Upwind levels likely reflected biomass burning that is prevalent in the surrounding informal settlements; levels at the e‐waste and downwind sites also reflected contributions from biomass combustion and traffic. The highest PM levels occurred in evenings, influenced by diurnal changes in emission rates, atmospheric dispersion and wind direction shifts. We demonstrate that moderate cost instrumentation, with some modifications, appropriate data cleaning protocols, and attention to understanding local sources and background levels, can be used to characterize spatial and temporal variation in PM levels in urban and industrial areas.Air quality information is scarce in low‐ and middle‐income countries. This study describes the application of moderate cost approaches that can provide spatial and temporal information on concentrations of particulate matter (PM) needed to assess community and occupational exposures. We evaluated PM levels at the Agbogbloshie e‐waste and scrap yard site in Accra, Ghana, and at upwind and downwind locations, obtaining both optical and gravimetric measurements, local meteorological data and satellite aerosol optical depth. Due to overload issues, the gravimetric 24‐hr samplers were modified for periodic sampling and some optical data were screened for quality assurance. Exceptionally high concentrations (e.g., 1‐hr average PM Ambient particulate matter was monitored onsite, upwind and downwind of an e‐waste site in Ghana using gravimetric and optical measurements2.5 levels by 57 μg/m3 over upwind levels of 31 μg/m3, and some exceptionally high levels were measuredE‐waste site emissions increased 24‐hr PMModerate cost methods can measure air quality and source impacts given attention to study design, sampler performance and local influences These activities can pose environmental and occupational health and safety concerns, particularly in low and middle income countries (LMICs) and informal settings where controls and inspections are lax or absent Ackah, . RecycliAgbogbloshie in central Accra, Ghana has been a hub for large scale e‐waste, automobile and scrap recycling for nearly 15 years. Currently, 4,500–6,000 workers at the site use rudimentary techniques to recover valuable materials   is shown in Figure 2 area, which lies within Accra, Ghana’s capital, with 2.27 million inhabitants. In addition to the scrap yard and recycling activities, the site contains a health clinic, a technical training center for e‐waste and scrap workers, a football field, mosques, extensive informal housing, workshops for motorcycle, car and electronics repair, commercial cooking, and metal fabrication .Agbogbloshie is the informal name for an area about 1 km from central Accra and adjacent to the South Industrial Area that contains the Agbogbloshie e‐waste site and scrap yard. The site is bounded by the Abossey‐Okai Road, the Odaw River, Cemetery Drain, and the Ring Road West. This 0.365 kmThe e‐waste site is adjacent to densely populated residential and commercial areas, including a large market, informal housing to the south, commercial offices and industrial areas to the N and E, vegetable markets to the E, and food markets to the NE. Markets , commercial cooking, metal working, and banking services are adjacent to the e‐waste site.3 of material; one report suggested that 40,000 m3 of silt was deposited annually in the basin  and government reports Awiah, . For floGambeta, .2.2We utilized “area” or fixed site monitoring with goals of measuring upwind, on‐site and downwind concentrations to understand impacts at the waste site adjusted for upwind or “background” levels, and to assess impacts at downwind locations. Our goal was to collect 24‐hr samples at the three sites simultaneously every sixth day to characterize 1‐hr, 24‐hr and long term PM levels, and to evaluate spatial differences and temporal patterns.Monitoring site locations were selected by considering e‐waste site activities , distance from the e‐waste site, prevailing wind directions, and the ability to obtain electrical power, rain shelter, site security and access, and permissions from property owners and operators. The siting process involved multiple site visits and consultations with leaders of the e‐waste workers, commercial and governmental facilities, and others. We obtained electrical power at two sites and installed a 12 V 80 A‐hr photovoltaic system  at the upwind site. Site locations are mapped in Figure At the e‐waste site (site 2), sampling equipment was placed near the ceiling (~2 m height) of a 2‐wall metal shed that served as a meeting area of e‐waste leaders Figure . This shThe upwind site (site 1) was 1.35 km SSE of site 2, located at an inoperable pump station/sand filter at the Korle Lagoon Figure . The pumThe downwind site (site 3) was 0.48 km NE of the e‐waste site. Monitoring instruments were mounted on a third floor balcony wall (~6 m above the ground) of a 3‐story building (Letap Jewelries Limited Building) used for pharmaceutical production (first two floors) and apartments , size selective samplers , and personal sampling pumps  operating at 10 L/min. Although the pumps had internal batteries, external power packs were used to allow 24‐h sampling. Near‐continuous measurements (every 1 min) were obtained using 5‐channel optical particle counters  operating at 2.83 L/min. External power also was used with these samplers. Temperature and relative humidity were measured every 1 min using logging instrumentation .We monitored PM, temperature and humidity using portable instruments placed in custom fabricated metal “cages” that were open on four sides and locked for safety and security Figure . Integra2.5 pumps were set to 10 L/min using a flowmeter  connected to a HEPA capsule filter , and instrument clocks were synchronized. OPC instrument flows were checked and adjusted to 2.83 L/min. After sampling, flow rates were measured again, filters were removed from the sampler cassette using clean forceps, folded in half (exposed side closed), and placed in individual poly bags until weighing. All filters were stored in a clean, sealed container until gravimetric analysis. In the laboratory, filters were weighed before and after sampling using a microbalance  after 48‐hr conditioning at 25 ± 1 C, RH = 33 ± 2%, and deionizing for 30 min. Newly weighed filters were placed in individual labeled poly bags.Pumps were programmed to sample for a 24‐hr period, and deployment and retrieval of instruments mostly occurred from 10:00 to 16:00. Prior to sampling, new filters were installed, PMPilot deployments showed that the integrated samplers were significantly overloaded, which was observed visually Figure ; overloa2.4https://www.ncdc.noaa.gov). The airport site is 12.1 km from the coast and 10.2 km NE of the e‐waste site. Terrain within 900 m of the airport is open; apartments and some midrise commercial buildings are within 1 km.Local meteorological variables were measured using a weather station  placed immediately SSW of site 1 at 4.5 m above ground level (36 m msl), 2.5 m above a ~ 2 m concrete wall that enclosed the sand filter. This site is 1.2 km from the coast (Gulf of Guinea). The surrounding area was largely flat and free of obstructions other than the filter enclosure and dredge spoils heaped in adjacent fields starting late 2017. 1‐min data from the upwind site were collected from 7/30/17 to 12/31/18, although the wind vane sensor failed on 8/28/18. We also obtained 2017–2018 hourly surface observations from Kotoka International Airport  from NOAA , as described in the SI.To help indicate the possible presence of Harmattan dusts, we examined daily maps of aerosol optical depth (AOD) over the region from November 2017 to February 2017 using both the MODIS dark target algorithm and the combined value‐added AOD (CVA‐AOD)  activities included colocation of sampling instruments, use of standard datasheets, and flow checks before and after deployment. For gravimetric samples, we used filter blanks, a minimum of two replicates of gravimetric measurements with an acceptance criterion of 10 μg, confirmation of flow volumes using the pump’s totalizer within 10%, and exclusion of PM masses over 600 mg that indicated an overloaded sampler. Due to power interruptions, sampler problems and field logistics, some sampling periods did not reach 24‐hr; we excluded periods shorter than 75% of the goal (<18 hr). Filters were handled using forceps and powder‐free gloves; different forceps were used for the oiled impactor substrate disc to avoid cross‐contamination. Filter blanks were subjected to same analyses as samples. To assess weighing accuracy, certified 200 mg standards were weighed at beginning and end of each weighing session, and after every 1210 and within 2% for PM2.5. While these instruments have a large dynamic range, very high concentrations can produce coincidence error that biases measurements, e.g., multiple small particles appear as a single larger particle that then overestimates mass in larger size channels. There is no specific threshold where coincident error becomes critical (the manufacturer did not provide guidance); our experience with the selected instrument suggests that biases start around 2000 μg/m3. Very high humidity also can bias results (as discussed later). Considering the 144,579 1‐min PM10 measurements collected, the top 10 values ranged from 4,926 to 11,084 μg/m3, 86 measurements (0.06%) exceeded 3,000 μg/m3, and 365 (0.25%) exceeded 2000 μg/m3. We considered OPC data as potentially biased if PM10 exceeded 2000 μg/m3. When aggregated to 1‐hr averages, this omitted 7 (0.3%) of the 2,327 hourly averages and dropped the maximum 24‐hr PM2.5 average from 865 to 522 μg/m3. As discussed later, these exclusions affected only the highest PM measurements. We also checked agreement with the gravimetric measurements, and did not apply correction factors to the optical measurements.For optical measurements, daily flow and zero checks were performed using a flowmeter connected to a HEPA capsule filter. Colocation tests showed average agreement within 6% for 1‐hr averages of PM2.62.5 and PM10; the coarse fraction (PM2.5–10) was also determined. Optical and gravimetric PM data were compared for the same periods using correlations and scatterplots; again, at least 80% overlap of hourly data were required in these comparisons. We calculated descriptive statistics for the 1‐hr and 24‐hr data and used probability, trend plots with third order polynomial curves to fit the data, and pollution roses to assess distributions, site differences, wind direction, and time‐of‐day patterns. To provide a single “best” estimate of concentration increments over background (site 1) levels, we combined 24‐hr averages from gravimetric and optical instruments, and estimated standard deviations using Gaussian quadrature. Meteorological data was converted to 1‐hr averages, and after ensuring comparability between the sites, the 2‐site average of hourly wind speed and direction was used to obtain a nearly complete meteorological record. Data were converted to the SCRAM format to generate wind roses using WRPLOT  to summarize wind speed and direction statistics.Hourly concentrations were calculated from OPC data if at least 80% of the 1‐min observations were available and valid, and 24‐hr averages were calculated if at least 80% of hourly averages were available. Analysis focused on PM33.1Most of the year, this coastal area receives moist maritime air originating over the Atlantic Ocean with little variation in daily temperatures, although cloud cover and rainfall varies. As discussed later, high pressure systems above the Sahara Desert can give rise to dusty Harmattan winds from November to February  were rare (<0.6%). The wind field rotates 75‐90o during the day: from midnight to noon, winds are westerly at lower speeds ; in the afternoon (12:00–17:00), velocity increases (5.62 m/s) and direction transitions to southerly; and in the evening (18:00–23:00), winds return from the SSW and velocity