3 .15N values , although a post-hoc Tukey test detected that 4th instars from Site 1 were slightly depleted in 13C relative to 4th instars from Site 2 (Tukey HSD p = 0.0480). The corresponding ANOVA model for δ15N showed no trophic separation between instars or sites .This study compared 4th and 5th instar larval bodies and silk glands collected from Sites 1 and 2, as part of the artificial substrate sampling-based life history analysis . Results13C and 15N, relative to bodies (δ13C t(15) = 7.18, p < 0.00001; δ15N t(15) = 3.68, p = 0.0022). These results were consistent with contrasts addressing the discrepancy that resulted in two silk gland samples from 4th instars of Site 2 . Within Site 1, 5th instar larval silk glands were enriched in 13C by approximately 9.4% (silk gland δ13C vs. mean body δ13C = 21.64 vs. −23.10) and contained approximately 2.5× more 15N by mass than 5th instar body samples (silk gland vs. bodies δ15N = 1.70‰ vs. 0.68‰). Within Site 2, 5th instar silk glands were 2.6× enriched in 15N relative to bodies (silk gland vs. bodies δ15N = 1.75‰ vs. −0.67‰), and were approximately 9.1% enriched in 13C (average body vs. silk gland δ13C = −23.34 vs. −21.33‰). Silk glands from 4th instar Site 1 larvae were similarly (9.2%) enriched in δ13C (average body vs. silk gland δ13C = −23.74 vs. −21.85‰)  .13C and 15N relative to larvae  = −6.14, p < 0.00001). This strong pattern was also evident in contrasts between adults and 4th instars (t(16) = −4.37, p = 0.00048), and between adults and 5th instars (t(16) = −4.57, p = 0.00031). Within the sexes, δ13C and δ15N values were nearly identical between sites, as were between-sex δ15N values  = −2.28, p = 0.046). However, within sites, this effect was evident in Site 1 (t(10) −3.00, p = 0.040), but not in Site 2 (t(10) = −1.07, p = 0.34)  .Hydropsyche species within the range of H. alternans, and concluded that voltinism was “not immutable” in this group, with species showing variable numbers of annual cohorts in response to changes in food resource availability and water temperature. Our look at of the life history of H. alternans supports this observation, indicating that most, but perhaps not all, of the population in 2020 belonged to a cohort that overwintered in the 4th or 5th instars, pupated in the spring, and enclosed as adults between mid-June and late-August. Eggs laid on our samplers and black light sampling indicated that oviposition started around the summer solstice and peaked in the first weeks of July. Eggs on the samplers included hatched and hatching larvae at the time of collection  could reflect a dispersal away from the natal substrate and/or 1st instars remaining on the natal substrate and molting to the second instar before the samples were retrieved. Instar 3 was clearly under-represented, which could represent low mobility or low effectiveness of the samplers at attracting this life stage. Fourth instars were the most commonly collected larvae overall, indicating a relatively high mobility and attraction to the samplers. The much larger 5th instar larvae colonized the samplers at a lower rate than the 4th instars, but they were also quite mobile, which is consistent with past reports of high rates of drifting by late-instar hydropsychids [H. alternans in our study area overwinter in the 5th instar, which would likely require mobility to avoid advancing and shifting ice.So how did the nearly two thousand psychids ,72. Our H. alternans in Lake Superior is limited to observations made before shore ice forms when larvae can be found near the shore, but in low densities compared to the autumn months. Key questions remain about what they do when ice accumulations form, whether they remain active under ice, and if their strategy or strategies differ from those of stream populations, which are also unknown. This rudimentary state of scientific understanding of H. alternans overwintering is generally consistent with that of the winter biology of most of the benthic organisms of the Great Lakes. This is particularly true of the winter life on the bottom of Lake Superior, which remains largely unexplored [H. alternans larvae overwinter beyond this ice accumulation zone, which would allow them to remain active or at least surrounded by liquid water throughout the winter. Stream-dwelling Hydropsyche species within the range of H. alternans cease net-spinning activity during the winter, but continue to feed on epilithic organisms [H. alternans larvae feed throughout the winter, the staple diet of epilithic algae could also be subsidized by pulses of offshore energy, which could be more available at this time of year, due to increased nearshore-offshore mixing [Limnocalanus macrurus, the dominant copepod in the lake and perhaps the lake’s most abundant animal, loads the pelagic realm with nauplii during winter reproduction [H. alternans in Lake Superior present researchers with obvious technical challenges. Overcoming them may be the key to understanding their apparent divergence to post-glacial lacustrine conditions, and their continued dominance of rocky surf zone habitats in Lake Superior.Our understanding of the overwintering strategy of explored . Based organisms . If H. ae mixing ,76 The ae mixing  and zoopoduction . OpportuH. alternans at our sites extended over eight weeks. Adult hydropsychids typically live for one to two weeks on the wing [H. alternans has adult dispersal capabilities similar to those of its congeners H. hageni and H. phalerata in Lake St. Clair, which lies between Lakes Huron and Erie, most females oviposit close to their larval habitat, but long-distance dispersal over 5 km occurs within a subset of the population [H. pellucidula and H. siltalai also maintained low levels of long-distance dispersal. These observations indicate that Hydropsyche females can survey the landscape and underwater habitats from the air to identify specific habitat features required for larval development. Because females attach eggs directly to the substrates on which larval development begins [H. alternans include silt-free, rocky substrates under clear waters in forested watersheds within a dynamic glacial landscape. The extension of the basal lotic habitat search parameters to include large lake littoral zones allowed this lineage and a few others within the family to exploit ecological opportunities available to attached, suspension-feeding animals in the vast expanses of large lake shorelines that have periodically formed and disappeared within their range over the past 800,000 years .Larval crawling and drifting accounts for movement within habitat patches; for example, newly open retreat locations in the form of our plate samplers, but population persistence in isolated habitat patches such as exposed bedrock slabs along the shores of Lake Superior likely requires aerial dispersal and habitat recognition from above. The 2020 flight period of the wing ,80, indipulation . Sode anpulation  reportedt begins , the cueH. alternans began to crawl or dive into proglacial lakes to oviposit. Most female Hydropsychidae oviposit underwater, and many species are known to utilize widened, paddle-shaped segments on their middle legs to swim to substrates  [Being selective feeders, hydropsychids are also known to avoid ingesting mineral particles , yet mosoniidae) , which soniidae)   that line