Sypheotides indicus) in Gujarat, India. We analysed GPS locations from 10 lesser floricans deployed with GPS/GSM transmitters between 2020 and 2022. The average home range size (95% KDE) was 10.73 ± 10.70 km2 (mean ± SD), while the average core area (50% KDE) was 1.95 ± 1.56 km2 (mean ± SD). The monthly and daily distances covered were 286.29 ± 599.42 km and 10.11 ± 19.78 km, respectively. Our analysis indicated that suitable habitats and movement patterns were the most important factors explaining the variation in home range size. Specifically, our results suggest that lesser floricans prefer multi-use agro-grassland habitat systems with heterogeneous structures to accommodate different life history requirements. This preference may reflect the depletion and degradation of grasslands across the species’ range. Therefore, managing grassland habitats amidst croplands should be one of the key conservation strategies for the lesser florican.The home range of a species is determined by a complex interplay of extrinsic and intrinsic factors, which can have profound impacts on the species’ resource use. Understanding these dynamics is especially important for conserving critically endangered species. In this study, we used satellite telemetry to investigate the home range of the critically endangered lesser florican ( Home range size varies in mammals and birds, depending on factors such as body size and feeding strategy4. The variation in home range size is influenced by several intrinsic and extrinsic factors specific to each species or population, such as habitat type6, body weight and size4, breeding phase7, and population density9. Similarly, habitat use or selection is also expected to vary based on these factors.The home range is the area traversed by an individual in search of necessary resources for survival and reproduction10, are of particular interest in understanding their contribution to home range size, especially in birds that inhabit agro-grassland landscapes. Among the factors that determine home range size, food availability is reported to be the dominant and primary factor in birds11. For many bird species, agricultural areas provide food resources such as seeds and prey species associated with croplands, which make up a significant fraction of their diet13.Anthropogenic alteration in the landscape may significantly affect habitat selection and home range patterns in birds. Agricultural areas, which are home to over one-third of all bird speciesSypheotides indicus) in and around Blackbuck National Park (BNP) and Kutch Bustard Sanctuary (KBS), Gujarat. This species is endemic to the Indian sub-continent and is the world’s smallest bustard species. Historically, it was widely distributed, and breeding areas occurred in Nashik, Ahmednagar, and Solapur districts of Maharashtra, eastern Haryana, and the Kathiawar Peninsula, Gujarat14. However, it now breeds in isolated grassland patches in Gujarat, Rajasthan, Maharashtra, and Madhya Pradesh16. Habitat loss and seasonal variation in rainfall are the primary factors responsible for its declining population18. A recent survey18 covering most of its known breeding areas in western Madhya Pradesh, southern Rajasthan, and southern and eastern Gujarat and Maharashtra revealed an 80% population decline over the past three to four generations. Lesser florican is a local migrant in India whose movement patterns are linked to seasonal rainfall, and males are reported to show moderate fidelity to their breeding sites20.In this study, we used GPS/GSM transmitters to determine the home ranges, movement patterns, and factors affecting the home range size and habitat use in the critically endangered lesser florican  and soybean (Glycine max) are their preferred food sources23, while sorghum (Sorghum vulgare), rice (Oryza sativa), wheat (Triticum vulgare), mustard (Brassica campestris), maize (Zea mays), and cotton (Gossypium spp) are visited less frequently17.Generally, lesser florican are known to breed in grasslands with sufficient rainfall, ample ground cover, and moderately tall grasses22. As a result, these isolated patches serve as important habitats amidst croplands and may even represent the last remaining grasslands available to the species. Therefore, it is crucial to gain a better understanding of the habitat parameters within these fragmented patches that affect the species’ habitat selection.The preferred habitats of lesser florican are fragmented and patchily distributed throughout their range26. However, telemetry studies that have tagged a relatively small number of birds lack information regarding the variations in the home range of lesser floricans29. Therefore, we aimed to explain such variation in home ranges of lesser floricans with respect to their habitat and the number of days they were tracked. Our second objective was to investigate the influence of habitat variables on the occurrence of lesser floricans at a fine-spatial scale, and finally, we aimed to provide new insights into their daily movement and migration patterns. Thus, by exploring the interaction of different habitat variables on the home range and habitat use of lesser floricans, we could establish comprehensive space-use patterns to provide information for the future management and conservation of the lesser florican and its habitat.The size of a bird’s home range varies based on season, breeding status, and available habitats2 and 1.95 ± 1.56 km2, respectively  home range (95% KDE) and core area (50% KDE) of all tagged birds were estimated to be 10.73 ± 10.70 kmThe best model for predicting the occurrence of lesser florican included all habitat variables, namely open and dense scrub, human settlement, agriculture, grasslands, and night light pollution Tables , 4. AgriLesser floricans covered a monthly distance of 286.29 ± 599.42 km (mean ± SD) and a daily distance of 10.11 ± 19.79 km (mean ± SD)  travelled approximately 25 km southwards from Ratanpur village in the Vallabhipur taluka to Golrama village in the Umrala taluka of Bhavnagar district. It continued its southward journey in shorter distance segments of approximately 20 km until November 11th, 2022, using stopovers in 16 villages distributed in two districts of Gujarat and 15 villages located in three districts of Maharashtra , respectively and covered an average distance of 51.92 ± 12.02 and 116.85 ± 51.20 km per day during migration.beyt)  dispersed about 150 km towards the northern Kutch from KBS and settled ~ 5 km south to the India-Pakistan International Border on two small-sized islands , which showed that home range size is influenced by season and reproductive status26. Although our study focused only on male birds tracked for a single season, the incorporation of individual ID in the model improved the explained variance from 90 to 98%  and the African houbara (Chlamydotis undulata)36. Our findings align with a previous study27 that revealed males have smaller and overlapping home ranges during the breeding season. This behaviour is typical of species with exploded lek mating systems, as reported in the lesser florican15. Although four telemetry studies (including this one)29 have investigated home range dynamics in lesser florican using satellite telemetry, our study provides novel insights into the spatial and movement ecology of this species by tagging a larger number of birds.The lesser florican, known for its exploded lek mating system37. These patches of habitat are crucial for lesser floricans as they provide important refuge and foraging grounds36. Our study found that lesser floricans occurred in areas with low (0.40%) and high (0.80%) percentages of croplands, but the relationship was negative, with occurrences declining as the percentage of croplands increased  of night light   and Great Rann of Kutch (GRK), and highlights the importance of these areas as suitable habitats for the species. Our study also provides new insights into the daily movement patterns of lesser floricans, revealing increased daily and monthly distances travelled by some individuals in November, which may be related to migration.The daily movement and dispersal patterns of lesser floricans remain largely unknown, but initial attempts to study their ranging patterns were made by ringing a large sample of 489 males in the erstwhile Bhavnagar state of Gujarat between 1943 and 194945. Lesser floricans spend a substantial amount of time feeding, especially during the breeding season, when lean birds tend to forage throughout the day18.Despite recent studies shedding light on the migration patterns of lesser floricans, the factors that prompt migration in certain individuals remain unknown. As observed in our study, migration was characterised by shorter distance segments of 20–25 km towards south Gujarat, with both individuals crossing the Gulf of Cambay in one attempt and covering a distance of 90 and 125 km. After crossing the Gulf of Cambay, both individuals took only one day to cross Gujarat, settling for shorter durations in agricultural fields near water sources along their migration pathway. These daily movement patterns were consistent with the species’ feeding habits, which involved walking for 5 to 10 m and pausing at shorter intervals to scan the area for threats and preyIn conclusion, the study provides valuable insights into the migration and home range of the endangered lesser florican in a changing landscape. The findings highlight the importance of maintaining grassland habitats interspersed with short organic farmlands to accommodate the different life history requirements of the species. The study also emphasises the need for collaborative efforts and scientific management to mitigate threats such as powerlines and fences around the croplands, especially like fishing nets. With the continued decline of grassland habitats and the increasing pressure of anthropogenic activities, it is imperative to implement effective conservation strategies to ensure the survival of this species.One of the limitations of the scientific study was that only male birds were tagged, and further observations of the tagged birds in the field were missing, which limits the inferences that can be made about important life-history processes. Additionally, the radio transmitters were inactive during