Brassica napus in relation to salinity tolerance. Free, conjugated and bound polyamines were analyzed, and changes in their accumulation were discussed with literature data. The most remarkable differences between the corresponding osmoprimed and unprimed seeds were visible in the free (spermine) and conjugated  fractions. The arginine decarboxylase pathway seems to be responsible for the accumulation of PAs in primed seeds. The obvious impact of seed priming on tyramine accumulation was also demonstrated. Moreover, the level of ethylene increased considerably in seedlings issued from primed seeds exposed to salt stress. It can be concluded that the polyamines are involved in creating the beneficial effect of osmopriming on germination and early growth of Brassica napus seedlings under saline conditions through moderate changes in their biosynthesis and accumulation.Currently, seed priming is reported as an efficient and low-cost approach to increase crop yield, which could not only promote seed germination and improve plant growth state but also increase abiotic stress tolerance. Salinity represents one of the most significant abiotic stresses that alters multiple processes in plants. The accumulation of polyamines (PAs) in response to salt stress is one of the most remarkable plant metabolic responses. This paper examined the effect of osmopriming on endogenous polyamine metabolism at the germination and early seedling development of  Soil salinity is an increasingly severe problem worldwide, posing a severe threat to food security due to its negative impact on agricultural productivity and sustainability. The area under soil salinity is gradually spreading on all continents. According to data provided by the Food and Agriculture Organization via the Global Map of Salt-affected Soils (GSASmap) tool, more than 3% of global topsoils and more than 6% of global subsoils are affected by salinity or sodicity . Soil saAn excess of soluble salt induces a double constraint on plants: first, a decrease in external water potential compromises water uptake (osmotic component), and second, the accumulation of toxic ions induces nutritional disorders . AccordiSeed priming is a presowing seed treatment that relies on controlled, partial seed hydration, leading to the activation of early germination events, however, insufficient for radicle protrusion through the seed coat. Since the completion of germination does not take place, seeds are still desiccation tolerant and can be dried back to their original moisture without loss of viability. Depending on the chemical nature of the priming agent, several seed priming methods are distinguished, including hydropriming, osmopriming, hormone priming, and others ,15. OsmoTriticum aestivum L.) [Oryza sativa L.) [Zea mays L.) [Glycine max L.) [Sorghum bicolor L.) [Brassica napus L.) seed germination in optimal conditions [Numerous studies showed multiple benefits of seed priming, including improved rate, percentage, germination uniformity, and seedling ability to emerge under salinity conditions. The alleviation of salt stress effects and increased tolerance induced by a priming treatment were reported in several crops, such as wheat (ivum L.) ,18 rice tiva L.) , maize (mays L.) ,21, soyb max L.) , sorghumolor L.)  and otheolor L.) . Our prenditions ,25 as wenditions . Rape isnditions . Plants nditions .Polyamines are organic cations found in various organisms, performing diverse biological functions . PutrescThe diamine putrescine is produced either from ornithine or from arginine through the action of ornithine decarboxylase  or arginine decarboxylase , respectively. Spermidine and spermine are then synthesized from putrescine through the addition of aminopropyl groups transferred from decarboxylated S-adenosylmethionine (dcSAM) in reactions catalyzed by spermidine synthase (EC 2.5.1.16) and spermine synthase (EC 2.5.1.22). DcSAM is produced from S-adenosylmethionine (SAM) by S-adenosylmethionine decarboxylase (EC 4.1.1.50) . SAM is Brassica napus seeds under salinity stress [This work aimed to better understand the beneficial effect of osmopriming on seed germination by estimating the modulation of the endogenous polyamine accumulation pattern during rape seed germination under salt stress. To gain more insight into PA metabolism, ODC and ADC activities were determined. As changes in PA metabolism may affect the production of ethylene, ethylene emission was also analyzed. This paper extends our previous paper related to osmopriming improved germination of y stress .2O (UP11H2O) and 16 h in 100 mM NaCl (UP16NaCl), which corresponds to reaching 1% of germinated seeds under the conditions mentioned above. Early visible radicle protrusion of primed seeds germinating under both salinity and control conditions occurred after 7 h of imbibition  (d), i.e., seeds soaked in priming agent (PEG) and dried to initial moisture content, and unprimed dry seeds (UPd) were used as a control.In the experimental layout used in this paper, the primed and unprimed seeds germinating on water or NaCl were collected at the same stage during germination. This experimental design gives a chance to identify changes linked to priming treatment but not directly connected with the more advanced development of germinating primed seeds. Thus, to perform analyses on primed and unprimed seeds at the same developmental stages, unprimed seeds were collected after 11 h of germination in Hctively) . The priBrassica napus seeds exposed to salt stress.To investigate whether osmopriming confers tolerance to salt stress through the action of polyamines, we used the high-performance liquid chromatography (HPLC) method for the quantification of putrescine, spermidine, and spermine in free, conjugated, and bound forms during germination of primed and unprimed d, P7H2O, P7NaCl) (11H2O vs. UP16NaCl). Comparing seeds that underwent priming treatment with untreated ones, osmopriming led to a rise of the free putrescine level during germination in optimal conditions (by 30%) but had no impact on seeds exposed to salt stress.During germination of primed seeds under both optimal conditions and in the presence of NaCl, no changes in the content of free putrescine were observed as compared to primed dried seeds—its level remained the same in all tested samples (P P7NaCl) . In unprThe changes of both free spermidine and spermine levels followed a broadly similar trend during seed germination under optimal conditions and during exposure to salt stress B,C.16NaCl) were characterized by statistically significantly lower free spermidine and spermine content than unprimed seeds germinating on water (UP11H2O) and seeds in the dry state.The salt stress did not differentiate the level of free spermidine and spermine in primed seeds—the contents of these polyamines remained at the same level as in the case of primed seeds germinating under optimal conditions as well as in dry seeds. A different tendency was noticed in unprimed seeds, in which exposure to the stress caused a decrease in the level of free spermidine and spermine. Unprimed seeds germinating under salinity conditions (UP2O7H) as unprimed seeds (UP11H2O). Under salt stress, there was a noticeable difference in the free spermidine and spermine accumulation between primed and unprimed seeds (16NaCl).Osmopriming treatment decreased the content of free spermidine and spermine in dry seeds but had no further impact on their level during postpriming germination under optimal conditions and in the presence of salt stress. The same contents of free polyamines characterized the osmoprimed seeds germinating in the presence of water  nor in the presence of NaCl (UP16 NaCl vs. P7NaCl).The highest concentration of conjugated spermine was noticed in dry seeds without differences in its content between UPPd seeds F. DuringNo modification of bound putrescine G was recThe pattern of changes in bound spermidine H and boud vs. UPd, P7H2O vs. UP11H2O, and P7NaCl vs. UP16NaCl).Concerning bound spermine I, the hiThe effect of priming treatment on ODC and ADC is presented in Unprimed seeds germinating under optimal conditions did not differ in tyramine level from unprimed seeds in a dry state but exhibited a lower level than unprimed seeds exposed to salt stress . The accOur results did not show any obvious differences in ethylene production during the first hours of germination between primed and unprimed seeds, both germinating in the presence of water or NaCl . HoweverPolyamines are involved in a myriad of processes in plants, including germination. Seed germination is an extremely dynamic process that requires metabolism activation, biogenesis of organellar structure, mobilization of stored substances, cell elongation, replication, and cell division. Literature data indicate the role of polyamines in a wide range of biological processes during plant development, from germination to flowering . In mostBrassica napus, we noticed an increase only in Put accumulation in seeds after priming. However, priming influences the ratio of the free, bound, and conjugated fraction of polyamines, which may affect their metabolic activity [The impact of seed priming on the polyamine metabolism has not been studied in detail, so far. Our results enhance the hypothesis about the function of polyamines in the acquisition of stress tolerance by plants, which, from an agrotechnical point of view, is extremely important at germination and early seedling development. Basra et al.  reportedactivity . It has activity . Arabidopsis thaliana, Malus domestica, Cedrela fissilis, and sweet corn, where Spd content was also higher than other PAs [Arabidopsis, the addition of exogenous Put (but not Spd or Spm) resulted in the inhibition of germination [d) as well as in unprimed seeds germinated on water (UP11H2O), whereas the lowest PA ratio was observed in unprimed seeds germinated under salt stress , the increase in Put level was accompanied by the upregulation of ADC activity, whereas in unprimed seeds germinated under salt stress, it was with higher ODC activity  gene expression and downregulation of proline dehydrogenase (PDH) gene expression involved in Pro degradation. The interaction between polyamines and signaling molecules, such as Heedlings . Exogenoeedlings . Furthereedlings . In the stresses ,33,70. Tstresses . The rolstresses  and Bailstresses . Hydrogestresses , an incrd et al.  and Pauld et al. , who demIn the present research a similar trend was also noticed for tyramine , which iBrassica napus seedlings observed under saline conditions takes place through changes in these two fractions, i.e., free and conjugated because in the case of bound polyamines there were no differences between the corresponding variants of primed and unprimed seeds  were surface sterilized and primed in PEG 6000 solution  for 7 days at 25 °C in the darkness on Petri dishes lined with 3 layers of filter paper wetted with PEG, according to the procedure described in our previous studies [d). This priming protocol was the most effective in terms of improved germination. Primed dried seeds (Pd) were collected for germination or frozen in liquid nitrogen and stored at −80 °C.The seeds of  studies ,25,26. Ad) and unprimed dry (UPd) seeds were placed in plastic Petri dishes  on the top of the three layers of filter paper wetted with 10 mL of deionized water or 100 mM NaCl and germinated in darkness at 25 °C. Primed seeds germinating for 7 h in H2O  were collected and frozen in liquid nitrogen and stored at −80 °C for future use.The primed dried (P2O (UP11H2O) and 