much of the shoreline at Site 3. The deep roots of A. breviligulata typically support extensive growths of mycorrhizae [H. alternans larvae are in their final instar and accumulating resources for pupation and reproduction in the spring. Dune hyphae and other surf-delivered terrestrial subsidies could represent important sources of high-quality food for H. alternans larvae facing a long winter of predictably harsh environmental conditions.Although we do not know the taxonomic identity or identities of the fungal hyphae consumed by Site 3 larvae, the likely source of this material was the eroding beach dune face found just up-current from where we collected, as can be seen in orrhizae , which lorrhizae . This suorrhizae . In the The specimens that we used for gut content analyses were collected in October, when surf-producing storms are frequent. We do not have the wave height data for these sites, but past conditions can be assessed to a general level with wind speed and direction data from a NOAA Buoy, 60 km north-northeast of the research area . Conditions in the two days leading up to the Site 4 collection date (6 October 2017) featured sustained strong west winds in the 50 km per hour range. All of our sites are sheltered from west winds , so on tAmmophila breviligulata, also have periodic access to these and other materials that accumulate in swash zones and that become mixed into shoreline waters. Therefore, we did not expect that the significant difference in δ13C between larvae from Sites 3 and 4 was necessarily related to the presence of hyphae in the guts of the Site 3 larvae. We do suspect, however, that hyphae, which had recently been consumed by 76% of the H. alternans larvae at Site 3 . Similar to dune grass, red pine needle δ13C is approximately −28‰ [13C of −27, the δ13C of some of the common fungal hyphae in this habitat would be approximately −21, or perhaps a few mil higher, as a result of hexose conversion to lipids, which would be expected to discriminate against 13C [13C −21.3 average for Site 3 larvae. This larval average may simply reflect a balance between benthic and pelagic production, but it is plausible that larval δ13C at Site 3 was in part also a reflection of these terrestrial energy subsidies which were based on the amount of direct, up-current location during our most common surf-producing storms.The apparent source of the hyphal energy delivered to the surf zone in Site 3 was photosynthesis by beach vegetation, most likely by the deep and extensively rooted izes 13C . This ulizes 13C ,96. The bstrates . Publish to −27‰ ,98. The ely −28‰ , and theely −28‰  to be −2H. alternans occupies the typical lotic Hydropsychidae niche within the surf zone food webs. Antocha crane flies, which specialize on epilithic algae, and Hydra, which are suspension-feeding benthic carnivores. Our analysis of 3rd instar larvae from Site 3 indicated that they may have been less carnivorous than the much larger 4th and 5th instar larvae  comprise roughly equal fractions of glycine, serine, and a combination of isoleucine, leucine, and valine [15N during trophic transfers, which is commonly assessed using δ15N bulk analyses. However, the relative amounts of 15N atoms that become sequestered into individual amino acids varies considerably. So too do the relative amounts of 13C in amino acid precursors. The major silk protein constituents glycine and serine exhibit strong positive δ15N and δ13C fractionation [Silk production has likely evolved multiple times within Insecta, allowing a myriad of lineages to address ecological challenges using various forms of this lightweight, strong, sticky, elastic, and waterproof wonder fiber . For hyd tissues . This obd valine . In geneionation ,105. Isoionation . Therefoionation .15N enrichment that we observed for adults relative to larvae , lake trout , and lake whitefish (Coregonus clupeaformis). We hope that the stable isotope values presented here for larval and adult H. alternans from Lake Superior will be useful in future attempts to clarify the roles that these common caddisflies and other native surf zone insects play in nearshore food webs.The physiological processes that occur during pupation produce major changes in isotopic values. Most of the energy and amino acids required for these biosynthesis activities is produced by the catabolism of larval proteins, while fat reserves from larval feeding are reserved for adult activities ,107. Theo larvae  likely rpupation . The dept energy ,107,109.Hydropsyche larva in a dredge sample taken from 26 m deep water along the north shore, approximately 200 km northeast of our research area. The presence of Hydropsyche larvae in water so deep in Lake Superior is intriguing and begs for further exploration, but perhaps even more notable was a statement by Chief Zoologist Sidney I. Smith regarding the discovery. He reported that “the larvae, pupae, and sub-imago of the same or closely allied species were found in great abundance in stomachs of white-fish taken at Sault Sainte Marie” [Coregonus clupeaformis) and Hydropsyche alternans, illustrates the potential importance of net-spinning caddisflies and other native aquatic insects in the Lake Superior food web. We are still in the discovery phase of understanding most aspects of the ecology of most of these species. Barton and Hynes conducted the most comprehensive study on the nearshore insects of the Great Lakes to date. They considered this work to be “a preliminary step toward expanding our knowledge of the benthos of large lakes to include the unique habitat found in such bodies of water—namely the continuously wave-swept shores of the Great Lakes.” [The earliest record of net-spinning caddisflies in Lake Superior was the capture during the 1871 United States Lakes Survey of a single e Marie” . The preH. alternans [H. alternans to exploit isolated pockets of relatively productive riverine and wave-swept lacustrine habitats within the vast, harsh, and dynamic glacial landscape.The high-latitude, periglacial conditions experienced by the westernmost documented population of lternans  . We fear that this and possibly other yet to be discovered tributary populations are poised to expand downstream to surf zone habitats. It is unclear whether environmental conditions in Lake Superior have thus far presented insurmountable challenges to invading mussels, or if Dreissena is slowly spreading [Hydropsyche alternans to thrive in the last remaining stronghold of the surf zone insects of the Laurentian Great Lakes.Many of the historical collection records presented in  mussels . The roc estuary  indicatepreading ,118. Whapreading , and tha