decreases (4.58 m/s). This pattern is highly consistent except from June through August when winds are southwesterly with little diurnal variation and speeds increase (5.04 m/s), due in part to upwelling and cooler water in the Gulf of Ghana that increase the sea‐land temperature differential, and the northernmost movement of the low pressure intertropical convergence zone (ITCZ), which alters the NE trade winds.Figure In Accra, the consistent wind patterns suggest that plumes from the e‐waste site will disperse N during the afternoon, NE from evening to midnight, and E from midnight to noon. Higher concentrations can occur at night with greater atmospheric stability that decreases dispersion. Later, we show that PM levels at the downwind site (NE of the e‐waste site) increase in the evening, suggesting contributions from the e‐waste site.3.22.5 collected, 61 passed QA checks and 43 had corresponding and valid OPC measurements. These 24‐hr average measurements showed reasonable agreement with the optical measurements, e.g., R2 = 0.47 , probably due to the ability to maintain flows, avoid filter overload with shorter sampling periods, and avoid periods with very high RH (only daytime sampling was used). Calculating the bias between gravimetric and optical measurements as 100% [1 – Copt/Cgrav], where Copt and Cgrav are the average of paired optical and gravimetric measurements, respectively  during a 24‐hr period when the RH averaged 92%. Otherwise, screening by humidity did not change agreement between optical and gravimetric measurements, though it is seen in some of the 1‐hr data (discussed below). Potentially a large fraction of PM at Agbogbloshie is organic, suggested by the black color on filters and impaction substrates, the poorly controlled combustion sources, and source apportionments in the literature (described later), and thus is relatively hydrophobic. Our ability to investigate RH biases was constrained since we compared 24‐h PM measurements, and RH stayed in a fairly narrow band.High RH may bias optical measurements, particularly for hygroscopic particles, which can demonstrate an exponential increase in hygroscopic growth at high RH, e.g., >85%  and 1‐hr PM10 reached 908 μg/m3 (665 ± 203 μg/m3). These statistics excluded 5 hours with 1‐min PM levels over 2000 μg/m3. Including these  data would have increased 1‐hr PM2.5 levels to 865 μg/m3 (average: 545 ± 223 μg/m3), and 1‐hr PM10 levels to 2,138 μg/m3 (average: 1109 ± 569 μg/m3). During this period, QA checks were not met for the corresponding filter‐based measurement (sample U0026). At site 2 during this period, optical measurements were unavailable and the gravimetric measurement (M0043) had flow discrepancies and an overloaded filter. At site 3, levels were fairly typical, e.g., 1‐hr PM2.5 levels reached 143 μg/m3 (average: 108 ± 15 μg/m3) and PM10 reached 418 μg/m3 (average: 345 ± 34 μg/m3), and the gravimetric measurement (L0043) was excluded due to (minor) flow discrepancies; if accepted, the concentration would have been 88 μg/m3. The meteorology during this period was not unusual; winds shifted from the SW to the W and wind speeds were low (1.5–3 m/s) during the 5 hours with the highest PM levels, however, RH averaged 89% and reached 94%. Other days with very high PM levels at site 1 showed similar patterns, e.g., on Feb. 8, 2017 from 1:00 to 8:00, 1‐hr PM2.5 levels averaged 423 ± 72 μg/m3 at site 1; 74 ± 21 at site 2, and 36 ± 11 μg/m3 at site 3, and again, winds were light and easterly, and RH was high (average 89% and up to 94%). As discussed later, these high measurements did not occur during Harmattan dust events.The highest PM levels in the study occurred at the upwind site for a 12‐hour period starting 23:00 on Jan. 14, 2018 when 1‐hr  PMThe pattern of nighttime, high and isolated PM events at site 1 with slight E winds is strong evidence of impacts from nearby fires in the spoil piles: most piles were W of the site, fires were sometimes very close (<10 m) to the sampling site, and pile heights approached the sampling height. Time lapse photography at this unoccupied site could have confirmed these emissions, as shown elsewhere  for the gravimetric data and 79 ± 33 μg/m3 (N = 81) for the optical data; PM10 levels  averaged 210 ± 91 μg/m3 (N = 81). Concentrations varied widely, e.g., 1‐hr PM2.5 ranged from 7 to 523 μg/m3, and 24‐hr levels from 23–192 μg/m3 (N = 81). These levels are well over 24‐hr and annual average WHO and Ghanaian standards and guidelines. Probability plots of 1‐hr PM2.5 and PM10 concentrations suggest lognormal distributions for PM2.5 and PM10 at sites 2 and 3  and highest at site 2 (e‐waste); the PM10 plot shows that concentrations were similar at the three sites up to the ~90th percentile. While the distributional analysis uses unmatched data and sites have varying number of measurements, it shows the high concentration events that occurred at site 1 after October 2017.Statistics of the 24‐hr data are presented in Table 2.5 levels between the three sites for the period prior to October 26, 2017 when concentrations averaged 31, 88 and 57 μg/m3 at sites 1, 2 and 3, respectively  and 26 ± 21 μg/m3 at the downwind site (site 3). PM2.5–10 increments were small, 16–18 μg/m3, thus differences between sites for PM10 were mainly due to PM2.5. Increments based on medians or matched data  were similar. Following October 26, 2017, site 1 levels generally exceeded those at the other two sites, likely reflecting dredging and spoil pile burning discussed earlier. For this later period, modest or negative increments were estimated.The trend plots Figure  show strSC,X for stability class SC and downwind distance X. A height change of ΔH (m) changes the concentration by a factor of 1 ‐ exp{− (ΔH)2/2}. For example, for a distance of 100 m and stable conditions (class F), σF,100 is 2.3 and 7.5 m for rural and urban terrain, respectively; under more common neutral stability (class D), σD,100 is 4.7 and 13.8 m (determined using AERSCREEN). The terrain at Agbogbloshie is urban, thus, the latter dispersion coefficients apply. Monitoring site heights were from 2 to 6.5 m above ground level. A height change of 4 m under stable conditions yields a concentration change of 25% for a source that is 100 m distant  and 4% for a source that is 300 m distant . Under neutral stability, changes are 8 and 1%, respectively, for sources 100 and 300 m distant . Atmospheric conditions change hourly, and conditions are often unstable and well mixed during the day, which would reduce differences. Site‐specific conditions will determine impacts. At site 1 (upwind), most sources were 100–300 m distant or further, except in late 2017 when smoldering and burning windrows of dredged materials were proximate and nearly at monitoring height (6.5 m) when some of the highest concentrations were observed. At site 2 (e‐waste site), the monitor was 2 m above ground (near breathing height). This site is surrounded by e‐waste activity, e.g., dismantling occurred within 50 m and burning within 300 m; this site would (as was intended) capture high concentrations. Site 3 was ~0.5 km from (most) e‐waste activities, but near a busy road. The monitoring height of 6 m would not significantly decrease concentrations attributable to the e‐waste site, but would likely lower contributions from nearby traffic.Monitoring height can affect concentration measurements, particularly if monitoring sites are near sources and the atmospheric is stable, which tends to limit vertical mixing. Such effects may be predicted using dispersion models such as AERSCREEN . As discussed later, prevailing winds shift from the south in the afternoon to the southwest in the evening, which can bring plumes from e‐waste burning in area 1 (which often continues in the evening) and also from cooking using biomass fuels in the extensive informal settlements located south and west of site 2 , suggesting burn area 1 or other local activities. At site 3, both median and 90th percentile levels (again neglecting NE and E arms for 90th percentile PM10) were highest from the SW, also suggesting e‐waste emissions.“Pollutant roses” plotting median and 903.610 ranged from 92–544 μg/m3), Harmattan dusts were not indicated for several reasons: few hours of N to E winds occurred on these days; PM levels or increases across sites were not comparable  site, located ~1 km N of site 2, and 93 μg/m3 at Graphic Road, a roadside site ~460 m ESE of site 3. In 2017, PM10 averaged 137 μg/m3 at SIA and 237 and 188 μg/m3 at Graphic Road for 2015 and 2017, respectively. We obtained comparable levels at sites 2 and 3 , although site 3 was ~62 m upwind (SW) and sheltered from traffic on Graphic Road by a large building, while the EPA site was in the middle of this 4‐lane road  and the highest levels near busy roads, e.g., Weija Junction averaged 155 and 245–295 μg/m3 for PM2.5 and PM10, respectively.The Ghanaian EPA started monitoring in Accra around 2011 and by 2015, 24‐hr PMd Figure . Figure 2.5 and PM10 measurements collected daily while walking 8–9 km paths over 1 week periods in four neighborhoods had geometric means of 35 and 86 μg/m3 in Asylum Down and 21 and 49 μg/m3 in Jamestown, the closest neighborhoods to Agbogbloshie; levels were higher along large roads and near roadside biomass fires (trash and stoves)  measurements collected every 6th day at four roadside locations  averaged 167 (Kaneshie) to 228 (Weija) μg/m3; the Graphic Road site  averaged 182 μg/m3  and PM2.5–10 averaged 138 ± 245 μg/m3 ; during Harmattan conditions (defined by Si levels >10 μg/m3), PM2.5 increased to 97 ± 89 μg/m3  and PM2.5–10 to 389 ± 395 μg/m3 ; a principal components analysis showed few differences between Harmattan and non‐Harmattan periods, and apportioned PM2.5 to crustal (38–39%), vehicles/biomass (24–38%), and industrial (16–33%) sources, while PM2.5–10 was apportioned to crustal (41–45%), vehicles/biomass (18–20%), industrial (15–17%), sea spray/other (6–13%), and sand  sources. From February 2008 to March 2009, 24‐hr (12‐hr during the Harmattan due to filter overload) samples were collected and analyzed for mass, carbon and metals at Ashaiman, a city  located ~30 km E of Accra and ~10 km from the industrial port city of Tema; PM2.5 averaged 22 μg/m3 , and sources identified using positive matrix factorization (PMF) were diesel (18%), soil/dust (18%), gasoline (16%), fresh and aged sea salt (16 and 6%), industry (11%), biomass (9.5%), and 2‐stroke engines (5%) , respectively; averages were 89 and 138 μg/m3; the highest levels occurred during NE Harmattan winds; and sources determined using PMF were Harmattan dusts, e‐waste burning, resuspended soil and dust, industry, vehicles, sea spray, biomass burning and oil burning.Three studies used receptor modeling to apportion PM sources in Ghana. For a 1‐yr period starting February 2006, gravimetric samples were collected and analyzed for PM, metals and black carbon at Kwabenya, an outlying suburb located ~20 km NE of Accra’s center  and one site in Cotonou, Benin (300 km ENE) using weekly gravimetric sampling at traffic and urban sites reported lower PM2.5 levels, averaging 28–32 μg/m3, but higher levels, 145 μg/m3 in an Abidjan market with ~25 wood‐burning fireplaces that smoked meat and fish or roasted peanuts  was 48 μg/m3 measured in a residential area (Danosoman) by EPA in 2015; we measured comparable levels, 44 ± 23 μg/m3, at the upwind site. Of note, 24‐hr PM2.5 levels at even these sites exceeded the WHO and Ghanaian EPA guidelines . Local impacts due to e‐waste, biomass burning, industry, traffic and Harmattan dusts can be sizeable and add to these levels, further degrading air quality.The main PM sources in Accra, as elsewhere in the region, are biomass burning, traffic, industry/energy, and Saharan dust  industrial zone recycles ~2 million tons of e‐waste annually, PM2.5 measured using high‐volume sampling at two sites 400–500 m distant averaged 38–49 μg/m3 (N = 7 per site) in summer and 109–154 μg/m3 (N = 6) in winter , higher than