the night, and therefore, important information about dispersal patterns and migration stopovers during the night is missing. The generalisation about their movement and migration patterns could not be ascertained based on the small sample size. The monitoring period of the birds was also relatively short, and a larger sample size, longer tracking periods, and more individuals should be monitored to reduce the bias in home range estimation, as recommended by other studies.Lesser florican individuals were tagged in two distinct sites in Gujarat, India: (1) the Blackbuck National Park (BNP), located in the Bhavnagar district, and (2) the Kutch Bustard Sanctuary (KBS) and Naliya Grasslands, situated in the Abdasa taluka of the Kutch district Fig. . BNP and2  , Montagu’s harrier (Circus pygargus), Hen Harrier (Circus cyaneus), and Pallid harrier (Circus macrourus). During the monsoon season, BNP has the highest concentration of lesser florican individuals in India.BNP is situated in the semi-arid grasslands and scrubland of the Bhavnagar district in Gujarat, India, covering a total area of 34.08 km2, is situated between 23° 11′ N and 68° 44′ E in Abdasa taluka, on the western boundary of the Kutch district  bird species found in Gujarat53. Naliya grassland is also an Important Bird Area (IBA) site of Kutch, belonging to the Criteria A1 category, and supports a rich diversity of avian fauna, including the critically endangered Great Indian Bustard (Ardeotis nigriceps)49. Furthermore, it is the sole location in India that harbours three species of bustards: the Great Indian Bustard, Asian houbara (Chlamydotis macqueenii), and lesser florican49. In the past, approximately 60 displaying male lesser floricans have been observed in this area16.KBS, covering an area of 2.08 kmict Fig. . The Kut28. In the second phase (2021–2022), an additional ten male lesser floricans were tagged with satellite transmitters, resulting in a sample of 12 individual birds. The characteristic breeding habit of male performing their courtship display from a specific place in their black breeding plumage makes them conspicuous and easy to detect. While females remain in their usual camouflaged plumage and are also skulker, decreasing their probability of being detected. As trapping a skulker female of a critically endangered species on her nest may disturb her breeding success, we intentionally avoided this to adhere to conservation ethics. Additionally, due to the short breeding season, we had a limited timeframe for trapping these birds. Therefore, only males had the highest chance of being captured. Of these, six were deployed with solar-powered GPS-GSM transmitters in BNP. Out of the four birds tagged in and around KBS, three were deployed with GPS-GSM transmitters while one individual was tagged with the solar-powered PTT satellite transmitters , while the remaining eight transmitters, weighing 10 g each, from Ornitela, UAB, Vilnius, Lithuania, used the GSM network (cellular phone) to transmit data. While satellite transmitters offer the advantage of providing near-real-time acquisition of location data from tags placed almost anywhere on the globe, they come with the drawbacks of high cost and substantial battery power consumption28. One individual (LFM2) was caught in a fishnet in an agricultural field and later died due to sustained injuries  and Microsoft Excel (Version 2302 Build 16.0.16130.20298). The transmitters recorded the location of each individual bird every 30 min over a 24-h cycle.55 in R56. To define the total home range size, we used 95% kernels (95% KDE), and for delimiting the core areas or most intensively used areas, we used 50% kernels (50% KDE). We determined the maximum area used by individuals using a minimum convex polygon at 95%. The smoothing parameter for all home range estimations was set to the reference bandwidth ‘href’58. To analyse the variation in home range size, we used linear models (LM) with fixed effects. Since none of the tagged birds were female, nor did all tagged birds migrate, we analysed the variation in home range size in relation to the area of each particular habitat type within home range polygons, the number of tracking days for each bird, mean monthly and daily distance covered by each bird. We calculated monthly and daily distances covered using the Tracking Analyst tool in ArcGIS 10.8.1 . The best model was selected based on the lowest Akaike Information Criterion (AIC)60, and linear models were calculated using the “lme4” package in R61.We estimated home ranges using kernel density estimators (KDE) and minimum convex polygons (MCP) with the R package “adehabitatHR”31. Before our analysis, we prepared the data for GLM modelling in the following way. We created a circular buffer of a 200 m radius around each presence location and generated twice the number of pseudo-absence locations using ArcGIS 10.8.1. We were primarily interested in assessing the fine-scale habitat use of lesser floricans and thus chose a 200 m spatial scale corresponding to the fourth-order habitat selection as described by Johnson62. The fourth-order habitat selection corresponds to the actual sites of resource utilisation such as food, nests, and shelter within the home ranges of the species62. Thus, a 200 m radius buffer provided an appropriate spatial extent to assess the fine-scale habitat use of lesser florican. We ensured that each pseudo-absence location was at least 200 m away from each presence location to avoid pseudo-replication. We also applied a spatial filtering of 200 m to the presence location data to reduce spatial autocorrelation, which is an inherent bias in spatial data due to the non-independency of variables sampled at nearby locations. We tested the random distribution of presence locations using Global Moran’s I63 in ArcGIS 10.8.1 and retained an equal subset of pseudo-absence locations to counteract the issues caused by unbalanced prevalence . GLM provides a simple interpretation of the binary data (lesser florican presence–absence) because the coefficients represent the simple log-odds of the event occurring, which was important in our case as we were interested in determining the influence of individual predictors on the probability of lesser florican occurrence. On the other hand, multinomial models are complex in interpretation as coefficients represent the log-odds of one category relative to a reference category. Our dependent variable was lesser florican presence–absence data, while four habitat categories  and two disturbance factors (night light and human settlement) served as explanatory variables. We specifically considered these habitat variables as they are reported from other studies to be strong indicators of lesser florican presencehttps://essd.copernicus.org/). There is a piece of growing evidence that night light interferes with bird navigation65, limits dispersal66, and may change the broad-scale distribution of birds67. All analyses pertaining to home ranges and statistical tests were performed using the R statistical programming language56 (http://www.r.project.org), and spatial data and temporal data, including daily movement and annual migration patterns, were analysed using Arc GIS 10.8.1.The landscape composition or LULC map of the Saurashtra region was originally obtained from Bhaskaracharya National Institute for Space Applications and Geo-informatics (BISAG-N), Gandhinagar, Gujarat. The original LULC map consisted of 18 classes for the whole Saurashtra region. We clipped the LULC to our Area of Interest (AOI) corresponding to the home rang polygons of the lesser floricans. We reclassified the LULC classes for our AOI into four major habitat types namely open scrub, dense scrub, grasslands, and croplands. We further undertook ground surveys to correctly assign each LULC class to the reclassified habitat types. In the next step, we further reclassified each habitat category on a continuous scale using the reclassify tool in ArcGIS 10.8.1 . During the reclassification process, we assigned a value of 1 to the habitat category of interest and 0 to others to obtain a reclassified raster layer with raster values on a continuous scale. We used focal statistics and extracted values to the points within a 200-m radius of each lesser florican presence-absence point. Copernicus Night light pollution data was downloaded from  as a test subset and the remaining as a training subset. We fitted a model on a training subset and evaluated it on the test subset to validate the predictions of the training model. To construct regression models, we used the training subset and specified the following equation:60, and the final model was validated using a separate test data subset. Model accuracy was evaluated using misclassification error, model specificity, model sensitivity, and area under the curve (AUC) (see Supplementary The minimum Akaike Information Criterion (AIC) was used for model selectionAll scientific research activities involved in this study were carried out after receiving approval from the competent authority , Government of India (Letter No. F.No. 1-27/2020 WL dated 14th July 2020 and F.No. 1-27/2020 WL dated 10th September 2021). All experimental methods and works were carried out in accordance with relevant guidelines and regulations suggested by the Principal Chief Conservator of Forests (Wildlife) & Chief Wildlife Warden, Gujarat Forest Department, Government of Gujarat. Technical experts, experienced bird trappers, handlers, qualified and experienced veterinarians carried out the tagging work. Throughout the trapping and tagging process, every possible measure was implemented to mitigate disturbance and prioritise the well-being of the birds. Moreover, the study is reported in accordance with ‘Animal Research: Reporting of In Vivo Experiments’ (ARRIVE) guidelines as applicable.The photographs in the Supplementary Information (SI) were taken and are owned by the Wildlife Division in Sasan-Gir. The corresponding author has the copyrights to these photographs and they are being shared for publication in an online open-access format as well as for offline printing. Permission was obtained to publish the images under open-access license of Creative Commons BY 4.0 DEED.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.