Brain function is dependent on the ability of neural circuits to remodel synaptic connections in response to internal and external stimuli. Historically, brain plasticity studies focused on excitatory synapses, with inhibitory connections considered largely stable. However, there is growing evidence that inhibitory synapses undergo short- and long-term forms of plasticity through a variety of pre- and postsynaptic mechanisms. Activity-dependent regulation of inhibitory transmission by Hebbian mechanisms has been well documented. On the other hand, homeostatic plasticity of inhibitory synapses has been demonstrated under physiological, pathological, pharmacological, and drug abuse conditions. These diverse forms of inhibitory synaptic plasticity represent an important source of neuronal network reorganization, and their crosstalk with excitatory synaptic plasticity maintains the brain's overall excitatory/inhibitory balance. Despite this growing experimental evidence of inhibitory synapse plasticity, several key issues remain.Fish and Joffe and Speigel and Hemmings review the functional diversity of the main classes of cortical and hippocampal interneurons: parvalbumin INs, somatostatin INs, 5HT3a serotonin receptor INs, calretinin/vasoactive intestinal peptide INs and neurogliaform/ivy cells. These articles describe neurophysiological properties, sites of synaptic connections, and expression of molecular targets in these GABAergic cells. They also discuss the different IN plasticity mechanisms induced by anesthetics and alcohol. Garcia DuBar et al. examined the synaptic connectivity of INs within the pontine circuit that coordinates arousal and voiding behaviors associated with micturition. The authors demonstrate that only somatostatin INs contribute to behavioral modulation, supporting the heterogeneity of GABAergic cell populations and indicating that they participate in specific brain circuitry. Finally, by investigating the biophysical and pharmacological properties of the δ-containing subtype of the GABAA receptor, Shu et al. highlight the importance of different GABAA receptor subtypes in inhibitory plasticity. Given the wide variety of INs, the intricacy of their microcircuits, and the importance of GABAA receptor subtypes, more studies are needed to fully elucidate the mechanisms of inhibitory plasticity during development and in adulthood.In this collection, two papers by Melkumyan and Silberman which examines the opposing effects of chronic alcohol on glutamatergic and GABAergic neurotransmission in the central amygdala. These authors introduce new players (astrocytes and microglia) and molecules (TNFα and IL-1β) and conclude that further studies are needed to determine the contribution of all these cell types, receptors and neuromodulators to the balance of excitation/inhibition neurotransmission. Similarly, the articles by Chapman et al. and Tipton and Russek review the interplay of plasticity between excitatory and inhibitory synapses. They describe how the induction of inhibitory LTP causes excitatory LTD and reduces excitatory neurotransmission. Conversely, they explain that stimulation-induced excitatory LTP dampens proximal inhibitory synapses (LTD). Thus, convergence of glutamatergic and GABAergic signaling mechanisms may allow for coordinated receptor plasticity and balancing of excitation/inhibition. They also identify calcium as a master regulator of synaptic crosstalk, and describe the importance of posttranslational modifications of the receptors and scaffolding proteins to coordinate the interplay of glutamatergic and GABAergic synapses. Since the homeostatic balance between excitation and inhibition is important for brain functioning, future studies should explore the imbalance of inhibitory and excitatory synaptic plasticity during disease.The importance of this topic is highlighted in the review by Speigel and Hemmings explain that anesthetics cause temporary cognitive dysfunction in adults and long-term effects in neo- and pre-natal babies due to disruptions in IN plasticity and development. Neonatal propofol exposure causes parvalbumin and somatostatin IN hypoactivity and vasoactive intestinal peptide IN hyperactivity, suggesting differential cellular effects. Similar to anesthetics, varied sensitivity to acute and chronic alcohol exposure has also been observed, as discussed by two reviews in the collection. Melkumyan and Silberman focus on alcohol's effects on distinct sub-regions of the central amygdala. The authors analyze the role of neuroinflammatory cells, the endocannabinoid system and different neuropeptides and neuromodulators, in the modulation of GABAergic and glutamatergic transmission by alcohol. The article by Fish and Joffe examines inhibitory microcircuits of the pre-frontal cortex as potential targets for the treatment of alcohol use disorder. Notably, both of these papers highlight the important of sex differences in alcohol's regulation of inhibitory synaptic plasticity and advocate for greater identification of sex-specific mechanisms that can be targeted for therapeutic drug development.Homeostatic disruption due to pharmacologically active compounds or drugs of abuse can induce plastic changes in inhibitory neurotransmission, as can long-term pathology. These alterations are associated with diverse neuropsychiatric and neurological disorders. Several papers in this collection address these issues in the context of therapeutic drug discovery. Mackenzie-Gray Scott et al. studied the correlation between hippocampal parvalbumin IN-generated gamma rhythmicity and amyloid beta plaques in a mouse model of Alzheimer's disease. The authors found no relation between gamma oscillations and plaque formation suggesting that hippocampal network activity is resilient. In contrast to Alzheimer's disease, the onset of epilepsy can occur during childhood or as an adult. Regardless of its genetic or acquired basis, inhibitory plasticity and shifts in the excitation/inhibition balance contribute to its pathophysiology, as reviewed by Tipton and Russek. The authors outline the plasticity mechanisms that control GABAA receptor function, modulation of inhibitory synapse formation and elimination, and the vulnerability of selective IN classes to seizures. Tipton and Russek highlight the importance of using single cell studies to identify selective IN vulnerability and develop more specific therapeutic approaches. They also propose identifying factors that make these cells resilient to the disease in order to promote survival of susceptible IN populations.Altered inhibitory synaptic plasticity has been associated with the cognitive decline that accompanies normal aging and in Alzheimer's disease. This Research Topic outlines recent progress and future directions in the field of inhibitory synaptic plasticity. We hope this collection will encourage further studies to elucidate the mechanisms of inhibitory transmission regulation, thus contributing to the development of new therapeutic interventions for neuropsychiatric and neurological disorders.