Rhizophagus irregularis and Funneliformis mosseae AMF species, were designed in 28S rRNA and large subunit (LSU) ribosomal RNA, respectively, and droplet digital PCR protocol for absolute quantification was set up. A higher number of DNA copies of both fungal species were detected more frequently in symptomatic than asymptomatic vines. Our study suggests a relationship between esca and native AMF in grapevine. These results underline the importance of native rhizosphere microbial communities for a better knowledge of grapevine esca disease.In this work we analyzed the relationship among native arbuscular mycorrhizal fungi (AMF) and vine roots affected by esca, a serious grapevine trunk disease. The AMF symbiosis was analyzed on the roots of neighboring plants (symptomatic and asymptomatic to esca) in 14 sites of three vineyards in Marche region . The AMF colonization intensity, identified by non-vital staining, showed higher value in all esca symptomatic plants (ranging from 24.6% to 61.3%) than neighboring asymptomatic plants (from 17.4% to 57.6%). The same trend of Glomeromycota phylum abundance was detected by analyzing fungal operational taxonomic units (OTUs) linked to the AMF community, obtained by amplicon high throughput analysis of ITS 1 region. Overall, the highest amount of OTUs was detected on roots from symptomatic plants (0.42%), compared to asymptomatic roots (0.29%). Specific primer pairs for native  Rhizophagus irregularis copy number quantification [Esca, together with Eutypa dieback and Botryosphaeria dieback, are considered the most destructive trunk diseases of grapevine, and are a rapidly growing concern in all wine producing countries . This dification . ddPCR sfication . With refication .R. irregularis and Funneliformis mosseae selected as marker species in the vineyard soils among Glomerales order.The goal of this work was to investigate the relationships among of AMF native root symbionts and esca disease. To achieve this aim, several approaches were used: (i) evaluating total AMF colonization using non-vital Trypan blue (TB) staining; (ii) analyzing the global abundance of Glomeromycota phylum according the OTUs with ITS primers; (iii) developing and validating the ddPCR identification and quantification of The experiment was performed from January to July 2017 in the three commercial vineyards, located in Ancona district, Marche region, in central-eastern Italy. In detail:Vitis berlandieri × V. riparia) rootstock. The soil has a medium texture tending to be melted, with a scarce amount of organic matter. The morphology of the land is hilly, and the exposure of the vineyard is south/west. The roots of the selected vine plants were collected in January. Vyn_1—The vineyard, located in Castelplanio (AN), 43°50′30″ N–13°09′50.3″ E, was planted in 2004. The grape cultivar was ‘Verdicchio’ grafted onto Kober 5BB , site at 43°31′09″ N–13°28′47″ E at 463 meters above sea level (m a.s.l.), was planted in 2002. The grape cultivar was ‘Verdicchio’ grafted onto Kober 5BB rootstock. The soil has a medium texture tending to clay, fresh, fertile, deep and mainly grassy. The roots from the selected grapevine plants were collected in May. Vyn_3—The vineyard, located in Osimo (AN), site at 43°29′81″ N–13°26′12.83″ E, at (m a.s.l.) was planted in 2002. The grape cultivar was ‘Chardonnay’, grafted onto Kober 5BB rootstock. The vineyard is exposed to the south/east. The soil has a medium texture, with a high organic substance. Usually, the soil is ploughed both in the row and along the rows, with elimination of any infesting flora. The roots from the selected grapevine plants were taken in July.The investigated vineyards were not irrigated, the fertilizers were distributed in winter, and an additional green pruning was applied in spring and summer, as are the normal practices for the area. In all vineyards, an integrated pest management program was applied to control the main fungal diseases  and pests (moths).In each vineyard, fourteen sites were randomly selected. From each site, one symptomatic plant (I) and the neighboring asymptomatic plant (A) were selected for the study . In detaw/v) TB in lactoglycerol  for 20 min at 80 °C. Stained roots were observed with a light microscope, Nikon Eclipse E600 microscope . The degree of mycorrhizal colonization of each root segment and the abundance of arbuscules were estimated as described by Trouvelot et al. [m/lt) * 100; M% = (95n5 + 70n4 + 30n3 + 5n2 + n1)/lt; A% = a% * 0.01 M%. Where: lm is the total number of root segments in which mycelium were found; lt is the total number of the examined segments; n5 –n1 is the total number of root segments in which the degree of colonization intensity by mycorrhizal structures was 5-1, ; a% is related to absolute abundance of arbuscules for the segments in which arbuscules were present .For assessing the total AMF symbiosis, from each plant, secondary roots were collected from different points of plant’s roots system. Then, 50 sections of 1 cm-long pieces were randomly chosen and analyzed by non-vital staining with Trypan Blue (TB) . After ct et al. . In detaPhaeomoniella chlamydospora and Phaeoacremonium ultimum, performed by PCR [For each sample, two subsamples were obtained. From each subsample, 2 g of pooled roots ground in liquid nitrogen, then 200 mg of pulverized tissues were collected and put in 2 mL microcentrifuge tube for total DNA extraction. The CTAB (cetyl trimethyl ammonium bromide) procedure  modifiedd by PCR , was con2, dNTPs, PCR primers and the template DNA in the reaction buffer were 1.5 mM, 200 mM, 0.5 mM and 1 ng/μL, respectively. The amplified samples were checked on agarose gel, then the amplicons of asymptomatic and symptomatic samples were, respectively (or separately) pooled, obtaining two different samples. The two samples were purified using Wizard® SV Gel and the PCR Clean-Up System , then quantified with the Biophotometer (Eppendorf), in order to estimate the threshold of quantity  and quality . Total DNA extracted from all asymptomatic and symptomatic root samples, was amplified in total volume 25 μL using the primer pair ITS1F_KYO2 (5′TAGAGGAAGTAAAAGTCGTAA 3′) and ITS2_KYO2 (5′TTYRCTRCGTTCTTCATC 3′) , able tohttps://unite.ut.ee/; accessed 15 September 2021) pre-clustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic category to all OTUs at confidence threshold of 0.8. The RDP classifier uses the UNITE ITS database, which has taxonomic categories predicted to the species level.Fungal ITS1 amplicon pools related to symptomatic and asymptomatic plants were sent to GENEWIZ, Inc. , which provided library preparations, Illumina MiSeq sequencing and data analysis according to the manufacturer’s instructions of GENEWIZ Amplicon-EZ technology. Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH (1.9.6) against the UNITE ITS database  and F. mosseae (NCBI cod. FN377865.1), respectively, using Primer3web version 4.1.0. . The primers named RI/f (5′-GGCGTTATTGTCGCACCTAT3′), and RI/r (5′-CCTTGGTTTTTCAAGGGTCA-3′), that amplify a 248 bp PCR fragment, were developed for R. irregularis species and primers named FM/f (5′-CCTATGGATCCCCCTTTTGT3′) and FM/r (5′-AGATGCTGCAGAAGGCAAAT3′), that amplify a 190 bp PCR fragment, were developed for F. mosseae species.Specific primers were selected according to 28S rRNA and large subunit (LSU) ribosomal RNA genes for R. irregularis and F. mosseae in the root samples, their ability and specificity to detect the species present in the root samples was validated according to qPCR protocol followed by amplicon sequencing analysis. The qPCR analysis was performed according the Landi et al. [Before using the primers in ddPCR technology, for absolute quantification of i et al.  protocolTo validate the homology with the AMF species, the sequence analysis of qPCR amplicons obtained from RI/f-r, primers by I1 and A5 plant roots (named S1_V1 and S5_V2 samples) and FM V/f-r primers by I4 and A10 plant roots (named S4_V1 and S10_V3 samples) were performed. The fragments were sequenced by Genewiz  and subjected to bioinformatic analysis. Sequence similarity searches were performed using Blast analysis in NCBI. R. irregularis qPCR fragment S1_V1 (from 3.5 × 10−8 to 3.5 × 10−11 ng/reaction) and F. mosseae, qPCR fragment S10_V3 (from 2.8 × 10−8 to 2.8 × 10−11 ng/reaction) were amplified alone or spiked with DNA from Quercus ilex, holm oak, AMF nonhost roots . In addition, several dilutions  of DNA from roots of esca symptomatic plant I10 and asymptomatic plant A7 were also analyzed . DNA from grapevine leaf tissue was included as negative control. The same sample dilutions were tested in the ddPCR setup, with the exception of the spiked with 5 ng/reaction of DNA from holm oak. After sequencing and qPCR validation, a serial dilution of qPCR fragments amplified from root samples generating a qPCR standard curve was performed. Both the limits of detection (LOD) estimated from analysis of number of positive replicates, , and posR. irregularis and F. mosseae 28SRNA PCR fragment alone or combined with DNA from AMF holm oak nonhost roots, and of the I10 and A7 grapevine roots samples, previously described and analyzed on qPCR assay, were tested. The analysis was carried out in 20 μL of reaction mixture including 1× QX200 ddPCR EvaGreen supermix (Bio-Rad), 0.3 or 0.5 μM each primer. Each 20 μL reaction mixture was transferred into the DG8 cartridge. Next, Droplet Generation Oil (Bio-Rad) was added to the cartridge which was placed into the QX200 Droplet Generator™ (Bio-Rad). After droplet generation, the mixtures were carefully transferred to a ddPCR™ 96-well PCR plate (Bio-Rad) after which the plate was sealed at 180 °C using PX1™ PCR plate sealer (Bio-Rad). The amplification was performed in the thermal cycler, ICycler (Bio-Rad), with a ramp rate of 2 °C/s with the following protocol: 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 30 s and 58 °C for 1 min. The enzyme was deactivated at 4 °C for 5 min followed by 90 °C for 5 min. Droplets were read in a QX200 Droplet reader (Bio-Rad) after which the ddPCR data were analyzed using QuantaSoft Version 1.6.6. The script analyzed the data of the signals exported from the QuantaSoft software, with its automatic threshold defined or with a selected, manually defined, threshold applied. This incorporates the calculation of the basic parameters of the ddPCR ; the mean copies per partition and the total volume of the partitions measured, as defined by the digital MIQE guidelines [The ddPCR assay was performed using QX200 Droplet Digital PCR system . With the aim to setup and validate the ddPCR assay, serial dilution of both, idelines . Two post-test. Differences were considered significant when p < 0.01. The same data sets were tested for correlations using the Pearson’s coefficient (r) p < 0.05. The difference among AMF symbiosis in the esca symptomatic and asymptomatic plants according to the vineyards were statistically analyzed . The arcsin square root transformation was used to normalize the percentage ratios data prior to statistical analysis. The data set of AMF colonization was subjected to one-way ANOVA for mean comparisons, standard deviation (SD) and significant differences calculated according to Duncan’s Multiple Range Test, p < 0.05. Data sets according to AMF colonization of symptomatic and asymptomatic plants, detected by non-vital staining, were analyzed according to Student’s paired R. irregularis and F. mosseae for each plant were statistically analyzed . Data set of ddPCR absolute quantification was subjected to one-way ANOVA for mean comparisons, standard deviation (SD) and significant differences calculated according to Duncan’s Multiple Range Test, p < 0.05.For qPCR set-up using I10 and A7 root samples and spiked samples see   and arbuscular abundance (A%),  than the neighboring asymptomatic plants  of around of 100%. Relating to each of the 14 sites identified in the