This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2 Heart failure affects millions of people worldwide and causes high morbidity and mortality ,2. Most Through the past two decades, cell therapy has been found to be a potential new treatment option for patients with ischemic heart failure. Treatments using mesenchymal stromal cells (MSCs) are the furthest in clinical translation, where the first phase III study was recently concluded (NCT02032004). However, a great challenge in the field of cardiac MSC therapy is the lack of understanding of the mode of action (MoA). It has been proposed that the cells mainly exert their function through paracrine actions, leading to improved angiogenesis and decreased fibrosis ,5,6. HowA recent study by Vagnozzi et al. found the regenerative effect of MSCs to be attributed to sterile inflammation and immune interaction occurring within the first days following treatment. Vagnozzi et al. applied the cells one week after myocardial infarction (MI) , which iUsing a rat model of chronic ICM, our aim was to identify early changes in cardiac cellular subpopulations and transcription in response to a well-characterized and pure cryopreserved allogeneic rat adipose tissue-derived stromal cell (ASC) product.Rat ASCs were harvested from subcutaneous fat of male Lewis rats. The phenotype of the ASCs was determined by flow cytometry on the basis of the recommendations of the International Society for Cellular Therapy (ISCT) ,14. Not Since the cells were to be injected immediately upon thawing, resembling clinical applications, the parameters for recovery and function were assessed following cryopreservation and thawing. Recovery of the cells was measured in percentages as the number of ASCs during thawing compared to the number frozen. A mean recovery of 76.0 ± 13.9% was observed.Adherence was measured in percentages as the number of ASCs attached to the bottom of the culture flasks 5 h after seeding compared to the number of live ASCs seeded. The mean adherence of the ASCs was 86.9 ± 4.3%.The ability of the ASCs to secrete vascular endothelial growth factor (VEGF) was confirmed using ELISA.The ASCs were deemed negative for aerobic and anaerobic bacteria using the BactAlert system. Furthermore, the products did not contain any rodent pathogens based on an extensive IDEXX IMPACT V PCR profile analysis  and was negative for Mycoplasm .These results demonstrated that the cryopreserved allogeneic rat ASCs were pure and responsive upon thawing and adhered to criteria for clinical cell products.Female Sprague–Dawley rats  were used in this study. The use of male ASCs from Lewis rats rendered the treatment allogeneic with MHC mismatch based on information from the vendor. The animals were acclimatized for two weeks prior to any operations.18F]-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT), and animals were randomized to receive saline, ASCs, or no injections  and regular qPCR as well as Y-chromosome identification by qPCR. The injection method was verified in a pilot experiment using bioluminescence imaging and transfected human ASCs  artery. At week 4, the infarct area was assessed using -FDG was used to assess infarct area. The animals had been given minimal amounts of food for 5 h prior to the scans in order to deplete the glucose in the blood. During cannulation of the tail vein for IV access, the blood glucose was measured using FreeStyle Precision (MediSense). To decrease circulating free fatty acids and shift the energy consumption of the heart to glucose, 15 mg/kg Acipimox  was injected subcutaneously 10 min prior to injection of [18F]-FDG. A PET scan was performed after a circulation time of 60 min.[PET listmode files were histogrammed into a single time frame before being reconstructed using OSEM3D/OP-MAP reconstruction protocol. Inveon Acquisition Workspace was used to reconstruct PET and CT images, and Inveon Research Workspace  was used for the co-registration of PET and CT images.The static FDG images were analyzed using Corridor4DM version 2017 . The images were oriented automatically into a short axis and vertical and horizontal long axis. The myocardium was automatically outlined, and the uptake was compared to a normal database created from other Sprague–Dawley rats. The infarcted areas were defined as areas with uptake values lower than 2 SDs from the normal database at the given location. The data output was infarct area percentage of total left ventricle as a summation of the AHA 17 segments model. All orientations and outlines were reviewed by experienced observers blinded to the treatment.6 cryopreserved ASCs were thawed at room temperature; then, they were centrifuged and resuspended to reach a concentration of 1 × 107 ASCs pr. mL CryoStor10 in the syringe. The injection needle was aligned with the transducer, after which the heart was localized in short axis view. The 30 G needle  was inserted in the myocardium through the intercostal space, with needle penetration of the myocardium being confirmed by 2–3 arrhythmic ECG peaks. Injection was performed anterior and lateral/inferior to the MI and visualized as white microbubbles in the myocardium on the echo image. The needle was retracted, and the animals were observed after the procedure. The operator was