at a reference site   , and the contribution of mechanical processes  for coarse PM emissions .Several studies have reported PM levels at e‐waste sites. In Moradabad City, India, which has extensive and illegal waste recycling activities, ambient PM2.5 levels at site 3 exceed background levels (at site 1) by 26 μg/m3  contribute to morning and late afternoon concentration peaks associated with rush‐hour, and cooking using biomass fuels increases in the evening. Other sources have intermittent or irregular emissions, e.g., open burning emissions depend on the fuel, fire stage, wind speed, accelerant, etc. Second, while highly variable, atmospheric dispersion is generally much reduced after sunset as the ground cools (reducing the boundary layer height and mixing), which can greatly elevate concentrations from nearby ground‐level sources  and the PM2.5/PM10 fraction reached ~80%, compared to 50–60% in non‐Harmattan season ; use of upwind sites to assess background, e‐waste and downwind impacts; documentation of “microinventories” around monitoring sites that might affect PM levels and interpretations; examination of regional sources and seasonal variation by obtaining sufficient observations and by examining other monitoring and satellite data; collection and analysis of local meteorological information to select monitoring sites and understand collected measurements; and analyses that applied rigorous QA checks, provided appropriate averaging, identified potential outliers, examined temporal, spatial and directional patterns, and used robust statistics.We recognize limitations of the study. Due to logistical issues, equipment failures, QA checks, and other reasons, our data record had gaps and coverage was incomplete, particularly during the Harmattan season. Upwind, on‐site and downwind levels cannot be fully captured using only three monitoring sites for such a complex environment as Agbogbloshie. The difficulties in establishing appropriate sites should not be underestimated. Continuous monitoring would enable other types of analyses. . RH measurements at each site might aid detection of RH‐induced artifacts. While perhaps a minor issue in Accra given the very consistent wind patterns, analysis during precipitation events, the Harmattan season, and other periods could be informative. Chemical analysis of the PM, and collection of additional pollutants, would aid source identification.5We characterized ambient PM levels at the Agbogbloshie e‐waste recycling site and nearby communities using gravimetric and optical instruments and several analysis techniques. Levels at the site were significantly elevated over the background level, which itself was often high, mainly due to biomass burning and vehicle emissions. Very high PM levels, which sometimes overloaded the instruments, typically occurred in the evening due to nearby waste and biomass fires. Overall, we demonstrate that low to moderate cost instrumentation, with some modifications in hardware, appropriate data cleaning, and attention to understanding local sources, meteorology and background levels, can be used to characterize the spatial and temporal variation in PM levels in complex urban and industrial areas. The dearth of air quality information in low‐ and middle‐income countries (LMICs), where pollution levels often far exceed air quality standards, can be addressed using such approaches.The authors declare no conflicts of interest relevant to this study.Supporting Information S1Click here for additional data file.


Micronutrient sensing is critical for cellular growth and differentiation. Deficiencies in essential nutrients such as iron strongly affect neuronal cell development and may lead to defects in neuronal function that cannot be remedied by subsequent iron supplementation. To understand the adaptive intracellular responses to iron deficiency in neuronal cells, we developed and utilized a Stable Isotopic Labeling of Amino acids in Cell culture (SILAC)-based quantitative phosphoproteomics workflow. Our integrated approach was designed to comprehensively elucidate the changes in phosphorylation signaling under both acute and chronic iron-deficient cell models. In addition, we analyzed the differential cellular responses between iron deficiency and hypoxia (oxygen-deprived) in neuronal cells. Our analysis identified nearly 16,000 phosphorylation sites in HT-22 cells, a hippocampal-derived neuronal cell line, more than ten percent of which showed at least  Iron deficiency (ID) is one of the most prevalent micronutrient deficiencies, affecting approximately 30% of pregnant women and pre-school age children worldwide, and it causes poor long-term neurodevelopment outcomes and increased risks of psychiatric disorders in later life ,2. Iron The ability of cells to detect nutrient deficiencies such as iron constitutes an early cellular response and plays a critical role in maintaining normal physiology and developmental activity ,14,15. SIn the present study, we applied a system-wide phosphoproteomic screening in combination with Stable Isotopic Labeling of Amino acids in Cell culture (SILAC)  to identAll experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee. Brain ID was induced in rat pups using an iron-deficient diet regimen described previously . In brie13C615N4-l-arginine and 13C615N2-l-lysine (heavy). The cells were labeled respectively in the light and heavy media for more than 6 generations before additional treatment. Cells were maintained in a humidified incubator at 3 °C and 5% CO2. For cell treatments, deferoxamine  was solubilized in DMSO. Upon treatment, the final concentration of DMSO was 0.7%. For acute iron deficiency treatment, HT22 cells were treated with 100 µM deferoxamine for 6 h. For chronic iron deficiency treatment, HT22 cells were treated with 10 µM deferoxamine for 24 h. For the hypoxia treatment, HT22 cells were incubated at 37 °C for 6 h in a hypoxia chamber . For the normoxia treatment, HT22 cells were incubated at 37 °C for 6 h in a humidified incubator .HT22 cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin . For SILAC labeling, the HT22 cells were maintained in DMEM for SILAC , which was supplemented with 10% dialyzed fetal bovine serum (FBS) (Gibco), 1% penicillin–streptomycin, 25 mg/500 mL proline, and 50 mg/500 mL l-arginine and l-lysine (light) or For phosphoproteomic analysis, SILAC-labeled HT22 cells were harvested at approximately 80% confluency by washing twice with cold phosphate-buffered saline  and subsequently adding boiling lysis buffer (6 M guanidine hydrochloride (GuHCl), 100 mM Tris, pH 8.5, protease inhibitor, phosphatase inhibitor) directly to the plate. Cells were collected by scraping the plate and immediately boiled for an additional 10 min followed by micro tip sonication. The lysate was subjected to high-speed centrifugation for 10 min.For WB analysis, HT22 cells were lysed in ice-cold RIPA buffer , 0.5% NP-40, and 0.5% sodium deoxycholate) with freshly prepared complete cocktail protease inhibitor .4P2O7, 2 mM Na3VO4, 1% Triton x-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) with complete protease inhibitor cocktails (Roche) using a Kimble pellet pestle motor . Then, lysates were sonicated using a Bioruptor Pico , centrifuged at 10,000 rpm 5 min, and stored at −80 °C until use.Flash-frozen hippocampus was lysed in 300 µl cytoskeletal protein lysis buffer , 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM NaF, 20 mM NaProtein concentration of the cell lysate was determined using Bradford assay (Thermo). Proteins were suspended in SDS loading buffer and boiled for 8 min. Then, 30 µg of proteins were separated in SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane. Blotted membrane was blocked in 5% non-fat skim milk (BD) diluted in TBST . After blocking, the membrane was incubated with primary antibody overnight and washed with TBST at least three times and then incubated with horseradish peroxidase (HRP)-linked secondary antibody  for >2 h and washed with TBST. The signal was developed with Luminata Crescendo Western HRP Substrate  and captured on X-ray film. Primary antibodies in this study include the Anti-O-Linked N-Acetylglucosamine antibody [RL2] , anti-Tubulin , and anti-HIF1a . Blot densitometry quantified with ImageJ. O-GlcNAc and HIF1a signal was normalized against tubulin.To induce cellular iron deficiency, 700,000 HT22 cells were seeded onto a 60 mm plate . At 70% confluency, cells were incubated in DMEM growth media with 10 or 100 µM iron chelator deferoxamine (DFO) for 8, 24, and 96 h. The cellular iron deficiency was assessed by the upregulation of transferrin receptor (TfR1) using Taqman gene expression assay  for Real-time PCR. In brief, following a PBS rinse, RNA was isolated from adherent cells with 0.5 mL RNA lysis buffer  and purified following manufacturer’s protocol (Invitrogen). Then, 1.0 µg of total RNA was used for cDNA synthesis . Then, cDNA was diluted 10× and quantified for TfR1 levels using a QuantStudio 3 Real-Time PCR system . Samples (n = 3/treatment) were analyzed in duplicates. TATA-box binding protein  or rps18 (Mm02601777_g1) was used as an internal control. The expression of either internal control gene was not changed by DFO treatment.w/w ratio. Proteins were reduced and alkylated with tris(2-carboxyethyl)phosphine (TCEP) (5 mM) and iodoacetamide (5 mM) followed by blocking with cysteine (5 mM). Protein sample was diluted to 1 M GuHCl using 50 mM Tris, pH 8.5. The sample was adjusted to pH 8.0 with 5 mM ammonium bicarbonate for tryptic digestion. Protein was digested with trypsin  in an enzyme/protein ratio of 1:50 (w/w) overnight at 37 °C and further digested with trypsin for 2 h at a ratio of 1:100 (w/w) at 37 °C. The sample was centrifuged at 2000 rpm for 10 min, and the resulting peptide mixture was concentrated using reversed-phase Sep-Pak C18 Cartridge . Peptides were eluted off the Sep-Pak with 1.2 mL 80% acetonitrile (ACN). The ACN was removed by vacuum centrifugation. Then, the peptides were stored at −80 °C.Protein concentration was estimated by Bradford assay . The heavy and light SILAC-labeled lysates were mixed in a 1:1 First, 4–5 mg of peptides were resuspended in 10 mM ammonia formate (pH 8.0) and fractionated using a Waters XBridge peptide BEH C18 column  on an Agilent 1100 HPLC system  operating at a flow rate of 1 mL/min with 2 buffer lines: buffer A  and buffer B . Peptides were separated by a linear gradient from 3% B to 35% B for 45 min followed by a linear increase to 95% B for 8 min and decreased to 3% B for 2 min and maintained for 5 min. Fractions were collected at 60 s intervals for a total of 4 concatenated fractions. Samples were lyophilized and desalted using Thermo Pierce peptide desalting spin columns. Ten percent of the peptide samples were saved for quantitative proteome analysis, and the remaining was used for phosphopeptide enrichment.Phosphopeptides from 1 mg of each peptide fraction were enriched using the High-Select Fe-NTA phosphopeptide enrichment kit from Thermo Fisher Scientific . Eluted peptides were dehydrated using a speed-vac and desalted using homemade C18 Stage-tips.v/v) and loaded onto a self-packed capillary HPLC column  heated at 55 °C. Peptides were separated by the Proxeon nLC system at a flow rate of 300 nL/min with a gradient consisting of 79 min of 5–22% HPLC buffer B , 11 min of 22–32% buffer B, and 10 min of 32–95% buffer B.Mass spectrometry (MS) experiments were performed on an Orbitrap Fusion mass spectrometer (Thermo Scientific) connected to an online Proxeon Easy nLC 1000 Nano-UPLC system (Thermo Scientific). Peptides were resolubilized in HPLC buffer A  of 30%, fragment ions were acquired in the linear ion trap with an isolation window of 1.6 m/z.Precursor ions were ionized using electrospray and detected by the orbitrap at a resolution of 120,000 at 200 Mass spectrometry data were analyzed with MaxQuant software (version 1.5.3.12). Peptides were identified using the integrated Andromeda search engine with default