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Each P2XR is activated by a distinct concentration of extracellular ATP, spanning from high nanomolar to low millimolar. P2XRs are implicated in a variety of physiological and pathophysiological processes in the cardiovascular, immune, and central nervous systems, corresponding to the spatiotemporal expression, regulation, and activation of each subtype. The therapeutic potential of P2XRs is an emerging area of research in which structural biology has seemingly exceeded medicinal chemistry, as there are several published P2XR structures but currently no FDA-approved drugs targeting these ion channels. Cryogenic electron microscopy is ideally suited to facilitate structure-based drug design for P2XRs by revealing and characterizing novel ligand-binding sites. This review covers structural elements in P2XRs including the extracellular orthosteric ATP-binding site, extracellular allosteric modulator sites, channel pore, and cytoplasmic substructures, with an emphasis on potential therapeutic ligand development.Extracellular ATP is a critical signaling molecule that is found in a wide range of concentrations across cellular environments. The family of nonselective cation channels that sense extracellular ATP, termed P2X receptors (P2XRs), is composed of seven subtypes (P2X The sevischemia . Individischemia . The bin K+ ions . The res1), smooth muscle cells , sensory neurons , epithelial cells , and immune cells (P2X4 and P2X7) (7 significantly improves the symptoms and neuropathology of AD (1), hearing loss (P2X2), asthma (P2X3), vascular inflammation (P2X7), and cancer (P2X7)  . Accordigy of AD . Severalr (P2X7) . As a rer (P2X7) .1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/5, P2X4/6, and P2X4/7 , defined the architecture of the extracellular and transmembrane regions for this receptor family (3 (hP2X3), giant panda P2X7 (pdP2X7), and rat P2X7 (rP2X7) receptors in successive structural studies  domain, the dolphin’s tail, is composed of two α-helices  that span the membrane, connecting the extracellular and cytoplasmic domains . Togethe3 . The P2X3 ATP-bound open state structure and the full-length rP2X7 structures highlight aspects of intracellular elements . The most significant variations between P2XR sequences lay in their cytoplasmic termini, which are known to play a critical role in receptor desensitization, trafficking, and signaling  and its uniquely large cytoplasmic domain, referred to as the cytoplasmic ballast   in zfP2X4 that makes no contacts to ATP, or a phenylalanine (F288) in rP2X7 that interacts with C5 on the ribose of ATP. These minor variations in the principal residues of the orthosteric binding site between subtypes seem unlikely to fully explain the dramatic differences in ATP sensitivity, suggesting there are other factors at play.The published structures of hP2Xing site . A closeing site , 3C–E (Hing site . Despitetivation . A compactively) , 3. The 3, zfP2X4, and rP2X7 are strikingly different  reveal these high-affinity antagonists bind at the orthosteric binding site in a Y-shaped pose, distinct from the U-shaped pose of ATP (3) are theorized to confer high specificity of TNP-ATP for hP2X3 and hP2X1, as this pair of key residues is only conserved in these two P2XR subtypes  is a high-affinity negative allosteric modulator of hP2X1 and hP2X3 (proposed to bind the same site as gefapixant), but does not effectively antagonize other subtypes  provides a structural template for the development of P2XR subtype-specific antagonists to this novel site.One of the visualized allosteric sites in P2XRs was revealed in the structure of hP2Xfapixant . This sifapixant , 6A,B. Tfapixant . Negativportance . It is tportance , 6B. Morsubtypes , 6B. Intsubtypes . The cha7 provide a view of another allosteric site present in P2XRs -1,3-dihydro-2H-benzofuro -1,4-diazepin-2-one (5-BDBD) is reported to bind to P2X4 at the equivalent allosteric site  , but a glutamine in hP2X2 and a lysine in hP2X5 and hP2X6 . This conserved charge at the lateral extracellular fenestrations could be targeted by designing ligands that prevent ion entry, regardless of whether the pore is open or closed. Such a ligand would need to selectively bind and block each extracellular fenestration, which are large surface domains with substantial void space in the ATP-bound open state  and epithelial Na (ENaCs) . Ions fl (ENaCs) , 7, 8 (S (ENaCs) . To valis signal . Glutamanditions . The resnd hP2X6 . In hP2Xen state , 7A. Thien state . These s7 (2+-binding site seen in the tick P2X structure (AmP2X)  similar to the Zn2+-coordinating residues  of AmP2X  . Of the of AmP2X . The act ligands .3 and 0.1 Å at S339 in rP2X7 (3 and 2.5 Å in rP2X7) , occurs through another movement of the TM2 helices, whereby disassembly of the cytoplasmic cap leads to a new gate near the center of the transmembrane region at V334 in hP2X3 (radius of 1.5 Å). This new gate is too narrow to allow passage of hydrated sodium ions  at the symmetry axis formed by a set of six TM helices consisting of two antiparallel helices from each protomer , 8A. In in rP2X7 . Interesn rP2X7) . Desensiium ions   . Conside3  at the appropriate point in post-translational processing. A greater understanding of the basic biology of P2X7 maturation is necessary before such a pharmacological strategy can be attempted.Within each protomer of P2Xtoylated . The paltoylated . When thsubtypes . While tsubtypes . Modificignaling . As a reignaling . Given t7, the residues of which are conserved in hP2X7, is quite intriguing , has associated guanine nucleotide exchange factors (GEFs) that facilitate GDP release  may be applied to study and therapeutically target P2X7-mediated signaling  and >500-fold less effective against rP2X7  structurally solved in complex with its target receptor to high-resolution. This process will be repeated iteratively until a threshold of affinity and specificity is reached (The feasibility of SBDD has dramatically increased with access to more advanced computational tools and the availability of structures for therapeutically relevant membrane proteins, largely due to the cryo-EM resolution revolution . With thompounds . Then, s reached . Even th reached . Cryo-EM7 cytoplasmic domain provided valuable insights, unexpected findings raised even more questions about its biological function. Most importantly, we have yet to fully understand how to selectively modulate the seven homotrimeric P2XRs or how effective such modulators would be against heterotrimeric P2XRs, considering their pharmacological complexity. The recent advancements in cryo-EM make this technique ideally suited to study P2XRs, including novel receptor structures and ligand-bound receptor complexes. While P2XRs are implicated in a host of physiological and pathophysiological conditions found within the cardiovascular, central nervous, and immune systems, there are no FDA-approved drugs targeting this receptor family. The success of antiplatelet drugs which antagonize the related P2Y GPCR family exemplifies the therapeutic value of targeting purinergic signaling (The substantial body of work characterizing P2XR structures has provided invaluable information on the biology and molecular pharmacology for this receptor family, however there remain many unanswered questions. Little is known about the structure and function of P2XR cytoplasmic domains and even less about heterotrimeric P2XRs. While the structural determination of the P2Xignaling . To this