16 h in 100 mM NaCl (UP16NaCl), which corresponded to reaching 1% of germinated seeds under the conditions mentioned above [d, UPd, P7H2O, UP11H2O, P7NaCl, and UP16NaCl. The experimental layout of ethylene measurement has been further expanded by unprimed and primed seeds germinating for 24 h in 10 mL of H2O and 10 mL of 100 mM NaCl.The imbibition time of 7 h corresponds to the achievement of 1% germinated primed seed under both salinity and control conditions, representing end of seed germination sensu stricto (time point just prior to radicle emergence) . To perfed above . To analExtraction of free, conjugated, bound polyamines (PAs) and tyramine was conducted according to the method described by Quinet et al.  with sli4, vortexed vigorously, and then left to stand for 1 h in an ice bath, mixed regularly. The extract was centrifuged at 23,100× g for 20 min at 4 °C. The supernatant was used for dansylation or the extraction of conjugated PAs. The pellet was used to extract bound PAs.Seed samples frozen in liquid nitrogen (500 mg FW) were ground to a fine powder with 500 µL 4% ice-cold HClOThe supernatant obtained from the free PAs extraction (200 mL) was mixed with 200 mL of 12 N HCl and vortexed for a few seconds at a slow speed. To perform acid hydrolysis, samples were heated at 110 °C for 16 h in hermetically closed glass bottles. HCl was then evaporated from the bottles at 80 °C, and the residue was suspended in 200 μL of 10% PCA and used for dansylation.4, vortexed vigorously, and centrifugated at 23,100× g for 20 min at 4 °C. The supernatant was discarded, and the procedure was repeated a second time. After washing, the pellet was suspended in 500 µL of 1 N NaOH, vortexed vigorously, and left to stand for 30 min in an ice bath, mixed regularly. The extract was centrifuged at 23,100× g for 20 min at 4 °C. The collected supernatant was hydrolyzed according to the procedure described in the section on conjugated polyamine extraction.The pellet, which remained after the extraction of free PAs, was washed by adding 500 µL 4% HClOPolyamine extracts were derivatized with dansyl chloride as described by Lefèvre et al.  and anal2 using (C14) labeled substrates (measuring CO2 evolution from the decarboxylation reaction). Protein content was measured by the protein-dye binding method according to Bradford [Extraction and assay of ADC and ODC enzymes were performed according to Quinet et al. . Enzyme Bradford , using b−1 for 15 min for 71 h.The ethylene production was measured using ethylene detector ETD-300  according to Critescu et al. . Seeds sp < 0.05. Results that showed no statistically significant differences are marked with the same letter.All analyses were conducted at least in three independent experiments . Germination tests were carried out on ten replicates of 100 seeds. The statistical deviation of the mean values was calculated using one-way analysis of variance (ANOVA) and the Tukey–Kramer multiple comparison test. The means were considered as significantly different at The present results shed a new light on the mechanisms underlying the osmopriming-induced improvement of seed germination and salt stress tolerance. The above findings are summarized in


This paper aims at an in-depth and comprehensive analysis of mechanical and microstructural properties of AISI 316L austenitic stainless steel  produced by laser powder bed fusion (LPBF) additive manufacturing (AM) technology. The experiment in its first part includes an extensive study of the anisotropy of mechanical and microstructural properties in relation to the built orientation and the direction of loading, which showed significant differences in tensile properties among samples. The second part of the experiment is devoted to the influence of the process parameter focus level (FL) on mechanical properties, where a 48% increase in notched toughness was recorded when the level of laser focus was identical to the level of melting. The FL parameter is not normally considered a process parameter; however, it can be intentionally changed in the service settings of the machine or by incorrect machine repair and maintenance. Evaluation of mechanical and microstructural properties was performed using the tensile test, Charpy impact test, Brinell hardness measurement, microhardness matrix measurement, porosity analysis, scanning electron microscopy (SEM), and optical microscopy. Across the whole spectrum of samples, performed analysis confirmed the high quality of LPBF additive manufactured material, which can be compared with conventionally produced material. A very low level of porosity in the range of 0.036 to 0.103% was found. Microstructural investigation of solution annealed (1070 °C) tensile test samples showed an outstanding tendency to recrystallization, grain polygonization, annealing twins formation, and even distribution of carbides in solid solution. Additive manufacturing (AM) has experienced unprecedented development in recent years, and it has gained a regular role among production technologies. AM allows the application of completely different, very complex, and non-traditional CAD approaches to the design of parts that are otherwise very limited by conventional manufacturing technologies. The growing availability of additive systems really opens the door to modern design and manufacturing tools, such as rapid prototyping, rapid manufacturing, or rapid tooling . FunctioWith laser powder bed fusion (LPBF) technology, units of material feedstock in the form of powder are distributed layer by layer in a powder bed and fused in desired regions together layer by layer using the thermal energy of a scanning laser beam. Layers of powder are applied to the desired thickness by a recoater. LPBF is single step AM intended for processing, with rare exceptions, of single material ,4,5,6. LDespite all the advances in additive technologies and the growing number of applications, many attempts to qualify load-bearing structural parts are a very difficult challenge, and many parts in the aviation sector do not pass certification tests ,20,21,223–108 K/s [As a result of the rapid heat-up, melting, and solidification (extremely high cooling rate of 10–108 K/s ) a fine –108 K/s ,31. The –108 K/s ,10,11,32–108 K/s  found hiStainless steel AISI 316L is widely used in many industries such as aerospace, medical, automotive, and energy, both conventionally produced and printed as well. Due to high corrosion resistance, higher strength, biocompatibility, and easy post-processing 316L SS has been long used on orthopedic implants and prostheses . Its lowConsidering these points, this study aimed to consider the effect of part orientation on anisotropy of mechanical properties and the microstructure of 316L SS. In addition to anisotropy, the influence of the process parameter “focus level” for two values was also evaluated. Analysis of mechanical properties was supported by optical microscopy and scanning electron microscopy for fracture surface investigation, providing an exceptionally comprehensive study. In addition, tendency to grain boundary formation during solution annealing was observed.Stainless steel powder labeled Concept Laser CL20ES  is commercially available and designed for LaserCUSING LPBF additive machines of the same manufacturer. CL20ES is an austenitic stainless steel with chemical composition equivalent to W.Nr. 1.4404, AISI 316L. CL20ES is low carbon stainless steel suitable for welding and subsequent heat treatment due to sensitization resistance. The chemical composition of pre-alloyed CL20ES powder is given in Test samples were built on a Concept Laser M2 Cusing additive manufacturing system . M2 Cusing is a middle-class machine with a 250 × 250 × 280 mm build envelop. Test samples were sintered in two batches using a standard and verified set of parameters for CL20ES material recommended by the manufacturer and only virgin powder. The processing parameters used to prepare samples are as following: layer thickness—30 µm; powder size range—10–40 µm; exposition of plane (core)—200 W, 800 mm/s; exposition of contours—180 W, 1600 mm/s; beam diameter—0.15 mm; beam overlap—15%; chessboard pattern 5 × 5 mm, scan rotation between layers—90°.Gór et al.  publisheParameter focus level was the only variable on the process parameters side: FL = −3 mm and FL = 0 mm. Parameter focus level (FL) describes the position (offset) of the laser beam focusing level with respect to the level of melting. In the case of CL20ES FL = −3 mm is used as a standard value.Test samples were stress-relief-annealed immediately after printing in a Nabertherm N31/H  electric furnace without a protective atmosphere according to the following regime: heating to 550 °C in 3 h, dwell of 6 hours at 550 °C, slow cooling in furnace.Mechanical anisotropy in relation to the orientation of the test specimen;Effect of the “focus level (FL)” parameter on the mechanical properties and integrity of the samples.The scope of the experiment was designed to assess two main phenomena:Tensile test bars and V-notched bar impact testing samples were distributed on 2 test building platforms.  . The ASTType 1: VERTICAL BUILD; notch plane of symmetry identical with the level of melting.Type 2: HORIZONTAL BUILD; notch plane of symmetry perpendicular to the level of melting; the notch bottom lies in one level.Type 3: HORIZONTAL BUILD; notch plane of symmetry perpendicular to levels of melting; the notch bottom line crossing levels of melting.V-notched bars were printed as 10 × 10 × 55 mm blocks with additional stock per surface. Test samples were subsequently ground  to meet dimensions, roughness, and GPS requirements of ASTM E23-07a specifications . Charpy The tensile tests were performed using LabTest 5100 SP1 servo-hydraulic high force universal testing equipment with a maximum load of 100 kN . Tests were carried out according to EN ISO 6892-1  at room 2) was calculated according to the formula KCV = KV/S0, where S0 is the minimum cross section under the notch. All tests were carried out at room temperature (20 °C).V-notched bars were tested on a Matest H060N  mechanical Charpy pendulum impact test device in accordance with ISO 148-1  using 15Roughness and contour measurements were performed on a MahrSurf XCR 20  tactile contour measuring station. Straightness of the tensile test bar was investigated for vertical and 45° orientation tensile bars on an evaluation length of 50 mm.With respect to anisotropy expected based on literature review, Brinell hardness was measured. Using load force of 187.5 kp (1839 N), a quenched ball of 2.5 mm in diameter was indented to the material (designated HBS 2.5/187.5 acc. to EN ISO 6506-1 ) on a WoMicrostructural observation required preparation of metallographic samples. Hot mounted samples were ground using SiC abrasive of different FEPA grits in steps of 80, 120, 320, and 500, and polished diamond pastes of different particle sizes, namely 9 µm + rubber disc, 3 µm + fine cloths disc, and 1 um + fine cloths disc. A Keyence VHX 6000  digital microscope with coaxial lighting was employed for polished and etched conditions of samples. Etching was performed electrolytically by 10% oxalic acid and the mixture HNO3 (50%) + ethanol (50%), which helps to suppress the internal grain microstructure and on the contrary highlight grain boundaries.The scope of the experiment enabled anisotropy of the material in three strategic orientations to be evaluated: horizontal (0°), tilted (45°), and vertical (90°). In the majority of publications and materials of powder manufacturers’ datasheets, the mechanical values obtained from vertically printed test bars are given, so vertical test samples were considered as reference average values in this experiment as well—UTS = 607 ± 1MPa; YTS = 476 ± 20 MPa; E = 44 ± 2%. From the point of view of tensile strength properties, tilted 45° samples are more suitable and showed a 14% higher UTS (689 ± 2 MPa) and a 16% higher YTS (550 MPa). At the same time, a lower elongation was observed by relative decrease of 16% E (37 ± 2%). Horizontal samples exhibited better values in all tensile parameters, as UTS was higher by 10% (666 ± 1 MPa), YTS by 15% (548 ± 23 MPa), and E by 2% (45 ± 2%). In all orientations, lower scatter of UTS results were observed, while higher standard deviations were achieved for elongation to fracture. The results are clearly presented in The experimentally measured values were partly in agreement with the already published studies ,27,33, aVertical samples made it possible to quantify the effect of the focus level parameter on tensile properties. Focusing the laser directly into the melting levels (FL = 0 mm) provided and indistinct increase of UTS by 1.2% (615 MPa) and a decrease of E by 1.25% (43%). The change in elongation was insignificant, while the yield strength showed a significant and repeatable improvement of 7.3% (511 MPa).A comparison of the mechanical properties of AM produced AISI 316L and conventionally produced steel is often misleading, as it is very important whether typical or threshold values permitted by the EN 10088-3 specification  are used3–108 K/s [Austenitic stainless steels are characterized by a decrease of plasticity reserve, a decreasing elongation, and a hardness increase with increasing strength ratio (i = YTS/UTS), printed parts not excluded. The YTS/UTS ratio for conventionally produced and annealed parts ranges from approximately 0.4 to 0.55, depending on the level of strain hardening and dislocation density. For AM parts, the high density of dislocations is mainly caused by high cooling rates 10–108 K/s ,30,31 an–108 K/s  reported2) were determined through a Charpy pendulum impact test (2). Two levels of melting were torn apart theoretically in this case, so reduced coherency and lower values were expected. Horizontal bars of TYPE 2 and TYPE 3 exhibited the same average impact toughness in both remaining notch orientations.Notch impact energy KV (J) and impact toughness KCV (J/cmact test . The wor2). Theoretically, this configuration should concentrate more energy directly into the melting level, which should result in a wider meltpool at the expense of the penetration depth and heating of the previous layers. Direct confirmation of this theory by measuring the meltpool width on metallographic samples was not reliable due to the overlap. For this purpose, it would be necessary to create and evaluate single laser track samples as published in [The influence of the process parameter focus level proved to be more significant against the influence of the position. The FL = 0 mm configuration on Type 1 notch bars parameter led to an increase in fracture toughness by 48%  on the upper shelf of the S-curve.Although the fracture was obviously ductile on the macroscopic scale in SEM observations, there was a mix of intergranular and transgranular mode on the fracture surface . IntergrAverage Brinell hardness was almost identical in both measurement directions, parallel to melting levels –215 ± 3 and perpendicular to melting levels217 ± 11 HBS 2.5/187.5, respectively see . From thFL −3 mm, indentation parallel to melting levels −230 ± 8 HV 05;FL −3 mm, indentation perpendicular to melting levels −229 ± 6 HV 05;FL 0 mm, indentation perpendicular to melting levels −235 ± 6 HV 05.The microhardness matrix was applied to metallographic sections to evaluate structural stability and hardness uniformity. Matrix 3 × 5 indents were defined, and outstanding measurement stability was achieved. Microhardness values at the edge of the solidification front of each linear laser passage and values inside the melted regions differed only minimally and unsystematically, regardless of both the FL parameter and orientation:Considering the test load, such a low variation of measurement is difficult to achieve even for conventional material. The direction of the indentation vector relative to the melting level did not affect the measured values, while the FL = 0 mm configuration showed a slight increase at the microstructural level. All measurements showed a higher hardness in the as built condition compared to conventional AISI 316L material in solution annealed delivery conditions.Tensile tests were accompanied by fractography analysis using scanning electron microscopy (SEM). Fracture surfaces bore the marks typical of ductile materials: dimple morphology, significant necking phenomenon, and angle between the necked surface and axis of the test bar strongly distinct and close to 45°. The fracture itself was of a pure ductile nature. Unmelted spherical powder particles could also be seen on the fracture surface see . The preNo obvious signs of oxidation were observed on powder particles by SEM or on particles trapped on metallographic sections, despite the less protective atmosphere generated by the nitrogen generator. These signs could probably be observed with repeated use of the powder. Pauzon et al.  describeOn the contrary, spherical pores did not exhibit this relation. The probable reason for this difference is that spherical pores are formed primarily in the melt pool by trapping a gas into the molten metal. Spherical pores on fracture surfaces were elongated see .Interpretation of the LPBF material structures is often challenging due to different appearances of the melting level section (top surface) and lateral sections as well as for unprecedented high melting and cooling conditions resulting in non-equilibrium structure condition. In addition, the lateral sections can differ from each other according to the angular rotation of the laser scanning direction. This can be neglected for the most common orthogonal rotation (90°). In any case, the rotation causes the successive layers to have differently oriented but periodically repeating melt pools.Polished conditions of metallographic samples in both top and lateral sections revealed very low porosity levels. The very low frequency of spherical pores testifies to the suitable setting of power parameters with scanning speed. Suitable combinations of laser power and scanning speed to achieve low levels of porosity are well described in . The larNon-spherical pores often retain unmelted or partially melted particles in their cavities. By comparing 2 was in the central part. Pores and voids were sorted by size to create a distribution curve as shown in Porosity analyses were performed using Keyence analytical software on three top sections in different heights of spare tensile test bars . The area of 9.72 mmThe obtained results are not entirely consistent with expectations based on process knowledge. As the built progresses layer by layer, the rubber blade of the recoater wears, which usually leads to uneven application, scratches in the applied powder surface, and the formation of larger non-spherical pores, which were not present in this case. Although there was an overall higher rate of porosity in top sections, small sized spherical pores predominated.Etched micrographs at lower magnification showed solidification tracks created after individual passes of the laser beam. Laser tracks are displayed in The lateral section was composed of hatch overlapped melt tracks of semi-circular shape that represented melt pool cross-sections with very clear melt pool boundaries. Melt pool width and depth ranged between 90 and 150 µm. Across melt pools, epitaxial growth driven by heat flow in the build up direction appeared on a macro-scale. The reason for that growth was partial remelting of previous layers.Based on the analysis of metallographic sections in high magnification on SEM, the authors in ,27,54 deIn the previous section, a typical inconsistency of the macrostructure of the LPBF manufactured material and anisotropy of mechanical properties were found. This anisotropy persisted even after the recommended stress relief annealing (550 °C/6 h). The structure retained a fine solidification character, and no grain growth occurred.(a)The clamping part not subjected to previous deformation ;(b)The proximity of the fracture surface with maximum deformation .For comparison, the damaged samples after the tensile test were used for subsequent solution annealing according to the regime . The subject of interest included two areas:In the as built + stress relief condition, a considerable elongation of the melt pools at the point of failure was evident, which was in accordance with the high elongation measured during the tensile test. After solution annealing, full polygonization of the grain was found, and in these specific lateral sections, the microstructure was practically indistinguishable from conventional material. Annealing twins appeared with great frequency in both areas. The area near the fracture surface had a significantly higher deformation energy and higher density of dislocations, which was the driving force of recrystallization and nucleation of new grains. Grains in the range of ASTM number G8 to G10 were found near the fracture surface and G4 to G7 in undeformed areas, according to ASTM E 112 specifications. According to the Hall–Petch relationship, a growth in grain size contributes to the significantly lower yield and tensile strength. Based on the Hall–Petch relationship, lower yield strengths can be expected for recrystallized material. Significant migration of meltpool boundaries to polygonized grain boundaries in a relatively short time of 10 minutes indicates high recrystallization kinetics. It is highly probable that the steel will have very good technological properties during soldering and brazing.Fine-grained carbides were evenly distributed in the structure. Precipitation of carbides at the boundaries and sensitization of the microstructure to intergranular corrosion were not found. In both areas, the annealing twins were clearly visible, as in conventionally-produced material see . DespiteThe anisotropy of tensile properties persisted in as-built material after stress relief annealing. Tilted (45°) samples showed 14% higher Rm, 16% higher YTS, and 16% lower E. Horizontal samples exhibited the best tensile properties—10% higher UTS, 15% higher YTS, 2% higher E against baseline.The shifting of focus level to the level of melting (FL = 0mm) brought an indistinct increase of Rm by 1.2% and a 1.25% increase of E, but a significant increase of YTS by 7.3%.Depending on the orientation and process parameters, the YTS/UTS ratio in the range of 0.7 to 0.85 was found.2) were achieved in the case of orienting the notch plane of symmetry into the melting level. Theoretically, two subsequent melting levels were torn apart in this case. Considering real coherency between melting levels, results were in line with expectations.Although the effect of notch orientation to melting levels was not significant based on the results, the lowest values of impact toughness KCV (127 J/cm2) compared to baseline.The impact toughness KCV of FL = 0 mm vertical samples was higher by 48% . From this point of view, consistent results of macrohardness and microhardness (229–230 HV0.5) could be observed.Using both hardness measurement methods, F= 0 mm samples exhibited higher values .Fractography analysis was