vineyards, all the esca symptomatic plants showed higher significative native mycorrhizal colonization related to both mycelium colonization (M%)  to 48.4% (Vyn_2) a, while A total of 66,352 quality reads were obtained from asymptomatic and symptomatic pool samples, grouped into 241 OTUs for taxonomic affiliation with Ascomycota, Basidiomycota, Cercozoa, and Glomeromycota, at phylum level. Other different OTUs sequences generically assigned to fungi kingdom were also detected a. From tR. irregularis, while the primers FM/f-r, amplify for a singular amplicon of 80.5 °C TM, specific for F. mosseae. Sequence analysis of the PCR amplicons indicated as isolates S1_V1 (NCBI accession number MK513942) and S5_V3 (MK513943) were homologous to >98% of the reference sequences related to 28S rRNA gene of R. irregularis, NCBI accession number HF968988.1, and FJ235574.1. In detail, for R. irregularis primers, we identified a 248-bp that included the nucleotide substitutions of A→G; G→A; G→C; A→G; . Blast analysis of the nucleotide sequences indicated as isolates S4_V1 (MK513940) and S10_V3 (MK513941) revealed a high nucleotide identity with the reference sequences related to the LSU gene of F. mosseae  The F. mosseae primers identified a 190-bp that included the nucleotide substitutions of A→C ; CT→GC  , according to primers RI/f-r, specific for 77865.1) .R. irregularis ranging from 22.3 + 0.12 quantification Cycle (Cq) for the 3.5 × 10−8 ng/reaction, to 31.6 ± 1.1 Cq for the 3.5 ×10−11 ng/reaction. Really, the probability of replicates’ detection of the last dilution was lower than 95%, underlining the limit of detection of the gene , to 32.8 ± 0.6 Cq (2.8 × 10−11 ng/reaction)  . The qPCfication .The analyses of four-point serial dilutions related to I10 and A7 grapevine samples root indicate a total inhibition of qPCR amplification testing 100 and 50 ng DNA/reaction for both species, while a positive sample was detected at 5 and 0.5 ng DNA/reaction but not for all replicates .R. irregularis species and about 5 to 3020 copies/20 μL/PCR reaction for F. mosseae  showed a good degree of linearity (R2 > 0.996) in the range of about 8 to 2940 copies/20 μL/PCR reaction for  mosseae . No ddPCR. irregularis and F. mosseae, with detection from 50 to 0.5 ng DNA of roots/reaction, while the amounts of DNA from roots per reaction showed saturation reached at 100 ng of DNA  for both g of DNA a,b. In t0 μL/PCR . No amplF. mosseae ranged from 29.5 ± 3.53 copies/20 μL (20 μL reaction was related to 50 ng DNA of grapevine roots) (plant A1), to 87 ± 4.2 copies/20 μL (plant A3), while in symptomatic plants from 47.5 ± 2.12 copies/20 μL (I1 plant), to 157 ± 15.5 copies/20 μL (I4 plant), were detected , to 400 ± 69.8 copies/20 μL (A2 plant), while in the roots from symptomatic plants the amount ranged from 92.5 ± 7.7 copies/20 μL (I3 plant), to 358 ± 9.9 copies/20 μL (I2 plant)  b. OveralF. mosseae ranged from 24.5 ± 4.5 copies/20 μL (A6 plant), to 92.5 ± 7.7 copies/20 μL (A8 plant), in esca asymptomatic plants, and from 22.7 ± 3.3 copies/20 μL (I6 plant), to 95.7 ± 6.3 copies/20 μL (I7 plant) in symptomatic plants  to 358 ± 14 copies/20 μL (A8 plant), were detected in the esca asymptomatic plants, and from 233.5 ± 19 copies/20 μL (I7 plant), to 410 ± 10.6 copies/20 μL (I6 plant) in asymptomatic plants , to 57.3 ± 1.8 copies/20 μL (A11 plant) in the esca asymptomatic plants, while it ranged from 36.5 ± 14.8 copies/20 μL (I10 plant), to 71.5 ± 4.9 copies/20 μL (I11 plant) in the symptomatic plants , to 355 ± 50.2 copies/20 μL, (A9 plant), while in symptomatic plants ranged from 61.5 ± 4.9 copies/20 μL (I12 plant), to 706 ± 37.4 copies/20 μL (I10 plant)  b. OveralGlomeromycota phylum, which includes all AMF species [The management of esca must be integrated with an interdisciplinary approach . Since t species , in the R. irregularis and F. mosseae species [R. irregularis and F. mosseae were detected in all analyzed plants. Although a great variability was found in the vineyards, the species R. irregularis is more often more present than F. mosseae. However, our data suggest higher gene copy numbers of both species in symptomatic plants than asymptomatic ones for most of all the analyzed sites or vineyards, but these numbers were not necessarily statistically significant for all cases. AMF in nature may differ in the amount of benefit they provide to hosts, but it is impossible to indicate any single partner as universally ‘beneficial’ or ‘not beneficial’ given the elevated complexity of interactions and conditions implicit in microorganism community. Our work highlights that there was no species-specific relationship among AMF and symptomatic or non-symptomatic esca plants, but that the highest amount of AMF symbiosis observed in symptomatic plants involves the whole mycorrhizal community and their rhizosphere relationship.To analyze if this result was AMF species-specific, we have set up a method to quantify the native species among those commonly associated with the roots of the grapevine as  species ,27,55 us species . In our F. mosseae in comparison to other AMF species [New studies are now required to understand what appears to be a paradox. Usually, clear relationships between root-associated fungal communities’ composition and plant health status were demonstrated in several plant-mycorrhizae-pathogen interactions involving both woody perennial crops and annual crops ,57,58,59 species ,62. Howe species ,63 or fu species , on leavR. irregularis has reduced both the number of root lesions and the severity of diseases caused by black foot disease pathogens [Ilyonectria spp., and changes in the function of the rhizosphere microbial community [I. liriodendra, a weak pathogen known for causing black foot disease, and R. irregularis AMF species, the amount of both AMF and pathogen were highest, unlike the results seen in control plants inoculated with one of the two. Further, the presence of the mycorrhizal fungus did not enhance the physiological parameters of the infected plants [Phytophthora fragariae [Fusarium oxysporum conidia [Concerning grapevine trunk diseases, the inoculation of the roots with the athogens . AMF havommunity . Howeverd plants . By contd plants . These rd plants . Similard plants ,68,69. Od plants . Then, td plants . On the d plants ,73,74. Ad plants ,38. The d plants ,14. Amond plants . In escad plants . Other wragariae , the rooragariae . In myco conidia . Then, t conidia . A syner conidia . Somethi conidia . Then, sThis investigation carried out on natural mycorrhizal state of plants affected by esca strengthens the knowledge on how the diseased plant interacts with beneficial microorganisms, modifying their concentration. In synthesis, the analysis of the AMF native symbiosis in grapevine roots showed higher levels of total mycorrhization in the symptomatic plants than in the asymptomatic plants. However, the protocol developed in this work according the ddPCR technology emphasizes that this difference is not associated with individual mycorrhizal species, but instead the entire AMF community contributes to this result. In this work we have focused on the esca symptoms–AMF relationship in grapevine roots. The investigation of entire microbial community involved in the grapevine roots, symptomatic and asymptomatic to esca, will be able to clarify many aspects. This work contributes to a better understanding of the interactions between native AMF and the fungi related to esca, and it underlines the role of mycorrhizae as bioindicators of the soil changes taking place in the rhizosphere. These results underline that the interactions between plants and their rhizosphere microbial communities are an important point to consider for the development of strategies for the sustainable management of grapevine esca disease.


Despite the continuous developments in pharmacology and the high therapeutic effect of new treatment options for patients with hematological malignancies, these diseases remain a major health issue. Our study aimed to synthesize, analyze in silico, and determine the biological properties of new melphalan derivatives. We obtained three methyl esters of melphalan having in their structures amidine moieties substituted with thiomorpholine (EM–T–MEL), indoline (EM–I–MEL), or 4-(4-morpholinyl) piperidine (EM–MORPIP–MEL). These have not yet been described in the literature. The in vitro anticancer properties of the analogs were determined against THP1, HL60, and RPMI8226 cells. Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity , and their ability to induce apoptosis . Changes in mitochondrial membrane potential were examined using the specific fluorescence probe JC–1 . The EM–T–MEL derivative had the highest biological activity, showing higher cytotoxic and genotoxic properties than the parent drug. Moreover, it showed a high ability to induce apoptosis in the tested cancer cells. This compound also had a beneficial effect in peripheral blood mononuclear cells (PBMC). In conclusion, we verified and confirmed the hypothesis that chemical modifications of the melphalan structure improved its anticancer properties. The conducted study allowed the selection of the compound with the highest biological activity and provided a basis for chemical structure-biological activity analyses. Blood cancers are currently among the most serious medical problems. Recent data have shown that one in eight cancer cases worldwide come from blood cells, bone marrow, or the lymphatic system. Malignant hematopoiesis, such as leukemia and lymphomas, is divided into more than 100 subtypes with varying survival rates ,3,4.Treatment of MM involves a combination of drugs with distinct mechanisms of action, including immunomodulating drugs, proteasome inhibitors, monoclonal antibodies, alkylating agents, and histone deacetylase inhibitors. Despite these numerous modern therapies, high doses of melphalan followed by autologous stem cell transplantation remain key in treating MM patients eligible for transplantation. Melphalan is a bifunctional alkylating agent. Each of the two bis-2-chloroethyl melphalan groups forms carbonium intermediates that cause alkylation by covalent bonding with nitrogen atoms in the DNA of the guanine molecule at the 7-position. This leads to cross-links between the two DNA strands, preventing transcription and DNA replication and inhibiting the cell cycle. By targeting highly proliferating cells, including malignant plasma cells, this drug causes cell death .A major limitation of chemotherapy, including melphalan treatment, is the lack of drug selectivity, which has adverse effects on healthy tissues. High doses of anticancer drugs also weaken patients’ immunity by affecting the body’s biomolecular defense system . The theOur previous studies  have shoSamples of melphalan derivatives used in biological research were obtained by three-stage chemical synthesis from commercially available substrates .The first step in the synthesis involved the reaction of commercial melphalan (MEL) with a 50% molar excess of N-formylmorpholine dimethylacetal (DMA–MOR) in methanol at room temperature. The morpholine derivative of melphalan (MOR–MEL) obtained was the starting material for the further synthesis of all new derivatives.MOR–MEL was then reacted with a 100% molar excess of the appropriate amine (thiomorpholine (T), indoline (I), or 4-(4-morpholinyl) piperidine (MORPIP)) in methanol at room temperature to obtain the appropriate amine derivative of melphalan . Initially, we intended to obtain a thiazolidine derivative of melphalan in place of MEL–MORPIP, but during the experiments, it turned out that MOR–MEL did not react with thiazolidine (TZ). Attempts to change the reaction conditions and the synthesis method were unsuccessful. Therefore, it was decided to discontinue further work and replace the thiazolidine derivative with 4-(4-morpholinyl) piperidine.In the last stage, the amine