blinded to treatment group.Treatment was performed using the Vevo 3100 Preclinical Imaging Platform. Prior to ASC or saline treatment, animals were anesthetized, hair was removed as mentioned previously, and analgesic treatment with buprenorphine was administered subcutaneously. A vial of 3 × 10® Lumina XR  and Living Image® software v.4.3.1 (PerkinElmer), and the distribution of cells was assessed. Subsequently, the animals were euthanized, and the hearts were excised and cut into 3 parts to assess the location of the cells in the myocardium  at −80 °C until usage. The remaining heart tissue was stored in Tissue Storage  on ice until dissociation.At day 3 or 7 following treatment, rats were euthanized, and the hearts were extracted and perfused using isotonic saline , after which atrias were cut off. For RT2 on a 4 °C surface plate (Medite), whereupon an enzyme mix from a Neonatal Heart Dissociation Kit, mouse, and rat (MACS Miltenyi Biotec) was prepared and added according to the manufacturer’s protocol. The tissue was dissociated under 200 rpm agitation (Heto) at 37 °C for 45 min, after which the cell suspension was diluted in PBS, filtered through a 70 μm filter , and centrifuged. Following centrifugation, hemolysis was performed using VersaLyse  according to the manufacturer’s instructions. Upon hemolysis, the cell suspension was gently filtered through a 70 μm filter and resuspended in PBS.For the dissociation of heart tissue, the tissue was cut into pieces of 1 mmAfter the isolation and dissociation of the heart tissue, cells were stained with FVS-780, washed in FACS-PBS, centrifuged, and resuspended in 100 μL FACS-PBS for staining with initially titrated antibodies .Finally, a FACSLyrics (BD Biosciences) flow cytometer was used for acquisition. The protocol was generated with automatic compensation using beads (BD Biosciences) and Fluorescence Minus One Controls. Samples were acquired on a medium flow rate, reaching a maximum event rate of 10,000 events/s until at least 500,000 events were acquired. Debris, dead cells, and doublets were excluded from the final data analysis, using FlowLogic software (Inivai), and gating was performed as illustrated in ® DNA/RNA/miRNA Universal Kit (Qiagen) on a QIAcube (Qiagen), following the manufacturer’s protocol for large samples (Qiagen). RNA purity was quantified using NanoDrop (Thermo Scientific). Synthesis of cDNA was performed on 0.5 µg RNA using the RT2 First Strand kit (Qiagen) according to manufacturer’s protocol on a Veriti 96-well fast thermal cycler (Applied Biosystems).Prior to DNA and RNA purification, frozen rat heart tissue was lysed and homogenized using a Precellys Lysing Kit (Bertin Instruments). Up to 30 mg of frozen tissue sample was transferred to a 2 mL Precellys tube containing ceramic beads, after which 600 μL Buffer RLT Plus (Qiagen) and 6 μL 14.3 M β-mercaptoethanol were added. Samples were lysed and homogenized for 20 s using a Precellys 24 Tissue Homogenizer (Bertin Instruments) and then centrifuged briefly, after which a 600 mL lysed sample was used for purification. Total RNA was automatically purified from the lysed sample using an AllPrep2 Profiler PCR Arrays (Qiagen) were designed and used for analysis of the 84 most relevant genes associated with cardiac regeneration (Custom RTneration . The rea2 profiler results was done using QuantiTech primers (Qiagen) in regular qPCR experiments. The amplification reactions were run in duplicates according to the manufacturer’s instructions. A two-step amplification cycle was performed with initial denaturation at 95 °C for 15 min followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 30 s. Finally, a melting curve analysis was performed to detect potential primer dimers.Verification of the RTAnnotation was performed on upregulated genes using PANTHER  on a Gene Ontology database of biological processes complete (Released 2 July 2021) against the reference list on Rattus norvegicus.n = 4). The amplification reactions were run in duplicates, in a final volume of 25 μL per reaction. Each reaction contained 600 nM of both forward and reverse primer, 50 ng DNA, and SYBR Green Mastermix (Qiagen). Primer sequences were obtained from Puppi et al. (qPCR was performed to detect the expression of the sex-determining region-2 on the Y-chromosome (Sry-2), originating from the injected ASCs  was used to analyze the RT2 Profiler PCR Array data. Cq values of target genes were normalized to the geometric mean of the included reference genes. Then, differences in expression levels were calculated using an independent samples t-test of 2-ΔCq values between the groups. For all statistical analyses, a p-value < 0.05 was considered statistically significant. Results are expressed as mean ± standard deviation (SD).Statistical analyses were performed in SPSS software version 25, and graphical depiction of data was performed in GraphPad Prism 9 with standard errors. A Shapiro–Wilk test was used to test the normality of data and Levene’s test was used to assess the homogeneity of variance. Data on cell populations from the ASC and saline groups within the same day after treatment were compared. If assumptions were met, Student’s