settings against the UniProt database for mouse at a 1% false discovery rate (FDR). Carbamidomethylation of cysteine residues was set as fixed, whereas the acetylation of protein N-termini, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine were specified as variable modifications. For SILAC quantification, the multiplicity was set at two with heavy labeled Arg10 and Lys8 selected.2 ratio ranges: less than −1, −1 to 0, 0 to 1, and greater than 1, respectively. We performed statistical enrichment analysis for each quantile using a hypergeometric test with the following R packages: GO.db, GOstats, and org.Mm.eg.db. We performed enrichment analysis for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Pfam domains, and Gene Ontology—biological process, molecular function, and cellular compartment. We calculated the −log10 of the p-value outcome of the enrichment and normalized them to calculate the z-score. The p-value cut off for significance is 0.05, and the z-score cut off for significance is 1.2. For each category, we used one-way hierarchical clustering  of the annotation based on the z-score in Genesis software.To carry out clustering analysis, we divided the data into four quantiles according to their normalized SILAC Heavy/Light (H/L) ratio. The quantiles were divided into four logwww.phosphosite.org) [2 transformed, and the average log2 ratio of the kinase targets was calculated. Next, we performed bootstrapping-based random sampling. For k target sites of a specific kinase quantified in this study, we randomly selected k quantifiable phosphorylation sites from our phosphorylation dataset, and the average log2 ratio of the randomly selected phosphorylation sites was calculated. Then, the random selection process was repeated 1000 times. The average log2 ratios from these random selections will form a normal distribution based on the Central Limit Theorem. Finally, from this normal distribution, we calculated the z-score for the average log2 ratio of the specific kinase target sites, and the z-score was termed as the Kinase Activity Score described below. An in-house developed Perl script was applied to perform Kinase Activity Profiling Analysis.To perform Kinase Activity Profiling Analysis, we first downloaded the kinase-substrate annotation database from PhosphoSitePlus (ite.org) . Then, wS stands for the average log2 ratio of normalized phosphorylation target sites of a specific kinase, Ave and Std stand for the average and standard deviation of the normal distribution formed by the average log2 SILAC H/L ratios of 1000 random samplings of k phosphorylation sites from the phosphorylation dataset, while k equals the number of target sites for a specific kinase quantified in the study.TfR1) as a marker indexing cellular iron deficiency [TfR1 mRNA levels increase in response DFO treatment  ,35,36, o8, Arg10) or “light”  amino acids for at least six generations. Two sets of heavy-labeled cells were treated with DFO to induce acute and chronic ID. The light-labeled control HT22 cells were incubated with the vehicle (DMSO) for 6 or 24 h, respectively. For the hypoxia treatment, heavy labeled HT22 cells were incubated at 37 °C for 6 h in a hypoxia chamber , while the light-labeled control cells were incubated at 37 °C for 6 h under the normoxia treatment . Proteins isolated from pairs of “light” and “heavy” cells were mixed equally and proteolytically digested with trypsin  annotations through one-way hierarchical clustering and to compare the differential enrichment of phosphoproteome under each treatment . To accoAcute and chronic ID as well as hypoxia induced both common and disparate effects on protein phosphorylation in neuronal cells, including known hypoxia- and ID-sensitive phosphorylation targets. Both ID and hypoxia lead to increased phosphorylation of HIF1a S652 and BCL2 interacting protein 3 (BNIP3) T86. The phosphorylation of HIF1a S652 by mitogen-activated protein kinase (MAPK) promotes HIF1a nuclear localization and transcriptional activity ,42,43. BID resulted in increased phosphorylation of proteins that regulate iron-related cellular processes and enzyme activities. Acute ID was characterized by a significant increase in cellular metal metabolism and protein metalation of the metallo-molybdopterin complex. Similarly, chronic ID increased proteins involved in the intracellular sequestering of iron ions and metal incorporation into the metallo-molybdopterin complex. Both acute and chronic ID lead to a significant increased phosphorylation of Gephyrin, which is a key enzyme in the biosynthesis of the molybdenum cofactor, at S336 and S338. Phosphorylation of both S336 and S338 occurs at known regulatory sites of Gephyrin-dependent neurotransmission ,46.Both ID and hypoxia induced significant changes in the phosphorylation of proteins in the metabolic pathways. Hypoxia increased pyruvate dehydrogenase E1 alpha 1 (PDHA1) phosphorylation at T231 (ratio 9.8), while chronic ID decreased PDHA1 phosphorylation at S300 (ratio 0.5). Both T231 and S300 of PDHA1 are regulatory sites of dehydrogenase activity and mitochondria respiration ,48. In aThe phosphorylation of proteins mediating cell division processes was significantly decreased under hypoxia but increased under ID. Hypoxia led to the decreased phosphorylation of proteins involved in mitotic spindle assembly and elongation, cell cycle checkpoint signaling, and regulation of mitotic recombination. These include the structural maintenance of chromosomes protein 1A (SMC1A) S358 (ratio 0.4), S360 (ratio 0.5), nucleophosmin (NPM1) S258 (ratio 0.3), and DNA topoisomerase 2-binding protein 1 (TOPB1) S863 (ratio 0.5). An exception was an increase of phosphorylated protein involved in mitotic spindle assembly checkpoint CDK5 regulatory subunit-associated protein (CDK5RAP2) S196 (ratio 1.6). In contrast, ID increased the phosphorylation of proteins involved in mitotic DNA replication checkpoint processes. SMC1A phosphorylation at S358 was increased under acute ID (ratio 9.7), and S360 phosphorylation increased under chronic ID (ratio 8.2). TOPB1 phosphorylation at S863 was also increased under acute and chronic ID .DNA repair is a critical cellular pathway maintaining genome integrity. Hypoxia significantly decreased the phosphorylation of proteins in both homologous recombination and non-homologous end-joining pathways, including DNA repair protein RAD50 (RAD50) S236 (ratio 0.4), Replication protein A1 (RPA1) S186 (ratio 0.2) and T189 (ratio 0.2), and exonucleases and endonucleases in DNA repair pathways. In contrast, the phosphorylation of proteins in multiple DNA repair pathways was significantly increased under chronic ID, including mismatch repair, nucleotide excision repair, and homologous recombination. RAD50 phosphorylation at S237 was increased under acute ID (ratio 25.0) and increased at T690 under chronic ID (ratio 6.7). Chronic ID also increased RPA1 S186 phosphorylation (ratio 4.4).The inflammatory response is intimately linked to iron and oxygen homeostasis and physiology ,50. AcutID led to an increased phosphorylation of epigenetic modifiers. Chronic ID increased the phosphorylation of histone H3 lysine demethylase 5C (KDM5C) S301 (ratio 1.7), S317 (ratio 2.2) and 4B (KdDM4B) S972 (ratio 2.1) and decreased the phosphorylation of histone lysine N-methyltransferase ASH1L T726 (ratio 0.5) and SETD2 S890 (ratio 0.5). While they altered histone-modifying proteins to a lesser extent, acute ID and hypoxia uniquely led to significant phosphorylation changes of proteins involved in the regulation of DNA methylation and methyl-CpG binding. Acute ID increased the phosphorylation of cytosine-specific methyltransferase (DNMT1) S140 (ratio 2.9), which is a known alpha serine/threonine-protein kinase (AKT) substrate and a negative regulatory site , but it k number of phosphorylation sites  and calculating the average SILAC ratio (log2 transformed). We included only phosphorylation sites with SILAC ratios that could be normalized by corresponding protein ratios. The sampling process was repeated 1000 times, and the average ratios of each random sampling formed a normal distribution based on the Central Limit Theorem. Then, we can calculate a z-score for the specific kinase based on the average SILAC ratios (log2 transformed) of experimentally measured kinase target sites and the normal distribution of averaged SILAC ratios from random sampling. We considered the kinase-specific z-score as the Kinase Activity Score. Positive scores indicated an increase of the phosphorylation level of the identified target sites of the kinase upon the treatment compared to the overall phosphorylation proteome changes under the same treatment, while negative kinase activity scores indicated otherwise. In addition, score absolute values also correlate with the confidence of the measurement, as kinases with more quantified target sites may have a more confident measurement of activities and therefore higher absolute score values. The clustering of the Kinase Activity Score of each kinase allowed us to compare the kinase activation profiles under different treatments. Our data revealed distinct kinase activity profiles under acute and chronic ID as well as hypoxia  with an online portal was limited to the analysis of human kinases and proteomes. To perform kinase activity analysis of our dataset, we developed and utilized a sampling-based statistical approach, termed Kinase Activity Profiling Analysis (KAPA), to evaluate the site-specific changes in the abundance of kinase targets that can be applied to any species with available kinase-substrate databases. For any given kinase, the method applies bootstrap strategy by randomly selecting  hypoxia A.Chronic ID shared a more similar kinase activation profile with acute ID but still has unique features. Similar to the acute treatment, chronic ID led to the activation of AKT, c-Jun N-terminal protein kinase (JNK1), and AMPK pathways. In addition, chronic ID activated the Protein Kinase A (PKA) pathway while downregulating the Mitogen- and Stress-activated protein kinase 1 MSK1 and Ribosomal S6 kinase (RSK) signaling pathways, including RSK2 and p90RSK. Both MSK1 and RSKs play important roles in the MAPK pathway. In contrast, acute ID activated MSK1 and RSK2 pathways. Acute ID specifically activated specific isotypes in the Protein Kinase C pathways including PKCD, PKCB, and PKCH and downregulated cell-cycle-related CDK1 as well as PKN1, which is a negative regulator of AKT kinase. In contrast to ID, hypoxia activated PKN1 kinase, while it significantly suppressed the JNK1 pathway in neuronal cells. Both hypoxia and chronic ID showed a suppression of mTOR and Erk1 pathways B but actThe predicted increase of AMPK activity led us to evaluate its effect on downstream AMPK targets. AMPK is known to phosphorylate and thereby inhibit glutamine-fructose-6-phosphate transaminase 1 (GFPT1) enzymatic activity ,55,56. WEarly-life ID results in long-lasting abnormal neurocognitive outcomes in humans . Using aHere, we present the first systematic and quantitative approach to elucidate the changes in phosphorylation dynamics following hypoxia as well as acute and chronic ID in a neuronal cell line. We assessed over 15,000 phosphorylation sites in HT22 cells. Our SILAC-based quantification of biological replicates showed excellent reproducibility and demonstrated high confidence in the reliability of the quantitative analysis, providing a rich resource for the community to study iron and oxygen-dependent phosphorylation signaling in neuronal cells. Importantly, our study revealed rapid and specific changes in global phosphorylation signaling under each condition.The observed increase in the phosphorylation of proteins involved in metal ion homeostasis is consistent with a previous study showing that DFO treatment decreased the response of genes responsible for dendritic and synaptic development . GephyriGiven that iron and oxygen are key substrates for multiple proteins in cellular metabolic