derivatives of melphalan (MEL–(T/I/MORPIP)) were esterified. Ester synthesis was carried out with 2,2-dimethoxypropane (DMP) in an acidic environment (approximately 10% addition of concentrated hydrochloric acid) to give the corresponding amine derivatives of melphalan methyl esters in the form of thick oils free of amines. All three amines were then converted to a hydrochloride form with a 5 M solution of hydrogen chloride gas in ethyl acetate.Because of these structural changes, their molecules were much less polar than melphalan itself. This was confirmed by HPLC analysis , which sTo determine whether the test compounds belonged to the druglike group, the rules of Lipinski and the definition of Veber were used. According to the five Lipinski rules, a drug candidate should meet the following criteria: molecular weight (MW) ≤ 500 Da; lipophilicity described as logP ≤ 5; number of hydrogen bond acceptors (HBAs) ≤ 10; and number of hydrogen bond donors (HBDs) ≤ 5 . The datAll the test compounds complied with Lipinski’s rules and had only one violation of Veber’s rules. The number of rotational bonds was slightly greater than the number specified in Veber’s rule. We also considered that none of the new compounds was a substrate for P-glycoprotein.2; solubility (INSOLU), log S not higher than 6; saturation (INSATU), fraction of carbons in the sp3 hybridization not less than 0.25; and flexibility (FLEX), no more than nine rotatable bonds.To illustrate the distribution of the most important physicochemical properties in the body, we also prepared radar charts  from theFor all the compounds tested, five of the six properties analyzed were located in the pink area, which proved their optimality and compliance with the properties typical for compounds belonging to the group of druglike compounds. Based on these properties, it was also concluded that the test compounds were predicted not to be orally bioavailable because they were too flexible. Intravenous administration would be recommended.50 parameter  was observed in human acute monocytic leukemia cells. The IC50 concentration of this derivative in this cell line was nearly 10 times lower than that of melphalan. In HL60 cells, the IC50 value of the EM–T–MEL derivative was approximately five times lower than that of the unmodified compound. Cells of the multiple myeloma line also showed high  cytotoxic activity after incubation with EM–T–MEL. At the same time, a decreased cytotoxic effect of EM–T–MEL was observed against peripheral blood mononuclear cells (PBMC). This analog was approximately 2.5 times less cytotoxic to PBMCs than melphalan. The cytotoxicity of EM–MORPIP–MEL was similar against THP1 and higher against HL60, RMPI8226, and PBMCs compared to the parent drug. Because of this, EM–MORPIP–MEL was eliminated from further analyses. One concentration per cell line was selected for testing of the next parameters: RPMI8226, 3 µM; HL60, 0.7 µM; THP1, 0.3 µM, which were the same as in previous studies evaluating the biological properties of melphalan analogs +) in the positive ESI mode. The results of the measurements were processed using the MassLynx 4.1 software (Waters) incorporated with the instrument (see High-resolution mass spectrometry (HRMS) measurements were performed using a Synapt G2–Si mass spectrometer (Waters) equipped with an ESI source and quadrupole-time-of-flight mass analyzer. The mass spectrometer was operated in the positive ion detection mode. The optimized source parameters were: capillary voltage 3.2 kV, cone voltage 30 V, source temperature 110 °C, desolvation gas (nitrogen) flow rate 600 dmment see .Synthetic procedure and analytical data of this compound -3-[4-[bis(2-chloroethyl) amino] phenyl] propanoic acid) come from: Ireana Oszczapowicz, Małgorzata Łukawska, Joanna Tobiasz, Anna Porębska, Agnieszka Owoc. Nowe pochodne melfalanu, sposób ich wytwarzania, zawierający je środek farmaceutyczny oraz zastosowanie medyczne. PL220880B1, p. 9, example 3. (available in polish language version only).N–(dimethoxymethyl)morpholine was added to this solution, and the mixture was stirred until the substrate disappeared . Then, the mixture was evaporated to dryness, and 4 ml methanol was added, followed by 35 ml of diethyl ether. The precipitated product was then filtered, washed with diethyl ether, and dried under vacuum. The expected product was obtained after recrystallization from the methanol/diethyl ether mixture and had the following characteristics. Mass: 310 mg (0.77 mmol). Yield: 77%. Off–white crystalline powder. 97.0% (HPLC). RRT (in ref. to MEL): 1.154, 1.201IR (cm−1): 1697 (>C=N–). 1H NMR : 9.30 , 7.74 , 7.06 , 6.71 , 3.90 , 3.77 , 3.70 , 3.28 , 3.15 –CO2H), 2.78 . Elemental analysis: calc. C—53.85%, H—6.28%, N—10.47%; found: C—53.65%, H—6.16%, N—10.27%.The reaction was carried out in an argon atmosphere. First, 305 mg (1 mmol) of melphalan was dissolved in 30 mL of methanol. Then, 195 mg (1.2 mmol) of New Melphalan Derivatives—General Procedure:First, 1.61 g (4.0 mmol) of MOR–MEL was dissolved in 240 ml of methanol. Then, 8.0 mmol of the appropriate amine was added to this solution, and the mixture was stirred 18–22 h (depending on the amine) at room temperature (TLC control). Next, the mixture was evaporated to dryness, obtaining the crude product as an oil that was then purified by preparative HPLC, leading to the amine derivative of melphalan .(g)/ethyl acetate to obtain the hydrochloride that precipitated from the solution. The solid was filtered off, washed with cold ethyl acetate, and dried under vacuum at 30 °C to obtain the final product.Then, 1 mmol (418–485 mg) of the melphalan amine derivative, 10 mL DMP, and 1 mL concentrated HCl were stirred for one hour at room temperature. The mixture was evaporated to dryness, and the resulting oil was purified by column chromatography to obtain the appropriate methyl ester with a purity of over 95% (HPLC). The free amine was then dissolved in 5 mL 4 M HClThe following compounds were synthesized in this manner:d20: −37.94° . IR (cm−1): >C=O (1743), >C=N– (1682). 1H NMR : OCH3 , CchiralH , CHAHBCchiralH , CHAHBCchiralH , C5aromH+C5′aromH , C6aromH+C6′aromH , N–CH2–CH2–Cl , N–CH2–CH2–Cl , –N=CH– , C2indH , C3indH , C4indH+C6indH , C5indH , C7indH . 13C NMR : OCH3 (52.51), >C=O (170.4), C3 (36.40), C4 (124.10), C5/C5′ (130.50), C6/C6′ (111.80), C7 (145.15), N–CH2–CH2–Cl (51.96), N–CH2–CH2–Cl (40.96), C2ind (48.07), C3ind (27.24), C3aind (132.50), C4ind (126.05), C5ind (127.65), C6ind (111.11), C7aind (149.98). HRMS: calc. for C23H28N3O2Cl2: 448.156; found: 448.155.Off-white crystalline powder. Yield: 25% . Purity: 98.80% (HPLC). RRT: 1.690. Mp.: decomposition below the melting point. [α]d20: –78.57° . IR (cm−1): >C=O (1748), >C=N– (1703). 1H NMR : OCH3 , CchiralH , CHAHBCchiralH , CHAHBCchiralH , C5aromH+C5′aromH , C6aromH+C6′aromH , N–CH2–CH2–Cl , –N=CH– , NCH2CH2S , NCH2CH2S , 4H). 13C NMR : OCH3 (52.49), >C=O (170.40), C2 (61.45), C3 (36.25), C4 (124.10), C5/C5′ (130.64), C6/C6′ (111.80), C7 (145.19), N–CH2–CH2–Cl (52.04), N–CH2–CH2–Cl (41.09), –N=CH– (155.13), NCH2CH2S , NCH2CH2S . HRMS: calc. for C19H28N3O2Cl2: 432.128; found: 432.127.Off–white crystalline powder. Yield: 30% . Purity: 96.27% (HPLC). RRT: 1.482. Mp.: decomposition below the melting point. [α]d20: –74.39° . IR (cm−1): >C=O (1744), >C=N– (1696). 1H NMR : OCH3 , CchiralH , CH2CchiralH , C5aromH+C5′aromH , C6aromH+C6′aromH , N–CH2–CH2–Cl , N–CH2–CH2–Cl , –N=CH– , NpipCHAHB , NpipCH2CHAHB , NpipCH2CH2CH , NCHAHBCH2O , NCH2C2HO . 13C NMR : OCH3 , >C=O , C2 , C3 , C4 , C5/C5′ , C6/C6′ , C7 , N–CH2–CH2–Cl , N–CH2–CH2–Cl , –N=CH– , NpipCH2 , NpipCH2CH2 , NpipCH2CH2CH , NCH2CH2O , NCH2CH2O (63.10). HRMS: calc. for C24H37N4O3Cl2: 499.224; found: 499.224.Off–white crystalline powder. Yield: 41% . Purity: 97.85% (HPLC). RRT: 1.105. Mp.: decomposition below the melting point. [α]http://www.swissadme.ch/, accessed on 5 December 2021) according to rules described in [A QSAR analysis was performed to see whether the new compounds met the criteria for druglike compounds. To provide a global pharmacokinetics profile of the test molecules, we used the freely accessible SwissADME web tool (ribed in .® CCL–155™)), promyelocytic leukemia cells (HL60 (ATCC® CCL–240™)), and acute monocytic leukemia cells (THP1 (ATCC® TIB–202™)) were obtained from the American Type Culture Collection . PBMCs were isolated from buffy coat purchased from the Central Blood Bank . PBMCs were isolated with Histopaque 1077  by density gradient centrifugation at 300× g for 30 min at 22 °C. The final concentration of lymphocytes was estimated by trypan blue  exclusion assay. All investigated cells were suspended in RPMI 1640 medium supplemented with 1% phytohemaglutinin (only in PBMC growth medium), 10% fetal bovine serum, penicillin (10 U/mL), and streptomycin (50 μg/mL) in standard conditions: 37 °C, 100% humidity, and an atmosphere of 5% CO2 and 95% air. Cell viability was systematically controlled using trypan blue . In all experiments, cells in the logarithmic phase of growth were used when their viability was above 95%.Myeloma cancer cells  and its new derivatives  were investigated with the use of resazurin sodium after 48 h of incubation with the test compounds. Living cells were metabolically active and had the ability to reduce resazurin via mitochondrial reductase to a highly fluorescent dye, resorufin. The amount of product formed was directly proportional to the number of viable cells, while the intensity of the color produced was a quantitative measure of cell survival.4 cells/well and incubated with various concentrations of test compounds at 37 °C. After 48 h of incubation for each of the wells, resazurin solution  was added, and the samples were incubated for 90 min. Fluorescence measurement was performed at ~530 nm excitation and ~590 nm emission using a Fluoroskan Ascent FL plate reader .Cells were grown in 96-well black plates at 1.5 × 10PI is negatively charged and penetrates only cells with damaged cell membranes, which allows the identification of necrotic cells or cells in the late stages of apoptosis. Hoechst 33342, on the other hand, freely penetrates the intact membrane of living and early-apoptotic cells, thus enabling their identification. As a result of the dye penetrating the intact biological membranes, the DNA of the cell nucleus is stained a light blue color. The dye fluorescence intensity is related to the degree of DNA packing, which allows, on the basis of the intensity of fluorescence of the fluorochrome in the cell nucleus, distinguishing strongly fluorescent apoptotic cells containing highly condensed chromatin from weakly fluorescing living cells containing looser chromatin. The simultaneous use of two fluorescent dyes (PI and Hoechst 33342), of which the mechanisms of penetration into the cell and the fluorescence spectra are different, allowed the identification of four types of cells in the sample: live cells , early apoptotic cells (bright light blue fluorescence), late apoptotic cells (pink-purple fluorescence), and necrotic cells (intense red fluorescence).5 cells in 1 mL of PBS. The fluorescent dyes Hoechst 33342 (0.13 mM) and PI (0.23 mM) were added and incubated for 10 min in the dark. The cell suspension was placed on a microscope slide. At least 300 cells per sample were counted in triplicate under an Olympus fluorescence microscope using an NB filter. The percentage of the particular cell types was determined from the total number of cells.THP1, HL60, and RPMI8226 cells were incubated with the test compounds for 4, 24, and 48 h. After an appropriate incubation time, cells were removed from the culture dishes, centrifuged, and suspended at a final concentration of 1 × 10Visualization of cells stained with annexin V–FITC and PI was applied according to the protocol of the manufacturer, BioVision Incorporated (annexin V–FITC apoptosis kit). This method is a useful tool for