performed using both scanning electron and optical microscopy. Fracture surfaces of tensile test samples contained typical features of ductile cracking with significant necking, high angled neck surface, and fine dimple morphology of the fracture. Several defects were observed on the metal-clean fracture surfaces—secondary cracks, voids, deformed spherical pores, non-spherical pores, and unmelted powder particles.Porosity investigation was performed in three levels along vertical tensile test samples with very promising results in the range of 0.036% to 0.103%. Across all samples according to the pore size distribution curve, more then 95% of total pore area was created by spherical pores smaller than 25 µm.Microstructural investigation of solution annealed (1070 °C) tensile test samples showed outstanding tendency to recrystallization, grain polygonization, annealing twins formation, and even distribution of carbides in solid solution.The purpose of the study was to analyze mechanical and microstructural properties of AISI 316L  manufactured using LPBF additive technology. In addition to a complete analysis of samples made with standard process parameters, the effect of focusing level shifting to the melting level was evaluated, and differences from baseline were reported. In the case of mechanical properties, vertical test samples loaded in the building direction and printed with standard parameters were considered as baseline . The findings led to the following conclusions:As mentioned above, the vast majority of studies ,40,41,52In addition to a comprehensive description of the anisotropy of mechanical properties, the presented article indicates the influence of the focus level parameter on the mechanical properties and the influence of plastic deformation on recrystallization processes and grain growth. Both of these influences have an indicative character, and in the further development it would be very appropriate to quantify these phenomena in several levels. At the same time, it would be good to exclude the persistence of anisotropy of mechanical properties in solution annealed conditions.LPBF technology is revolutionary and undoubtedly belongs to the fastest growing industries. The performed analysis confirms the high quality of additively manufactured material, which can be compared with conventionally produced material. In the development phase of new products, it is necessary to consider real mechanical properties and their anisotropy, as well as their modification through process parameters. The situation is even more serious because there is a tendency to use additive manufacturing for topological optimization tasks and the creation of lightweight structures. It is detailed knowledge of mechanical properties and its anisotropy that opens the possibilities of advanced topological optimization in relation to the production process and part orientation.


Background: Research on supplementing feed with rice husk activated charcoal was carried out to determine the effect of variations in the concentration of rice husk activated charcoal on the growth and histological features of the giant trevally Caranx ignobilis intestine.Methods: This study used an experimental method with a completely randomized design consisting of six treatments and four replications, including adding activated charcoal to feed at concentrations of 0%, 1%, 1.5%, 2%, 2.5%, and 3% for 42 days. The measured parameters included daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE), survival rate (SR), length of foveola gastrica, width of foveola gastrica, length of intestinal villi, and width of intestinal villi. Data were analyzed statistically using one-way analysis of variance and Duncan’s test.Results: The results showed that supplementing fish feed with rice husk activated charcoal at different concentrations significantly affected the values of DGR, AGR, FCR, FE, SR, length of the foveola gastrica, length of the villous intestine, and width of the villous intestine, but did not significantly affect SGR or foveola gastrica width.Conclusion: The 2% rice husk activated charcoal treatment showed the best results for all parameters. Caranx ignobilis) is a commercially valuable fish species widely distributed in Indonesian waters3. This fish is a top predator in coral reef ecosystems with a relatively long predicted age of 25 years and can grow to a size of 165 cm and 87 kg, making giant trevally (C. ignobilis) the largest species in the Carangidae family4. However, fishing continues to increase without conservation efforts, causing the fish population to decline in the last few decades7. Therefore, efforts towards fish domestication are needed to reduce wild fishing. However, several obstacles have been encountered during domestication, such as slow growth of fish due to underdeveloped feed technology in this species.Giant trevally (C. ignobilis) culture is dependent on trash fish  because the fish grow faster than when using commercial feed. However, the costs incurred to provide trash fish are relatively expensive and not balanced with the selling price of fish at harvest. In addition, trash fish are not always available and can carry diseases. Therefore, giant trevally (C. ignobilis) feed technology needs to be assessed immediately, through the compilation of appropriate feed ingredients so that it can support the nutritional needs of the fish. One of the technologies that has the potential to be applied is supplementing the feed with activated charcoal.The development of giant trevally 26, olive flounder 27, Striped catfish (Pangasiaodon sp.)28, nile tilapia (Oreochormis niloticus)8, african catfish (Clarias gariepinus)29, gilthead seabream (Sparus aurata)30, and beluga sturgeon (Huso huso)31. A study on supplementing coconut shell activated charcoal, mangrove wood, rice husk, and oil palm shell in feed on intestinal growth of giant trevally (C. ignobilis) indicated that 2% activated rice husk charcoal had an optimal effect on growth of intestinal tissues32. However, the appropriate concentration to supplement rice husk activated charcoal in feed for giant trevally (C. ignobilis) has not been reported.Several studies have reported applying activated charcoal in fish, such as supplementing activated charcoal in feed on the growth of fugu rubripes B = Feed treated to contain 1% rice husk activated charcoalC = Feed treated to contain 1.5% rice husk activated charcoalD = Feed treated to contain 2% rice husk activated charcoalE = Feed treated to contain 2.5% rice husk activated charcoalF = Feed treated to contain 3% rice husk activated charcoalC. ignobilis) juveniles  were collected from local fishermen in Ulee Lheue Village, Banda Aceh City, Indonesia. The fish was acclimatized in the maintenance pond at Ujung Batee Brackish Aquaculture Fisheries Center for 2 weeks.A total of 360 giant trevally (C. ignobilis) were chosen randomly, and the total weight and length of the fish were measured. The fish were distributed into 24 plastic containers (48 × 43 × 70 cm) with a water volume of 75 liters (15 fish/container). The fish were fed the experimental food twice daily at 7 am and 5 pm for 42 days.Juvenile giant trevally , specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE), survival rate (SR), length of the foveola gastrica, width of the foveola gastrica, and length of the intestinal villi. The biometric measurements, including the length and width of the foveola gastrica and intestinal villi, were made following the methods of AmiriWhere Wo is initial fish weight (g); Wt is fish weight at the end of the study (g); t is the duration of the study (days). L1 is the initial length of the fish (cm), L2 is the final length of the fish (cm), Δt is the rearing period (days). Nt is the number of fish at the end of maintenance, No is the number of fish at the beginning of maintenance.et al.37. The stomach and intestines were removed with tweezers, and placed in a bottle containing 4% formalin for 1 week. Histological sampling was carried out using the paraffin method based on Osman and Caceci38. Samples were dehydrated through a gradient alcohol series  and dehydrated in xylol. The stomach and intestinal samples were embedded in paraffin blocks. The paraffin blocks were cut 6-mm thick and stained with hematoxylin and eosin. The length and width of the histological preparations were measured using a binocular microscope  which was projected onto a screen. Treatment of the experimental animals followed the guidelines for the use of animals in research at Syiah Kuala University (Ethic Code No. 958/2015).Gastric and intestinal samples were excised at the end of the study. Four fish were taken randomly for each replication of the treatments. Fish were anesthetized in 30 ppm clove oil, and the belly of the fish was dissected following the procedure of PurushothamanThe research parameters were analyzed statistically using one-way analysis of variance to detect differences followed by Duncan’s multiple range test. P < 0.05 was considered significant. All data were analyzed using SPSS software version 20 . Qualitative  data of the stomach and intestine were analyzed descriptively.The active rice husk charcoal supplement had a significant effect on DGR and AGR, but did not significantly affect SGR . The higC. ignobilis) juveniles. The best FCR was observed in the 2% rice husk activated charcoal treatment with a value of 1.257, indicating that 1.257 kg of feed is required to increase fish weight 1 kg. The highest FCR value was detected in the 2.5% rice husk activated charcoal treatment at 1.922. Furthermore, the best FE and SR values were also observed in the of 2% rice husk activated charcoal treatment with 80.264% and 88.33%, respectively, while the lowest FE and SR values were observed in the 2.5% rice husk activated charcoal treatment at 52.114% and 65%, respectively , FE (P<0.001), and SR (P<0.002) of giant trevally (ctively .C. ignobilis juveniles). The highest average length and width of the foveola gastrica were observed in the 2% rice husk activated charcoal treatment with values of 171.574 µm and 120.409 µm. respectively, while the shortest average length of the foveola gastrica was detected in the control treatment and the shortest foveola gastrica width was observed in the 1.5% rice husk activated charcoal treatment  on the length of the foveola gastrica in giant trevally (eatment .C. ignobilis) intestinal villi. The longest mean length and width of intestinal villi were observed in the 2% rice husk activated charcoal treatment at 64.027 µm and 16.672 µm, respectively. The shortest mean length of intestinal villi was detected in the 2.5% rice husk activated charcoal, while the shortest mean width of intestinal villi was observed in the 1% rice husk activated charcoal treatment  the length and width of the giant trevally (eatment .C. ignobilis) juveniles, which enhanced the immune system, leading to a high SR. According to Prescottet al.39, activated charcoal functions as a bacterial endotoxin absorbent, which inhibits absorption of nutrients. In addition, Mulyono and Wibisono40 reported that activated charcoal absorbs ammonia (NH3), which is a toxic substance. Kutluet al.41 stated that adding activated charcoal to feed accelerates the healing process of the mucosa by eliminating intestinal pathogenic bacteria. Furthermore, Thuet al.27 reported that activated charcoal plays a role reducing intestinal surface pressure by removing and absorbing gases and poisons that occur along the intestine, so that nutrient absorption is optimal.Supplementing fish