pathways , hypoxiaWhile oxygen and iron regulation share similar metabolic and metal ion homeostasis regulatory control, numerous pathways diverge between hypoxia and ID. In contrast to observed downregulation under hypoxia, ID led to an increased phosphorylation of proteins involved in cell cycle regulation, inflammation, and DNA damage and repair. Hypoxia decreased the phosphorylation of proteins mediating the DNA damage response (DDR) pathways, including both homologous recombinant (HR) and non-homologous end-joining (NHEJ). In contrast, chronic ID increased the phosphorylation of proteins in DDR pathways including HR, nucleotide excision repair (NER) and mismatch repair (MMR), and p53 signaling.Based on the phosphorylation dynamics under these treatments and existing knowledge on the kinase–substrate relationship, we developed an algorithm (KAPA) to predict the profiles of kinase activity and changes in response to each treatment. The algorithm enables species-independent evaluation of the site-specific phosphorylation dynamics from known kinase substrates. In agreement with our phosphorylation dynamics analysis, KAPA showed distinct changes in kinase activity under each treatment. Acute and chronic ID exhibited more similar profiles compared to the hypoxia treatment and therefore were clustered together. Interestingly, ID led to the activation of AKT, JNK1, and AMPK pathways that are involved in the regulation of energy homeostasis in neuronal cells. These changes further corroborate previous finds of altered mTOR signaling in a neuronal-specific ID mouse model . On the ERK1 kinase activity was differentially altered by hypoxia and ID in HT22 neuronal cells A,B. Of nO-GlcNAc modification of proteins is an important signaling mechanism that closely links to glucose metabolism in neuronal cells. The dynamics of this post-translational modification is regulated by a single pair of enzymes O-GlcNAc transferase and O-GlcNAcase . O-GlcNA


Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3β4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and β4 subunits on lupeol binding with nAChRs. Double mutants of α3β4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3β4 nAChRs. A characteristic of α3β4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3β4 nAChRs. The interaction between lupeol and α3β4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3β4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3β4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3β4 nicotine acetylcholine receptor using the oocytes of  Cardiovascular disease (CVD) is a major global cause of mortality and of global interest due to the increasing trend of cardiovascular risk factors. Cardiovascular risk factors include hypertension, dyslipidemia, being overweight, obesity, and diabetes. Among them, hypertension is the strongest or one of the strongest risk factors for almost all different types of CVD ,2,3. Hyp+ and Ca2+ to flow into the intracellular region when the channel is opened n/([k]n + [x]n), where Vmax is maximal currents, x is the concentration of acetylcholine or lupeol, k is the concentration of lupeol that corresponded to half the maximal current, and n is the Hill coefficient. The data difference between control and cotreatment of lupeol was statistically analyzed using paired and unpaired Student’s t-tests. T-test values of p < 0.05 were considered to be statistically significant.All experiment data were expressed as means ± standard error means (SEMs). Concentration–response currents for acetylcholine and lupeol were plotted by Sigma plot and fitted to the Hill equation using Origin software 8.0 . The equation formula was y = VACh compared with the treatment of ACh (100 μM) alone, like official antagonist MEC (n = 4–5 from three different frogs). The inhibition percentage of IACh was 97.9% ± 2.0% and 64.5% ± 1.8% for MEC and lupeol, respectively  alone showed no change in current, but cotreatment of lupeol (30 μM) with acetylcholine (ACh) of 100 μM significantly inhibited Iectively B. The inversible C. There max * [lupeol]n/([k]n + [lupeol]n), where Vmax is the maximal current, k is the concentration of lupeol that corresponds to half the maximal current, and [lupeol] is the concentration of lupeol. Half-inhibitory concentration IC50 for lupeol was 15.4 μM (n = 10–13 from five different frogs). The Hill coefficient for lupeol was 1.17 ± 0.24. These data showed that lupeol exhibited inhibitory effects on nAChRs in a concentration-dependent manner.The concentration–response profiles of different concentrations of lupeol with acetylcholine revealed that cotreatments inhibited the expression of α3β4 nAChRs in a concentration-dependent manner A,B. The n = 7–9 from four different frogs  of α3β4 nicotinic acetylcholine receptors, we analyzed IACh by treatments with 30 μM lupeol with different concentrations of ACh. The curves produced by the cotreatment of appropriate concentrations were similar in shape to the control, and the only difference was in the degree of inhibited current . Howev. Dascal .The α3 and β4 subunits of nAChR were mutated to investigate the relationship between receptor binding sites and lupeol. We employed covalent docking modeling to compare the wild-type and mutants of α3β4 nAChRs. Through best-fit analysis, it was possible to check the bonding position with a high probability in three dimensions . In Figu50), Hill coefficient (nH), and Imax values are presented in To find out whether inhibition applies in vitro rather than in silico, a mutant gene was obtained by point mutation, and a two-electrode voltage clamp (TEVC) was used to compare the inhibitory effect of wild types and mutants in acetylcholine and lupeol. All mutant genotypes confirmed binding with lupeol using TEVC. Among the mutants, double-mutant α3β4 (I37A/N132A) showed a reduced inhibitory effect (48%) compared to one-subunit mutants α3 (I37A) + Wild β4 (66%) and Wild α3 + β4 (N132A) (65%). Additional half-inhibitory concentration  cotreatment of acetylcholine and lupeol inhibited IAch-expressing α3β4 nAChRs in a reversible and concentration-dependent manner; and (3) inhibition by lupeol occurred in a voltage-independent and noncompetitive manner.In this study, we confirmed that: (1) cotreatment with lupeol and acetylcholine inhibited IThe mechanism of interaction of lupeol with the α3β4 nicotinic acetylcholine receptor has not been determined. There are several possibilities for the interaction of lupeol with α3β4 nAChRs. One possibility is that it may act as an open channel blocker of α3β4 nAChRs. As shown in ACh by interfering with the binding of acetylcholine to the binding site of α3β4 nAChRs. However, in our experiment data, we did not show any curve movement even though we had treated ACh with various concentrations of lupeol. Accordingly, lupeol may not act as a competitive inhibitor of α3β4 nAChRs.Another possibility is that lupeol inhibits IIn previous studies, nicotine ligands were commonly known to bind to alpha and beta subunits, and form ligand-binding sites ,21. We iα3β4 and α3β2 nAChRs are mainly expressed in the autonomic nervous system  accordinACh in receptor-expressing oocytes, thereby demonstrating that it functions in a concentration–response, noncompetitive, and voltage-independent manner. The inhibitory effect of lupeol can be utilized as a novel therapeutic approach for the antihypertensive targeting of α3β4 nAChRs. Furthermore, residues at specific positions in the α3β4 (I37A/N132A) subunits play a crucial role in the binding of lupeol to the receptor. It is hypothesized that the present understanding of the interaction between lupeol and α3β4 nAChRs should be helpful in pharmacological CVD studies.In summary, lupeol inhibited I


Computational docking simulation indicated that Van der Waals interactions between zerumbone and both the cholinesterases were the main forces responsible for its inhibitory effects. Furthermore, zerumbone showed the best physicochemical properties for both bioavailability and blood–brain barrier (BBB) permeability. Together, in the present study, zerumbone was clearly identified as a unique dual AChE and BChE inhibitor with high permeability across the BBB, suggesting a strong potential for its physiological benefits and/or pharmacological efficacy in the prevention of AD.Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) mediate the degradation of acetylcholine (ACh), a primary neurotransmitter in the brain. Cholinergic deficiency occurs during the progression of Alzheimer’s disease (AD), resulting in widespread cognitive dysfunction and decline. We evaluated the potential effect of a natural cholinesterase inhibitor, zerumbone, using in vitro target enzyme assays, as well as in silico docking and ADMET  simulation. Zerumbone showed a predominant cholinesterase inhibitory property with IC Alzheimer’s disease (AD) is a fatal neurodegenerative disease characterized by an irreversible degeneration of neurons, leading to the impairment of cognitive functions. Major pathological features of AD include the extracellular accumulation of amyloid-β peptide (Aβ) in senile plaques and the intracellular deposition of hyperphosphorylated tau protein in neurofibrillary tangles. Specifically, amyloid precursor protein (APP) proteolysis into Aβ by β- and γ-secretases, forms the initiating pathological event in AD . Acetylcholine (ACh), the most abundant primary neurotransmitter in the brain, is responsible for cholinergic transmission. Cholinesterase enzymes, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), catalyze the hydrolysis of Ach . In the The blood–brain barrier (BBB) modulates CNS homeostasis by severely controlling the transport of compounds to the brain . LimitedZingiber zerumbet (L) Smith. The compound has been found to exert a broad range of potential biological effects, such as hepatoprotective, antioxidant, immunoregulatory, anti-inflammatory, and anticancer activities  × 100, where C is the absorbance of control after 60 min of incubation, C0 is the absorbance of control at time 0, S was the absorbance of samples after 60 min of incubation, and S0 was the absorbance of the samples at time 0. To evaluate BACE1 inhibitory activity, a BACE1 FRET assay kit with a recombinant baculovirus-expressed BACE1 and a Rh-EVNLDAEFK-Quencher substrate was used . BrieflyTo determine enzyme selectivity, trypsin, chymotrypsin, and elastase were analyzed using N-benzoyl-L-Tyr-pNA, N-benzoyl-L-Arg-pNA, and N-succinyl-Ala-Ala-Ala-pNA as substrates, respectively. The reaction mixture was incubated for 20 min and the absorbance was measured at 410 nm .The cholinesterases inhibition kinetics of zerumbone were analyzed using Dixon and Lineweaver-Burk plots. The dissociation constant (Ki) was determined by interpretation of the Dixon plot, and Vmax and Km were calculated by Lineweaver–Burk plots, using the initial velocities obtained over a substrate concentration range . These khttp://biosig.unimelb.edu.au/pkcsm/). pkCSM is a novel method for predicting and optimizing small molecule ADMET properties which rely on distance-based graph signatures. The pkCSM tool adopted the concept of cutoff scanning to indicate 30 predictors, which were classified into five main categories: absorption (7 parameters), distribution (4 parameters), metabolism (7 parameters), excretion (2 parameters), and toxicity (10 parameters) [The pharmacokinetic properties including absorption, distribution, metabolism, excretion, and toxicity (ADMET) of zerumbone were calculated by pkCSM (ameters) .The Autodock Vina program (version 1.1.2) was used to predict the bound conformation and the lowest binding energy of the target enzyme–ligand complex. Pck software was conducted to search the binding pocket residues of enzymes. The chemical structure of zerumbone was drawn, displayed, and characterized using Marvin Sketch . The grid box of dimensions was set to x = 30 Å, y = 30 Å, and z = 30 Å. The Protein Data Bank (PDB) ID of 3D structures of human AChE and BChE were 4PQE and 