distinguishing live cells (not stained with any fluorochrome) from early apoptotic cells (stained with annexin V–FITC only), late apoptotic cells (stained with annexin V–FITC and PI), and necrotic cells (stained with PI only).5 cells/well and treated with the test compounds for 24 h. The cells were then washed with PBS and resuspended in 300 μL binding buffer. Then, 3 μL annexin V–FITC and 3 μL PI was added. The mixture was incubated for 10 min on ice. Samples were centrifuged, applied to chamber slides, and then visualized using fluorescence microscopy . Magnification was 200×.Briefly, cells were seeded at a density of 2.5 × 10The activation of executive (caspases 3/7) and initiating caspases  and internal (caspase 9) pathways) is widely known as a reliable indicator of cell apoptosis. DEVD–ProRed™, IETD–R110, and LEHD–AMC were used as fluorogenic markers for the activity of caspases 3/7, 8, and 9, respectively. Following caspase cleavage, the DEVD–ProRed, IETD–R110, and LEHD–AMC caspase substrates generate three different fluorophores, ProRed™ (red fluorescence), R110 (green fluorescence), and AMC (blue fluorescence), which can be easily monitored using flow cytometry.6), and then the compounds were added at the appropriate concentration. After an appropriate incubation time , cells were transferred to cytometric tubes, resuspended in a prepared working solution containing the appropriate substrate (50 µL of substrate into 10 mL assay buffer), and incubated for 30 to 60 min at room temperature, protected from light. Cells were analyzed using a flow cytometer  using the following wavelengths: caspase 3/7, ex: 488 nm, em: 620 nm; caspase 8, ex: 490 nm, em: 525 nm; caspase 9, ex: 355 nm, em: 470 nm. The minimum number of events recorded in the sample was 20,000. The results are presented as the median fluorescence in the corresponding channel: PE–Texas Red, FITC, DAPI as % relative to control.THP1, HL60, and RPMI8226 cells were seeded in plates (1 × 106) were centrifuged , and the culture medium with phenol red was discarded. PBS was added to the pellet, and cells were centrifuged. In the next step, cells were suspended in a working culture medium without phenol red with JC–1 (10 µM) and incubated at 37 °C for 30 min in the dark. The cells were then centrifuged and washed with PBS to remove the dye, which otherwise could have adsorbed on the microplate well plastic and distorted the measurements. Cells prepared in this way were seeded into 96-well microplates at 8 × 104 cells per well. The cells were incubated with the test compounds or CCCP, a mitochondrial uncoupling agent (20 and 100 µM), for 4–24 h. At the end of the treatment, the fluorescence of both JC–1 monomers and dimers was measured on a Fluoroskan Ascent FL microplate reader using filter pairs of 530/590 nm (dimers) and 485/538 nm (monomers). The results shown in the figures are expressed as a ratio of dimer fluorescence to monomer fluorescence in relation to the control fluorescence ratio, taken as 100%. The cells presented in the images were incubated with drugs for 48 h. JC–1 fluorescence was photographed immediately after drug treatment using an inverted Olympus IX70 fluorescence microscope .Cells collected from the culture vessel  and treated with MEL, EM–I–MEL, and EM–T–MEL at the indicated concentrations for 4, 24, and 48 h at 37 °C. The cells were washed with PBS, and the pellet was resuspended in 50 μL of 0.75% low-melting point (LMP) agarose in PBS, pH 7.4. The samples were applied to heated primary slides that had previously been coated with 1% normal-melting point (NMP) agarose. Slides prepared in this manner were then subjected to alkaline lysis  for a minimum of 1 h at 4 °C. Slides were then placed in developing buffer  for 20 min and then in electrophoresis buffer . Electrophoresis was conducted at 29 V and 30 mA for 20 min. Slides were then stained with DAPI (2 μg/mL) in the dark. Fifty randomly selected cells from each slide were analyzed using an Eclipse fluorescence microscope  attached to a COHU 4910 video camera  equipped with a UV–1A filter block and connected to the Lucia-Comet v. 6.0 image analysis system . The percentage of DNA in the comet tail is an indicator of DNA damage.The cells were plated in 12-well plates . Analysis of variance (ANOVA) with Tukey’s post hoc test was used for multiple comparisons. All statistics were calculated using STATISTICA . A Research in cancer biology has led to the elucidation of the mechanism of action of anticancer agents. It has also provided a basis for the successful design of new drugs. The development of new analogs of anticancer drugs is a complicated task. Applying chemical principles and analyzing chemical structures together with biological activity enables the synthesis of derivatives with higher biological properties than the parent drug. In vitro studies have confirmed the hypothesis that chemical modification of melphalan leads to significantly higher cytotoxic and genotoxic activities. These modifications include esterifying the carboxyl group and replacing the amino group with amidine containing a thiomorpholine residue with two heteroatoms (N and S) in its structure. The generation of apoptotic bodies and reductions in the mitochondrial membrane potential suggest that these compounds induce apoptosis, the preferred type of cell death. This studies allowed us to select EM–T–MEL as the most active analog. It also significantly deepened the knowledge on improving the biological activity of the currently used chemotherapeutic agent .


R201C induces cystic growth more effectively in ductal than acinar organoids, whereas KRASG12D is more effective in modeling cancer in vivo when expressed in acinar compared with ductal organoids. KRASG12D, but not GNASR201C, induces acinar-to-ductal metaplasia-like changes in culture and in vivo. We develop a renewable source of ductal and acinar organoids for modeling exocrine development and diseases and demonstrate lineage tropism and plasticity for oncogene action in the human pancreas.The exocrine pancreas, consisting of ducts and acini, is the site of origin of pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the genesis and progression of human pancreatic diseases, including PDAC, is limited because of challenges in maintaining human acinar and ductal cells in culture. Here we report induction of human pluripotent stem cells toward pancreatic ductal and acinar organoids that recapitulate properties of the neonatal exocrine pancreas. Expression of the PDAC-associated oncogene GNAS  •Generation and long-term maintenance of human acinar organoids from stem cells•R201C induces cystic growth more effectively in ductal than in acinar organoidsGNAS•G12D induces cancerous lesions more often from acinar versus ductal organoidsKRAS•G12D in acinar organoids induces an acinar-to-ductal metaplasia-like phenotypeKRAS G12D or GNASR201C induced cell-lineage-specific phenotypes and cell-state plasticity in culture and in vivo, identifying a renewable source of ductal and acinar organoids for modeling development and diseases of the exocrine pancreas.Huang et al. describe conditions for generation of pancreatic ductal and acinar organoids from human stem cells. Expression of KRAS KRAS mutations are observed in more than 80% of PanINs and associated with invasive PDAC  and acinar (~85%) epithelia. The most common type of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), is thought to arise from the exocrine compartment . Precursive PDAC , identifant KRAS , and adjKRAS, G12D is the most frequent  supports an acinar cell of origin for PDAC  to assume a duct-like state  are a powerful platform to generate multiple cell types in culture . We and ar cells , whetherPDX1-positive PP cells obtained with an established protocol  were useThe acinar organoids were small (~20–105 μm in diameter on day 16) with no visible lumen. In comparison, ductal organoids were large (~50–220 μm in diameter on day 16) and cystic with visible hollow lumens B. MorphoSOX9, HNF1B, CEL, PNLIP, CTRB1, and CTRC, showed trends in average expression across culture types that were in line with organoid differentiation (We used single-nucleus RNA (snRNA) sequencing as an unbiased assessment of the molecular differences between the two organoid lineages. Samples were multiplexed in the 10X Chromium system to obtain single-nucleus transcriptomes from PP cells and day 8 acinar and ductal organoids . Uniformntiation A but werRBPJL/RBPJ, an indicator of acinar specification  and the pancreatic acinar markers PTF1A and chymotrypsin C (CTRC) D demonstPDX1, CFTR, and NKX6.1 compared with duct-like organoids  and acinar  markers over time  or GNASR201C (a mutation observed frequently in IPMN) and induced gene expression on day 8 of the differentiation protocol   A. GNASR2rganoids B. Duct-lincrease C. Overexrganoids C. More trganoids D, demonsGNASR021C but not GNASWT expression increased cell numbers in organoid cultures . More than 30% of duct-like organoids expressing GNASR201C stained positive for Alcian blue , ductal , and acinar (PTF1A) epithelia  CRISPR Prkdc Il2r Gamma (NCG) mice (N = 12). We did not observe any outgrowth that stained positive for human KRT19 or class I major histocompatibility complex (HLA) or human nuclear antigen. However, two of eight mice sacrificed after 8 weeks and one of four mice sacrificed after 14 weeks showed abnormal regions representing proliferative glands and metaplasia, likely because of injection-induced reactions   B and S5C (p-ERK) . Express reports , expressp16INK4A E and S5Fsitivity A and 5B sitivity C, suggesd images D and 5E.le lumen D and 5E rganoids F. These KRASG12D, we assessed changes in expression of markers of PP , ductal , and acinar  epithelial cells. In duct-like organoids, KRASG12D upregulated PDX1, NKX6.1, and SOX9 and downregulated and CFTR, suggesting change toward a progenitor state  staining. AB signals were increased by KRASG12D and enhanced further by TGF-β (KRASG12D-expressing duct-like organoids with low frequency (~2.5%), and this was lost in TGF-β-treated organoids. MUC2 expression was not detected in acinus-like organoids  ; without KRASG12Dexpression) or DOX-containing food  and monitored by palpation twice a week. All mice were sacrificed 8 weeks after transplantation.To investigate KRASG12D exhibited a wide spectrum of growth patterns, and three had no growth. Four had lesions with large, convoluted, dilated ductal structures lined with epithelium showing mild to moderate dysplastic changes, analogous to IPMN in humans  associated with IPMN neoplastic lesions. Expression of GNASR201C in acinar cells failed to induce expression of MUC2, which is consistent with a previous study where acinus-specific expression of GnasR201C in mouse models was not sufficient to initiate PDAC Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contact, Senthil K Muthuswamy  mice of 6-8 week old were purchased from Charles River . Results for each experimental group are summary of two to three independent operations using independent organoid cultures.Animal experiments performed in this study followed protocols approved by the institutional animal care and use committee at Beth Israel Deaconess Medical Center . Male immunodeficient NOD CRISPR Human embryonic stem line HUES8 was generated at Harvard University and has a normal 46 XY karyotype . Induced pluripotent stem cell lines 11 and 13 were generated by the Melton laboratory at Harvard University. Donor for line 11 is a 26 year old healthy male. Donor for line 13 is a 27 year old healthy female. pINDUCER21 (Addgene #46948) was used for transgene expression.Pancreatic progenitor cells were derived from Hues-8 cells by Dr. Douglas Melton’s group, following a previously published protocol . The PP1∗π∗Area/Perimeter2.For phase contrast imaging, organoids were seeded in Falcon culture chamber slide and images were taken every two days using 10X objective. For