The effects of post heat treatment atmosphere on microstructure and corrosion resistance of duplex stainless steel welded joints were investigated. Post weld heat treatment (PWHT) was carried out with and without protective atmospheres. Nitrogen and argon are used as protective gases individually. Detailed microstructure examination  demonstrates that nitrides precipitates are highly observed in the welded zones for nitrogen protected samples. An observed drop of ferrite volume fraction in post weld heat treated samples compared with welded samples without heat treatment leading to corrosion resistance enhancement of heat treated welded joints. An exception for using nitrogen as heat treatment atmosphere a decreased corrosion resistance of weldments is investigated due to nitride precipitates. An increase in the weld zone hardness for post weld heat treated samples compared with base alloy. The initial hardness of duplex stainless steel was 286 Hv while average hardness of weld zone was 340, 411, 343, and 391 Hv for as welded, PWHT using air, argon, and nitrogen atmospheres, respectively. Weld zone hardness increased to 33, 44, 20, and 37%. A significant decrease in the ultimate tensile strength and elongation after PWHT. The initial Ultimate tensile strength duplex stainless steel base material was 734.9 MPa while Ultimate tensile strength of the welded joints was 769.3, 628.4, 737.8, and 681.4 MPa for the following conditions: as welded, PWHT using air, argon, and nitrogen atmospheres, respectively. The chemical composition for duplex stainless steel (DSS) contains Cr, Mo, Ni, and N, in addition, the alloying elements distribution of duplex stainless steel (DSS) are inhomogeneous, whereas Cr and Mo are leads to an increase in the ferrite volume fraction, Ni and N increase the austenite volume fraction. The key factor to influence ferrite volume fraction and intermetallic phases precipitation (harmful phases)such as the sigma (σ) phase, chi (χ) phase, secondary austenite (γ2), nitride (CrN and Cr2N), carbides (M23C6) are annealing temperature, cooling rate, solidification after the welding process8 and heat input10.Duplex stainless steel (DSS) is the most suitable metal to use in severe environments such as deep-sea pipelines to transfer petroleum material, seawater desalination, reactors, petroleum tanker, oil refinery chemical, and petrochemical industries owing to its outstanding corrosion resistance and high strength11. DSS after welding shows three different zones in chemical composition of ferrite and austenite phase which consequently lead to different corrosion resistance. The welding influence not just be restricted to chemical composition, affect also the volume fraction of ferrite because of heat13. Whereas Nilsson14 indicated the multi pass welding allows to form excessive amount of secondary austenite Thus, it leads to low corrosion resistance in the weld zone.The welding process is a basic and indispensable process in the industry. It’s a heat treatment process that results in three zones: base material (BM), heat affected zone (HAZ), and weld zone (WZ) each17. An attempt has been made to investigate the effect of filler metal on solidification, microstructure, and mechanical properties of the dissimilar weld between super duplex stainless 2507 and high strength low alloy API X70 pipeline steel by Khan et al.15. They concluded that the 309L filler weld’s microstructure is composed of skeletal ferrites in the austenite matrix, whereas the 2594 filler weld has multiple reformed austenite embedded in the ferrite matrix. Moreover, Ramkumar et al.18 investigate the weldability, metallurgical and mechanical properties of the UNS 32750 super- duplex stainless steels joints by Gas Tungsten Arc Welding (GTAW) employing ER2553 and ERNiCrMo-4 filler metals. They recommended the using of ER 2553 for welding super-duplex stainless steel because the enhancement of mechanical properties of welded joints employing ER 2553 compared with welded joints employing ER NiCrMo-4. They attributed this comparative improvement of mechanical properties to the presence of sufficient amounts of ferrite, allotriomorphic and the austenite in the form of wedge shaped widmanstätten and as Intergranular precipitates in the weld zone employing ER2553.Furthermore, the selection of the welding electrode is of paramount importance in controlling the microstructure of the welding area and thus on the properties after weldingBased on these studies the need for controlling the weld zone microstructure is an important concern. After welding processes, the most famous and important way for microstructure improvement is the post weld heat treatment (PWHT). For proper (PWHT) the following variables should be controlled: heating temperature, holding time, cooling rate, and process atmosphere (protection gas). Post weld heat treatment (PWHT), improper annealing temperature, protective gas, and slow cooling rate promotes formation of intermetallic precipitation (harmful phases) which depends on presence of Cr, Mo, and C.Several studied were carried out concerning the PWHT of welds in general and duplex stainless steel weldments in particular.21. On the other hand, Shen19 indicated that the optimal annealing temperature for DSS without the presence of intermetallic precipitation is between 1050 and 1100 °C. Whereas Zhang8 clarified the effects of short time heat treatment after welding at  1020, 1050, 1080, 1100 and 1150 °C and confirmed the optimal annealing temperatures  are 1050 °C and 1080 °C. In addition, the highest pitting corrosion resistance was at annealing temperature 1080 °C for 3 min.Several studies indicate that the appropriate temperature for annealing is between 1000 and 1200 ℃ followed by water quenching23. While that the cooling rate during PWHT is an important concern that the slower cooling rate after solution treatment of welded joints leads to the formation of harmful phases25.During PWHT the time of annealing and cooling rate should be considered. Several studies indicate the effects of Solution Annealing Time (holding time) and confirmed that increasing the annealing time leads to a decrease in corrosion resistance4, and consequently low corrosion resistance and deteriorated mechanical properties27.Furthermore, several studies showed sigma phase enriched with Cr, Mo, and presence of intermetallic phases (harmful phases) make duplex stainless steel was prone to Embrittlement21. From this point of view, there is great importance in choosing the optimal conditions for PWHT.Therefore, the PWHT procedure must be observed carefully to avoid the formation of harmful phases. On the other hand, the proper PWHT can enhance the corrosion resistance of the duplex stainless steel weldments due to the increasing of austenite volume fractionBecause few studies were carried out on the effect of heat treatment atmosphere on the microstructure of DSS welded joints, therefore, it was excited to study in details effect of different protective atmospheres (argon and nitrogen) during PWHT on microstructure, mechanical properties, and corrosion resistance of DSS weldments. Furthermore, nitrogen low cost gas compared to argon gas.In this paper, the post weld heat treatment was performed at 1050 °C followed by water quenching to avoid formation of harmful phases  which deteriorate microstructure, mechanical properties, and corrosion resistance.In the present work, DSS plates were welded using different welding processes at the same joint with a groove angle of 60°. Figure Welding was carried out on industrial S32205 DSS hot rolled plates with 8.5 mm thickness and adopted dimensions of 100 × 100 mm (length × width). The welding process was carried out in root with shielded metal arc welding (SMAW) with Filler metal E2209-16 whereas the filling and capping were welded using gas tungsten arc welding (GTAW) with Filler metal ER2209 seen in Fig. 16.These filler metals were chosen because it has a similar chemical composition to that of the base metal (2205 DSS) seen in Table Also, shielding metal arc welding (SMAW) was used to substitute the backing gas and ensure high-quality welding in the filling and capping. The process parameters for both shielding metal arc welding (SMAW) and gas tungsten arc welding (GTAW) are shown in Table After welding the cap and root were removed using a milling machine with cutting fluid, then the samples were cut using a wire cutting machine.To investigate the effect of heat treatment atmosphere after PWHT on the microstructure evolution, the pitting corrosion resistance, mechanical properties, and the secondary phase precipitation, the samples were annealed at 1050 In addition, to observe the microstructure an optical microscope (OM) was used, and the specimens were polished with diamond paste to 0.25 µm and ground from 400, 600, 800, 1000, 1200, 1500 to 2000 grit SiC abrasive paper successively. Then electrolytic etching in potassium hydroxide (KOH) solution (20 g potassium hydroxide (KOH) and 100 mL deionized water applying 7 V for 10–15 s) was used.28. Oxalic acid also was used to show the secondary austenite or intermetallic.Potassium hydroxide (20% KOH solution) for electrolytic etching was used because it has excellent ability to highly attack different phases  and highly distinguish between them by good contrast of gray, white and dark, respectively, based on optical metallography. This was used to calculate the volume fraction of austenite and ferrite by Image J software. MATLAB software was used to calculate volume fraction of ferrite, austenite and secondary phases clearlyTensile tests were carried out to observe ultimate tensile strength (UTS), proof stress (PS), elongation, and tensile coefficient.Air, HArgon, and HNitrogen) been performed was tested force of 1 kg, and the test force duration time was 15 s. Hardness values were measured in three zones  and average values were taken.The Vickers hardness test was performed in the polished samples , platinum foil used as a counter electrode (CE), and saturated calomel electrode (SCE). In addition, the specimens were polished with diamond paste to 0.25 µm and ground from 400, 600, 800, 1000, 1200, 1500 to 2000 grit SiC abrasive paper successively and an electrochemical corrosion solution was used from 3.5% NaCl.Figure EDS reveals that high amount of nitrogen content and correspondingly, shows decreased Chromium content is existed in selected analyzed EDS point (chromium nitride), see Fig. air, Hnitrogen, and Hargon, respectively. It is noticed in the as welded sample (W) exhibits different types of austenite. They are called grain boundary austenite (GBA), Widmanstattten austenite (WA), and Intergranular austenite (IGA). The GBA grows at the ferrite grain boundaries, then WA grows from GBA, also IGA nucleates at ferrite grain that contains high Ni concentration as shown in Fig. 512. Also, the secondary austenite appeared in microstructure, the clear evidence of secondary austenite is EDS results which reveal that a high nickel, calcium, aluminum, and oxygen contents. Correspondingly, shows drastically decrease Chromium and molybdenum contents in the selected analyzed EDS point as seen in Fig. 29.Nomenclatures for microstructure of weld zones of as welded is (W) and the three post-weld heat treated symbols during air, nitrogen and argon are HOn the other hand, a high percentage of ferrite 59%) was existed in as-welded condition sample as seen in Fig. 9% was ex14.Figure 14.Figure Figure argon). Microstructure consists of F (Grey), A (columnar in white), IGA (fine structure). Figure Figure Nitrogen). It consists of few amounts of F (Grey), A (coarse and columnar in white), IGA (fine structure) and nitride precipitates (black points). Figure Figure Figure Austenite (open color and more white), light dark as in left side is Ferrite.Air) of non-protected sample with PWHT process which contains F (light dark), A (white). Figure Air). It consists of Ferrite and Austenite.Figure Argon) protected with Argon gas during PWHT process which contains F (dark), A (white). Figure Argon). It consists of Ferrite (dark) and Austenite (Grey).Figure Nitrogen) protected with Nitrogen gas during PWHT process which contains F (grey), A (white). Figure Nitrogen) in details. It consists of Ferrite (dark) and austenite (Grey).Figure Air, HArgon, HNitrogen) samples by image J software. A clear reduction of austenite fraction and consequently increases of ferrite content is observed in the (W) sample compared the heat treated samples. and in order of samples from where the highest of austenite volume fraction is  respectively.Figure 30. Moreover, one of the most important factors that may control the HAZ microstructure is reheating process due to multiple passes welding techniques31.Figure 33. In addition, there are different types to describe Unmixed zone (UZ), first one is island while the second one is peninsula in addition third one is filler deficient beach33. Figure Because of the difference between filler metal and base metal in the melting point macrosegregation is formed near the fusion boundary. Macrosegregation takes different forms such as Transition zone (TZ), Unmixed zone (UZ), and Partially mixed zone (PMZ). Several studies indicate that nickel mixing during the welding process leads to the formation of a gradient microstructure near the fusion boundary (HAZ–WZ interface)Nitrogen) reveals the lowest austenite content of weld zone. Furthermore the highest austenite content of weld zone is observed in the sample without protected atmosphere heat treatment (HAir sample). Furthermore, that austenite volume fraction of Heat affected zone of (HAir and HNitrogen) samples was convergent.Figure 34. The microstructural investigations for heat treated samples indicate that using argon as controlled atmosphere results in no second phase precipitation whereas second phase precipitation is observed in microstructure of sample under