1P0I, respectively. The crystal structure of zerumbone was gained from PubChem (CID 5470187) .All data were represented as the mean ± standard deviation (SD) in three independent experiments, each performed in triplicates. Statistical analyses were determined using Duncan’s multiple range tests using the Statistical Analysis System (SAS), version 9.3 .Z. zerumbet in the range of 35.5% to 84.8%. The chemical structure of zerumbone is represented in 50 values  (50 14.47 ± 0.33 µM). In considering the wide IC50 ranges of published natural AChE inhibitors as positive controls, including flavonoids, coumarins, alkaloids, terpenoids, and polyphenols (from 0.28 µM to >200 µM); zerumbone was identified as a potent AChE inhibitor. The unique chemical structure comprising of an α, β-unsaturated carbonyl group at the C-8 position may account for the high inhibitory potency of zerumbone against cholinesterases. α-Humulene, a structural analog lacking only the concerned carbonyl group in zerumbone, was virtually inactive in oxidative stress related inflammation and cancer cell proliferation, suggesting that the α, β-unsaturated carbonyl group in zerumbone might have a crucial role in interacting with the targets [Zerumbone is a sesquiterpene compound found in the essential oils of ctively) . The com targets .To assess the enzyme specificity of zerumbone, its inhibitory activities on trypsin, chymotrypsin, elastase, and β-secretase 1 (BACE1) were evaluated. Zerumbone did not exhibit any inhibitory activity on the above-mentioned enzymes, suggesting that the compound is selective and specific against AChE and BChE .As shown in Computational docking analysis of zerumbone with the target enzymes predicted the conformation that best fits the binding sites and estimated the stability of the ligand−enzyme complexes. To better understand the biological activity of zerumbone against AChE and BChE, a docking simulation was conducted. The lowest binding energies of zerumbone were −8.0 kcal/mol with AChE and −7.6 kcal/mol with BChE, suggesting a high affinity for both enzymes . In the AChE contains two substrate binding sites, namely, the catalytically active site (CAS) that is responsible for neurotransmitter degradation, and the peripheral anionic site (PAS) that is responsible for the non-cholinergic functions of AChE. The PAS binding site stimulates the Aβ aggregation and senile plaques formation, and thus increases the toxicity of Aβ. The present molecular docking results demonstrated that zerumbone occupies the PAS region of AChE, including TYR72, ASP74, TYR124, TRP286 and TYR341, which is consistent with the in vitro experimental results, indicating zerumbone to be a non-competitive inhibitor. Moreover, the zerumbone−BChE complex was found to be stabilized by the formation of Van der Waals interactions with ASP70, TRP82, PHE329, TYR332, TRP430, and HIS438. The important active site residues of BChE were divided into three groups, including catalytic triads , acyl binding pockets , and a choline binding pocket (TRP82). Interestingly, zerumbone bound closely with one of the catalytic triads (HIS438) and the choline binding pocket (TRP82) of BChE, implying that the mode of action is competitive.−6 cm/s and human intestinal absorption >30%). The skin permeability value (−2.06 log Kp) of zerumbone, which defines the rate of a chemical passage through the stratum corneum, was comparable with that of the standard value (−2.5 log Kp), showing its potential as a lead structure and justifying its drug-likeliness behavior [The designing of novel anti-AD candidates requires substantial attention to their pharmacokinetic properties. ADMET parameters were evaluated to assess the bioavailability of zerumbone. As shown in behavior .In distribution properties, volume of distribution (VDss) was considered. VDss represents the total volume required by the drug concentration to be uniformly distributed to give the same concentration at the target site as in the plasma . Higher The BBB and CNS permeability values were also calculated for zerumbone and compared with the respective standard values. It has been reported that candidates with a >0.3 log BB value have the potential to pass the BBB, while those with a less than −1 value are poorly distributed to the brain. In addition, candidates that have > −2 log PS value are considered to have penetrated the CNS, while those with < −3 value have difficulty in penetrating the CNS. Fortunately, zerumbone was observed to be blood−brain positive (0.52 log BB) and CNS permeable (−2.65 log PS). 50) of zerumbone did not fall within the range of toxicity. The potential relevance of zerumbone was observed in several previous in vivo studies which demonstrate the administration of the compound did not produce significant acute toxicity [The non-mutagenic and non-toxic behavior of zerumbone was confirmed from the predicted Ames toxicity. Zerumbone did not act as a substrate or inhibitor for the hepatic cytochrome P450 isoenzymes. Further, no hepatotoxicity characterized by disrupted normal liver functions was observed. The predicted median lethal concentration  attenuated cognitive impairments and memory deficits related to scopolamine, an anticholinergic agent, in Sprague–Dawley rats . AnotherThe multifactorial etiology of AD indicates that a multi-targeted approach might be more advantageous than a single-targeted approach. The current study is the first investigation to demonstrate the potential anti-AD efficacy of zerumbone as a strong dual inhibitor of both AChE and BChE with different modes of action. Moreover, zerumbone exhibited optimal pharmacokinetic profiles as well as relatively low toxicity in silico, suggesting that the compound could be considered as a lead compound for the prevention of AD.AChE and BChE form one of the therapeutic targets for the management of cognitive dysfunctions in AD. Due to the unique dual enzyme inhibition conferred by zerumbone, the compound has distinctive advantages over the previously reported single enzyme-targeted inhibitors. Furthermore, our positive results of ADMET prediction for zerumbone support its potential implication as a preventative agent in AD.


Calanus finmarchicus during the summer solstice period in the Arctic, with the lowest diel oscillation and the highest altitude of the sun's position. Here we reveal that in these extreme photic conditions, a widely rhythmic daily transcriptome exists, showing that very weak solar cues are sufficient to entrain organisms. Furthermore, at extremely high latitudes and under sea-ice, gene oscillations become re-organized to include <24h rhythms. Environmental synchronization may therefore be modulated to include non-photic signals . The ability of zooplankton to be synchronized by extremely weak diel and potentially tidal cycles, may confer an adaptive temporal reorganization of biological processes at high latitudes.Solar light/dark cycles and seasonal photoperiods underpin daily and annual rhythms of life on Earth. Yet, the Arctic is characterized by several months of permanent illumination (“midnight sun”). To determine the persistence of 24h rhythms during the midnight sun, we investigated transcriptomic dynamics in the copepod   •C. finmarchicus during the midnight sun periodTranscriptomic rhythms persist in •Daily transcripts dominate in ice-free environments and at lower latitude•Ultradian transcripts are revealed under sea-ice and at higher latitude•Reorganization of the transcriptome might be an adaptation to under-ice tidal cues Microbiology; Systems Biology; Transcriptomics Zeitgeber (time-giver) although other clocks are known to synchronize to other monotonous cycles . Endogenous clocks are of adaptive advantage because they enable organisms to anticipate and prepare for predictable environmental changes by temporally organizing short- and long-term biological processes  sea-ice-free and a northern (82.5° N) sea-ice-covered station in the Barents and Arctic Seas respectively.In order to determine the persistence of daily rhythms during the midnight sun, we investigated South and North respectively) on the 30° E longitude, within nine days of the summer solstice  was analyzed on the transcriptomic level for each time point and station using RNA sequencing as described in We sampled copepods at 74.5° N  and 82.5° N   oscillated in Antarctic krill Euphausia superba during an Antarctic summer day  and 12 634  rhythmically expressed genes at ectively . Represemmer day . In the mmer day , we showtebrates  or terretebrates  in tempeNorth, where they were 2.1 times less numerous than at South  is greater at South than at North  were specific to each station , with 12 101 transcripts being exclusively rhythmic at South and 6 690 exclusively rhythmic at North (South) and ice-covered (North) ecosystems, leading to differential physiological requirements. For example, in ice-covered areas where phytoplankton in the water column is scarce, the sea-ice algae community is a critical food source for copepods  an increase of ultradian oscillations across all biological processes examined, except “DNA repair”, occurred at North, explained by both station-specific and common rhythmic transcripts switching from daily at South to ultradian at North and; (3) the time of peaks of expression (phases) according to daily or tidal cycles is specific to each station.Gene ontology (GO) analysis revealed that rhythmic transcripts at both latitudes were particularly common to metabolic and cellular process, signaling, response to stimuli, localization or biological regulation A. To gainscripts  were selnscripts B to get nscripts . Examplenscripts , we noteSouth to ultradian ones at North   are identified , while the daily transcripts are in the majority in genes exclusively rhythmic at South (excl. S) for these functions . In contrast enolase transcription is ultradian at both stations (South and at high tides at North). This gene encodes for a protein involved in glycolysis and has been shown to have a peak of expression during the night in the Antarctic krill in the lab under L/D exposure (no tides), corresponding to the high level of activity and oxygen consumption in this species in the field  E. These at South D to 42%/h , salinity or temperature  gene cycles. Entrainment of the circadian (or other) clock(s) and clock-controlled genes may therefore be modulated to include other, non-photic signals  . Intereshe tides . While uperature . Thus, tion rate , as suggC. finmarchicus transcriptomes, presenting both daily and ultradian oscillations, as well as a differential daily / ultradian ratio between stations, were in accordance with the bimodal oscillations of circadian clock transcripts  controls different layers of regulation to provide robust timing cues at the cellular and tissue level. Here we identified temporal patterns in periodic gene expression by measuring mRNA accumulation. However, the temporal regulation is a dynamic process, including regulation of posttranscriptional mechanisms such as translational efficiency or protein accumulation . Thus, flaura.payton@uni-oldenburg.de).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Laura Payton (This study did not generate new unique reagents.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA628886) and in the figshare collection 5127704 (https://doi.org/10.6084/m9.figshare.c.5127704). All the scripts supporting the current study are available from the corresponding author on request.The transcriptomic data sets generated during this study are available in the NCBI Bioproject PRJNA628886 (All methods can be found in the accompanying