each time point, three independent cultures were imaged with at least 100 organoids imaged per culture. For analysis of phase contrast images, CellProfiler program  was usedFor direct immunostaining, organoids were seeded in Falcon culture chamber slides. At the time of analysis, organoids in chambers were washed once with PBS then fixed with 4% PFA for 40 minutes. After fixation, organoids were washed three times with PBS-glycine (100mM glycine) with 10 minute incubation each wash. Organoids then were permeabilized by 0.5% Triton X-100 of 5 minute incubation. After permeabilization, organoids were blocked using blocking buffer  for one hour. For primary antibody staining, antibodies were diluted with the blocking buffer and organoids were incubated with the dilution for 2-4 hours at room temperature then washed three times with blocking buffer (10 minutes incubation per wash). For secondary antibody staining, goat anti-mouse IgG or goat anti-rabbit IgG antibodies from Invitrogen were diluted at 1:400 in blocking buffer then incubated organoids with the dilution for 1.5 hour. After the secondary staining, organoids were washed with PBS three times and stained with DAPI for 5 minute. Slides were mounted with DAKO fluorescence mounting media. Alcian blue staining were performed using the Alcian Blue (pH2.5) Stain Kit . Imaging of slides were conducted under Zeiss LSM880 confocal system, using 20X objective or a Keyence BZ microscope. Image analyses were performed using CellProfiler.2O for 10 minute per incubation. Antigen retrieval were done by incubating slides with citrus buffer at high pressure heating for 15 minutes. After this, slides were washed twice with PBS then blocked with blocking buffer for one hour. Primary staining and secondary staining were done by incubating slides with antibodies for one hour at room temperature, with three washes in between using blocking buffer. After secondary staining, slides were washed with PBS three times and stained with DAPI for 5 minutes. Slides were mounted with DAKO fluorescence mounting media. Slides were imaged using Zeiss LSM or Keyence Vision System. Antibodies used: SOX9, 1:200, #AB5535, Millipore ; CAII, 1:100, #sc-133111, Santa Cruz; PTF1A, 1:100, #s25-763, BD ; CTRC, 1:100, #11456-T24, Sino Biological; CPA1,1:100, #10504-RP02, Sino Biological; Muc2, 1:100, #sc7314, Santa Cruz; Muc5AC, 1:100, #sc33667, Santa Cruz; phosphor-ERK, 1:200, #4370, Cell Signaling; KRT19, 1:200, #ab9221, Abcam;For preparation of paraffin blocks, organoids in chamber slides were fixed with 4% PFA for 2 hours then embedded with Histogel in cryomold. Histogel capsules were transferred into cassette and processed at BIDMC histology core using standard paraffin embedding protocols. For staining of slides, paraffine sections of 5 μm thick were incubated with xylene 2x5 minutes then rehydrated in 100%, 95%, 75% ethanol and HFor western blotting, organoids were seeded in 6 well plates. At the time of analysis, culture media were aspirated then 1ml ice-cold PBS was added into one well. Pipette several times to physically disrupt the Matrigel and transfer the slurry into 15ml conical tube. Centrifuge tubes at 3500 rpm x 5minutes. Aspirate the supernatant carefully to not disrupt Matrigel layers. Add 1ml of ice-cold Cell Recovery Solution (Corning) and pipette several times then incubated tubes on ice for 1 hour. After the incubation, centrifuge tubes at 3500rpm x 5 minutes and aspirate the supernatant carefully not to disrupt cell pellets. Add 1ml of PBS to wash the pellet then centrifuge tubes at 3500rpm x 5 minutes and aspirate the supernatant carefully not to disrupt cell pellets. Pellets were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Protein concentration of cell lysates were determined by Bradford assay and lysates were subject to standard procedures of western blotting. Antibodies use: Sox9, 1:500, #AB5535, Millipore; CAII, 1:500, #sc-133111, Santa Cruz; PTF1A, 1:500, a gift from Dr. Christopher Wright’s lab (Vanderbilt University); CTRC, 1:500, #11456-T24, Sino Biological.Organoids were seeded in 6 well plate. At the time of analysis, culture media were aspirated then 1ml ice-cold PBS was added into one well. Pipette several times to physically disrupt the Matrigel and transfer the slurry into 15ml conical tube. Centrifuge tubes at 3500 rpm x 5minutes. Aspirate the supernatant carefully to not disrupt Matrigel layers. Add 1ml of ice-cold Cell Recovery Solution (Corning) and pipette several times then incubated tubes on ice for 1 hour. After the incubation, centrifuge tubes at 3500rpm x 5 minutes and aspirate the supernatant carefully not to disrupt cell pellets. Add 1ml of PBS to wash the pellet then centrifuge tubes at 3500rpm x 5 minutes and aspirate the supernatant carefully not to disrupt cell pellets. Pellets were lysed with enzyme analysis buffer  with no protease inhibitors supplemented. The protein concentration of cell lysates was determined by Bradford assay. Enzyme activities were analyzed using Lipase Activity Fluorometric Assay Kit  and Carbonic Anhydrase Activity Assay Kit , following the instructions by the manufacturer.Organoids were seeded in 12 well plates. At the time of analysis, culture media were aspirated and 0.5 mL of ice-cold Cell Recovery Solution (Corning) was added into one well. Pipette several times to physically disrupt the Matrigel and transfer the slurry into 1.5ml microcentrifuge tube and incubate on ice. After 30 minutes, centrifuge the tubes at 8000rpm x 5 minutes. Aspirate supernatant then add 250 ul Trizol (Invitrogen). Lyze pellets for 15 minutes at room temperature. Purification of total RNA was done using Direct-Zol RNA kits (ZymoResearch). cDNA was synthesized using SuperScript IV First-Strand Master kit (Invitrogen). Gene expression was quantitated by quantitative PCR using PrimeTime qPCR Probe Assays kit.Mutant genes were cloned into pINDUCER21 (Addgene #46948) and lentivirues produced were concentrated by ultracentrifugaton at 25,000rpm for 2.5 hours at 4 degrees. Viruses were resuspended in PBS. 6-well plate was coated with Matrigel by incubating with 5% Matrigel for 1 hour then aspirate the solution to air dry. 500,000 pancreatic progenitor cells with culture media were seeded in one well and allowed to grow overnight. On second day, lentiviruses were mixed with culture media supplemented with 4ug/ml polybrene and 2ml solution was added into each well. To improve infection rate, plates were centrifuged at 3500rpm for 30 mins. The plates were placed in incubator overnight. On third day, cells were digested with Accutase (Sigma), collected and seeded in 3D. For induction of transgene, doxycycline was added into culture media at final concentration of 1ug/ml.G12D expression were digested with STEMxyme I (1mg/ml) for 1.5 hours then dissociated with Accutase (Sigma). Dissociated cells were counted and resuspended in 90% Matrigel with a final density of 10 million cells/ml. Orthotopic mouse pancreas transplantation was conducted following the protocol approved by the institutional animal care and use committee at BIDMC. 6-8 week old male NCG mice  were purchased for this study. Mice were anesthetized by isoflurane and a 1.5 cm incision was cut at the left upper abdominal flank. The pancreas was carefully pulled out, and 50 ul of cell suspension was injected at the tail of the pancreas using a 29G needle. After injection, the pancreas was gently placed back into the peritoneal cavity, and the wound was sutured. Meloxicam was used as analgesics. Mice were fed with regular food or diets with doxycycline . Eight weeks after injection, mice were sacrificed following approved protocol, and the pancreas was collected then processed as paraffin blocks.Day 16 organoid cultures with or without KRasFor mammary fat pad transplantation, experiments were performed following the protocol approved by the institutional animal care and use committee at BIDMC. 6-8 week old female NCG mice  were purchased for this study. At the time of operation, Mice were anesthetized by isoflurane and a 1.5 cm incision was cut on the skin near No.9 and No.4 glands. A 29G needle was used to inject 50ul of cell/Matrigel suspension into mammary fat pad, at density of 500,000 cells/gland. After injection, the wound was sutured and Meloxicam was used as analgesics. Mice were fed with regular food or diets with doxycycline . Eight weeks after injection, mice were sacrificed following approved protocol and pancreas was collected then processed as paraffin blocks.2 3mM) were added to one well of 6-well plate with organoid culture. Slurries were collected then centrifuged at 800 g for 3 minutes at 4 degrees. Supernatant was discarded and cell pellets were incubated with 2 mL of citric acid-Triton buffer  for 3 minutes on ice then centrifuged at 800 g for 3 minutes at 4 degrees. Supernatant was discarded and pellets were resuspended with 2 mL of citric acid-based buffer  then pass through a 35 μm cell strainer. The flow-through was centrifuged at 800 g for 3 minutes at 4 degrees. The supernatant was discarded and pellets were resuspended with 0.5 mL of storage buffer , 40% Glycerol). Nuclei in storage buffer were transferred to 1.5 mL microcentrifuge tubes and froze at −80 degrees.Isolation of single nuclei from organoids were performed based on a published protocol . 2ml of https://github.com/chris-mcginnis-ucsf/MULTI-seq). The quality of all libraries was assessed using the High Sensitivity DNA kit for the Agilent 2100 Bioanalyzer. cDNA and barcode libraries were multiplexed and submitted to the UCSF Center for Advanced Technology for sequencing on a NovaSeq 6000 SP flow cell, yielding 518M reads.Frozen nuclei pellets were thawed in 50 μL 10% PBS-BSA, pelleted , and washed with 1 mL 2% PBS-BSA. Nuclei were counted, pelleted again, and resuspended at 5x10ˆ6 nuclei/mL in cold PBS. ~200k nuclei were aliquoted from each sample for labeling with MULTI-seq barcodes . After lhttps://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest), using the option “–include-introns” to enable detection of unspliced mRNAs. Using the Seurat v3.2 R package (https://github.com/chris-mcginnis-ucsf/MULTI-seq) to assign each nucleus to its sample of origin. Doublets, unclassifiable nuclei, and samples with insufficient nuclei recovery were removed from the dataset, yielding a final dataset of 4,450 nuclei. From these we captured an average of 959 UMIs and 780 genes per nucleus. Mitochondrial genes were removed from the dataset to mitigate biases arising from differences in nuclei isolation between samples. Expression data were normalized with SCTransform . Pearson correlation coefficients were calculated pairwise between acinar organoids, ductal organoids, and progenitors using the average expression of all genes in the dataset computed by Seurat’s AverageExpression function.http://singlecell.charite.de/pancreas/ (As a reference point against which to compare our organoids, single-nucleus RNA-seq data of the human neonatal pancreas was downloaded from ancreas/  and filtINK4A expression, Mann-Whitney test was used for testing the statistical significance and p = 0.05 was used as the threshold. Details for chart representation and experimental replication can found in figure legend.For data regarding RNA expression quantitated by qPCR, enzyme activities, protein marker expression detected by immunofluorescent staining, Alcian blue staining, cell number changes, percentages of organoids with lumen changes, t test was used for testing the statistical significance and p = 0.05 was used as the threshold. For data regarding organoid size and form factor changes, Ki67 expression, p16Single nuclei sequencing results were analyzed using the Seurat package in R language. Statistical significance of differences in gene expression was tested using the Wilcoxon Rank Sum test and p = 0.05 was used as the threshold. Details can be found in experimental procedures.