nitrogen atmosphere. Also, the unprotected sample (air is the furnace atmosphere) gives intermetallic precipitates somewhat similar microstructure of protected one with nitrogen. Nitrogen gas is classified as an inert gas and probable interaction with duplex stainless steel during heat treatment is out of the question. On the other hand, Brunzel et al.35 studied the effect of nitrogen gas on iron alloys during annealing. They noticed an increasing of nitrogen content (in the form of nitrides) in steel after annealing process compared with the starting material before annealing. Also, they concluded the intensity of the effect of nitrogen gas to form nitrides depends mainly on temperature–time parameters of the process, and the composition of the atmosphere, as well as the contents of alloying elements in the steel. In the present work, nitride precipitates are detected in the ferrite regions for samples heated without controlled atmosphere and that heated using nitrogen controlled atmospheres whereas sample treated in the argon controlled atmosphere reveals no precipitation. This is agreeing with the results of Brunzel et al.35 where nitride precipitation is observed. Interaction of molecular nitrogen and the iron alloys obeys the reaction:The effect of post weld heat treatment on the properties of duplex stainless steel welded joints was studied by several researchers. In the present wok two types of controlled atmospheres are used individually that are argon and nitrogen compared with non-protected sample. Most of heat treatment processes are used argon as controlled atmosphere and the using nitrogen as controlled atmosphere for post weld heat treatment of duplex stainless steel is nearly not found in the publications. Using nitrogen (purity 95%) instead of argon as controlled atmosphere has an economic considerations because nitrogen gas is less expensive than argon gasN2 is the partial pressure of nitrogen in the atmosphere.That has an equilibrium constant:36. Moreover, the solubility of nitrogen in austenite is greater than that in ferrite and with increasing of temperature the nitrogen solubility decreases in austenite and increases in ferrite37.The amount of nitrogen dissolved in the iron depends on the temperature, nitrogen partial pressure, and the form and contents of alloying elements in the alloy in equilibrium with the gaseous atmosphereTensile testing was performed to observe ultimate tensile strength (UTS), proof stress (PS), elongation, and tensile coefficient. Figure Air, HArgon, HNitrogen) samples. Results indicate the ultimate tensile strength of base metal was 734.9 MPa while an observed increase in UTS reached 4.7% and 0.4% for as welded and HArgon samples respectively. The ultimate tensile strength of as welded and HArgon samples was 769.3 MPa and 737.8 MPa respectively Fig. Air and HNitrogen samples respectively. The ultimate tensile strength of HAir and HNitrogen samples was 628.4 MPa and 681.4 MPa seen in Fig. Figure Air, HArgon, and HNitrogen samples respectively shown in Fig. Furthermore, proof stress (PS) were 554.3, 574.3, 512, 561.7, and 512.3 MPa for base metal, as welded, HFigure Air, HArgon, and HNitrogen samples. Tensile coefficient was 104.7, 85.5, 100.4, and 92.7% for as welded, HAir, HArgon, and HNitrogen samples respectively.Figure Air and HNitrogen samples. This reduction of ductility may be attributed to the formation of nitrides in HAir and HNitrogen sample. In addition, harmful phases make duplex stainless steel was prone to Embrittlement4 consequently low corrosion resistance and deteriorated mechanical properties27. Confirms it engineering stress–strain curves seen in Fig. Air and HNitrogen samples due to formation of nitride. Correspondingly, an observed drop in HArgon sample compared to as welded sample. Bhanu32 indicated that the PWHT can negatively affect the ductility. Therefore, samples can be classified after tensile test to ductile and brittle seen in Fig. Figure Air, HArgon, and HNitrogen were 340, 411, 343, and 391 respectively see Fig. Air, HArgon, and HNitrogen) samples respectively as shown in Fig. Air, HArgon, and HNitrogen) samples respectively. Table Figure 19 and change in chemical composition at both ferrite and austenite phases  samples, polarization curves indicate exciting things. Although there is no any obvious intermetallic in HArgon sample and precipitates (Nitride) presence in weld zone of HAir sample. The highest corrosion resistance was  respectively seen in Fig. 38. As presence high content of nickel and nitrogen in austenite lead to high corrosion resistance in austenite compared ferrite seen in Table 27. So it was necessary to investigation volume fraction of ferrite and austenite. Zhang et al.28 concluded that PWHT for duplex stainless steels weldments increases the austenite volume fraction  leading to the improvement of corrosion resistance of the welded joints that is agree with the results of this article. But presence Nitrides significantly in Nitrogen sample and secondary austenite in W sample was main reason of increase corrosion rate in this samples39. As corrosion current density is the main factor to determine corrosion rate. So increase corrosion current density lead to fast corrosion rate. Table Air, HArgon, HNitrogen, W) respectively.Figure PWHT enhance the grain refining and increasing of austenite fraction in the weld zone and HAZ.Using of nitrogen as protection gas during heat treatment leads to the formation of nitride precipitates the same result is also obtained when the specimens heat treated without protected gas whereas the using of argon gas during heat treatment gives no nitride precipitation.Among with heat treated specimens using different furnace atmospheres the specimen that heated with using nitrogen during PWHT has the highest ferrite volume fraction (lowest austenite volume fraction) compared with specimens that using argon and air during heat treatment.A significant drop of UTS and ductility are observed after PWHT especially when using air and nitrogen as heat treatment atmospheres.Air and HNitrogen welded joints, and this may be due to nitride precipitates.Higher Vickers hardness values are observed for HNitrogen sample) and as welded (W sample) owing to nitride precipitation and secondary austenite respectively.Reduction of corrosion resistance is noticed for post weld heat treatment using nitrogen (HFinally, it is not recommended to perform PWHT for duplex stainless steel weldments if the mechanical properties are of great importance, while if the chemical properties and corrosion resistance are required to be improved, it is preferable to perform PWHT because it improves the corrosion resistance of duplex welds.The following are important findings from this study on the effect of heat treatment atmosphere on the microstructure of duplex stainless steel welded joints:


The pyruvate dehydrogenase complex serves as the main connection between cytosolic glycolysis and the tricarboxylic acid cycle within mitochondria. An infant with pyruvate dehydrogenase complex deficiency was treated with vitamin B1 supplementation and a ketogenic diet. These dietary modifications resolved the renal tubular reabsorption, central apnea, and transfusion-dependent anemia. A concurrent metabolome analysis demonstrated the resolution of the amino aciduria and an increased total amount of substrates in the tricarboxylic acid cycle, reflecting the improved mitochondrial energetics. Glutamate was first detected in the cerebrospinal fluid, accompanied by a clinical improvement, after the ketogenic ratio was increased to 3:1; thus, glutamate levels in cerebrospinal fluid may represent a biomarker for neuronal recovery. Metabolomic analyses of body fluids are useful for monitoring therapeutic effects in infants with inborn errors of carbohydrate metabolism. The Apgar scores were 4 and 8 at 1 min. and 5 min., respectively. The patient exhibited severe hypotonia and required mechanical ventilation for 4 days, and she remained in the neonatal intensive care unit for 33 days.The laboratory test results are summarized in the Since a subgroup of PDHAD patients have been reported to respond to vitamin B1 supplementation ,11, the The patient's psychomotor development was delayed. At 6 months of age, she fixated and followed objects and cooed. She required multiple antiepileptic medications for a symptomatic West syndrome. At 18 months of age, the patient was able to continue the ketogenic diet without adverse effects. She acquired head control and visual pursuit but was unable to sit without support.33.1PDHA1: chrX(GRCh37):g.19369472G > T, NM_001173454.1:c.479G > T, p.. Sanger sequencing confirmed the results (http://gnomad.broadinstitute.org/). The result of an in silico analysis using PolyPhen-2 was “deleterious,” and highly conserved among many non-human species [Our research protocol was approved by the ethics committee of Keio University School of Medicine, Tokyo, Japan . Written consent to performing a genetic analysis was obtained from the parents. Peripheral blood samples were obtained from the patient and her biological parents. DNA was extracted by the standard phenol extraction method. A whole genome sequencing in trio identified a de novo heterozygous missense mutation in  results . This mu species . The PRO species . Accordi species , the mut3.2To assess the impact of the ketogenic diet on amino acid and central carbon metabolic pathways, urine, CSF, and arterial whole blood samples were collected when the diagnosis was made, after the start of vitamin B1 supplementation, 7 days after starting the 2:1 ketogenic diet, and 10 days after switching to the 3:1 ketogenic diet. The serum was separated by centrifugation after the blood clotted. To avoid metabolite degradation, all samples were immediately frozen at −80 °C until analyzed. Quantitative capillary electrophoresis-electrospray ionization mass spectrometry  was used to measure the metabolites as previously described .The serial metabolomic serum, CSF, and urine analyses at the multiple time points during diagnosis and after the start of vitamin B1 supplementation and ketogenic diet confirmed the diagnosis of PDHAD. The ketogenic diet induced robust metabolic changes outside the TCA cycle but not within it A. First,N-acetyl-β-D-glucosaminidase and β-2-microglobulin, which are known biomarkers of renal tubulopathy. However, most urinary amino acid concentrations including that of leucine, which is known to have an inhibitory effect on pyruvate oxidation, remained at least two-fold above the normal values for newborn female infants [The urinary metabolome analysis showed that vitamin B1 supplementation reduced the total amount of urinary amino acids , and this trend was accelerated by the initiation of the ketogenic diet B. The im infants  before a4In this study we performed serial metabolomic analyses of body fluids in an infant with PDHAD before and after the start of vitamin B1 supplementation and a ketogenic diet. Vitamin B1 improved the patient's renal tubular reabsorption, and the ketogenic diet resolved her central apnea and anemia. The ketogenic diet increased the total amounts of substrates in the TCA cycle, and the increases were in proportion to the ketogenic ratio. These findings suggested improved energetics in the renal, nervous, and hematopoietic systems. Taken together, the results obtained in this study provide molecular and clinical evidence that the combination of vitamin B1 supplementation and a ketogenic diet improves mitochondrial energetics in multiple organ systems.The early institution of appropriate dietary and pharmacologic interventions is critical in treatable inborn errors of metabolism, and rapid and precise genetic diagnosis by whole genome sequencing is increasingly being utilized to make it possible to do so . A recenVitamin B1 supplementation appeared to restore the renal mitochondrial energetics in the present patient. Indeed, the renal proximal tubules contain an abundancy of mitochondria second only to that in the central nervous system . We specOne potential biomarker for neuronal activity that we identified in the analyses in this study was glutamate. Glutamate is a major excitatory neurotransmitter in the central nervous system and functions as a metabolic hub between carbohydrate and amino acid metabolisms . DecreasIn conclusion, vitamin B1 supplementation and a ketogenic diet improves mitochondrial energetics in multiple organ systems in PDHAD. The changes in carbohydrate and amino acids in body fluids, i.e., serum, CSF, and urine, in our patient were in proportion to the ketogenic ratio of the diet and paralleled the patient's clinical improvement. Rapid genetic diagnosis combined with therapeutic monitoring by means of metabolomic analyses is recommended in infants with inborn errors of carbohydrate metabolism, including PDHAD.10.13039/100009619Japan Agency for Medical Research and Development under Grant Number JP22gk0110038 (to TT), Japan Foundation for Pediatric Research, Grant No. 21-007 (to TT), Keio Gijuku Academic Development Funds (to TT), Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (to TT), Japan Society for the Promotion of Science under Grant-in-Aid for Challenging Exploratory Research, Grant Number JP22K19494, and Mother and Child Health Foundation (to TT). This research was also supported by World Premier Institute of BioQ2 for 10.13039/501100001697Keio University (to MS), and 10.13039/100009619Japan Agency for Medical Research and Development - Moonshot Project for Microbiome (to MS).This research was supported by Eri Ogawa drafted the first manuscript. Takako Hishiki, Noriyo Hayakawa, Hisato Suzuki, Kenjiro Kosaki, Makoto Suematsu reviewed and critically revised the manuscript. Toshiki Takenouchi conceived and organized the entire project and edited the final article.The authors have no conflicts of interest directly relevant to the content of this article.