feed with different concentrations of rice husk activated charcoal had significant effects on DGR, AGR, FCR, FE, SR, length of the foveola gastrica, length of the villous intestine, and width of intestinal villi, but had no significant effect on SGR or the width of the foveola gastrica. The 2% activated charcoal treatment revealed the best results for all parameters, including DGR (0.359 g/day), SGR (1.492%/day), AGR (0.092 cm/day), FCR (1.257), FE (80.264%), SR (88.33%), foveola gastrica length (171.574 µm), foveola gastrica width (120.409 µm), villous bowel length (64.027 µm), and intestinal villous width (16.672 µm). These results indicate that supplementing with 2% rice husk activated charcoal was a catalyst for absorption of feed nutrients by giant trevally  better than the other treatments, thereby reducing the quantity of feed (FCR) given to the fish. This shows that the 1.5%–2% rice husk activated charcoal treatment functioned effectively and efficiently, resulting in higher fish weights with less feed compared to the other treatments. In addition, the SR was maximum in this treatment, with a value of 83.33% in the 1.5% rice husk activated charcoal treatment and 88.33% in the 2% rice husk activated charcoal treatment. However, different results were reported by AdemolaC. ignobilis) juveniles. The length of the foveola gastrica was positively correlated with increasing concentrations of rice husk activated charcoal in feed from 0%–2%, but the size decreased at concentrations of 2.5% and 3%. Although there was a decrease in the length of the foveola gastrica in the 2.5% and 3% activated rice husk charcoal treatments, the decrease was not significantly different from the longest foveola gastrica value in the 2% rice husk activated charcoal treatment. Piraratet al.8 reported that exceeding the optimum concentration of activated charcoal in feed does not have a positive effect on the development of digestive organs and interferes with absorption of nutrients from feed.Adding activated rice husk charcoal to the feed significantly affected the length of foveola gastrica, but did not affect the width of the foveola gastrica in giant trevally  fed 2% activated charcoal developed an epithelial surface layer covering the entire foveola gastrica. This reinforces the conclusion that growth of the foveola gastrica occurred optimally with the 2% active rice husk charcoal supplement.43 and Mekbungwanet al.44 reported that adding activated charcoal to feed increases the growth and intestinal function of land and aquatic animals and improves FE. Thus, growth of intestinal villi is one of the determinants of nutrient uptake during fish digestion.C. ignobilis). The 2% active rice husk charcoal treatment revealed the best results for all research parameters, including DGR (0.359 g/day), SGR (1.492%/day), AGR (0.092 cm/day), FCR (1.257), FE (FE) 80.264%), SR (88.33%), foveola gastrica length (171.574 µm), foveola gastrica width (120.409 µm), villous bowel length (64.027 µm), and villous bowel width (16.672 µm).The results showed that supplementing the feed with different concentrations of activated rice husk charcoal significantly affected the values of DGR, AGR, FCR, FE, SR, length of foveola gastrica, length of villous intestine, and width of the intestinal villi, but had no significant effect on SGR or width of the foveola gastrica in giant trevally , specific growth rate (SGR), absolute growth rate (AGR), and feed efficiency (FE) ofCaranx ignobilis,https://doi.org/10.6084/m9.figshare.13094930.v249.Figshare: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevallyThis project contains the feed conversion ratio (FCR) and survival rate (SR) of Caranx ignobilis.Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the Caranx ignobilis” has contributed useful information for improvement of giant trevally culture through the development of a potential sustainable fish feed product. I am of the opinion that the manuscript is already worthy of indexing in its current form. Authors have generally addressed issues highlighted in previous review reports. However, having said that there are three points that I would like to suggest for authors to consider – also commented in previous review:The manuscript entitled “Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestine from giant trevally, Page 3. Please include at the end of the Introduction “Thus, this study aims to identify the appropriate concentration of rice husk activated charcoal supplement in fish feed which will produce optimal effect on growth parameters. We hypothesise that optimal growth can be achieved at a specific concentration”. Page 4. Please state the management regime to monitor (measuring pH etc) and ensure satisfactory water quality  after the following sentence on this page “ The fish were fed the experimental food twice daily at 7 am and 5 pm”. Page 5. It would also be appropriate to add that the data was tested for homogeneity in the data analysis although it appears that they were homogeneous, based on the tabulated results.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Aquacuture and Ecological GeneticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The citations used on this works were closely related and were within the last ten years. This research was carried out accurately and meets scientific principles. The methods and data analysis used were appropriate to the type of research. The details of the methods and analysis used in this work were replicable, however there were some details missing. For example, the composition of the feed was not fully explained. The data was analysed using appropriate statistical analysis that produced reliable and accurate results. The conclusions were drawn from accurate data produced through appropriate methods and analysis. Overall, this research has been carried out in accordance with scientific principles and produced results that can be scientifically justified.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquaculture, fish health includes immunostimulants feedI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Caranx ignobilis” provides basic information for feed development and aquaculture of giant trevally.Based on my opinion, the manuscript entitled “Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestine from giant trevally, However, the manuscript writing and the presentation of data are not enough to index in its current form. I suggest the authors to use the broken-line analysis for predicting the optimal level of rice husk activated charcoal. More details and considering comments of this manuscript are listed below.General comments:Please provide common name of fish species, followed by its scientific name in parenthesis. The scientific name of species must be used the full name in the first mention and then use the abbreviations of generic name in the other places.There are several errors relating to the use of capital/lowercase letters, italics, units, symbol, brackets and scientific names. Please carefully check the consistency of writing throughout the manuscript.Some sections contained errors from the use of abbreviations. If all parameters were already abbreviated, so that the subsequent part must be explained using abbreviations. Please carefully check throughout the manuscript.Please replace “%” by “g/kg” for feed ingredients.Specific comments:Introduction: lack of hypothesis and objective.Methodology section should be re-categorized as time and site, charcoal preparation and activation, feed preparation, feeding trial , histological investigation and data analysis. In addition, it is suitable to replace the treatment titles from “A-F” to “0-3%”.Please replace fish flour and shrimp flour by “fish meal” and “shrimp meal”, respectively.Methods to collect uneaten feed and water management  should be given.How to prepare the fish before sampling? Were they starved?Please provide ID or reference number for animal ethics.For parameters indicating growth and feed utilization, it is necessary to provide the references for each equation; these equations are fact and all literatures are not original.Table 1: 1) delete the column “protein content ingredients (%)”; 2) provide detail for the premix composition; and 3) provide proximate chemical composition of basal diet. In addition, this table must be re-written since only one feed formulation was used for all six experimental feeds. The authors should explain the formulation of “basal feed” and then included with varying levels of rice husk activated charcoal (0-3%).For data analysis: Did you test the normality and homogeneity of each of your datasets? Were the data transformed before conducting the statistical analysis? In addition, I suggest the authors to use the broken-line analysis for predicting the optimal level of rice husk activated charcoal.Tables 2 and 3 should be grouped as only one table, as well as for Tables 4 and 5.The results section should be re-written and shortened. It is not necessary to add any value in text again since the readers can access all the values directly from tables or figures. Moreover, some result explanations were not supported by the data in the tables; please carefully check.The references were not well checked and contain many errors, in both content and format. Please carefully check the references section.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquatic animal nutrition, digestive enzymes in aquatic animals, aquafeed chemistryI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Bone cancer pain (BCP)–depression comorbidity has become a complex clinical problem during cancer treatment; however, its underlying molecular mechanisms have not been clarified. Several long noncoding RNAs (lncRNAs) have been demonstrated to be promising therapeutic targets in depression, but research on the role of lncRNAs in BCP–depression comorbidity has been limited. Therefore, high-throughput RNA sequencing was performed to detect differentially expressed profiles in the amygdala of a BCP–depression rat model in this study. We detected 330 differentially expressed mRNAs (DEmRNAs) and 78 differentially expressed lncRNAs (DElncRNAs) in the BCP–depression comorbidity model and then verified the expression of six DEmRNAs and six DElncRNAs with the greatest degrees of difference by RT-qPCR. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that differentially expressed genes were strongly enriched in inflammatory and immunologic systemic responses. Then the nuclear factor kappa B (NF-κB) signaling pathway and the Th17 differentiation pathway showed significant differences, as determined by Western blot analysis. Finally, we constructed a protein–protein interaction (PPI) network to explore the potential regulatory mechanism of DEmRNAs. In conclusion, our study reveals a new resource for the understanding of dysregulated lncRNAs and mRNAs in BCP–depression comorbidity and provides novel potential therapeutic targets for further approaches. Primary and metastatic cancers that invade the bone are frequently associated with severe and intractable bone cancer pain (BCP) . BCP canLong noncoding RNAs (lncRNAs) are highly conserved RNAs of more than 200 nucleotides in length . Gene reTo evaluate current therapies and develop new medical treatments, we performed transcriptome sequencing of the contralateral amygdala of rats with BCP–depression comorbidity to investigate differentially expressed profiles of lncRNAs and mRNAs and attempted to provide new information for the comorbidity of BCP and depression through the functional analysis of differentially expressed genes (DEGs).Female Sprague Dawley rats 150–180 g) were provided by the Experimental Animal Center of Fujian Medical University and group-housed in cages at 22 ± 2 °C with a 12 h/12 h alternating light/dark cycle and free access to food and water. Our experimental methods were approved by the Experimental Animal Care and Use Committee of Fujian Medical University  and complied with the guidelines for pain research on laboratory animals [0 g were 7 cells/mL for inoculation. Animals were intraperitoneally injected with 2% phenobarbital sodium (40 mg/kg) for deep anesthesia. The right hind paw of the rats was sterilized three times with a 7% tincture of iodine and shaved above the tibial plateau using a hair clipper, after which an incision was performed to expose the tibia. Next, the right tibial plateau was drilled with a 50-μL microsyringe, and the marrow cavity was slowly injected with 10 μL of MRMT-1 carcinoma cell suspension (or 10 μL of D-Hank’s solution without cells in sham rats) through the hole. Bone wax was used to seal the needle hole, and the incision was interruptedly sutured after washing with 75% ethanol.As previously described ,20, MRMTTibial X-ray and hematoxylin and eosin (H&E) staining were performed 14 days after BCP modeling to determine bone destruction and evaluate tumor growth.Mechanical withdrawal threshold (MWT): The MWT was assessed by measuring the paw withdrawal mechanical threshold (PWMT) using von Frey filaments at day 0 before the operation and on days 7, 14, and 21 after the surgery. Each rat was placed in a transparent box (26 cm × 12 cm × 12 cm) and habituated for 30 min before the test; then, the MWT was measured with von Frey filaments ranging in force from 0.4 g to 26 g  stabbing perpendicularly to the hind paw, following the methods a previous study . A positSucrose preference test (SPT): Rats were adapted to drinking sucrose water before the operation, and the baseline sucrose preference was then assessed. After fasting for 24 h, rats were given a preweighed bottle of 1% sucrose water and drinking water within 1 h. The position of the bottle was changed every 30 min. Finally, the liquid consumed from both bottles was determined by weighing the remaining liquid. Sucrose preference (%) was calculated as sucrose water consumption divided by total liquid consumption. Rats with a preoperative SPT index greater than or equal to 65% were included.Forced swimming test (FST): Rats were separated into a cylindrical container (20 cm in diameter and 50 cm in height) filled with water (24–26 °C) and subjected to adaptive swimming for 15 min during the pretest. After 24 h, the rats were put back into the water tank. The rats were adapted to swimming for 1 min, and the immobility time within 5 min  was recorded by video. Two researchers blinded to the group assignment were trained to keep track of the resting time by a manual stopwatch. The results are the averages of the two researchers’ measurements. The water in the tank was changed after each rat. The test took place between 8 a.m. and 4 p.m.On the 28th day after surgery, all rats were thoroughly anesthetized and sacrificed mechanically by decapitation. The contralateral amygdala was quickly isolated, and meninges were removed; then, the fresh samples were snap-frozen in liquid nitrogen and preserved at −80 °C.Total RNA was extracted utilizing a TRIzol reagent kit  in accordance with the manufacturer’s protocol. The quality of the RNA was evaluated on an Agilent 2100 Bioanalyzer  and verified using RNase-free agarose gel electrophoresis. Following the extraction of total RNA, eukaryotic mRNA was enriched with oligo(dT) beads and fragmented by fragmentation buffer. Next, the enriched mRNA was reverse transcribed into cDNA with randomized primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTPs, and buffer. Subsequently, the cDNA fragments were purified using a QiaQuick PCR extraction kit  and end-repaired; poly(A) was added, and the fragments were ligated to illumina sequencing adapters. PCR amplification products were selected by agarose gel electrophoresis and sequenced using an Illumina NovaSeq6000 from Gene Denovo Biotechnology Co. .The sequence data were validated using a real-time quantitative PCR system . Briefly, total RNA was extracted as described above and reverse-transcribed according to the manufacturer’s instructions . The primer sequences are listed in After sacrificing the rats of both groups, the amygdalae were removed quickly. The tissue was immediately frozen and lysed in RIPA buffer containing protease and phosphatase inhibitors . The concentration of protein lysates was determined by a BCA protein assay kit (Beyotime). The proteins were transferred to polyvinylidene fluoride membranes after separation by electrophoresis  and were then incubated with primary antibodies after blocking with 5% nonfat dry milk in TBST. The primary antibodies included rabbit anti-TGF-β , rabbit anti-IL-6 , rabbit anti-NF-κB p105/p50 , rabbit anti-NF-κB p105/p50 (phospho S337) , anti-IκBα , and anti-IκBα (phospho S36) , which were used according to standard procedures. The blots were visualized by ECL , and quantification analysis was performed .By comparing transcripts in known protein databases, two software programs, CNCI Utilizing Sequence Intrinsic Composition to Classify Protein-Coding and Long Non-Coding Transcripts  (versionhttp://www.geneontology.org/ in 1 June 2021), the numbers of genes were computed for each term, and the GO terms significantly enriched in the DEM compared with the genome background were determined by a hypergeometric assay. The calculated p-value was corrected by the FDR, assuming FDR = 0.05 as the threshold, and the top 25 terms  of each class were displayed above the bar graph. KEGG is the main publicly available database of pathways [GO has three ontologies: molecular function (MF), cellular component (CC), and biological process (BP). First, all DEGs were mapped to GO terms within the GO database . For the MWT data, a two-way ANOVA with repeated measures was used to detect differences between the two groups, followed by Bonferroni post hoc analysis for multiple comparisons. For SPT, FST, and all biochemical data, a Student’s t-test was used to compare the differences between groups. A value of p < 0.001).As shown in Rats that underwent the SPT and FST before surgery were subjected to the SPT and FST again 25–26 days after surgery, and the results of the SPT showed a decreased preference for sucrose on postoperative day (POD) 25 A, while The expression patterns of mRNAs and lncRNAs in the amygdala of BCP–depression and sham rats were evaluated. The transcripts in each sample were distributed almost equally on all chromosomes, and the upregulated genes constituted the majority of the transcripts A. All trp-value < 0.05, FDR < 0.05, and fold change >2 or <−2. Finally, 330 DEmRNAs (248 with upregulated expression and 82 with downregulated expression) and 78 differentially expressed lncRNAs (DElncRNAs) (34 with upregulated expression and 44 with downregulated expression) were revealed by comparing the quantification of gene expression across the two groups. Moreover, there was a clear distinction shown in the hierarchical classification of mRNA and lncRNA expression between the BCP–depression and sham rats . The quantitative PCR findings were in line with those of RNA-sequencing  and 6 DElncRNAs (3 upregulated and 3 downregulated) to conduct RT-qPCR to validate the reliability of our RNA-sequencing results on independent samples  and IκBα protein in NF-κB signaling and the TGF-β and IL-6 proteins in Th17 differentiation, were identified and selected to test the expression levels by Western blotting. The results showed that the protein expression levels of the phosphorylated NF-κB1 p105/p50 subunit and the phosphorylated IκBα were significantly increased compared with those of the sham group, while their total protein levels were minimally changed. TGF-β and IL-6 are necessary proteins during Th17 cell differentiation, and their expression levels were substantially increased .Moreover, the PPI network was constructed using the top 200 DEmRNAs corresponding proteins and associated interactions . A totalIn this study, we observed both BCP and depressive behaviors after implantation of MRMT-1 carcinoma cells. As the central site of depression and pain, the amygdala integrates and modulates the nociception signal and brings forth extensive alteration on molecular, cellular, and functional levels in the BCP–depression comorbidity condition. Given that the biological mechanisms of most lncRNAs remain unknown, coupled with the limited number of studies about their roles in BCP–depression comorbidity, RNA sequencing was conducted to assess the overall view of gene expression changes in the current study, which detected 330 DEmRNAs (248 with upregulated expression and 82 with downregulated expression) and 78 DElncRNAs (34 with upregulated expression and 44 with downregulated expression).Several lncRNAs have been functionally characterized in previous research on pain or depression, such as XIST, uc.48+, NEAT1, and NONRATT007487.2 in neuropathic pain ,27 and DMounting evidence highlights the involvement of inflammatory/immune responses and their relationships in depression and pain research ,37,38. DAs common neural mediators exist between pain and depression, it is assumed that inflammation induces a complex network of immune-to-brain signaling that regulates behavioral production during illness . ClinicoConsequently, Th17 cells, one of the differentiated subtypes of CD4+ T cells, can infiltrate the spinal cord through BCP and cause an imbalance in the Th17/Treg cell ratio in the spinal cord . CoincidSimilarly, as NF-κB is a key transcription factor that regulates the expression of inflammatory and immune response genes , the actUsing PPI network analyses, we identified five hub proteins: MMP-9, CCND1, NFκB1, IL-1β, and SOCS3. The results concerning these hub proteins are mostly in agreement with previously reported experimental studies, which found that they may play a pivotal role in the inflammatory effects of BCP–depression comorbidity. For instance, the activation of MMP-9 in the brain led to increased BBB permeability, which is the beginning of the invasion of peripheral immune cells into the CNS ,55. In aThrough sequencing, we found that these lncRNAs were mainly enriched in the inflammatory and immunological pathways. However, the role of certain lncRNAs in BCP–depression comorbidity was not specifically discussed, which is a limitation of our study. Another limitation of our research that should be addressed in future work is the lack of mechanistic studies. In addition, it should be recognized that although we implemented rigorous quality control at each of the key milestones and the results have been confirmed by conventional reverse transcription-quantitative polymerase chain reaction, many factors have the potential to influence the quantification of RNA sequences. Consequently, a strict experimental check is essential to obtain precise conclusions.In conclusion, this is the first study providing abnormal lncRNA and mRNA expression profiles in the amygdalae of rats with BCP–depression comorbidity. Our data indicate that the differentially expressed genes are predominantly enriched in the inflammatory and immunological pathways. Our study may provide novel insights and lay a theoretical foundation for future research on the pathogenesis of BCP and depression comorbidity.