In modern societies, people are regularly exposed to artificial light . Dim light in the evening (DLE) imposes an artificial extension of the solar day, increasing our alertness before bedtime, delaying melatonin timing and sleep onset, and increasing sleepiness in the next morning. Using laboratory mice as a model organism, we show that 2 wk of 4-h, 20-lux DLE postpones rest–activity rhythms, delays molecular rhythms in the brain and body, and reverses the diurnal pattern of short-term memory performance. These results highlight the biological impact of DLE and emphasize the need to optimize our evening light exposure if we are to avoid shifting our biological clocks. Opn4−/− mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions―due to artificial light exposure―are experienced by the majority of the populace on a daily basis.Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in  Light exerts profound effects on physiology and behavior, synchronizing biological rhythms to the light/dark cycle (LD) as well as directly modulating alertness and sleep , 2. In m3Per1,2 and Cry1,2 are blunted under 20:4 LD  = 64.31, P < 0.001], whereas activity midpoints and offsets were delayed by 2.57 ± 0.37 and 2.52 ± 0.83 h . Changes in activity midpoints and offsets, which occurred hours after the DLE, suggest that these are not the acute effect of activity suppression by DLE. Furthermore, under constant darkness (DD), activity rhythms in mice with prior exposure to DLE free ran at later clock times than mice with prior exposure to LD , with an overall phase difference of ∼4 h . The persistence of DLE-induced phase delay under DD confirmed that these effects were not the result of the suppression of locomotor activity by light.To examine the effects of 20-lux DLE during Zeitgeber times (ZT)12 to 16 on circadian rhythms, locomotor activity phases  were determined from passive infrared sensor (PIR) recording across each 24-h cycle. Among the three phase markers, activity offset is known to show the highest variability ; this isF = 1.66, P = 0.23; F = 0.06, P = 0.81; and F = 2.68, P = 0.13, for onsets, midpoints, and offsets, respectively].As the oestrous cycle in female mice (which lasts for 4 to 5 d) has been shown to affect wheel-running activity rhythms , we asseOpn4−/−) mice of both sexes. Under DLE, activity onsets, midpoints, and offsets of Opn4−/− mice were delayed by 2.74 ± 0.18, 2.32 ± 0.95, and 1.61 ± 0.61 h . The difference in activity offsets under 16:8 LD versus DLE, 0.64 ± 0.36 h, was not significant (P > 0.09). Comparisons between WT and Opn4−/− mice did not reveal any genotype difference in phasing  . Similarly, effects of 16:8 LD on sleep were stronger relative to DLE . Under 16:8 LD, mice slept more and exhibited longer sleep bouts during ZT8 to 16 and ZT20 to 24, but they slept less and showed shorter sleep bouts during ZT0 to 4 . There was no significant difference in sleep between WT and Opn4−/− mice . Thus, DLE exerts weaker effects on locomotor activity rhythms and sleep than a long day.When the intensity of the 4-h evening light is the same as the prevailing light phase, this would become a long-day photoperiod. We compared locomotor activity rhythms and sleep under 16-h day/8-h night cycles (16:8 LD) versus DLE. Under 16:8 LD, activity onsets and midpoints were further delayed by 2.21 ± 0.49 and 0.99 ± 0.18 h relative to DLE . In the dorsal hippocampus, the peak of normalized Per2 expression was shifted from ZT14 to ZT8 . In addition, centers of gravity (CoG)—providing an estimate of the acrophase (φ) of the molecular rhythm . When pooled across peripheral tissues, one-sample Student’s t tests (two-tailed) showed that mean ∆φ values of Rev-erbα (2.07 ± 0.57 h) and Dbp (1.81 ± 0.51 h) were different from the value of 0 .Misalignment of circadian rhythms is accompanied by changes in clock gene expression throughout the body . To invend Gapdh ; validathe night . Some ofhe night A. Peaks r rhythm –55—were Hmgcr, Hdac3, Ccrn4l, Pparγ, Npc1, and Cyp7a1) was also examined. Notably, DLE delayed the circadian phase of the cholesterol synthesis gene Hmgcr: it peaked at ZT8 under LD  as reported in a previous study  = 5.037, P = 0.008]. The mean ∆φ value of all six hepatic genes under investigation was 2.35 ± 0.95 h .The expression of six hepatic genes involved in adipogenesis and lipid/glucose metabolism  and period length were indistinguishable between groups . However, the acrophase of Dbp bioluminescence rhythm was delayed by an average of 2.34 h in mice with prior DLE exposure ; this is consistent with our previous findings  = 0.038, P = 0.85; Time of Day × Repeated Testing interaction F = 0.60, P = 0.45], demonstrating the stability of this behavioral rhythm under LD.Given the DLE-induced delay in circadian physiology of ∼2 h, we examined if there was any consequence for short-term memory processes at 2 h into the light phase (ZT2) and 2 h after the light phase (ZT14). We used the spontaneous recognition memory task A 58), w, w58), w59findings . RepeateF = 9.88, P = 0.01; Lighting × Time of Day × Type of Stimulus interaction F = 0.13, P = 0.73; F = 6.21, P = 0.032]. By contrast, under DLE performance was better at ZT14 than at ZT2 . This resulted in enhanced performance at ZT14 under DLE . The improved test performance at ZT14 was due to reduced familiar object exploration . In the sample phase, there was a suggestion of elevated exploratory activity at ZT2 but reduced exploration at ZT14 under DLE . However, these effects did not reach significance  and did not relate to the pattern of exploratory activity in the test phase .To examine the effect of DLE, another group of mice first received recognition trials at ZT2 and ZT14 under LD and at the same time points following 2 wk of DLE exposure A. In theR2, between prior sleep and performance, and R2 reached an asymptotic value of ∼0.9 at t = ∼2 h . Notably, there was a strong linear relationship between preceding 2-h sleep and performance at the group level (Right). When all individual cases were considered , linear mixed-effects models . The bootstrap 95% CI of the effect of sleep was , which did not overlap with zero. By contrast, sleep history did not bear any relationship to sample exploratory activity , which in turn was unrelated to test performance ; 95% CIs of these effects,  and , respectively, overlapped with zero. Together, our data suggest that prolonged wakefulness at night is associated with a decline, whereas DLE-induced sleep is associated with a facilitation in short-term memory process.We then explored if the reversal of memory performance was related to changes in sleep history under DLE. To address this question, we determined the amount of sleep from time windows of varying widths (from 10 min up to 3 h in 10-min increments) preceding each recognition trial. Adopting a wider time window increased the goodness of the linear fit, up level C, Right.s models  confirmeF = 60.23, P < 0.001]. Under LD, there were more cFos+ cells in the SCN at ZT2 than at ZT14 , but an opposite, nocturnal pattern was observed in the mPFC , indicating that SCN and mPFC were out of phase under LD. Importantly, the diurnal pattern in the SCN under LD was attenuated but remained significant under DLE  due to increases in cFos+ cell count at ZT2 and ZT14 : there was an increase in the number of cFos+ cells at ZT2 but reduced signals at ZT14 . In the POA, there was an increase in the number of cFos+ cells at ZT14 . By contrast, in the LHA, there was a decrease in cFos+ cell count at ZT14 . In addition, cFos+ cell counts in the LHA and DMH were positively related to each other and to the cortex but not to the POA . Thus, cFos signals in the LHA/DMH versus POA were modified in a way that corresponds to their established roles in regulating wakefulness and sleep, respectively.The preoptic hypothalamus (POA) is known to regulate body cooling and promote sleep –67, wher68F = 0.51, P = 0.50; Lighting × Time of Day interaction F = 1.08, P = 0.33; SI Appendix, Fig. S11, Upper] or in the VTA/SNc .Exposure to long-day photoperiods for 1 to 2 wk is known to decrease the number of dopamine tyrosine-hydroxylase–expressing (TH+) cells in the paraventricular nuclei of the hypothalamus (PVN), increasing behavioral immobility and reducing exploratory activity in rats and mice , 21. SimSI Appendix, Fig. S2); 3) delays molecular clock rhythms in peripheral tissues  delays locomotor activity onsets, midpoints, and offsets ; 2) prom tissues ; 4) reve tissues ; and 5)  tissues . DLE-indesponses –44. Howeesponses . HoweverSI Appendix, Table S2. However, it should be noted that the effects of long photoperiods on TH reduction in previous studies have used more extreme 19:5 LD conditions  is suggestive of some plasticity because weakened dorsal–ventral SCN coupling can maximize adaptability of the SCN to the extended day  for human phase-shifting responses to 6.5-h light varies between 12.54 (460 nm) to 13.07 (555 nm) log quanta , they are equally compatible with an account based on sleep homeostasis (process S); it could be argued that DLE promotes sleep, postponing the accumulation of sleep pressure in the next morning, without affecting the phase of the internal clock. While our data are compatible with a change in either process C or S, in reality, these processes are intricately related  and 12 Opn4−/− mice  obtained from crossbreeding heterozygous Opn4+/− mice (IMSR_JAX:000664) were used in subsequent experiments. In the DD experiment in which in vivo hepatic bioluminescence and locomotor activity were examined, 16 Albumin-Cre;DbpKI/+ liver reporter mice (9♀ and 7♂) were used  Act 1986 under Project License PE4ED9D2C/PP0911346 and Personal Licenses I869292DB and IDB24291F. The DD experiment involving Albumin-Cre;DbpKI/+ mice was conducted at Smith College, Massachusetts in accordance with the standard of the Association for Assessment and Accreditation of Laboratory Animal Care International.A total of 111 mice were used in this study. In experiments examining home cage activity and sleep, there were 12 WT mice of C57BL/6 background , the difference between short and long moving averages (Δi) was determined, and the product of Δi−1 and Δi was calculated. The daily onset of activity was the time point at which the short moving average first exceeded the long moving average, that is, when Δi−1Δi < 0 and Δi−1 < 0, whereas daily activity offsets were the time points at which the short moving average dropped below the long moving average, that is, when Δi−1Δi < 0 and Δi−1 > 0. Activity midpoints were defined as the time points at which total activity in the preceding 8 h was the same as total activity in the subsequent 8 h (i) for each time bin and calculated the product of Δi−1 and Δi. Activity midpoints were estimated as the time points at which Δi−1Δi < 0 and Δi−1 < 0. Activity onset, midpoint, and offset calculations were conducted in R (RRID: SCR_001905).Raw PIR data with a temporal resolution of 10 s were first smoothed with a 1-h moving average across the entire recording period. Following ref. uent 8 h . To implMice were housed under LD for 4 wk; half of the animals were then exposed to DLE, while the other half remained under LD. After 2 wk of exposure to DLE, animals were killed by cervical dislocation at ZT2, ZT8, ZT14, or ZT20. Samples from the heart, liver, adrenal gland, and dorsal hippocampus  were collected and flash frozen