The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers. The protein capsids of many viruses are able to self-assemble from individual capsid building blocks (CBBs) under appropriate conditions. In vivo, some virus capsids are assembled without the participation of the viral nucleic acid, which is later encapsidated in an independent reaction; other virus capsids are co-assembled with the viral nucleic acid to directly yield virions . HoThe finding of conditions for in vitro assembly of nucleic acid-free (empty) capsids from a number of different viruses has opened up the possibility of using them as nanocarriers or nanocontainers for a plethora of biomedical or nanotechnological applications. A general strategy involves the encapsidation of specific molecules or macromolecules during the in vitro assembly of either natural or engineered viral capsids . ThFor papillomavirus capsids, conditions have been found for the very efficient (~60–100%) self-assembly of structurally correct capsids in vitro ,14. HoweThe capsids of parvoviruses, including the minute virus of mice (MVM) ,26 may bThe authentic MVM capsid is made from two variants (VP2 and VP1) of the same capsid protein, which have an identical sequence and fold but differ in that VP1 has a longer N-terminal extension. VP2 alone is required for self-assembly, as the MVM capsid can be assembled from 60 identical VP2 subunits, both in transfected cells  and in tIn this study we have analyzed the effects of several different conditions on the fidelity and efficiency of self-assembly of the VP2-only MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is a remarkably faithful process, irrespective of the conditions used, and that its efficiency can be increased under certain conditions of pH, ionic strength, and/or macromolecular crowding.2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.6) at 30,000 rpm in an SW40 rotor for 5.5 h at 10 °C. Purified VLP suspensions were extensively dialyzed against PBS, concentrated by ultrafiltration using an Amicon-Ultra4 device of 100 kDa cutoff , and stored at 4 °C. VLP purity, integrity and monodispersity were analyzed by polyacrylamide gel electrophoresis and transmission electron microscopy (TEM). Because of the very large amount of VP2 and VLPs produced in insect cells, the two-step ultracentrifugation approach yielded highly pure preparations. MVM capsid protein concentration was determined by UV spectrophotometry.MVM capsids made of 60 VP2 capsid protein subunits (termed here VLPs) were expressed, assembled in insect cells, and purified as described previously ,38. In eSamples were deposited on ionized Formvar/carbon-coated copper grids , incubated for 3 min and washed 3 times for 30 s with distilled water. Negative staining was carried out using incubation for 45 s in a 2% (weight/volume) uranyl acetate (Electron Microscopy Sciences) solution. The samples were then dried and visualized in a JEM-1010 electron microscope (JEOL) at magnifications ranging from 25,000× to 120,000×. Images were taken with a TemCam-F416 camera (TVIPS). Samples from each experiment were processed strictly in parallel, to allow a direct comparison of the data obtained.Purified native VLPs obtained from cells as described above were disassembled into free capsid building blocks. Purified VLP samples at a total protein concentration of 0.5–0.6 mg/mL were incubated at 25 °C in PBS pH = 7.6 in the presence of 3.5 M guanidinium chloride (GdmHCl) with agitation (350 rpm) in a Thermomixer Compact  for a specified amount of time  until VLP disassembly was nearly complete, as determined by TEM.VLP reassembly from capsid building blocks obtained by the disassembly of purified VLPs as described above was triggered by fast dialysis of 150 µL samples for 12–25 min (unless otherwise noted) in Slide-A-Lyzer cassettes of 0.5 mL capacity and 10000 MWCO . Residual GdmHCl concentration after dialysis was estimated using a spectrophotometry-based approach . DiffereThe relative number of complete VLPs as a function of experimental conditions and time was estimated by counting the particles present in several  EM fields of a defined size (1.23 × 1.23 μm), and averaging the results. Particle counting was usually carried out using a home-made macro developed in Fiji . In ordebN, dN and rN are the average number of complete VLPs counted, respectively, before disassembly was triggered by adding GdmHCl (bN), just after the disassembly reaction was ended  and after the reassembly reaction was ended (rN). dD is the dilution factor due to the addition of a volume of concentrated GdmHCl to trigger VLP disassembly. rD is the dilution factor due to the addition of a volume of a particular solution to determine VLP reassembly under certain specified conditions.The reassembly efficiency was calculated as follows:The chemical stability of the MVM VLPs under different pH and ionic strength conditions in different buffers  was tested by the addition of a GdmHCl solution to a final concentration of 3.75 M and incubation of the sample at 25 °C. Small aliquots were taken at different times and the percent remaining intact VLPs under each condition was estimated by TEM, as described above.Models of MVM CBBs were built using the structure and symmetry matrices corresponding to the PDB entry 1Z14 . Atomic Unless indicated otherwise, in this study the reassembly efficiency of MVM VP2-only capsids (VLPs) was defined in terms of the final yield of free VLPs assembled from capsid building blocks that had been obtained by disassembly of native VLPs. This is a demanding definition: even if 100% of the possible VLPs were correctly reassembled, the reassembly efficiency thus defined would be lower if some of them dissociated or became aggregated before the product was analyzed.To determine the reassembly efficiency under each tested condition we used as a starting point a previously described experimental setup ; see MetA and aftTo start the reassembly reaction, most of the GdmHCl was rapidly removed by dialysis under the conditions chosen for VLP reassembly C. DialysThe total number of free reassembled VLPs and the reassembly efficiency were estimated first as a function of capsid protein (VP2) concentration at 25 °C in PBS pH = 7.6 . ConcentThe main reason behind this decrease in reassembly efficiency  at the higher protein concentrations was traced to the formation of VLP aggregates . These aA propensity for aggregation was also observed for monodisperse native VLPs that had been correctly assembled in cells, and that had not been disassembled and reassembled in vitro. After storage for long enough at relatively high concentrations  at either 25 °C or 4 °C in PBS pH = 7.6, intact, free VLPs formed aggregates similar to those observed after VLP reassembly in the same buffer. VLP aggregation was generally observed, irrespective of the buffer in which the particles were stored or reassembled. Those buffers included phosphate-based buffers from pH 6.0 to 8.0 containing from 0.15 to 2.0 M NaCl, and 75 mM Tris-HCl pH = 6.8, 250 mM NaCl. The stickiness of native MVM VLPs led also to the clustering of latex beads, due to adsorption of VLPs to the beads and to each other . The aboA test was then carried out to see whether the reassembly efficiency could be increased by using a macromolecular crowding agent to increase the capsid protein (VP2) chemical activity (“effective concentration”) without actually increasing the number of protein molecules in the sample volume. Ficoll-70 was chosen as the crowding agent because of its low viscosity, a size comparable to those of many proteins, and its low tendency to interact specifically with proteins and other solutes .The relative reassembly efficiency in the presence or absence of 50 g/L Ficoll-70 at 25 °C in PBS (pH = 7.6) was compared as a function of reaction time . The resNext, tests were carried out to see whether the reassembly efficiency could be increased by using temperatures lower or higher than 25 °C. After the reassembly reaction was essentially complete (the VLP yield reached a plateau) the reassembly efficiency was similar at 25 °C or 37 °C, and somewhat lower at 4 °C or 15 °C .Electrostatic screening of protein–protein interactions by increasing the ionic strength may heavily influence interactions between capsid proteins and/or VLP aggregation. Thus, the effect of ionic strength on VLP reassembly efficiency was tested by increasing the NaCl concentration in the buffer from 0.15 M up to 2 M A. The reWe then asked whether the higher reassembly efficiency found at 0.5 M NaCl correlated with some change in the values and distribution of the surface electrostatic potential on the VLP surface, including the intertrimer interfaces. Simple surface electrostatic calculations  revealedChanges in the protonation state of acidic or basic side chains on the VLP surface or at the intertrimer interfaces could also influence VP2–VP2 interactions and/or VLP aggregation. Thus, we compared the reassembly efficiency within a pH interval from 6 to 8 A. The reThe stability of many viral capsids is pH-dependent. Thus, a higher capsid stability at slightly acidic pHs could contribute to the higher reassembly efficiency determined for MVM VLP at acidic pH, compared to neutral or slightly basic pH. To test the pH dependency of VLP stability, a dissociating agent was added (3.75 M GdmHCl), and the disassembly rate of purified VLPs as a function of pH was compared in two different buffers with a different ionic strength . The VLPAs for ionic strength, we asked whether the higher reassembly efficiency and particle stability at slightly acidic pH correlated with some change in the values and distribution of the surface electrostatic potential on the VLP surface, including the intertrimer interfaces. Surface electrostatic calculations  revealedWe then asked whether the increases in reassembly efficiency at relatively high ionic strength or acidic pH relative to physiologic ionic strength or neutral or basic pH were additive. The reassembly efficiency in PBS was compared at pH = 7.6, 0.15 M NaCl, pH 7.6, 0.5 M NaCl; pH 6.6, 0.15 M NaCl; and pH 6.6, 0.5 M NaCl . As expeVariations in some conditions tested for MVM VLP assembly, including capsid protein concentration, the absence or presence of a macromolecular crowding agent, ionic strength, or pH, led to 2- to 3-fold increases in reassembly efficiency. However, the maximum absolute reassembly efficiencies thus achieved were never higher than 35–40%. From these observations alone it could be thought that, in vitro, the