Congenital self‐healing reticulohistiocytosis of Hashimoto and Pritzker (CSHR) is a rare, benign form of Langerhans Cell Histiocytosis (LCH) that presents at birth and involutes by 6 months of age. We present an atypical case of CSHR with the first onset at 7 months of age, treated with surgical excision. Congenital self‐healing reticulohistiocytosis (CSHR) presents at birth; however, in this case, it is acquired at 10 months of age which is atypical. CSHR is benign and it must be differentiated from malignant form of Langerhans Cell Histiocytosis. These myeloid neoplasms of dendritic cells are characterized under electron microscopy by the presence of Birbeck granules resembling that of “tennis rackets.” LCH has been categorized into four main clinicopathologic types: congenital self‐healing reticulohistiocytosis of Hashimoto‐Pritzker disease (CSHR), multifocal multisystem LCH, unifocal LCH, and multifocal uni‐system LCH Table . MultifoCSHR was first described in 1973 by Hashimoto and Pritzker. This entity is unique from the other variations of LCH due to its benign nature and presentation at birth with involution in the first few months of life.2A 10‐month‐old male patient born full term via vaginal birth, otherwise healthy, presented to the dermatology clinic with several 4–8 mm, pink, shiny, grouped, raised, dome‐shaped, umbilicated papules with a central dell located on the right anterior proximal arm present for three months. The clinical differential diagnosis included molluscum contagiosum and multiple epidermal inclusion cysts. Hydrocortisone 2.5% cream was prescribed to be applied twice daily to the affected area for treatment of molluscum dermatitis.At one‐week follow‐up, the lesion had spread and the scattered, pink papules had coalesced into a larger confluent plaque Figure . An 8 mmAt one‐month follow‐up, our patient was noted to have a pink to brown papule adjacent to the biopsy scar on the right anterior proximal upper arm. Despite the benign nature of CSHR, a surgical excision with clear margins was performed to ensure that there would be no recurrences given the atypical presentation at 7 months of age in our patient. The histology of the surgical excision had the same features as that of the previous biopsy. Hence, we concluded that the recurrent growth was that of CSHR. Our patient was instructed to have clinical follow‐up with dermatology and oncology. Oncology evaluation and subsequent 3‐month follow‐up showed no recurrences, further supporting the diagnosis of CSHR. Three years post‐op, our patient remains well without lesions.3Congenital self‐healing reticulohistiocytosis (CSHR) differs from other forms of Langerhans cell histiocytosis (LCH) because of its benign involuting nature and presentation at birth rather than in early infancy or childhood. CSHR commonly presents with a solitary red‐brown papule or nodule with a button‐like central erosion.While the various forms of LCH share similar histopathological findings, their pathogenesis has not been clearly elucidated. Eosinophilia, as well as CD1a+ and S‐100+ stains, are common presentations of LCH. Other immunostains for LCH include IFN‐ γ, Ki‐67, PHH3, and E‐cadherin. IFN‐γ, which is not expressed by benign Langerhans cells, is present in all forms of LCH, thus exacerbating recurrence and spread in the benign forms.CSHR is a congenital, self‐healing disorder that does not require medical treatment, but rather close clinical follow‐up after diagnosis so that malignant LCH is excluded. Lesions can be multiple or solitary, though this does not ultimately affect involution. However, in the event of malignant LCH, treatment is necessary. Treatment for LCH includes prednisone and chemotherapeutic drugs such as vinblastine.With complete excision removal of the CSHR, our patient's lesion did not recur at the two‐month follow‐up nor at the six‐month follow‐up with dermatology and oncology; follow‐up with dermatology and oncology are important nonetheless. Full workup to evaluate for systemic involvement is integral for the diagnosis of CSHR especially in our case due to the delayed onset beyond the congenital age, and also because the aggressive variants of LCH mimic CSHR histologically. Both concerns addressed above applied to our patient's case. CSHR rarely presents after birth, and recurrent cases may be characterized by malignancy.Our patient's unique presentation of CSHR with onset at 7 months of age raises a concern for unreported cases of CSHR that arise after birth. This issue is further exacerbated by the similar clinical and histopathological findings between CSHR and other forms of LCH. Thus, it has been speculated that CSHR may occur more frequently than reported.Christopher Yuki is the main author and did majority of the writing. Patrick Young helped write the paper. Dr. Ohsie is the pathologist who read the slides. Dr. Nguyen is the overseeing physician.The authors have no conflict of interest to declare.This case has not been presented in any prior conferences.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Gyrodactylus salaris, a fish parasite, GsaCAβ, has been investigated for its inhibitory effects with a panel of sulphonamides and sulfamates, some of which in clinical use. Several effective GsaCAβ inhibitors were identified, belonging to simple heterocyclic sulphonamides, the deacetylated precursors of acetazolamide and methazolamide (KIsof 81.9–139.7 nM). Many other simple benezene sulphonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 µM. The least effective GsaCAβ inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with KIs in the range of 16.9–24.8 µM. Although no potent GsaCAβ-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the inhibition profile of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.A β-class carbonic anhydrase  present in the genome of the Monogenean platyhelminth  Gyrodactylus salaris, GsaCAβ,Salmo salar) is particularly sensitive to this parasite, which produced catastrophic losses in fish farms in Scandinavian countries and elsewhere, starting with the 1970s,Schistosoma haematobiumSchistosoma mansoniWe have recently reported the cloning and characterisation of a β-class carbonic anhydrase  encoded in the genome of ,,2 hydration reaction, with a kcat of 1.1 × 105 s−1 and a kcat/mK of 7.58 × 106 M−1 × s−1. Furthermore, inorganic anions, a well-known class of CA inhibitors (CAIs),I of 81 µM) and sulphamic acid (IK of 6.2 µM) showed the most efficient inhibitory action2NH2 moiety found in the most investigated class of CAIs, the aromatic/heterocyclic sulphonamides and their isosteres ,CAs are well known drug targets for the management of human diseases1–24 and AAZ-HCT were commercially available, highest purity reagents from Sigma-Aldrich  or were synthesised as previously reportedCompounds Protein production was carried out according to the previously reported protocol2 hydration activity4 for maintaining constant the ionic strength, following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10–20 mM) were prepared in distilled-deionized water and dilutions up to 0.01 µM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, whereas the kinetic parameters for the uninhibited enzymes from Lineweaver-Burk plots, as reported earlier,An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO,GsaCAβ shows catalytic properties for the physiologic reaction similar to those of the slow human isoform hCA I, being however slightly less effective as a catalyst compared to hCA I . On the ,We have investigated the inhibition profile of GsaCAβ with a panel of sulphonamides and sulfamates  known to13 and 14, the deacetylated precursors of acetazolamide and methazolamide, which showed IK values of 81.9–139.7 nM, which is 5.1–5.6 times a better inhibitory activity compared to the clinically used derivatives AAZ and MZA  and heterocyclic derivatives. However, it seems that rather simple and elongated scaffolds lead to effective inhibition whereas the inclusion of bulkier substituents  is detrimental for the inhibitory activity.A rather large number of derivatives, such as 4–6, 8–10, 21, DCP, BRZ, TPM, ZNS, SLP; IND, VLX, CLX