on dry ice before being stored at −80 °C.Tissue samples were homogenized in 500 μL TRIzol (Thermo Fisher Scientific) and incubated at room temperature for 5 min before adding 100 μL chloroform (Sigma Aldrich) and spinning for 15 min at 12,000 RPM at 4 °C. After phase separation, the clear lysate containing RNA was removed and further purified using the RNeasy Plus Mini Kit (Qiagen). RNA was quantified using a Nanodrop1000 (Thermo Fisher Scientific).Per2, Bmal1, Rev-erbα, Cry1, Dbp, Hmgcr, Hdac3, Ccrn4l, Pparγ, Npc1, and Cyp7a1) was performed using TaqMan Gene Expression Assays (Thermo Fisher Scientific) by the StepOnePlus Real-Time PCR System (Applied Biosystems); for the two housekeeping genes (Tbp and Gapdh), RT-qPCR was performed using Fast SYBR Green (Thermo Fisher Scientific). TaqMan Gene Expression Assays (Thermo Fisher Scientific) for the genes of interest were mm00478099_ml (Per2), mm00500223_ml , mm00520708_ml (Rev-erbα), mm00514392_ml (Cry1), mm00497539_ml (Dbp), mm01282499_m1 (Hmgcr), mm00515916_m1 (Hdac3), mm00802276_m1 (Ccrn4l), mm00440940_m1 (Pparγ), mm00435300_m1 (Npc1), and mm00484152_m1 (Cyp7a1). The primer sequences 5′ to 3′ for Tbp were TGGGCTTCCCAGCTAAGTTC (forward) and GGAAATAATTCTGGCTCATAGCTACTG (reverse), and for Gapdh, they were TGCACCACCAACTGCTTAG (forward) and GATGCAGGGATGATGTTC (reverse). During RT-qPCR, each 96-well plate was loaded with cDNA samples from all four time points from each lighting condition; cDNA samples from LD and DLE conditions were amplified on separate plates.Complementary DNA (cDNA) was synthesized from extracted RNA using SuperScript VILO Master Mix (Thermo Fisher Scientific), adding 250 ng RNA to the reaction where possible. RT-qPCR of 11 genes of interest (T) method ; validation of housekeeping genes is reported in SI Appendix, Supplementary Methods. Individual 2CT−Δ values were then divided by the highest mean 2CT−Δ value in each lighting condition to obtain 2CT−ΔΔ, indicating changes in mRNA level relative to maximal expression. In addition, CoG were obtained from the CircWave software . The extent of the phase shift, Δφ, was calculated from φDLE − φLD.mRNA levels of the genes of interest were quantified using the comparative threshold cycle (C) method . The 2−Csoftware –55. ThesAlbumin-Cre;DbpKI/+ mice expressing firefly luciferase in hepatocytes under the control of Dbp were used to measure hepatic Dbp bioluminescence signals in vivo , the order of recognition trials under LD was the following: ZT2 (odor), ZT14 (odor), ZT14 (object), and ZT2 (object); whereas for the remaining animals (subgroup 2), the arrangement was reversed: ZT14 (odor), ZT2 (odor), ZT2 (object), and ZT14 (object). Under DLE, the order of assessment was ZT2 (object), ZT14 (object), ZT2 (odor), and ZT14 (odor) for subgroup 1; for subgroup 2, the order was ZT14 (object), ZT2 (object), ZT14 (odor), and ZT2 (odor). The order of recognition assessment is summarized in SCR_014289), which tracked the position of the mouse's snout every 0.1 s and recorded stimulus exploration times. Test performance was expressed as the proportion of time spent with the novel stimulus. Rodents show the strongest novelty preference within the first 30 s of stimulus exploration , with average exploration times  similar to previous mouse studies  for 5 d. They then received object and odor recognition trials at ZT2 and ZT14 and at the same ZTs after 2 wk of exposure to DLE. During the sample phase of each trial, two identical replicates of an object or an odor cue were placed in the arena; the sample phase was terminated after 10 min. After a 3-min delay, novelty preference was assessed in the arena, now containing a third replicate of the familiar sample and a novel stimulus; the test phase was terminated after 3 min. For six of the animals  for at least 90 min, and incubated at 4 °C with rabbit anti-cFos , a molecular correlate of recent neuronal activity. In addition, for mPFC sections, the rat anti-Ctip2 antibody  was added to serve as a marker for deep cortical layers (layers five/six); for SCN sections, the mouse anti-GAD67 antibody  was added to serve as a marker for the SCN; and for other hypothalamic sections, the chicken anti-TH antibody  was added to mark hypothalamic TH-immunoreactive cell bodies and fibers. After 72 h of primary antibody incubation, POA, SCN, and LHA/DMH sections were washed in PBS and incubated at room temperature with Alexa Fluor 488 donkey anti-chicken, Cy3 donkey anti-rabbit, and Alexa Fluor 647 donkey anti-mouse secondary antibodies , whereas mPFC sections were incubated with Alexa Fluor 488 donkey anti-rabbit and Cy3 donkey anti-rat secondary antibodies . After 4 h of secondary antibody incubation, sections were washed in PBS. Finally, stained sections were mounted on glass slides and coverslipped with Vectashield . Processed sections were scanned using a confocal microscope  under 10× and 20× objective lenses. Identical settings were used to acquire images of each region from the four different conditions, and cFos-immunoreactive nuclei were quantified in Fiji ImageJ  and 4% paraformaldehyde (PFA). The brain was extracted from the skull, kept in 4% PFA overnight, rinsed with PBS the next day, and transferred into 30% sucrose solution for cryopreservation. After 2 to 3 d, brains were sectioned coronally at 50 μm on a freezing microtome (Leica). Based on Franklin and Paxinos’ atlas , coronali ImageJ .SCR_002865) and R (RRID: SCR_001905). α = 0.05 was adopted in all analyses unless otherwise specified. To analyze behavioral data , we conducted within-subjects and split-plot ANOVAs with Lighting, Time of Day, and Type of Stimulus as within-subjects factors and Sex and Genotype as between-subjects factors; for locomotor activity under DD, we conducted between-subjects ANOVAs with Prior Lighting as a factor. To analyze molecular data (clock gene expression and cFos+ cell counts), Lighting × Time of Day between-subjects ANOVAs and Lighting × Time of Day × Region split-plot ANOVAs were conducted. Significant interaction effects in most cases were followed up with simple effect analyses in SPSS (df) whenever the assumption of sphericity was violated  (IBM; RRID:  in SPSS . For maiviolated ; these Fels in R , with prhe model . The 95%Supplementary File


In this study, rats received PNI surgery on their right lower legs followed by LIFU stimulation of the L4–L5 section of the spinal cord for 4 weeks, starting 3 days after surgery. We used the 50% paw withdraw threshold (PWT50) to evaluate mechanical allodynia. Western blotting (WB) and immunofluorescence (IF) were used to calculate the expression of phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and KCC2 in the L4–L5 portion of the spinal cord after the last behavioral tests. We found that PWT50 decreased (P < 0.05) 3 days post-PNI surgery in the LIFU− and LIFU+ groups and increased (P < 0.05) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased (P < 0.05) and that of KCC2 increased (P < 0.05) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 (P > 0.05) was unaffected. Our study showed that LIFU could effectively alleviate NP behavior in rats with PNI by increasing the expression of KCC2 on spinal dorsal corner neurons. A possible explanation is that LIFU could inhibit the activation of the CaMKIV–KCC2 pathway.Effective treatment remains lacking for neuropathic pain (NP), a type of intractable pain. Low-intensity focused ultrasound (LIFU), a noninvasive, cutting-edge neuromodulation technique, can effectively enhance inhibition of the central nervous system (CNS) and reduce neuronal excitability. We investigated the effect of LIFU on NP and on the expression of potassium chloride cotransporter 2 (KCC Neuropathic pain (NP) is defined as pain originating from primary lesions and dysfunction of the somatosensory system, either at the peripheral or central level . Many st2) in the spinal cord plays an important role in NP. KCC2 is an ion transporter protein present in mature neurons of the central nervous system (CNS) and can remove Cl− from the cytoplasm to the extracellular space i) in nerve cells is upregulated, thereby reducing the inhibitory effect of the neurotransmitter γ-aminobutyric acid (GABA) , F [ab′]2 fragment [Alexa Fluor 488 Conjugate]; anti-mouse IgG [H + L chain], F [ab′]2 fragment [Alexa Fluor 594 Conjugate]) were used for incubation at RT in the dark for 2 h. After three 10 min washes with PBS, we incubated the sections with 4′,6-diamidino-2-phenylindole . Images were captured under a fluorescence microscope . We used ImageJ software  to quantify the density of positive regions.For IF, each slice was washed in phosphate-buffered saline (PBS) for 10 min at RT and then incubated with 5% goat serum and 0.03% Triton X-100 in 0.1 M PBS for 2 h. Then, we incubated the slices in primary antibodies against KCC50, WB, and IF data. P < 0.05 was considered statistically significant.Data are presented as mean ± standard deviation (SD). We used SPSS version 23.0  for all statistical analyses. GraphPad Prism software version 8.0  was used to generate graphs. After verifying that all data were normally distributed, we used one-way analysis of variance (ANOVA) to analyze PWTWe saw no swelling or nuclear fragmentation of neurons, neutrophil infiltration, or bleeding under cross-sectional magnification , ×100 of50 to assess the effect of LIFU stimulation on PNI rats at different times. One day before LIFU stimulation, PWT50 had significantly decreased from 25.3 ± 1.2 g (LIFU− group) and 25.4 ± 1.1 g (LIFU+ group) to 6.6 ± 4.6 g and 7.7 ± 3.6 g, respectively (P <0.05), but there was no statistically significant difference between the two groups (P > 0.05). After LIFU stimulation, PWT50 gradually increased, eventually becoming higher in the LIFU+ group (12.1 ± 5.0 g) than in the LIFU− group (6.1 ± 2.2 g) after 3 weeks of LIFU stimulation (P < 0.05) and remaining stable to the end of LIFU stimulation. However, it was still lower in the normal and sham operation groups (P < 0.05), which there was no significant difference . There was no difference between the normal and sham groups  cotransporter 2 (KCC2) is the only cationic chloride cotransporter expressed in mammalian neurons. It plays a prominent role in maintaining low [Cl−]i, which is necessary for the function of GABAA and glycine receptors (GlyRs) and for mediating spinal cord inhibition oxy], or DIOA), heat-evoked withdrawal latency and innocuous brush stimulation are significantly reduced  = 4 MPa; DC = 5%) that is used to irradiate muscle and kidney tissue in vitro, the temperatures of these tissues increase by 1.1°C and 0.7°C, respectively [As a noninvasive neuromodulatory method, LIFU has many advantages such as higher spatial resolution, greater penetration depth, and no tissue damage . As a noectively . In this2; and (iii) stimulation of the spinal cord with LIFU was safe.Overall, our study demonstrated that (i) LIFU stimulation of the spinal cord could effectively improve neuropathic pain behavior induced by peripheral nerve injury, which has potential value in the clinical treatment of NP; (ii) LIFU stimulation of the spinal cord might affect the expression of CaMKIV, CREB, and KCC2. Third, we found that LIFU could affect the expression of CaMKIV, p-CREB, and KCC2, but we failed to explore the specific mechanism by which it affected the expression of the above proteins. Thus, further experiments are needed.Our study had some limitations. First, we established only a short treatment period and did not evaluate the long-term efficacy of US therapy. Second, we selected only one time point at which to measure the expression of CaMKIV, p-CREB, p-ERK, and KCC2 in the spinal dorsal corner. Moreover, LIFU upregulated the expression of KCC2, possibly by inhibiting activation of the CaMKIV–KCC2 pathway.We found that LIFU could effectively alleviate NP in rats with PNI by increasing the expression of KCC