Salmo trutta) were previously described. To test the hypothesis regarding the origin of a new cell type following genome duplication, we analysed the gene and paralogue gene expression patterns of erythrophores in brown trout skin. In addition, the ultrastructure of both erythrophore types was precisely examined using transmission electron microscopy (TEM) and correlative light microscopy and electron microscopy (CLEM). Ultrastructural differences between the sizes of erythrophore inclusions were confirmed; however, the overlapping inclusion sizes blur the distinction between erythrophore types, which we have instead defined as cell subtypes. Nevertheless, the red spots of brown trout skin with subtype 2 erythrophores, exhibited unique gene expression patterns. Many of the upregulated genes are involved in melanogenesis or xanthophore differentiation. In addition, sox10, related to progenitor cells, was also upregulated in the red spots. The expressions of paralogues derived from two genome duplication events were also analysed. Multiple paralogues were overexpressed in the red spots compared with other skin regions, suggesting that the duplicated gene copies adopted new functions and contributed to the origin of a new cell subtype that is characteristic for red spot. Possible mechanisms regarding erythrophore origin are proposed and discussed. To the best of our knowledge, this is the first study to evaluate pigment cell types in the black and red spots of brown trout skin using the advanced CLEM approach together with gene expression profiling.Based on their cell ultrastructure, two types of erythrophores in the spotted skin regions of brown trout ( At least six main chromatophores with specific pigments are present in teleost fish: (1) melanophores , (2) xanthophores , (3) iridophores , (4) erythrophores , (5) leucophores , and (6) cyanophores 2. Mosaic chromatophores, containing more than one pigment (e.g. erythro-iridophores or cyano-erythrophores), and additional new pigment cell types and subtypes are also being described5. Deciphering the underlying mechanisms of diverse pigment patterns and pigmentations in adult fish specimens using genetic and developmental analyses has been challenging2. The list of genes known to affect pigmentation in teleost fish is constantly increasing but has mainly focused on skin pigmentation-related processes in zebrafish (Danio rerio)7.Teleost fish exhibit a variety of skin pigmentations and patterning. This great diversity of pigments, types of pigment cells (i.e. chromatophores), and pigment pattern is influenced by duplicate copies of pigmentation genesSalmo trutta and Salmo salar8. They are distinguished from xanthophores based on the absence of xanthosomes, which are present only in xanthophores, while carotenoid-containing inclusions are present in both cell types, but may differ in size.Red pigmentation in teleost fish is caused by erythrophores, red–orange pigment cells containing carotenoid pigments. Together with melanophores, xanthophores, and iridophores, erythrophores were found in the skin of some adult Salmonidae fish species: S. trutta4). Erythrophores with similar ultrastructural features as xanthophores, but containing only carotenoid vesicles and no xanthosomes, were found in the black spots and were designated as type 1 erythrophores. Type 2 erythrophores were more distinctive; they were densely filled with one type of round organelle of much larger size than carotenoid vesicles (named erythrosomes) and located only in the red spots of brown trout skin4. While type 1 erythrophores were sparse in black spots, in which melanophores predominate, type 2 erythrophores represented almost the only cell type in red spots, with fibroblasts and some isolated melanophores above them. Interestingly, erythrophores were not observed in marble trout , a phylogenetically closely related but phenotypically distinct trout species without spots but containing xanthophores, melanophores, and iridophores in the dark skin regions. It was proposed that melanophore-xanthophore and melanophore-erythrophore interactions play an important role in pigment pattern formation in trout4.Using transmission electron microscopy (TEM), we recently described two types of erythrophores in the spotted skin regions of brown trout  are dendritic cells specialised not only in the storage but also in the translocation of their numerous light-absorbing pigment organelles. Some studies even indicate evolutionary relationships between these cells . This demonstrates the existence of specific chromatophore cell types, subtypes, or states, and/or surrounding cells in red spots that may be involved in metabolic pathways or the specific communication between chromatophores and their surrounding cells9. Since many of these upregulated genes are involved in melanogenesis, we hypothesise that these genes might be involved in the origin of new cell types, subtypes, or states in brown trout. Genome duplication-derived paralogue gene copies might play an important role in this process.Recently, we revealed the expression of different biological/metabolic pathways in brown and marble trout skin as well as the differential expression of differently pigmented skin regions in brown trout using skin transcriptomic data9) in the red spots were analysed, and their expression in the different pigmented