and SAC. These compounds had KIs in the range of 1.63–9.1 µM. As above, they belong to a large number of diverse chemotypes in order to draw a rationalisation of their SAR. Saccharin, also being a medium potency inhibitor, is among the most selective ones for inhibiting GsaCAβ over the human isoforms  were detected so far.As seen from Gyrodactylus salaris, a fish parasite of salmon and other economically relevant aquaculture fish species, encodes for a β-class CA, GsaCAβ, which has been investigated here for its inhibition profile with sulphonamides/sulfamates, as a possible antiparasitic drug target. We identified several effective GsaCAβ inhibitors, belonging to simple heterocyclic sulphonamide derivatives, the deacetylated precursors of acetazolamide and methazolamide, which showed IK values of 81.9 − 139.7 nM. Many other simple benezenesulfonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 µM. The least effective GsaCAβ inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with IKs in the range of 16.9 − 24.8 µM. Although no GsaCAβ-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the SAR for inhibition of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.The Monogenean platyhelminth


Radiographic parameters and patient-reported outcomes, including leg and back pain visual analog scale (VAS) and Oswestry Disability Index (ODI), were assessed. Loosening signs were noted in 48 out of 552 pedicle screws. The screw loosening rate was higher in the FG TLIF (10.2%) than Ro TLIF group (4.3%). A significant correlation was found between screw loosening and age, the number of level(s) fused, and the ratio of the average distance from the pedicle screw to the upper endplate to vertebral body height. VAS-leg, VAS-back, and ODI showed significant improvements in both groups postoperatively . These results indicated that robot-assisted pedicle screw placement in TLIF had a lower screw loosening rate and similar patient-reported outcomes compared with the fluoroscopy-guided technique.Robot-assisted pedicle screw placement for spine surgery has become popular in recent years. This study compares clinical, radiographic outcomes and the screw loosening rate between robot-assisted and fluoroscopy-guided pedicle screw placement in patients who underwent transforaminal lumbar interbody fusion (TLIF). We retrospectively examined 108 patients with the degenerative lumbar disease who underwent TLIF. According to whether the robotic system was used, patients were assigned to either the robot-assisted (Ro TLIF,  Pedicle screw placement for spinal fusion surgery has been widely performed since its introduction by Boucher in the 1950s ,3,4,5, fThis was a retrospective single-center cohort study. This study was approved by the institutional review board of our institution . We identified all patients with the lumbar degenerative disease who underwent TLIF at our institution between May 2018 and September 2020. The operations were performed by three spinal surgeons with more than 20 years of experience as attending physicians. The inclusion criteria were as follows: (1) patients with the degenerative lumbar disease who underwent TLIF at our hospital, (2) 18 years old or older, and (3) regular follow-up at our outpatient department for at least 12 months. The exclusion criteria were as follows: (1) age < 18 years, (2) follow-up period < 12 months or loss to follow-up, and (3) underwent TLIF due to infection, revision, tumor, or trauma.The patients were assigned to the robot-assisted TLIF (Ro TLIF) group or the fluoroscopy-guided TLIF (FG TLIF) group, according to the method used for pedicle screw placement.Baseline data included age, sex, body mass index (BMI), diabetes mellitus (DM), diagnosis, number of level(s) fused, cage type (polyetheretherketone (PEEK) or trabecular metal (TM) cage), screw type (solid or hollow screw), surgical segment(s), estimated blood loss, surgical time, and length of hospital stay.The general steps of the FG TLIF procedure are as follows: A posterior midline incision was made. Paravertebral muscles were dissected from the spinous process, lamina, facet, and transverse process to expose the anatomical landmarks for pedicle screw entry points. Screw diameter was determined by pedicle size on preoperative MRI. An awl was used for entry point  followed by probing the pedicle tract to the vertebral body. Screw length was determined by measuring the probe length in the bone tunnel. Guide pins for pedicle screws were placed through the tract, and their positions were confirmed by fluoroscopy. Decompression was performed using laminotomy, laminectomy, foraminotomy, facetectomy, discectomy, or a combination of the above procedures according to clinical circumstances. Endplates were prepared, followed by the placement of an interbody device with bone grafts. Pedicle screws were inserted via guide pins with hollow screws or under direct vision with solid screws, and the rods were connected.® system  was used for the Ro TLIF procedure. The system is composed of a flexible robotic arm and a workstation  the flexible robotic arm was placed in the planned position  thposition g; then aL-spine anteroposterior and lateral plain radiographs were obtained on the first postoperative day, as per our standard protocol. We measured several parameters of each pedicle screw on the L-spine lateral view using the Picture Archiving and Communication System Ultraquery , including the distance from the pedicle screw tip to the upper endplate, pedicle screw to the upper endplate at the posterior body level, and pedicle screw tip to the vertebral body anterior cortex, vertebral body height, and vertebral body anteroposterior diameter . All parScrew loosening is defined as a minimal thickness of 1 mm radiolucent zone around the edges of pedicle screws on plain radiography ,15,16,17Patient-reported outcomes, including the back and leg pain visual analog scale (VAS) and the Oswestry Disability Index (ODI), were recorded on the day before surgery and at 1, 3, 6, and 12 months postoperatively.Further, complications, including urinary tract infection, surgical site infection, screw loosening, cage subsidence, and broken rod or screw, were noted.p < 0.05. All statistical analyses were performed using IBM SPSS Statistics, version 22.0 .Categorical variables between the groups were compared using the chi-square test. Continuous variables were compared using the Mann–Whitney U test. Differences in the patient-reported outcomes in the same group were calculated using the Friedman test. The association between screw loosening rate and potential risk factors was assessed using logistic regression analysis. A correlation analysis was conducted using the Pearson correlation coefficient to validate the intraobserver and interobserver reliability. Statistical significance was set at A total of 185 consecutive patients underwent TLIF at our facility between May 2018 and September 2020. Twenty-nine cases of revision and one case of infection were excluded. A total of 155 patients met our inclusion criteria and were assigned to the Ro TLIF or the FG TLIF group according to whether the robotic system was used for pedicle screw placement. Seven patients in the Ro TLIF group and 24 in the FG TLIF group had a follow-up period of fewer than 12 months and were excluded. Two patients in the Ro TLIF group and 14 in the FG TLIF group performed by two spinal surgeons less than 10 years of experience as attending physicians were also excluded. Finally, 29 patients were assigned to the Ro TLIF group, and 79 patients were assigned to the FG TLIF group.