The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development. Eze et al. use single-cell sequencing and immunohistochemical validation to create an atlas of early human brain development. In the telencephalon, they discover a diversity of progenitor subtypes, including two that are enriched in humans. The human brain consists of billions of cells across several functionally interconnected structures that emerge from the neuroectoderm. Many of these structures are substantially expanded or distinct compared to other mammals, particularly the cerebral cortex, the outermost layer of the human brain responsible for perception and cognition. These differences emerge at developmental stages before birth, and thus exploring the cell types in the developing human brain is essential to better characterize how cell types across the brain are generated, how they may be affected during the emergence of neurodevelopmental disorders and how human neural stem cells can be directed to specific cell types for modeling or treatment purposes.1. Later in development, across brain regions, a series of similar neurogenic and gliogenic processes give rise to the constituent cell types. However, at the molecular level, the sequence of events that leads to the emergence of these progenitor cells early in development is less well understood. The diversity of brain structures is known to emerge as a result of segmentation events that generate the prosencephalon, mesencephalon and rhombencephalon that are then further specified into the anatomical structures  that were dissected in this study2  has added substantially to our knowledge about cellular diversity and signaling networks, particularly during stages of peak neurogenesis. However, the first trimester of cortical development has not been described at this level of molecular detail, and important questions remain about the timing of neurogenesis, the presumed uniformity of the neuroepithelium and the signals that promote the transition to radial glia.The human cerebral cortex is more than three times expanded compared to our closest nonhuman primate relatives7. To identify the chief cell populations across brain regions and to compare them to one another, we sampled all available and identifiable structures, including the telencephalon, diencephalon, midbrain, hindbrain, cerebellum, ganglionic eminences, thalamus, hypothalamus and cortex  12 to 22, corresponding to gestational weeks 6–10. We also included cortical samples from one CS13 and one CS22 individual that were analyzed in a previous studyHOX genes (hindbrain)9; PAX7 (midbrain)10; GBX2 11; NKX2-1 12 and FOXG1 13) segregated these regions from one another as early as CS13. However, the cell type-defining gene expression programs were largely conserved across brain regions, resulting in subsets of progenitors or neurons that transcriptomically appear similar across regions  and the cortex specifically (when it was identifiable for subdissection). Clustering this data revealed minimal batch effects as most clusters contained contributions from multiple individuals, and clustering segregated the samples based on early and late first trimester stages Fig. . Each of16. Presumably these neurons were produced locally by direct neurogenesis from radial glia that occurs early in nonhuman models of cortical development as well as human cortical organoids17, and would be consistent with the observation that the IPCs that mediate indirect neurogenesis are nearly absent until later in the first trimester (after CS16). However, without lineage tracing, the inference that the earliest neuronal populations are a product of direct neurogenesis remains a working hypothesis. Subclustering of the neuronal populations resulted in subtype clusters, some of which were strongly enriched in either younger samples  or older samples (>CS16) .We also sought to describe the spatial organization of the cortical populations across development. Thus, we performed immunostaining validation in five individuals from the first trimester compared to an early second trimester sample at gestational week 14. We observed FOXG1 staining as early as CS16, but not earlier, as has been described previously26  were identifiable as early as CS14, but markers of maturing neuronal identity such as BCL11B (CTIP2) emerged after CS16. As expected, IPCs identified by TBR2 staining did not emerge until CS18 in the dorsal telencephalon28. This algorithm incorporates messenger RNA levels and inherent expression variability of individual cells to infer steady states that contribute to potential lineage relationships. Across cortical cell types, the most apparent lineage stream originated in progenitor cell populations from older samples and followed a trajectory through IPCs to neurons  of presumptive endothelial (FN1) and pericyte (RGS5) markers, but not markers of microglia identity (AIF1) or the marker of the mesenchymal population (LUM)  as an indicator of neuroepithelial cells, and SOX2 as a label for all progenitor populations, we saw small numbers of nestin-positive radial glia at CS14, with substantial upregulation by CS22 that simultaneously corresponded to a waning of ZO-1  and subclustered the remaining cells. The resulting analysis yielded nine subclusters all marked by strong VIM and SOX2 expression  immunoreactivityO-1 Fig. . By gestion Fig. . Each ofion Fig. . To examSOX2 (progenitor), TOP2A (dividing), LHX5-AS1 (previously uncharacterized cell population) and LUM . Comparison of these subpopulations to the nine progenitor subtypes identified across all samples demonstrated strong correspondence to these nine cell types, with additional heterogeneity in the CS12 and CS13 mesenchymal populations . NTRK3 was a highly specific marker of Progenitor Cluster 3, and intrigued us because it is commonly described as a TrkC receptor that enables survival of specific neuronal populations34 and has broad neocortical expression at later stages of mouse cortex development35. We observed that at early stages , NTRK3 broadly colocalized with SOX2-positive progenitor cell populations 43. Consistent with this observation, here we find that Notch 1 staining peaks and is largely restricted to the ventricular zone at CS14 and CS16  peaks at CS16 and diminishes subsequently  or Progenitor Cluster 7 (marked most highly by ID4). To test the hypothesis that these populations were underrepresented in the mouse data, we used immunostaining to explore the expression of the main progenitor population markers in the mouse. Indeed, no fluorescent in situ signal for C1orf61 or immunostaining signal for Id4 was identifiable in the mouse forebrain at TS14 or 16  to E11.5–E17.5 (ref. 47). In the mouse, Id4 peaks at E11.5 and is low after E13, whereas human ID4 is sustained in its expression pattern after CS13 . ID4-deficient mice exhibit impaired brain size and mistimed neurogenesis49, suggesting that earlier expression in human progenitors compared to mouse could indicate a potential mechanism by which early progenitors expand more rapidly in humans than in mice.To evaluate the similarities and differences between early forebrain development in human and rodent, we performed scRNA-seq of the mouse forebrain at embryonic days (E) 9 and 10, which correspond to Theiler stages (TS) 14 and 16. Although the onset of neurogenesis differs between human and mouse 16 Fig. –22. AlthS13 Fig. . By cont7). Using 108,593 cells derived from weeks 3 and 5 of this dataset, we explored the fidelity of organoid cell types to early human cortex. The preservation of excitatory neuronal identity mirrored that of later developmental stages . However, the remaining primary cell types had much lower fidelity in organoids (average of 0.28)  Fig. . These dHere, we present a comprehensive overview of scRNA-seq from the first trimester of human development. As these samples are rarely accessible, we seek to present both the single-cell sequencing as well as the immunostaining validation provided as part of this study as a community resource. Our analysis highlights the granular gene expression programs that emerge across brain structures and across cell types in the human neocortex as development progresses, and provides a dataset by which other model organisms and in vitro cortical organoids can be compared to their primary human counterparts. The tissues used for the purpose of this study are fragile, and dissections rely on morphological hallmarks without knowledge of expression patterns before sample collection. As such, the single-cell analysis becomes even more essential as a tool to explore and validate accurate regional identities. The gene signatures that we describe and validate may offer additional markers that can be used to mark these brain regions, as well as the transition from neuroepithelia to radial glia cell identity.Our analysis of several brain regions highlights many similarities across progenitors that were only mildly distinguished by the expression of regional-specific transcription factors. These similarities in gene programs across the developing human brain suggest that the process by which initially uniform stem cells give rise to neuronal and glia heterogeneity characteristic of the adult brain are parallel across brain structures and that insights from one region, including the degree of heterogeneity and the trajectories of differentiation, may be able to cross-inform identification of regulatory gene programs across the brain. We aspired to annotate specific gene programs that distinguish progenitor groups within the cerebral cortex. Instead, we observed a gradient of expression patterns, with clear neuroepithelial and radial glia cell populations on the ends of the spectrum, indicating that the transition from one population to another may be gradual rather than distinct. However, because our data are limited by the snapshot view of each timepoint sampled, and by imperfect representation of all possible timepoints along this continuum, this conclusion will need to be further explored through additional sampling, validation and eventual mechanistic examination.50. These cells may be secreting LUM as a structural component to support the physical formation of the telencephalon before the emergence of