skin regions in brown trout were compared. In addition, paralogue-specific expression of a set of DEGs was analysed, and different scenarios regarding erythrophore origin are proposed and discussed. To further and more precisely describe the ultrastructure of both erythrophore types in brown trout skin, TEM of the black and red spots in situ was also performed. Moreover, correlative light and electron microscopy (CLEM) was performed on both erythrophore types to unequivocally determine and compare their ultrastructure. To the best of our knowledge, this is the first study to evaluate pigment cell types in the black and red spots of brown trout skin using the advanced CLEM approach together with gene expression profiling.The aim of this study was to further analyse the gene expression pattern of erythrophores in brown trout skin and to test the hypothesis regarding the origin of new cell types following genome duplication. Therefore, a new set of candidate differentially expressed genes , although the size and thickness of CLEM samples were significantly smaller.acana, bicc2, cldn24, coch, csf1r1, fzd9b, kcnk3a, melA, pax7a, and scarb1), highly significant differential expression was detected in red spots compared with black spots and background colour regions (p < 0.001). Eight of these genes  were upregulated by ≥ five-fold in the red spots.Using qPCR, the expression of 12 genes was analysed in 24 brown trout samples . The qPCR results were consistent with the transcriptome sequencing data. All the genes were upregulated in the red spots, compared with the black spots or background colour regions, and followed the same expression trend: red spot > black spot > background colour region Fig. . For tenmc1r, ednrb, mitf, and sox10 from9) showed that the only enriched gene ontology (GO) term was transmembrane signalling receptor activity (GO:MF), when brown trout (S. trutta) was selected. When analysing orthologues in D. rerio, multiple enriched GO terms were connected with pigmentation .The enrichment analysis of 16 genes  or both Ss4R and fish-specific genome duplication (FSGD) events (Table nptx1).Blast hits of analysed transcripts and their locations are reported together with paralogue gene information Table . Except mitfa, sox10, and pax7a. Two copies of the mitf gene are known in zebrafish, mitfa and mitfb, as a result of FSGD. The Ss4R promoted the presence of four paralogues of mitf in the brown trout genome , 28, and 30 (both designated as mitf) .Three genes were selected for more in-depth analyses: pax7 (two copies of pax7a stemming from Ss4R and pax7b), whereas most teleost fish have two copies (pax7a and pax7b as a result of FSGD). Two paralogues of sox10 are also present in the brown trout genome (stemming from Ss4R), whereas no paralogue from FSGD is retained in teleost genomes. The original primers preferentially amplified one of the copies  and to analyse the paralogue expression profiles in differently pigmented skin regions in brown trout  between the two cell types. However, the present study has revealed that type 1 erythrophores can contain rare larger inclusions/vesicles (with diameters of up to 1 µm), which closely resemble the erythrosomes found in type 2 erythrophores , and that type 2 erythrophores contain also some smaller inclusions. These findings blur the boundary regarding cell inclusion size, which was the main criteria for distinguishing between the two different erythrophore types in our first study4. Therefore, we here question the validity of defining two types of erythrophores and rather propose describing them as cell subtypes or differentiation states11. To this end, the term subtype, instead of type, is hereafter used to differentiate between erythrophores originating from either black or red spots of brown trout skin.Previously, TEM revealed that erythrophores can be divided into two types: type 1 with carotenoid vesicles and type 2 with markedly larger erythrosomes (up to 1 μm in diameter)Subtype 2 erythrophores seem to be stabilized and firmly integrated into red spots by fibroblasts or fibroblast-like cells, which were well connected via numerous desmosomes as well with subtype 2 erythrophores. Subtype 2 erythrophores in red spots were better connected with cell junctions than subtype 1 erythrophores in black spots. This was noticed also during sample preparation for CLEM, where four subtype 2 erythrophores have remained connected whereas subtype 1 erythrophores were always solitary. Moreover, in red spots, subtype 2 erythrophores were surrounded by a prominent network of collagen fibres that presumably hinder the migration of erythrophores out of the red spots.9. With a more detailed analyses of our previous results and an additional assessment of differential gene expression using qPCR, the present study showed a unique pattern of gene expression in the red spots of brown trout, which are characterised by subtype 2 erythrophores.Multiple genes were found to be upregulated in the red spots compared with other skin regionsAlthough we speculate that the analysed genes are chromatophore-specific, it must be emphasized that the expression was analysed using the whole skin sample, including the epidermis and dermis with chromatophores and surrounding cells. Thus, both the type