® system. There were no significant differences in age, sex, BMI, DM, number of level(s) fused, surgical segment(s), cage type, preoperative pain VAS-leg, preoperative pain VAS-back, or preoperative ODI (Cohort characteristics are presented in tive ODI .p = 0.677), operative time for single-level TLIF , and length of hospital stay  between the FG TLIF and Ro TLIF groups. In the FG TLIF group, 14 patients had screw loosening, 2 had cage subsidence, 2 had superficial wound infection, 1 had urinary tract infection, and 1 had a broken rod during the follow-up period. In the Ro TLIF group, three patients had screw loosening, three had cage subsidence, two had superficial wound infection, and two had urinary tract infection. A total of 19 patients (24.1%) in the FG TLIF group and 7 patients (24.1%) in the Ro TLIF group had complications (some patients might have had two or more of the above complications), and no significant difference was found between the two groups (p = 1.000). The only complication requiring revision surgery in the patient was a broken rod in the FG TLIF group.Perioperative and postoperative outcomes are shown in p < 0.001). The ratio of the distance from the pedicle screw tip to the anterior cortex to the anteroposterior diameter of the vertebral body showed no significant difference between the FG and Ro TLIF groups .There was a total of 412 pedicle screws in the FG TLIF group and 140 screws in the Ro TLIF group. The ratio of the distance from the pedicle screw to upper endplate to the vertebral body height was higher in the FG TLIF group than that in the Ro TLIF group . The results are presented in During the follow-up period, 48 of 552 (8.7%) pedicle screws showed signs of loosening on plain radiography  ,15,16,17p < 0.001) (p = 0.100).Analysis of the screw loosening and non-screw loosening groups showed that the ratio of the distance from pedicle screw to upper endplate to the vertebral body height was higher in the screw loosening group than non-screw loosening group  . The ratThe intraobserver and interobserver ICCs were excellent (ICC > 0.9) for all measurements.The correlation between potential risk factors and pedicle screw loosening rate was established using a logistic regression model . The scrp = 0.868) and VAS scores for back pain  and leg pain  were not significantly different between the FG and Ro TLIF groups .VAS for back pain, VAS for leg pain, and ODI showed significant improvements in both groups postoperatively  than in the fluoroscopy-guided pedicle screw placement group 10.2%). Ohba, T. et al. reported an overall screw loosening rate about 15.2% (44 out of 290 pedicle screws) in patients who underwent minimally invasive lumbar or thoracic spinal stabilization . Other s.2%. OhbaTo the best of our knowledge, this is the first study to report the relationship between the distance from the pedicle screw to the vertebral body cortex and the screw loosening rate. We found that the ratio of the distance from the screw to the upper endplate to the vertebral body height positively correlated with the screw loosening rate. When using the fluoroscopy-guided technique, the probe tended to “slide” through the soft part of the pedicle, making it difficult to get closer to the upper endplate. Thus, the distance of the pedicle screw to the upper endplate might be larger when the screw is fixed on less dense bone, resulting in a higher loosening rate. With the robotic system, we could preoperatively design a pedicle screw trajectory closer to the upper endplate; thus, we were able to overcome this problem. In a cadaveric study, the novel hollow screw was less resistant to loosening when compared with a conventional solid pedicle screw . In our Patient-reported outcomes showed significant postoperative improvement in both Ro TLIF and FG TLIF groups. The complication rates were also similar between the two groups. Only one complication (broken rod) required revision surgery in the FG TLIF group. Kim et al.  found thThis study had several limitations. First, this was a retrospective study, and might have a selection bias. Second, only a limited number of patients were included in the Ro TLIF group. Third, robot-assisted pedicle screw placement was a newly introduced technique at our facility, and the learning curve was not evaluated. Finally, patients underwent operations performed by different surgeons, and different implants (pedicle screw and cage type) were used. Although similar procedures and techniques were preformed, experimental confounding effects may have existed.p < 0.05) improvement in all patient-reported outcomes.The screw loosening rate was lower in the robot-assisted TLIF group than in the fluoroscopy-guided TLIF group; this may be associated with placing pedicle screws closer to the upper endplate. While performing pedicle screw insertion in future operations, designing a screw trajectory closer to upper endplate may be considered. Although no differences were detected between the robot-assisted and fluoroscopy-guided TLIF patient groups at any of the follow-up periods, there was a highly significant (


Previous studies have shown that aging promotes myocardial apoptosis. However, the detailed mechanisms remain unclear. Our recent studies revealed that aging not only activates apoptosis, but also activates some anti-apoptotic factors. By quantitative phosphoproteomics, here we demonstrated that aging increases cytochrome c (Cytc) phosphorylation at threonine 50 (T50), a post-translational modification with unknown functional impact. With point mutation and lentivirus transfection, cardiomyocytes were divided into four groups: empty vector group, WT (wild type), T50E (as a phosphomimic variant), and T50A (non-phosphorylatable). TUNEL staining and flow cytometry were used to determine the apoptosis ratio in different groups after hypoxic/reoxygenated (H/R) treatment. The results showed that T50-phosphorylated Cytc suppressed myocardial apoptosis induced by H/R. Furthermore, Western Blot and ELISA measurements revealed that Cytc T50 phosphorylation inhibited caspase-9 and caspase-3 activity without altering caspase-8, BCL-2, BCL-XL, and Bax expression. In our study, we demonstrated that aging increases phosphorylation Cytc at T50 and this aging-increasing phosphorylation site can suppress H/R-induced apoptosis. Acute myocardial infarction is one of the leading causes of death worldwide. Thanks to the rapid development of interventional technology, the number of people dying from acute myocardial infarction has decreased significantly. On the contrary, more and more people are suffering from post-ischemic heart failure . Heart fSubstantial evidence indicates that apoptosis is the leading cause of ischemia-induced heart failure . Our preMore interestingly, our previous work demonstrates that aging not only stimulates pro-apoptotic pathways in cardiomyocytes, but also activates some anti-apoptotic factors , 9, 10. In the present study, we performed proteomics on myocardial tissue obtained from young and old mice. Compared with the young group, the expression of 88 proteins was up-regulated, and 80 were down-regulated in old mice. At a post-translational modification level, phosphorylation levels were up-regulated at 445 sites and down-regulated at 1526 sites in the old group. Phosphorylation level changes were widespread and stable in each sample. More importantly, these changes occurred in many essential apoptotic regulatory proteins, including MAPK14, Akt1, mTORC2, and p21. Upon further analysis of these results, changes in Cytc phosphorylation at T50 caught our attention as Cytc phosphorylation is involved in mitochondrial function . SurprisWe found a new aging-increasing phosphorylation site by proteomics, which may be associated with aging-related myocardial apoptosis regulation.in vivo and is closely related to apoptosis [P-value<0.05 as a cut-off, the change in the amount of differential modification >1.3 was considered as significantly up-regulated and less than 1/1.3 as significantly down-regulated.  