V. alginolyticus vaccine by culturing bacteria in a high concentration of magnesium to attenuate bacterial virulence. Furthermore, metabolomes of zebrafish immunized with the live-attenuated vaccines were compared with those of survival and dying zebrafish infected by V. alginolyticus. The enhanced TCA cycle and increased fumarate were identified as the most key metabolic pathways and the crucial biomarker of vaccine-mediated and survival fish, respectively. Exogenous fumarate promoted expression of il1β, il8, il21, nf-κb, and lysozyme in a dose-dependent manner. Among the five innate immune genes, the elevated il1β, il8, and lysozyme are overlapped in the vaccine-immunized zebrafish and the survival from the infection. These findings highlight a way in development of vaccines and exploration of the underlying mechanisms.Vaccines are safe and efficient in controlling bacterial diseases in the aquaculture industry and are in line with green farming. The present study develops a previously unreported approach to prepare a live-attenuated  Vibrio alginolyticus is an important pathogen to both aquatic animals and humans. In aquaculture, it infects a variety of aquatic animals, such as orange-spotted grouper  gene in Vibrio anguillarum attenuate bacterial virulence and subsequently protected rainbow trout (Oncorhynchus mykiss) against infection caused by the bacterium , is avirulent; SR-11 ΔsucCD mutant, unable to generate succinate from succinyl-CoA, is moderately attenuated; SR-11 ΔsdhCDA mutant, unable to generate fumarate from succinate, is slightly attenuated; and SR-11 Δmdh mutant, unable to convert malate to oxaloacetate, is highly attenuated  were generated through successive passage of virulent strains under specific conditions –23 or tracterium . Tricarbirulence , 28. Spetenuated , 28. Thu2+ participates in a multitude of essential processes. The majority of cytosolic Mg2+ is involved in various aspects of protein synthesis such as being a cofactor, a counter ion for ATP or neutralizing negative charges from phosphates present in the rRNA and then assemble ribosomes . V. alginolyticus is grown in 3% NaCl 0.5% yeast overnight and 1:100 using fresh 3% NaCl 0.5% yeast medium and grown at 30°C. For growth curve, OD600 of the bacterial cultures was measured in medium with 0, 0.78, 3.125, 12.5, 50 or 200 mM MgCl2, which were designed according to the event that 50 mM is approximately the MgCl2 concentration of coastal waters and then 50 time or divided by 4 for other concentrations. Notably, different from V12G01 used in a previous report . Zebrafish were reared in 25 L water tanks and each tank was equipped with closed recirculating aquaculture systems. These fish were cultured for two weeks before experimental manipulation and were fed twice daily.Zebrafish (~0.2g body weight), V. alginolyticus cultured with 200 mM MgCl2 (2 × 105 CFU) per fish. Spleens were collected at 48 h post-injection. Nine spleens were mixed for a sample. A total of ten samples were obtained for GC–MS analysis.For the metabolic profile of zebrafish injected by vaccine, zebrafish (n = 180) were randomly divided into two groups, control group (n = 90) and bacterial vaccination group (n = 90). The control group was injected with 5 μl of 3% saline per fish and the vaccination group was intramuscularly injected with 5 μl V. alginolyticus  per fish, respectively. Spleens were collected at 24 h post-injection. Nine spleens were mixed for a sample. A total of ten samples were obtained for GC–MS analysis.For the metabolic profile of survival and dying zebrafish from bacterial infection, zebrafish (n = 270) were randomly divided into two groups, control group (n = 90) and bacterial challenge group (n = 180). The control and bacterial challenge groups were intramuscularly injected with 5 μl of 3% saline and 5 μl g for 10 min, 10 μl of 0.1 mg/ml ribitol (Sigma Aldrich) as an analytical internal standard was added. The supernatant was concentrated for 4 h in a rotary vacuum centrifuge device, LABCONCO. The dried polar extracts were used for the GC–MS analysis. Ten biological samples with two technical repeats were separately used for the test and control groups.To prepare the fish sample for the GC–MS analysis, samples were quenched with 1 ml of cold methanol  and sonicated for 5 min at a 200 W power setting. After centrifugation at 12,000g for 10 min at 4°C. A sample analysis was carried out by Agilent 7890A GC equipped with an Agilent 5975C VL MSD detector . The injector temperature was kept at 270°C, and 0.1 μl aliquot was injected into a column. Temperature program of the GC oven was held at 85°C for 5 min, followed by an increase to 270°C at a rate of 15°C min and then held for 5 min. Helium was used as carrier gas and its flow rate was 1 ml/min. The MS was operated in a range of 50–600 m/z. For each sample, two technical replicates were prepared to confirm the reproducibility of the reported procedures.GC–MS analysis was carried out with a variation on the two stage techniques as described previously , 38. In via intraperitoneal injection. These fish were intramuscularly challenged with 5 μl V. alginolyticus  per fish.To investigate the effect of the TCA cycle on host survival, a total of 80 zebrafish were randomly divided into saline group (n = 20) and sodium malonate group (n = 60). For sodium malonate group, the fish were further randomly divided into three subgroups (n = 20). Each subgroup was reared in an individual tank. Control group was injected with 5 μl saline (0.85% sodium chloride) per fish, while the three subgroups were separately injected with 12.5, 25, and 50 mM sodium malonate, once per day for three days 2, 6.5 mM PMS, 0.2 mM TPP, 50 mM PBS, and 2 mM sodium pyruvate (for PDH) or 2 mM sodium α-Ketoglutaric acid (for α-KGDH). The reaction systems of SDH and MDH included 0.5 mM MTT, 13 mM PMS, 50 mM PBS, 5 mM succinate (for SDH) or 50 mM malate (for MDH). All the reactions were performed in a final volume of 200 μl in a 96-well plate. Subsequently, the plate was incubated at 30°C for 10 min for PDH, a-KGDH, SDH, and MDH. The optical absorbance was performed in a microplate reader  at 566 nm.The activity of pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (a-KGDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) was measured as previously reported with a modification . BrieflyV. alginolyticus cultured with 200 mM MgCl2 (2 × 105 CFU), respectively, each fish. L group, M group, and H group were intramuscularly injected with 5 μl V. alginolyticus  each fish. These zebrafish were collected at 24 h post-injection. For expression of genes encoding innate immune immunity, zebrafish were intramuscularly injected with 5 μl (50 or 100 μg) fumaric acid, once a day for three days, and collected from vaccination and bacterial challenge as described earlier. Expression of these genes was analyzed by the qRT-PCR as previously described . qRT-PCR was performed in 384-well plates with a total volume of 10 μl containing 5 μl 2 × SYBR Premix Ex TaqTM, 4.6 μl H2O, 0.1 μl cDNA template and 0.2 μl each of forward and reverse primers (10 mM). The reaction mixtures were run on a LightCycler 480 system . Data were shown as the relative mRNA expression compared with the β-actin gene by 2−ΔΔCT method. All qRT-PCR reactions were performed for four biological replicates. Gene-specific primers used for qRT-PCR are shown in For expression of genes encoding the TCA cycle, zebrafish (n = 252) were divided into five groups, control group (n = 24), vaccination group (n = 24), L group (n = 24), M group (n = 60), and H group (n = 120). Control and vaccination groups were intramuscularly injected with 5 μl saline solution and 5 μl escribed . The totV. alginolyticus, growth curve of V. alginolyticus ATCC33787 was determined in a gradient concentration of MgCl2. Different growth rate of the bacterium was observed in different MgCl2 concentrations, especially during the first 1–2 h. The growth rate of ATCC33787 from the fastest to the slowest were in 50 mM ≥ 12.5 mM > 3.125 mM > 0.78 mM > 0 mM > 200 mM MgCl2 . Nine spleens were pooled as one sample. A total of 240 aligned peaks were identified in each sample. After the removal of internal standard, ribitol, any known artificial peaks, and merge of the same compounds, 80 metabolites with reliable signals were identified in each sample. Ten samples with two technical repeats for each sample were included in each group, yielding 40 data sets. The correlation coefficient between technical replicates varied between 0.996 and 0.999, demonstrating the reproducibility of the data (http://www.genome.jp/kegg) and MetPA (http://metpa.metabolomics.ca), respectively, for pathway enrichment. A total of eight metabolic pathways were enriched, where the top three impactful pathways were TCA cycle, pyruvate metabolism, alanine, aspartate and glutamate metabolism  in survival and dying groups were shown as heatmap, where survival and control groups were clustered , respectively. As compared to the control group, seven metabolites were increased and eight metabolites were decreased in the vaccination group  was adopted to identify shared biomarkers between vaccination and survival groups. To do this, S-plot was used for identification of discriminatory variables. Cut-off values were set as greater or equal to 0.5 and 0.05 absolute value of covariance on group G. PCA a6 CFU, H), middle  and low  doses of ATCC33787 and the 200 mM MgCl2-prepared live-attenuated vaccine (vaccination) and saline solution was used control. They caused 15% survival  and 85% dying (dying-H), 60% survival  and 40% dying (dying-M), 100% survival , and 100% survival (vaccination), respectively. On the whole, higher expression of genes was detected in the three survival groups and vaccination group than the control, while lower expression of genes was measured in the two dying groups than the control. The high expression was ranked as survival-H > survival-M and vaccination > survival-L and low expression was listed as dying-H > dying-M , α-Ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) in the TCA cycle and pyruvate metabolism were measured. The activity of all enzymes was elevated in the vaccination group, survival group and reduced in the dying group D. Furthil1β, il4, il8, il21, tnf-α, c3b, tlr1, tlr3, nf-κb, and lysozyme were measured. Compared to the saline control, the vaccination group exhibited elevated expression of il1β, il8, and lysozyme but the rest of the genes remain unaffected .HL and X-XP conceptualized and designed the project. JY, X-LY and Y-BS performed experiments. JY and HL interpreted the data. HL and X-XP wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was sponsored by the Projects of Natural Science Foundation of China for International Cooperation and Exchanges NSFC  and the Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) (311021006).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.