of chromatophore and specific homo- and hetero-typic communication between chromatophores and their surrounding cells might lead to specific expression profiles for particular skin regions.ednrba, mitfa, fzd9b, and mc1r are involved in melanogenesis; pax7a and scarb are involved in xanthophore differentiation and carotene metabolism; and sox10 is involved in chromatophore differentiation from neural crest cells15. The lineage relationships between erythrophores (particularly subtype 2) and other cell types in salmonids are unknown. However, our results imply a very specific gene expression profile for erythrophores, with overexpressed genes associated with melanophores and partially xanthophores. Xanthophores have never been found in red spots, whereas individual melanophores are present above the erythrophore layer (4 and this study). Interestingly, mitfa, pax7a, and sox10 are essentially involved in the differentiation of pigment cells and can also be expressed in pigment cell precursors/progenitors in zebrafish17. A common chromatophore precursor or stem cell has been postulated18 and has been suggested to be located in the dorsal region of adult zebrafish19. It was also demonstrated that sox10 acts cell-autonomously in all pigment cell lineages20. Furthermore, it was hypothesised that in zebrafish, melanoblast- and xanthoblast-specific markers, such as mitfa, csf1r1, xdh, and pax7, are co-expressed in a sub-population of dorsal chromatophore precursors16. Interestingly, many of the genes that were identified in zebrafish as characteristic of chromatophore precursors were co-expressed and upregulated in erythrophore-containing red spots in brown trout.Among the tested genes upregulated in the red spots, 8 and are poorly characterised. Unlike xanthophores, which are present in all salmonid species and in all developmental stages, erythrophores are not found in all salmonid species nor in all stages of ontogenesis21. Interestingly, a similar situation was found in the Danio clade, for which the phylogenetic distribution of species with erythrophores suggests that this cell type may have independently appeared and disappeared multiple times24. Most adult Danio species have either xanthophores or erythrophores on their bodies23. Recently, Huang et al.24 described adult skin pigment pattern of D. albolineatus, where both cell types are present, but in distinct skin regions (bands) on body and fins, where they are the only pigment cell type present. Similarly, both cell types are present simultaneously also in brown trout, although they do never appear together (no direct cell–cell interactions exist between them4). While xanthophores are distributed across almost all skin regions in salmonids, erythrophores are mainly distributed within spots or lateral lines21.Erythrophores have rarely been described in salmonid species4 and this study). Some of the analysed genes were overexpressed also in black spots, in which subtype 1 erythrophores are present in a small proportion, compared with background colour regions. Together with two additional genes with almost identical expression patterns between black and red spots (gja5 and dct9), the gene analysis in the present study suggests at least partially similar expression patterns in both erythrophore subtypes. Nevertheless, the distinct expression profiles with highly upregulated genes in the red spots indicate a regulatory independence25 of subtype 2 erythrophores.Subtype 2 erythrophores are of special interest, since they are the predominant cell type in red spots, characterised by a particular cell ultrastructure and gene expression profile. However, our analyses could not determine the gene expression profile of subtype 1 erythrophores or xanthophores. These last two cell types are skin-region specific; however, they rarely undergo homotypic interactions but rather undergo heterotypic interactions with other cells (5. Iridophores obtained a particular morphology (organization of guanine-reflecting platelets) in zebrafish stripes and interstripes during their differentiation that depended on the presence or absence of melanophores. The iridophores of interstripes and stripes also exhibited distinct transcriptomic states, indicating that they represent different cell types5. A similar model of cell differentiation could also be proposed for erythrophores in brown trout. We suppose that cell communication with melanophores and particular paracrine signalling might promote differentiation into subtype 1 erythrophores in black spots, while homotypic cell communication with paracrine/autocrine signalling promotes differentiation into subtype 2 erythrophores in red spots.It has been shown already that pigment cells in zebrafish can differentiate into different cell types or states, based on their micro-environment28. To date, many studies have described the diverse mechanisms of cell communication between fish chromatophores and surrounding cells (and their influence on chromatophore distribution and pigment patterns), including gap junctions, potassium channels, long-range communication through cellular extensions , macrophage-mediated communication through airinemes, pseudopodia communication, and communication via diffusing signals (reviewed in27). Although many genes have