and increased apoptosis in T50A group when compared with WT group  is also closely related to apoptosis. Lee et al. isolated Cytc from cow heart and identified the phosphorylation site by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. Tyr-97 is the first phosphorylation site discovered on Cytc . The othIn conclusion, there are several innovations in this study. First, we observed that aging increases phosphorylation Cytc at T50. Second, this aging-increasing phosphorylation site can suppress H/R-induced apoptosis. In addition, this anti-apoptotic effect is mediated mainly through inhibition of downstream caspase-9 and caspase-3 activity. Based on our previous studies , T50-phoIn summary, our study proved a new phosphorylation site that is increased in the process of aging. More importantly, phosphorylation at this site can decrease the apoptosis ratio induced by H/R.Approved by the institutional ethics committee, this study was in compliance with the United States National Institutes of Health guidelines. Male C57 mice (aged eight weeks and 18 months) were anesthetized with sodium pentobarbital. Mouse hearts were harvested by exteriorizing the heart via a left thoracic incision.Heart tissue from young and old mouse were harvested and delivered to PTM-Biolab Co., Ltd.  for phosphoproteomic analysis. The quantitative study of phosphorylated proteomics used TMT labeling and phosphorylated enrichment techniques and high-resolution liquid chromatography-mass spectrometry.http://www.ebi.ac.uk/GOA/). Firstly, converting identified protein ID to UniProt ID and then mapping to GO IDs by protein ID. If some identified proteins were not annotated by UniProt GOA database, the InterProScan soft was used to annotate protein’s GO function based on the protein sequence alignment method. Then proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component, and molecular function.Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database .A special antibody was ordered from PTM-Biolab Co., Ltd. an antigen. Specific antibody response was examined with immunohistochemistry staining.Myocardial tissue and AC16 cells were homogenized in an ice-cold lysis buffer. After homogenization, the lysates were centrifuged. The supernatant was saved and separated by electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride-plus membranes. After blocking buffer, the immunoblots were probed with anti-T50 phosphorylation of Cytc, anti-Cytc, anti-Cleaved Caspase-3, anti-Caspase-3, anti-Cleaved Caspase-9, anti-Caspase-9, anti-BCL-2, anti-BCL-XL, anti-Bax, and anti-GAPDH antibodies overnight at 4° C, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour.in situ by the use of 3, 3 diaminobenzidine kit .Paraffin sections made from young and aged mouse hearts were used for immunohistochemistry assays to detect protein expression levels of T50 phosphorylation of Cytc proteins. In accordance with the manufacturer’s instruction, tissue sections stained immunohistochemically are determined separately by two pathologists using the indirect streptavidin peroxidase method. The primary antibodies against T50  and horseradish peroxidase-conjugated IgG were used in this study. Then, the proteins were visualized in vitro, we generated T50E for constant phosphorylation and T50A for non-phosphorylation. Three sequences of WT, T50E and T50A are as below. WT:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACACAGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. T50E:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACGAGGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. T5OA:ATGGGTGATGTTGAGAAAGGCAAGAAGATTTTTATTATGAAGTGTTCCCAGTGCCACACCGTTGAAAAGGGAGGCAAGCACAAGACTGGGCCAAATCTCCATGGTCTCTTTGGGCGGAAGACAGGTCAGGCCCCTGGATACTCTTACGCCGCCGCCAATAAGAACAAAGGCATCATCTGGGGAGAGGATACACTGATGGAGTATTTGGAGAATCCCAAGAAGTACATCCCTGGAACAAAAATGATCTTTGTCGGCATTAAGAAGAAGGAAGAAAGGGCAGACTTAATAGCTTATCTCAAAAAAGCTACTAATGAGTAA. With these sequences, construct lentivirus vectors, purchased from Shanghai Yibeirui Bio-pharmaceutical Technology Co., Ltd. .Three sequences of WT, T50E and T50A were synthesized after codon optimization, purchased from Sangon Biotech Co., Ltd. . To study the effect of T50 phosphorylation Human adult ventricular cardiomyocyte cell line AC16 was purchased from the American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37° C. In this study, four types of lentiviruses were used to infect AC16 cells. Infection efficiency was monitored by fluorescence microscopy 48 hours post lentivirus infection.In vitro cardiomyocyte H/R modelSeventy-two hours after cardiomyocyte transfection, all groups were treated with hypoxia (95% N2 and 5% CO2) for 6 hours , followed by normoxia  for 2 hours.Cardiomyocytes were cultured directly upon coverslips. TUNEL staining was performed per the manufacturer’s instructions . Total nuclei were stained by DAPI . Apoptotic index  was determined via blinded manner.After incubation for two days, the harvested cells were used for apoptotic determination by two methods. First, the apoptotic rate of transfected cells was evaluated by using Annexin V APC/PI apoptosis detection kit (KeyGEN) by following the manufacturer’s instruction and then analyzed using FACScan. All tests are carried out in triplicate. Second, by following the manufacturer’s instructions, Caspase-9 and Caspase-3 levels were detected utilizing human Caspase-9 ELISA kit (BC3890), Caspase-3 ELISA kit (BC3830) and Caspase-8 ELISA kit (BC3880) from Beijing Solarbio Science and Technology Co., Ltd. .The data were analyzed with Prism 8.0 . All values in the text and figures are presented as mean ± SD. Statistical differences were determined by Student’s t-test for comparison between 2 groups and ANOVA followed by Bonferroni multiple comparison test for comparison among ≥3 groups. Probabilities of.05 or less were considered statistically significant.The data used to support the findings of this study are available from the corresponding author upon request.in vivo or clinical significance should be explored in the future.In this study, phosphoproteomics were performed only in myocardial tissue from young and old mice, which reduces the targeting of the experiment. The relationship between T50 phosphorylation and mitochondrial release or caspase 9 interaction will be explored in the next step. In addition, the functional impact of Cytc T50 phosphorylation