the radial glial scaffold. In support of this hypothesis, we found that the ALX1-positive cells were in the presumptive stroma surrounding the cortex, while LUM was detected throughout the cortex. Although cortical organoids do not express ALX1 or LUM at early stages, their expression emerges after 7 weeks of development  by both single-cell sequencing and immunostaining, that comprised a large swath of the telencephalon at the earliest timepoints that we sampled. We hypothesize that the ALX1-positive cells may resemble previously described neural crest-derived cell populations that give rise to the meningesent Fig. , furtherLUM and ALX1 clusters, could be identified in mice  (RRID:SCR_015820) and deposited in the Neuroscience Multi-omic (NeMO) Archive (RRID:SCR_002001), Additional methodological details can also be found in the Nature Research Reporting Summary included in the g for 5 min and the papain was removed. Macaque tissue sections (E64) were a gift from A. Pollen (UCSF), originally a gift from A. Tarantal (UC Davis).Acquisition of all primary human tissue samples was approved by the UCSF Human Gamete, Embryo and Stem Cell Research Committee . All experiments were performed in accordance with protocol guidelines. Informed consent was obtained before sample collection and use for this study. First trimester human samples were collected from elective pregnancy terminations through the Human Developmental Biology Resource, staged using crown-rump length and shipped overnight on ice in Rosewell Park Memorial Institute media or in 4% paraformaldehyde. Samples were donated and deidentified for sex. Although we can infer sex for sequenced samples, we have not accounted for it because it is an unclear measurement during the first trimester. Fixed samples were used for downstream immunostaining and imaging. Mouse samples (CD-1 IGS Mouse) were killed at E9 and E10, and staged using the somite number. Mice were housed in shared housing, five mice to a cage with a 12 light/12 dark cycle and temperatures of roughly 18–23 °C (65–75 °F) with 40–60% humidity. Equal numbers of male and female embryonic mice were used. All mouse experiments were approved by and conducted according to the UCSF Institutional Animal Care and Use Committee (protocol AN078703-03A). Half of the mouse samples were randomly assigned for fixation in 4% paraformaldehyde and the remaining live samples were used for dissociation. Live samples were subdissected into identifiable regions and dissociated using papain  with DNase. Samples were minced and incubated in 1 ml activated papain for 15 min at 37 °C, according to the manufacturer’s instructions. Samples were then inverted for several times and incubated for an additional 15 min. The dissociated cells were centrifuged at 300Single-cell capture was performed following the 10X v.2 Chromium manufacturer’s instructions. Each sample was its own batch. For each batch, 10,000 cells were targeted for capture and 12 cycles of amplification for each of the complementary DNA and library amplifications were performed. Libraries were sequenced according to the manufacturer’s instructions on the Illumnia NovaSeq 6000 S2 flow cell (RRID:SCR_016387).15. Batch effects (as pertaining to day of capture) were removed by normalizing all cells in a batch to the most counts, and then multiplying by the median counts in that batch. These normalized counts were merged together across samples and log2 normalization was performed. Using default parameters of Seurat v.2, variable genes were identified. Batch was regressed out in the space of variable genes during scaling, again using default Seurat v.2 parameters. For three samples, there was only one individual per timepoint, which may have resulted in confounding age and batch, but because of the strong batch effects that result from 10X data, we moved ahead with the batch correction. In the space of the scaled, batch corrected variable genes, we performed principal component analysis. Significant principal components were carried forward, as identified using the calculation detailed in a previous publication. Using the RANN package, the top ten nearest neighbors were identified for each cell in the space of significant principal components. We used a custom script to calculate the Jaccard distance of these neighbors, and used igraph to perform Louvain clustering for each analysis. Clustering with batch effect correction was performed for each dissected area, and also without batch correction by each individual, both across all brain regions and within cortex only.scRNA-seq data were aligned to the GRCh38-0.1.2 reference genome, and cells were identified using CellRanger v.2 (RRID:SCR_017344). Quality control removed cells with fewer than 500 genes per cell and cells with greater than 10% mitochondrial content. Clustering and batch effect were performed as has been previously described in these established methodsCluster markers were interpreted and assigned cluster identity by using known literature cell type annotations, or by associating progenitor and neuronal genes to other identifiable details. Progenitors were distinguished as radial glia if they expressed neurogenic genes, else they were determined to be neuroepithelial  clusters from the data. After subclustering, neuronal populations were identified and removed iteratively until 54 subclusters were identified and did not include neuronal populations. These 54 subclusters were recombined by correlating marker genes to one another, and then performing a dendrogram cut to generate the nine clusters presented here.7. Briefly, cluster markers were generated for each dataset individually, and gene scores integrating the specificity and fold enrichment for each marker was calculated. A matrix for every marker gene and gene score was generated across all clusters and used for correlations. This was performed in the space of all mouse forebrain clusters as compared to primary human progenitor clusters  algorithms. Reads that passed quality control after clustering were used as input for the velocyto command line implementation. The human expressed repeat annotation file was retrieved from the UCSC genome browser (RRID:SCR_005780). The genome annotation file used was provided via CellRanger. The output loom file was used as input to estimate velocity through scVelo. For each individual analysis, cells were filtered based on the following parameters: minimum total counts ≥200, minimum spliced counts ≥20 and minimum unspliced counts ≥10. For the combined cortical analysis, the processed loom files for each individual analysis were combined to generate a new unique molecular identifier count matrix of 15,473 genes across 53,096 cells, for which the velocity embedding was estimated using the stochastic model. For the combined progenitor analysis, cells that were identified as progenitors were used to create the loom file. The loom files for each of the individuals were combined for a total count matrix of 14,207 genes across 30,562 cells for the velocity embedding using the same criteria. Each embedding was visualized using uniform manifold approximation and projection (UMAP) of dimension reduction.Primary human and mouse samples and organoids were collected, fixed in 4% paraformaldehyde, washed with 1× PBS and immersed in 30% sucrose in 1× PBS until saturated. Samples were embedded in cryomolds using 50% optimal temperature cutting (OCT) compound  and 50% of 30% sucrose in 1× PBS and frozen at −80 °C. All samples were sectioned at 16 μm onto SuperFrost Plus microscope slides. Citrate antigen retrieval  was performed for 20 min at roughly 95–100 °C. Slides were then washed with 1× PBS and blocked using 5% donkey serum, 2% gelatin, 0.1% Triton in 1× PBS for 90 min at room temperature. Primary antibody incubation occurred in blocking buffer overnight at 4 °C, and washed five times using 0.3% Triton + 10 mM glycine. Secondary antibody incubation occurred in blocking buffer for 2–3 h at room temperature. Primary antibodies used were TRKC ; ALX1 ; ID4 ; N-CADHERIN ; DLK1 ; DLK1 ; CROC-4 ; LUM ; ZO-1 ; SOX2 ; SOX2 ; CTIP2 ; KI67 ; HOPX ; HOPX ; TBR2 ; TBR2 ; NESTIN , DCX ; NEUN ; PAX6 ; FOXG1 ; SATB2 ; LHX5 ; REELIN ; Phospho-β-CATENIN ; Phospho-S6  and NICD/NOTCH1 . All secondary antibodies were AlexaFluor used at a dilution of 1:1,000. Secondary antibodies used were donkey antimouse 488 ; donkey antirabbit 488 ; donkey antichicken 488 ; donkey antichicken 594 ; donkey antimouse 546 ; donkey antimouse 594 ; donkey antimouse 647 ; donkey antimouse 680 ; donkey antirabbit 546 ; donkey antirabbit 594 ; donkey antirabbit 647 ; donkey antirat 594 ; donkey antirat 488 ; donkey antigoat 546 ; donkey antigoat 594 ; donkey antigoat 647 ; donkey antisheep 546 ; donkey antisheep 594 ; donkey antisheep 647  and donkey antiguinea pig 647 .C1orf61 amplification, targeting nucleotides 60–897 of NM_006365.3 (Advanced Cell Diagnostics Probe Design no. NPR-0003991); MEF2C amplification, targeting nucleotides 1058–2575 of NM_002397.4  and the protocol for BaseScope v.2 Assay  for chromogenic LHX5-AS1 amplification, targeting nucleotides 69–293 of NR_126425.1 (Advanced Cell Diagnostics Probe Design no. NPR-0003991).Primary fixed samples were treated using the protocol for RNAScope Multiplex Fluorescence Assay v.2  for Images were collected on the Leica SP8 (RRID:SCR_018169) inverted confocal microscope using a ×40 oil-immersion objective. Because of the scarcity of first trimester primary tissue, only one sample per panel was imaged. For each imaging panel, the parameters  for image acquisition was left constant for all samples. Images were later processed using FIJI Image J (RRID:SCR_003070).t-test was used in Supplementary Figs. No statistical methods were used to predetermine sample sizes. No randomization was used in this study. Distributions of the data were not tested. Data collection and analysis were not performed blind to the conditions of the experiments. The Wilcoxon rank sum test was used to calculate cluster markers within Seurat for a variety of analyses. A one-sided Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41593-020-00794-1.Supplementary InformationSupplementary Figs. 1–26.Reporting SummarySupplementary TablesSupplementary Tables 1–12.