self-assembly reaction that builds the MVM capsid is only moderately efficient, and not fully reversible. In fact, incomplete or aberrant MVM VLPs  were onlDifferent conditions were tested to attempt the disaggregation of the VLP clusters. Sonication did not achieve any substantial effect. Addition of a chaotropic agent (GdmHCl) at moderate concentrations (1.75 M) led to some disaggregation of large clusters into smaller clusters and individual free VLPs visualized by TEM, which again confirmed that the aggregates are made of fully reassembled VLPs. However, the addition of GdmHCl not only led to the release of some VLPs from the aggregates, but also to the dissociation of some of the free VLPs in the sample. Thus, the yields of free VLPs were not significantly increased (results not shown). Monodisperse suspensions of reassembled VLPs of MVM could be achieved only by elimination of the remaining aggregates through centrifugation.Extensive inspection using EM of the products of MVM VLP reassembly in vitro during this study did not reveal significant amounts of incorrectly assembled, aberrant viral particles under any tested condition of protein concentration, macromolecular crowding, temperature, pH, or ionic strength. These results provide evidence for a remarkable fidelity of MVM assembly that is virtually unaffected by changing conditions.The assembly of MVM and other parvoviruses inside infected mammalian cells is a complex process ,50. StabRecombinant VP2-only capsids (VLPs) of MVM and other parvoviruses (canine parvovirus (CPV) and porcine parvovirus) have been assembled in insect cells ,37,58,59Recombinant VLPs of MVM, AAV2 or human parvovirus B19 have been assembled in vitro starting from VP subunits ,60,61. DIn vitro assembly of the MVM capsid along a defined pathway  did not The above observations reinforce the notion that the higher complexity of parvovirus capsid assembly in mammalian cells arose because of the need to achieve during infection a regulated, highly efficient virus morphogenetic process in a compartmentalized, intricate cellular environment. In a highly dilute solution in the test tube under adequate conditions, the isolated, stable VP trimers were able to spontaneously and efficiently recognize each other with the correct orientation. The result is the faithful and efficient self-assembly of the capsid through a defined pathway (, and thiTo sum up, this study has defined conditions for the faithful and efficient in vitro self-assembly of MVM VLPs in the absence of additional factors that may be required for in vivo capsid assembly. These results encourage the use of MVM VLPs as nanocarriers for different compounds. It must be noted that the defined assembly conditions constitute a starting point that might have to be changed somewhat, depending on the influence that the desired cargo molecules could have on the intertrimer interactions. The high thermal, chemical and mechanical stability of these virus-based VLPs and their biological features, such as their nuclear tropism, make them suitable choices for targeted drug delivery or other biomedical or nanotechnological applications.


When studying unknown human remains, the estimation of skeletal sex and ancestry is paramount to create the victim’s biological profile and attempt identification. In this paper, a multidisciplinary approach to infer the sex and biogeographical ancestry of different skeletons, using physical methods and routine forensic markers, is explored. Forensic investigators, thus, encounter two main issues: (1) the use of markers such as STRs that are not the best choice in terms of inferring biogeographical ancestry but are routine forensic markers to identify a person, and (2) the concordance of the physical and molecular results. In addition, a comparison of physical/molecular and then antemortem data  was evaluated. Antemortem data was particularly beneficial to evaluate the accuracy rates of the biological profiles produced by anthropologists and classification rates obtained by molecular experts using autosomal genetic profiles and multivariate statistical approaches. Our results highlight that physical and molecular analyses are in perfect agreement for sex estimation, but some discrepancies in ancestry estimation were observed in 5 out of 24 cases. When unidentified human remains are recovered, forensic investigators are tasked with creating a biological profile to narrow down the pool of subjects in the research of missing persons and, eventually, to assist in identifying unknown individuals. In forensic anthropology, the four major pillars of the biological profile are skeletal sex, ancestry, age at death and stature. Skeletal sex estimation relies on determining morphological and metric dimorphic features within the pelvis ,2, the cObtaining a DNA profile to identify human remains is of limited use if antemortem data such as a direct reference sample  or a biological sample of the victim’s relatives  are not available for comparison. In such circumstances, as occurs, for example, in armed conflicts, other situations of violence or unidentified persons, obtaining additional information—such as, for instance, biogeographical ancestry (BGA)—from DNA analysis could be beneficial in providing support in the creation of the biological profile, directing investigative activity in search of family members of the victims, and assisting with the identification of unknown remains. Therefore, in this paper, a multidisciplinary approach  to infer the sex and biogeographical ancestry of different skeletons using routine forensic STR markers, such as autosomal and Y STR and mitochondrial profile, was explored. Recently, Yang et al.  also tesHere, the comparison of physical, genetic, and then antemortem data  was particularly beneficial to evaluate the accuracy rates of the biological profiles produced by anthropologists and classification rates obtained by molecular experts using autosomal genetic profiles and multivariate statistical approaches. Thomas et al. carried out two retrospective studies on accuracy rates for skeletal sex  and anceThe specimens selected were from 24 forensic cases of human skeletal remains investigated by the Laboratorio di Antropologia e Odontologia Forense (LABANOF) of the University of Milano between 1997 and 2018 . The remFor each case, a biological profile was created. Since this is a retrospective study, different methods were used according to the time of the analysis. In general, sex estimation was based on morphological traits of the cranium  and of tMolecular analyses were performed in two different laboratories: Parma Forensic Biological unit of Carabinieri and Forensic molecular anthropology unit of University of Florence.Sample Inspection and ProcessingAt first, each piece of evidence was roughly shattered, then externally cleaned to allow the selection and collection of a small bone fragment, and subjected to further decalcification before turning it to dust and proceeding with the forensic genetic inspections.Fragments of about 400 mg to 500 mg from each bone sample were treated with 1.5 mL of 0.5 M EDTA and incubated at 37 °C for at least 3 to 4 days until the bone was completely decalcified ,26.® DNA Investigator kit , according to the manufacturer’s instructions for the “trace protocol”, with a final elution in TE buffer set at 50 µL volume.A second overnight incubation step at 56 °C was then applied to the fully decalcified bone fragments after adding 100 μL of G2 lysis Buffer , 60 μL of Proteinase K (Qiagen) and 20 μL of dithiothreitol (Qiagen). The samples’ nucleic acids were then extracted with the EZ1® 7500 Real-Time PCR System and analyzed with the HID Real-Time PCR Analysis Software v 1.3.The extracted samples were quantified using the Quantifiler™ Trio DNA Quantification kit  run on the Applied BiosystemSTR Loci Amplification: Autosomal DNA and Y Chromosome Markers® Fusion 6C System  was used for the autosomal STR loci analysis, and the PowerPlex® Y23 System (Promega) for the Y Chromosome STRs.The extracted and quantified samples were then subjected to STR marker amplification, based on multiplex PCR systems available on the market: PowerPlexTyping of Both Autosomal and Y-Chromosome Markers® Genetic Analyzer 3500 XL , with the proper reagents and the suggested parameters as indicated by the manufacturer Promega for both the Powerplex® Fusion 6C System and the PowerPlex® Y23 System; data interpretation was supported by the analysis conducted with the GeneMapper® ID-X software v. 1.4 . The Y-chromosome haplogroup inference was determined with calculations performed on the free online tool NEVGEN Y-DNA Haplogroup Predictor , using general level for prediction.For typing the amplified genetic material, the capillary electrophoresis (CE) technique  was usedSample Preparation and DNA ExtractionBone samples were processed in the molecular anthropology unit of the University of Florence, a state-of-the-art facility dedicated to the analysis of degraded DNA samples.