In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants.  Gammacoronavirus in the order of Nidovirales. IBV is endemic in most countries around the world and causes huge economical losses in the poultry industry. Many different IBV strains circulate worldwide, which were recently classified into 32 phylogenetic lineages  [Infectious bronchitis virus (IBV) is an enveloped, positive-strand RNA virus, belonging to the genus The virion of IBV is composed of four structural proteins, including the nucleocapsid (N) protein, encapsidating the genomic RNA, and the envelope proteins membrane (M), envelope (E) and spike (S). The S protein of IBV is a homo-trimer, which is translated as a single polypeptide and subsequently cleaved by host proteases to generate two subunits, S1 and S2. These subunits remain non-covalently bound in the virion, where S2, the stalk of the spike, is anchored in the membrane and essential for virion–host membrane fusion. The S1 subunit forms the head of the spike protein, which is essential for receptor binding. IBV strains use  sialic acids  for viraSpikes of coronaviruses are heavily decorated with glycans on asparagine (N) residues , and conserved across different genotypes . GlycosyS1 of vaccine strain H52 was replaced by that of IBV-M41 (r-M41-S1) or by individual N-glycosylation mutants thereof (r-M41-N-#-A). While nine-out-of-ten recombinant viruses could be rescued, showing similar replication kinetics in chicken embryo kidney (CEK) cells compared to r-M41-S1, the receptor specificity of five mutants was changed, based on glycan ELISA and haemagglutination assays using chicken erythrocytes. This reveals that N-glycosylation of S1 in the virion has implications on receptor binding and specificity.To assess the role of N-glycosylation of the spike of pathogenic IBV-M41, we set out to rescue recombinant viruses using our previously published targeted recombination system , where tM41-S1 gene, RNA was isolated from a virus stock of IBV-M41  using the QIAamp viral RNA Mini Kit . Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit  with random hexamers according to the manufacturer’s protocol. PCR was performed with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using the primers listed in S1 domain of IBV-H52 for that of M41 in the plasmid used for targeted recombination, the M41-S1 PCR product and the previously generated plasmid containing the H52-S gene [PacI and SnaBI , ligated and subsequently transformed in HB101 E. coli. Sequences were confirmed by automated nucleotide sequencing . A step-wise ligation approach was used to obtain the H52 M41-S1 donor plasmid. To introduce mutations leading to individual N-to-A substitutions in N-glycosylation sites in the receptor binding domain (RBD) sequence, site-directed mutagenesis was performed using the primers listed in S1 gene with the introduced mutations were confirmed by automated nucleotide sequencing .To obtain the fragment coding for the 2-S gene  were digM41-S1 sequence  and r-M41-N-#-A representing N-glycosylation mutants, all in the IBV-H52 genetic background . The rescued r-M41 viruses were passaged twice in 8-day-embryonated chicken eggs (ECE) until embryo death was observed (maximum of 7 days post inoculation). Allantoic fluid was harvested and the sequence of the S1 gene was assessed by automated sequencing . Virus stocks were subsequently produced by inoculation of passage 2 (P2) virus (diluted 1 : 10000) in 8-day-ECE, and allantoic fluid was harvested 24 h later to use for subsequent experiments. Viral quantity was calculated based on RT-qPCR, as described previously [Targeted recombination was performed as described before . For eaceviously . PrimersAnti-IBV immunohistochemistry on chorioallantoic membranes (CAM), collected in P0, was performed as described previously  using a 5 cells per well onto glass coverslips. CEK cells were infected in triplicates with 1.5*106 virus particles based on the calculated RT-qPCR titre. Culture media samples were collected at 8, 24, 48 and 72 h post infection (p.i.), after which RNA was isolated and RT-qPCR was performed in duplicates as previously reported [P<0.05 between r-M41-S1 and IBV-M41wt.CEK cells were isolated from 18-day-ECE . Single-reported  using a CEK cells grown on coverslips were analysed by immunofluorescent staining as described previously . Antibod6 genome equivalents) were pre-treated with 50 mU ml−1 of Arthrobacter ureafaciens neuraminidase (AUNA)  for 1 h at 37 °C, before application to the coated plates and incubated for 2 h at room temperature. After washing with PBS, primary monoclonal mouse anti IBV-S1  was applied in phosphate buffered Normal Antibody Diluent (NAD)  for 1 h at room temperature. Dako Envision HRPO labelled polymer anti-mouse  at a 1 : 1 ratio (v/v) in NAD was added to the wells, and after 30 min the wells were washed with PBS. TMB buffer  substrate was used as peroxidase substrate to visualize binding, after which the reaction was terminated using 2 N H2SO4. FLUOstar Omega (BMG Labtech) was used to measure optical densities (OD450nm) and MARS Data Analysis Software for analysis of the data.Synthetic Neu5Acα2-3Galβ1-3GlcNAc-polyacrylamide  (0.5 µg per well) was coated in a 96-well maxisorp plate  overnight at 4 ˚C. This was followed by a blocking step using 3 % (v/v) BSA  in PBS/0.1 % (v/v) Tween. Virus dilutions were made in triplicate and added to the plates, followed by incubation for 2 h at room temperature. Where indicated, virus stocks  and/or chRBC’s were pre-treated with 5 mU ml−1 of AUNA  for 1 h at 37 °C before twofold serial dilutions of the virus were made in in PBS-/-. chRBC were applied to the (pre-treated) viruses in a 1 : 1 vol ratio. Haemagglutination titres were determined after 15 min incubation at room temperature.Chicken erythrocytes (chRBC) were collected from the brachial vein and collected in 3.2 % (v/v) sodium citrate (NaC) . chRBC were washed four times (1500 r.p.m. for 10 min) in PBS without calcium chloride and magnesium chloride (PBS-/-)  and diluted in PBS-/- to a concentration of 2 % (v/v). Virus stocks  to alanine (A) substitution . To this end, in the plasmids used to generate recombinant IBV-H52, the ies ml−1 , and use2 throughout the course of infection, whereas titres of r-M41-S1 were between 1×104 and 1×105 from 8 h p.i. onwards for both clones. IBV-M41wt was included as a positive control, which showed a significant higher viral load at 48 and 72 h p.i., potentially explained by the chimeric nature of the genetic background of the recombinant viruses and IBV-M41wt. These results show that all rescued recombinant viruses were able to enter CEK cells, and replication in CEK cells appears not to be affected significantly by N-glycosylation of the RBD of the IBV spike.To compare the growth kinetics of the rescued recombinant viruses, CEK cells were inoculated with r-M41-S1 and each of the rescued N-glycosylation mutants. After 8 h, infection of CEK cells was confirmed by observing viral nucleocapsid protein expression for all viruses in immunofluorescent staining , with thTo assess whether the rescue of nine-out-of-ten recombinant viruses was based on infection through binding of Neu5Ac, we investigated whether N-glycosylation mutants in the viral context retained affinity for this receptor. r-M41-N145A, N219A, N229A and N246A, were still able to bind Neu5Ac in a concentration-dependent manner, whereas for r-M41-N33A, -N59A, -N85A, -N160A and -N194A no binding to Neu5Ac was observed . InteracTo demonstrate and confirm the N-glycosylation mutants unable to bind Neu5Ac are dependent on a different (glycan) receptor, haemagglutination assays were performed using chicken erythrocytes (chRBC). These host cells display a variety of glycans on their surface , and areTaken together, we observed for r-M41-S1, as well as for r-M41-N145A, -N219A, -N229A and -N246A binding to Neu5Ac in ELISA , indepenHere we reveal that N-glycosylation of at least five sites in the spike protein of IBV-M41 affects the binding specificity for sialylated host glycans. The ability of recombinant viruses to recognize Neu5Ac is completely in line with binding of recombinant RBD proteins in ELISA , indicatNine-out-of-ten single substitutions in N-glycosylation sites in the spike protein of IBV allowed rescue of recombinant viruses. We cannot draw any definitive conclusion for the reason why r-M41-N126A could not be grown in embryonated eggs, but some speculations can be addressed. Previously, we observed that recombinantly expressed RBD-N126A lost its binding to chicken trachea tissue; however, this cannot be the sole explanation, as the same was observed for five other mutants  , which ain silico ligand binding predictions previously point towards involvement of amino acids S87, N144 and T162 of the M41-RBD [in vivo, which remains to be revealed.Recently the cryo-EM structure of IBV-M41 was resolved, revealing the complete spike of IBV-M41 is decorated with N-glycans to (at least) 20 asparagine residues . In our  M41-RBD . These aThe infectivity and growth kinetics of the recombinant viruses were not affected by N-to-A mutations in the RBD. Whereas wild-type IBV-M41 showed significantly higher titres than r-M41-S1, possibly due to its slightly different genetic background, the mutants showed titres comparable to r-M41-S1 at all timepoints . These rMany viruses are decorated with N-glycans on the virion surface, including the flaviviruses Dengue and Zika virus, influenza A virus, Ebola virus, HIV, Nipah virus and human coronaviruses . The gly


The sequence is well-dated from 12 convergent OSL dates. Thanks to the dating of the stratigraphic units and an OSL date from the layer , the artefacts are dated to the end of Pleistocene or Early Holocene. Palaeoenvironmental data suggest that the settlement took place within a mosaic environment and more precisely at the transition between the open landscape of savanna on the glacis and the plateau, and the increasingly densely-wooded alluvial corridor. These humid areas must have been particularly attractive during the dry season by virtue of their rich resources . The Fatandi V site constitutes the first stratified site of the Pleistocene/Holocene boundary in Senegal with both precise geochronological and palaeoenvironmental data. It complements perfectly the data already obtained in Mali and in the rest of western Africa, and thus constitutes a reference point for this period. In any case, the assemblage of Fatandi V, with its bladelets and segments and in the absence of ceramics and grinding material, fits with a cultural group using exclusively geometric armatures which strongly differs from another group characterized by the production of bifacial armatures, accompanied in its initial phase by ceramics (or stoneware) and grinding material.The end of the Palaeolithic represents one of the least-known periods in the history of western Africa, both in terms of its chronology and the identification of cultural assemblages entities based on the typo-technical analyses of its industries. In this context, the site of Fatandi V offers new data to discuss the cultural pattern during the Late Stone Age in western Africa. Stratigraphic, taphonomical and sedimentological analyses show the succession of three sedimentary units. Several concentrations with rich lithic material were recognized. An  The end of the Palaeolithic represents one of the least known periods in the history of western Africa, both in terms of its chronology and the identification of cultural entities based on typo-technical analyses of its industries . This isThose rare stratified sites, both studied and dated, suggest highly contrasted regional evolutionary trajectories: the transition from nomadic Palaeolithic populations, with a foraging subsistence mode, to semi-sedentary proto-Neolithic societies practising intensive selective foraging, and then to sedentary Neolithic populations producing their own food, seems to be increasingly complex and variable depending on the region . To giveFinally, the question of a parallel evolution over the entire Holocene period of two populations that may be identified either by the use of bifacial pointed tools, or by geometric and possibly microlithic tools, is again indirectly raised ,7,8. TheAcacia seyal, Balanites aegyptiaca and Combretum sp. as the dominant species.The site is located on the left bank of the river Falémé, some 100 