® 300 series). After brushing, each sample was irradiated with ultraviolet light for 45 min in a Biolink DNA Crosslinker . A minimally invasive approach was followed to recover approximately 50 mg of bone powder from petrous bones, as described by Sirak et al. [TM 4 Fluorometer (dsDNA High Sensitivity Kit).The outer layer of the bones was mechanically removed to remove potential contamination using a rotary sanding tool  from the NIST 1036 Revised U. S. Population Dataset . The Y-SAccuracy, sensitivity, and specificity metrics were estimated for all the developed PLS-DA models. Once developed, the models were tested to predict 24 samples for each marker under exam, i.e., STRs, Y STRs and mitochondrial markers.Skeletal sex and ancestry estimations classified the samples as follows: 13 European male individuals and 7 European and 3 African females. Evidence 10 was a human cranium, whose dimorphic traits were too ambiguous to express a conclusive sex estimation , whereas the estimated ancestry was European . Age at Autosomal STR typing was performed on all extracted samples, and complete profiles were obtained from 15 out of 24 samples . While partial profiles with a number of loci typed ≥13 were obtained from six samples . There were no results from 3 out of 24 samples . STR typing highlighted that 12 out of 24 skeletal remains belonged to individuals of the male sex and 10 were females. PLS-DA was performed on all STR profiles except for the three samples from which no results were obtained . In addilocus was missing.Y STR typing was performed on 12 male individuals and complete profiles were obtained from 7 out of 12 samples , 3, 4, 9, 21, and 24). Partial profiles with the number of loci typed being 17, 21 and 16 out of 23 were obtained from samples 12, 16 and 26, respectively, and 9 loci were typed from sample 5. No profile was achieved from sample 8. The Y haplogroup of each sample was assigned using the Nevgen Y-DNA Haplogroup Predictor tool and the geographical distribution is reported in MtDNA analysis was performed on all extracted samples, and complete profiles were obtained from 24 out of 24 samples. The 24 mitogenomes were sequenced to an average coverage depth of 507.40X (from 103.40X to 979.72X) .https://github.com/pontussk/PMDtools, accessed on 16 March 2022) was subsequently performed and the new consensus was used to estimate the haplogroup. Sequences were assigned to eight distinct haplogroups, as seen in table , they hIn conclusion, this study extended the research line of comparative studies on physical and DNA evidence for the sex and ancestry estimation of skeletal remains from casework. As seen here, physical and molecular sex estimations are confirmed to be highly reliable, with higher accuracy rates (100%) than those previously reported (94.7%) . It is tPhysical and molecular anthropology have been making advances in the field of forensic science and can now contribute to identifying missing persons by providing information about skeletal sex and ancestry through physical and molecular methods. Therefore, the present study evaluated the sex and biogeographical ancestry of different skeletons using physical methods and routine forensic STR markers associated with multivariate statistical approaches. A recent paper reported the challenges faced by anthropologists when studying a migrant population via SNP markers and the NGS (next-generation sequencing) approach, where such an agreement between disciplines was not observed . Here, t


Mapt gene was replaced with the human MAPT gene under control of the endogenous Mapt promoter. This model represents an ideal in vivo platform to study the function and dysfunction of human tau protein. Accordingly, a detailed understanding of the effects MAPT KI has on structure and function of the CNS is warranted. Here, we provide a detailed behavioral and neuropathological assessment of MAPT KI mice. We compared MAPT KI to wild-type (WT) C57BL/6j mice in behavioral assessments of anxiety, attention, working memory, spatial memory, and motor performance from 6 to 24 months (m) of age. Using immunohistological and biochemical assays, we quantified markers of glia , synaptic integrity, neuronal integrity and the cytoskeleton. Finally, we quantified levels of total tau, tau isoforms, tau phosphorylation, and tau conformations. MAPT KI mice show normal cognitive and locomotor behavior at all ages, and resilience to mild age-associated locomotor deficits observed in WT mice. Markers of neuronal and synaptic integrity are unchanged in MAPT KI mice with advancing age. Glial markers are largely unchanged in MAPT KI mice, but glial fibrillary acidic protein is increased in the hippocampus of WT and MAPT KI mice at 24 m. MAPT KI mice express all 6 human tau isoforms and levels of tau remain stable throughout adulthood. Hippocampal tau in MAPT KI and WT mice is phosphorylated at serine 396/404 (PHF1) and murine tau in WT animals displays more PHF1 phosphorylation at 6 and 12 m. Lastly, we extended previous reports showing that MAPT KI mice do not display overt pathology. No evidence of other tau phosphorylation residues  or abnormal conformations (TNT2 or TOC1) associated with pathogenic tau were detected. The lack of overt pathological changes in MAPT KI mice make this an ideal platform for future investigations into the function and dysfunction of tau protein in vivo.Tau is a microtubule-associated protein with a diverse functional repertoire linked to neurodegenerative disease. Recently, a human tau knock-in (MAPT KI) mouse was developed that may overcome many limitations associated with current animal models used to study tau. In MAPT KI mice, the entire murine  Tau is a microtubule-associated protein that is predominantly expressed in the nervous system where it plays a role in regulating cytoskeletal dynamics and signaling in the axon . Beyond MAPT gene on a mouse tau knockout background. These mice lack murine tau and overexpress all 6 human tau isoforms, resulting in neuropathological and cognitive deficits in later ages  animals. The advantages of these models are the use of unmodified WT tau expressed at physiological levels. However, the major limitations are differences between human and murine tau. Within the adult human brain, alternative splicing of the isoforms . Tau isoisoforms . In contisoforms . Thus, risoforms . The N-tisoforms . Taken tMapt gene was replaced with the human MAPT gene and is expressed under control of the endogenous murine Mapt promoter. MAPT KI mice express all six isoforms of tau in a ratio roughly equal to that of the adult human brain and show a normal subcellular localization of tau , increase amyloid-β deposition or modify markers of apoptosis and gliosis  mouse that overcomes many of the limitations of available tau models . Within n of tau . Humaniz gliosis . While tDue to the tremendous utility this model holds for the tau field, we believe it is essential to have an in-depth characterization of the MAPT KI mouse phenotype throughout their adult lifespan. Thus, the purpose of the current study was to provide a detailed behavioral and neuropathological assessment from 6 to 24 months (m) of age in male and female MAPT KI mice. To this end, we performed behavioral assessments of anxiety, attention, working memory, spatial memory, and motor performance over the adult lifetime of MAPT KI mice. Using both histology and biochemistry we quantified markers of glia , synaptic integrity, neuronal integrity and the microtubule system in adult MAPT KI mice. Finally, we extended previous reports by quantifying total tau expression, levels of tau isoforms, tau phosphorylation, and tau conformation over the adult lifetime of MAPT KI mice. This information can serve as a baseline comparison for future studies utilizing MAPT KI mice to study the tau protein.ad libitum. All studies were conducted in compliance with federal, state, and institutional guidelines and approved by the Michigan State University Institutional Animal Care and Use Committee.Breeding pairs of MAPT KI mice were obtained from Dr. Karen Duff at Columbia University with permission from the Saido group at the Riken Center for Brain Science. Breeding pairs were used to generate an in-house colony. For MAPT KI breeding, a single adult male mouse was housed with two adult female mice . Following birth of new litters, offspring were weaned from the Dam at day P20-P21. Experimental groups formed from in-house colonies were composed of animals from multiple litters. Adult male and female wildtype C57/BL6j  were obtained from Jackson labs at 6, 12, 15 or 20 m of age. The 15 m and 20 m-old animals were then aged to 18 m and 24 m, respectively, in our animal facility prior to testing. Animal weights at 6, 12, 18 and 24 m are shown in Individual groups of male and female MAPT KI and WT animals underwent a battery of behavioral tests at 6, 12, 18, and 24 m of age. Behavioral testing was performed at the same time of day for all cohorts. For all behavioral tests, animals were singly housed prior to the task and allowed to acclimate in the room containing the behavioral apparatus for at least 30 minutes (min) prior to testing. The room containing the behavioral apparatus was partially sound proofed and a white noise machine was used to eliminate extraneous noises that could affect the experiment. The experimenter was blinded to all treatment groups throughout the entirety of the behavioral testing and analysis.2 open field apparatus box with a video camera overhead. AnyMaze video tracking software (Stoelting Co.) was used to delineate the center (~28 cm2 square center of the apparatus) and peripheral (~6 cm wide zone surrounding the center zone) zones within the open field apparatus. AnyMaze software was used to track the movements of the animal’s head during a single 15 min trial. AnyMaze software was used to quantify total time in each zone as well as total distance traveled. Between each trial the behavioral apparatus was cleaned with 70% ethanol and allowed to completely dry to eliminate residual olfactory stimuli.Animals were placed individually within a 40 cm2 behavioral testing box with a video camera mounted above the box. Spatial cues were present on three of the walls of the behavioral apparatus to allow the animals to orient themselves during the task. During each trail, AnyMaze video tracking software was used to track the movement of the animal’s head and the amount of time the animals spent investigating each object (as indicated by time within object zones that were delineated within the AnyMaze software). Trial 1-training: animals were placed into the behavioral apparatus that contained 2 identical objects located in the two back corners of the apparatus . Animals were allowed to explore the objects for the 10-min trial and AnyMaze software was used to quantify the amount of time the animals spent exploring each object. Trial 2-OLT: One of the objects used in trial 1 was moved to a new corner  while the other object used in trial 1 remained in the same position. Animals were allowed to explore the objects for the 10-min trial and time spent exploring each object was quantified. Trial 3-NORT: The object that was not moved in trial 2, was replaced with a novel object. Animals were allowed to explore the objects for the 10-min trial and time spent exploring each object was quantified. In both OLT and NORT, a priori criteria were set such that mice must have a total exploration time of at least 20 s to be included in the analysis . Once all animals were loaded into the rotarod module, the protocol was initiated. The protocol begins at 4 RPMs and accelerates to a final speed of 400 RPMs over a 300 s time frame. The latency to fall for each animal was recorded. Between each trial the rotarod module was cleaned with 70% ethanol and allowed to dry to eliminate any residual olfactory stimuli.Memory deficits were investigated using the RAWM as previously described . The RAWAt the age of sacrifice, mice were administered an overdose of Fatal Plus solution (≥100 mg/kg).Mice were transcardially perfused with 