meters from the riverbank, between the villages of Alinguel and Goundafa (13°50’50.38”N / 12°09’55.92”W) . The contribution of cosmic radiation to the total dose rate was calculated using the equation established by Prescott and Hutton (1994) , of whicet al. (2016) ,,60,59,60et al.  for U3. The F12 sample coming from the in situ archaeological layer in the northern part of the site gives an age interval of 11,300–9,200 BCE at 68% confidence level  and places the human occupation on the Pleistocene/Holocene transition. It is also of interest to remember that the F10 sample alone, located 10 cm below the North Square concentration, gives an earlier chronological boundary at 10,800 BCE [12.8 ka] at 95% confidence level. Thus, considering F10 and F12 as framing the layer, a hypothesis to be considered with great caution is a date of the archaeological level between 10,800 and 8,300 BCE , or even between 9,900 and 9,200 BCE with a 68% confidence level . This would make it possible to attribute the occupation to the Early Holocene. However, considering the uncertainties associated with the use of a single date (without Archeophases) and a 68% confidence level, we will remain cautious in maintaining a chronological placement of the human occupation around the Pleistocene/Holocene boundary.The deposits of the three sedimentary units identified as U1, U2, and U3 would therefore be chronologically associated with the end of MIS2 and the Holocene. The application of the ArchaeoPhases package allows the estimation of intervals at 68% confidence level from 13,400 to 8,200 BCE  for U1, and from 10,900 to 7,500 BCE  for U2, which covers the The archaeological level is located above a sedimentary unit corresponding to the silty deposits attributed to the end of Pleistocene and the beginning of the Holocene  . The micThis erosion explains the establishment of a second sedimentary unit . This corresponds to silty-sandy sediments containing or covering archaeological artefacts . These sCommelina forskaolii   . Neverth11.5 ka] ,74,75. I11.5 ka] . It is iet al. 2013  and 10,900 to 7,500 BCE . The only OSL sample collected directly from the archaeological layer in the northern part of the site confirms this chronological framework and occupation at the end of Pleistocene or the very beginning of the Holocene: 11,300 to 9,200 BCE .The sample of lithic material from the very well-preserved concentration in the North square, supplemented by several artefacts from adjacent sectors, together show the excellent state of preservation of the artefacts. These artefacts were made almost exclusively out of a blueish-green grauwacke of high quality, despite the numerous fissures. The assemblage is mainly oriented towards the production of bladelets. Several methods have been identified but all of them are simple in their execution, with very short series of production. The produced blanks seem to be mainly thin, somewhat wide, rectilinear bladelets. These productions are associated with flake production, also of simple conceptual process. Several bladelet cores are occasionally recycled into flake cores, according to this principle. The tools are few in number, consisting of several roughly retouched blanks, but also of two segments (in stratigraphy) executed on a different, more chalcedoneous raw material. These segments seem to have been produced on bladelet or elongated blanks. These blanks are technically similar to those made from the bladelet productions identified in the assemblage, but their dimensions seem to be slightly larger. This observation and the difference in raw material raise the question of abandonment and export of segments made on site.chaînes opératoires. However, the blanks that could be retouched such as the segments we collected seem to be missing, and tools made of non-local raw material have been abandoned. While this appears to be evidence of knapping operations on the site of Fatandi V, it would be premature to conclude that a knapping workshop was present. Indeed, several factors complicate this interpretation. The concentrations of numerous artefacts may suggest intensive knapping activity. However, the raw material used is extremely fissured and many artefacts represent unused blocks or natural fragments and splitted debris. Moreover, since the industries were very simple and short series production, the waste thus seems overly abundant compared to the final result . It should also be noted that the stratified concentration in the North square, with its 729 artefacts longer than 2 cm, closely concentrated in an area less than 1m2, could represent a waste disposal zone rather than a true knapping area. The management of this space could therefore be essential to understanding the organization and functioning of the site. The presence of a small striated hematite pebble also raises questions: was it used for knapping purposes, or for another activity carried out at the site? The scarcity of the retouched tools raises the question of other activities that may have been performed at the site. Finally, as the raw material was not available in the immediate vicinity, the blocks of grauwacke had to be selected and transported to Fatandi from elsewhere, which would justify the site being something other than a knapping site. Only an extensive excavation coupled with a spatial analysis of the other well-preserved areas of occupation, as well as a micro-stratigraphic analysis of the knapping piles, could provide relevant information.The function of the site currently remains difficult to establish. The absence of fauna and organic remains is typical of West African contexts. Due to the sedimentary conditions, which are not favorable to the preservation of this type of remains, an important part of the information is lost. The presence of multiple lithic concentrations, composed of several hundreds or even thousands of artefacts, attests to nearly-complete Chaînes opératoires are also different with unipolar, bipolar and orthogonal series of debitage to obtain small flakes and bladelets. Abrupt retouching is made to produce numerous segments and points, never longer than 2 to 3 cm. These elements are associated with larger flakes made on siliceous rock and sandstone by hard percussion, and no ceramics or organic remains were found. Since certain technical differences are evident, in particular with regards to the type of blanks selected, as well as the types of armatures and their dimensions, we must remain prudent regarding a potential cultural link between Toumboura I and Fatandi V. But the use of segments and the absence of ceramics connect these two sites in a wide range.This site has produced first data relating to the occupation of the Falémé and, more generally for the basin of the river Senegal, during the end of Pleistocene and Early Holocene. It clearly features a technical tradition anchored in bladelet industries with geometric segments. As regards close sites for the same period, the site of Toumboura I, located about 12 km north of Fatandi V , yields th millennium BCE.The site of Ravin des Guêpiers, in the Falémé valley too, is chronologically earlier, attributed to mid-MIS 2, and technologically very different with very simple debitage methods . RecentlIn the Senegal valley, north of the country, a new site has also recently been identified, Ndiayène Pendao . Its arcnd millennium BCE [2nd mill. cal BC] at Fanfannyégèné 1 , this technical choice is initially made in the absence of ceramics or grinding material, as we also see at Iwo Eleru in Nigeria, Bingerville in the Ivory Coast, or at Damatoumou—Ounjougou in Mali [West African Microlithic Technocomplex [th millennium BCE [8th mill. cal BC], ceramics and grinding material (grindstone and hand grinders) are directly associated with a microlithic industry comprising segments [nd millennium BCE [2nd mill. cal BC] on the Senegalese coast or at Fanfannyégèné I in Mali  in Mali at Ravin de la Mouche—Ounjougou, or to the 9th millennium BCE [9th mill. cal BC] in Niger, as at the Saharan sites of Tagalagal and Temet. If the differences between the two groups are of a cultural nature and if the use of geometrics really exclude the production of bifacial armatures, these populations may evolve in alternation, as at the site of Le Promontoire—Ounjougou between the 6th and 4th millennium BCE [6th and 4th mill. cal BC] in Mali [rd millennium BCE [3rd mill. cal BC] [nd millennium BCE [2nd mill. cal BC], or north of Ounjougou, still in Mali  ,91 or at in Mali . On the  in Mali –95.The environmental data collected at Ounjougou and Fatandi could in theory suggest environmental factors as an element for distinguishing cultural practices and material industries. Nevertheless, throughout a large part of the Holocene, there is a real juxtaposition of Saharan and sub-Saharan populations with one or another of these kinds of armatures. They evolved either in alternation within the same region (as at Ounjougou), or in parallel . This allows us to consider here the hypothesis that two completely techno-culturally distinct populations coexisted at the very end of Pleistocene and during the Holocene in western Africa. This suggests eliminating the role of the environment in the mobility of these populations from the beginning of the Holocene. After the first phases, which remain to be studied, these populations were no longer dependent on a specific ecological niche. Of course, this opposition is macro-cultural and macro-geographic . This prThe Fatandi V site constitutes the first stratified site around the Pleistocene/Holocene boundary in Senegal with both precise geochronological and palaeoenvironmental data. It perfectly complements the data already obtained in Mali and in the rest of western Africa, and thus constitutes a reference point for this period at the end of Pleistocene and beginning of the Holocene. In any case, the assemblage of Fatandi V, with its bladelet debitages and segments and in the absence of ceramics and grinding material, is related to a group using exclusively geometric armatures, which strongly differs from another group characterized by the production of bifacial armatures, accompanied in its initial phase by ceramics (or stoneware) and grinding material. A cultural distinction is hypothesized. The available geographical and palaeovironmental data prevent any geographical determinism in the spatial distribution of these two groups. Moreover, the pattern of the excavated sites attributed to these groups in West Africa does not suggest any specific adaptation to environmental change. Thus, the distinction between two cultural groups could indicate a complex spatio-temporal pattern of cultural origin that remains to be studied and understood. We lack data to measure the significance of this cultural model, but this is probably a crucial question to better understand the settlement pattern of West Africa from the Late Stone Age to the Early Neolithic and the development of farming and herding practices. For the time being, the weakness of surveys and excavations focused on this type of well stratified and preserved archeological sites prevents a better understanding of this complex situation. Therefore, further fieldwork and archaeological research need to be supported to explain the transition from the Late Stone Age and foraging groups to the productive societies in West Africa.S1 Figet al. (2016) [The error is given to 1σ. The samples with an asterix are those that were processed by Lebrun . (2016)  and ment(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe equivalent doses are normalised with regards to the average of the estimations of different single-grain models. The error ranges or credibility interval are not represented here for sake of clarity. Blue circles: multi-grain CAM; green diamonds: single-grain CAM; green circles: baSAR-LogNormal_M; orange circles: baSAR-Normal; red diamonds: arithmetic mean.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(DOCX)Click here for additional data file.