0.9% saline containing heparin  followed by 4% paraformaldehyde. Brains were then removed and post-fixed in 4% paraformaldehyde overnight. For cryoprotection, brains were immersed first in a 15% sucrose solution until saturated, followed by a 30% sucrose solution until saturated. A freezing stage, sliding knife microtome was used to prepare six separate series of 30 μm coronal brain sections encompassing the entire rostral caudal axis of the brain.Mice were transcardially perfused with 0.9% saline containing heparin . Brains were then removed, and the hippocampus was immediately dissected and frozen on dry ice.Immunohistochemistry was performed using standard protocols . BrieflyImmunofluorescence staining using LiCor near-infrared secondary antibodies was performed using previously established protocols , 2018. Fg for 20 min at 4°C and the supernatant (S1) was collected while the pellet (P1) was further processed to obtain sarkosyl insoluble tau samples. P1 was homogenized in 5 volumes of brain pellet homogenization buffer  and centrifuged at 27,000xg for 20 min at 4°C. Sarkosyl was added to the resultant supernatant (S2) to yield a final concentration of 1% sarkosyl and was incubated at 37°C for 1 h. Following incubation, the sarkosyl - containing S2 fraction was centrifuged at 200,000xg for 1 h at 4°C. The resultant pellet (P3) was resuspended in brain pellet homogenization buffer and used for analysis as sarkosyl insoluble tau. Protein content was determined using a Pierce rapid gold BCA kit  according to the manufacturer’s instructions. For analysis of tau isoforms, S1 lysate samples were dephosphorylated overnight at 37° C using FastAP Thermosensitive Alkaline Phosphatase . For all samples, 20–25 μg of protein was mixed with 6x Laemmli sample buffer and heated to 98° C for 5 min. Proteins were separated by SDS-PAGE using AnyKD Criterion TGX gels  or 10% Criterion TGX gels (5671034) and transferred to BioTrace nitrocellulose membranes . Membranes were blocked in 2% non-fat dry milk in TBS prior to incubation overnight at 4° C in primary antibodies. Primary antibodies used were IBA1 , GFAP , Synapsin 1 , NeuN , myelin basic protein , PSD-95 , PHF1 , AT8 , pS422 , Tau5 , 3R tau , 4R Tau , Beta III tubulin (Dissected hippocampi were homogenized in 10 volumes (w/v) of brain homogenization buffer  containing protease  and phosphatase inhibitors  by sonication using 10 short pulses at power 2 (Misonix XL-200). Crude lysates were centrifuged at 27,000x1:10,000), MAP2  but does prevent the age-associated decrease in locomotor activity observed in WT animals.First, we performed the open field task as a measure of locomotor behavior and anxiety-like behavior . As a meMAPT gene KI does not affect spatial memory or object recognition.We next performed the object location task (OLT) and novel object recognition task (NORT). This task takes advantage of mice’s natural tendency to explore novelty and can serve as a metric of spatial memory and motivation that does not rely on extrinsic motivators (such as fear or reward). Figure As another index of spatial memory, we performed the radial arm water maze . In thisFinally, we performed the rotarod task as a metric of locomotor function. Here, animals are placed on a rotating rod that accelerates from 4- to 400 RPMs over a 5 min period. After training, the latency to fall is recorded over 4 trials and averaged. There was an age-associated decrease in the latency to fall in both WT and MAPT KI mice after 6 m of age . More spMAPT gene KI. To this end, we used a combination of semi-quantitative immunofluorescence and biochemistry to quantify markers of neurons (NeuN), pre- and post-synapses  as well as glia  in the hippocampus (Hp) and cortex (Cx) of MAPT KI and WT mice at 6, 12 and 24 m of age. Again, for all analyses we did not detect a significant difference between sexes within a given age and genotype  and PSD-95 (post-synaptic marker) in the Hp and Cx. MAPT gene does not alter glial numbers or glial activation compared to WT animals, and that both WT and MAPT KI mice show an age-associated increase in GFAP levels.We next performed the same combination of immunofluorescence quantification combined with biochemical quantification of glial cell markers. We quantified total tau protein and individual tau isoforms in the Hp across the adult lifetime of MAPT KI mice. To quantify total tau, we performed western blotting using the pan-tau antibody, Tau 5, using Hp S1 lysates from MAPT KI and WT animals at 6, 12 and 24 m of age. Finally, we investigated tau phosphorylation and abnormal tau conformations in MAPT KI and WT mice at 6, 12, and 24 m of age. To determine tau phosphorylation status, we performed IHC and western blotting for AT8 (phosphorylation within aa198-210) , pS422 , and PHFin vivo function and dysfunction of tau protein. The MAPT KI mouse expresses all 6 isoforms of human tau protein at physiological levels without the inherent drawbacks of transgenesis or overexpression. We believe this makes MAPT KI mice one of the best available animal models to study tau function in vivo. Due to the utility of this model, an in-depth characterization of the effects of MAPT gene KI was warranted. To this end, we have completed a battery of behavioral and neuropathological assays from 6 m to 24 m of age in MAPT KI mice and compared their performance to C57BL/6 J WT mice. Finally, we provided a detailed analysis of total tau expression, the relative expression of tau isoforms, as well as investigations into tau phosphorylation and abnormal tau conformations in MAPT KI mice. Taken together, we have confirmed and extended previous reports showing that KI of the human MAPT gene does not cause overt pathology or behavioral abnormalities.Tau is a functionally diverse protein that plays important roles in normal neurophysiology and human disease pathogenesis. Experimental research models are necessary to accurately dissect the We performed a detailed behavioral analysis that revealed MAPT KI mice show normal cognitive and locomotor behavior at all ages examined. In fact, MAPT KI mice display a resilience to the age-associated decreases in locomotor function that occurs in WT animals. More specifically, 24 m WT mice showed a significant decrease in locomotor activity in the open field task that was not observed in MAPT KI mice. In the rotarod test, MAPT KI mice had a significantly greater latency to fall from the bar at 18 m compared to WT animals, although this difference was abolished by 24 m of age. Several reports have previously linked alterations in murine tau expression to changes in locomotor activity. For instance, hTau mice exhibit increases in total distance traveled in the open field task . SeveralMAPT gene KI seems to merely preserve a normal locomotor phenotype throughout life, as opposed to increasing or decreasing locomotion. Published results and those presented herein suggest manipulation of tau can impact motor function, however, the underlying mechanisms are only partially understood. Motor impairment in most tau transgenic mice is an end stage phenotype resulting from degeneration of motor neurons following accumulation of pathological tau in the spinal cord , post-synapses (PSD-95), and neuronal cell bodies (NeuN) were unaltered compared to age matched WT animals and did not change with age in the Hp or Cx of MAPT KI mice. This agrees with previous reports that did not detect morphological changes in hippocampal or cortical thickness as measured by MRI in MAPT KI mice , and supter maze . Similarter maze . Thus, tAnalysis of glial markers revealed an age-associated increase in IBA1 in the Hp of MAPT KI mice, as well as an age-associated increase in Hp GFAP in both MAPT KI and WT mice. These results indicate a proliferation and/or activation of both microglia and astrocytes in the aged MAPT KI brain when compared to young MAPT KI mice. However, an important caveat to this conclusion is that, based on immunofluorescence analysis, GFAP levels were decreased in the Hp of MAPT KI mice compared to WT mice at 6 m of age, after which the increase in Hp GFAP in 24 m MAPT KI mice equalized levels of Hp GFAP between WT and MAPT KI mice. Furthermore, biochemical quantification of Hp GFAP revealed increased levels in 24 m MAPT KI and WT mice compared to 6- and 12 m MAPT KI mice, with no difference between genotypes at 24 m of age. An age-related increase in GFAP is a well-documented phenomenon. For instance, previous reports found increased GFAP mRNA in the Hp of 24 m rats compared to 6–7 m rats . This reAn interesting finding from the current work was the relatively high level of Ser396/S404 (PHF1) phosphorylated tau in both MAPT KI and WT mice. Previous reports revealed low levels of AT8 and pS422 tau, in addition to PHF1 tau, in MAPT KI mice . HoweverPrior to the generation of the MAPT KI mouse, the primary mouse model available that expressed all six human tau isoforms was the hTau transgenic mouse. This mouse expresses a human tau transgene from a PAC on a mouse tau knockout background , 2005. hMapt gene accelerates tauopathy induced by the injection of AD-tau seeds . Recently, tau seeding models have emerged as an alternative to transgenic or viral vector-based overexpression models of tauopathy. In these models, “seeds” of misfolded tau are injected into the rodent brain. The tau seeds are subsequently taken up into neurons where they template endogenous tau to take on the abnormal conformation, resulting in pathology . Tau seeau seeds . Finally KI mice . More re KI mice . These rIn conclusion, we have provided a detailed characterization showing that MAPT KI mice express all 6 human tau isoforms at physiological levels in the absence of behavioral or neuropathological abnormalities. Further, MAPT KI mice show signs consistent with normal brain aging in healthy individuals. The data presented herein can serve as a baseline for future studies utilizing this model to study the normal function of tau as well as the consequences of tau dysfunction.The original contributions presented in the study are included in the article/The animal study was approved by Michigan State University Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.MB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. SP: Formal analysis, Investigation, Methodology, Writing – review & editing. TS: Resources, Writing – review & editing. TCS: Resources, Writing – review & editing. TG: Formal analysis, Investigation, Methodology, Writing – review & editing. NK: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.