been characterised as specific to subtype 2 erythrophores, these genes that are upregulated in the red spots of brown trout could also be differentially expressed in the cells surrounding the erythrophores, and this could influence chromatophore distribution and pigment patterning via yet unknown mechanisms of communication. Surrounding cells may also directly affect the differentiation state of the two erythrophore subtypes and subsequently their gene expression. For example, melanophores may affect subtype 1 erythrophores, which may consequently never reach the final cell differentiation state that is presumably present in red spots.The role of the tissue microenvironment in pattern formation and maintenance has been highlighted multiple times30. The process of gene duplication is believed to be a major factor contributing to the generation of novel gene functions within a genome32. Following genome duplication, the functions of duplicated genes can either be retained, lost, or can diverge. In teleost fish, pigmentation genes have been preferentially retained in duplicate after FSGD, so that teleost fish have 30% more pigmentation genes than tetrapods33. It has been shown that large parts of the melanophore and xanthophore pathways are present in two copies in teleost fish33. It has also been concluded that FSGD has made an important contribution to the evolution of teleost-specific features of pigmentation, which include novel pigment cell types or the division of existing pigment cell types into distinct subtypes33. Furthermore, salmonids experienced another whole-genome duplication (Ss4R) about 80 million years ago34, resulting in multiple paralogues of pigmentation genes in their current genome. Given the above and based on our results, we suggest that the duplication events FSGD and/or Ss4R may have also promoted the emergence of new cell subtypes in brown trout when duplicated genes or regulatory regions underwent neofunctionalization. Although, subfunctionalization could also be considered as regulatory fate for gene duplicates, neofunctionalization was suggested as predominant evolutionary fate of genes after the whole genome duplication events in salmonids35. Detailed analyses of mitf paralogue gene and protein copies  and other cell types in salmonids are unknown. However, as we demonstrated, many differentially expressed genes in subtype 2 erythrophores (or in red spots) are involved in melanogenesis, which is characteristic of melanophores, and in xanthophore differentiation. Our detailed analysis of these genes and their transcripts revealed that two or even four paralogues are present in the brown trout genome; however, the expression of only one of them was analysed at first. Paralogues in salmonids are the consequence of two genome duplication events (FSGD and Ss4R). Thus, it is possible that erythrophores (subtype 2 or both subtypes) developed as a consequence of duplicated gene neofunctionalization, either from melanophores, xanthophores, or their common precursors/progenitors derived from neural crest cells. Differentiation into erythrophores might be further triggered by paracrine or autocrine signalling, promoting the expression of duplicated gene copies. Recently, Huang et al.37. It was even proposed that functional melanin and coloured pteridine pathways are present in both melanophores and xanthophores, and that this could enable chromatophore transdifferentiation (i.e. the transformation of one type into another)38. Furthermore, mosaic pigment cells containing more than one type of organelle, and mosaic organelles containing more than one type of pigment can be found in vertebrate skin39. For instance, in frogs, a mosaic of chromatophores containing melanosomes, erythrosomes, xanthosomes, and refractosomes was observed41. This suggests evolutionary relationships between these cells and possible transdifferentiation of already differentiated chromatophores8. In vitro and interspecies transplantation experiments demonstrated that amphibian xanthophores and iridophores can transform into functional melanophores and vice versa42. These findings highlight the proximity of the melanin and pteridine pathways, suggesting that, in teleost fish, established melanophores may also differentiate into xanthophores or erythrophores, or that xanthophores can differentiate into erythrophores, and vice versa. The differentiation of xanthophores into erythrophores would require even fewer changes in the function of genes and metabolism in addition to requirements for several genes in red or yellow coloration, as recently demonstrated in D. albolineatus24. Similarly, studies on medaka (Oryzias latipes) showed that a changed mode of interaction between only two genes (Sox5 and Sox10) affects the specification of pigment cell types and, by affecting the xanthophore progenitor, correlates with the evolution of a novel leucophore pigment cell type43. Differential expression of one or more paralogues might thus promote the differentiation of chromatophore precursors to a novel pigment cell subtype or state, e.g. erythrophores in brown trout.Nevertheless, similarities between the functions of melanophores and xanthophores in metabolic pathways and during development have also been demonstratedmitfa coded on Ch:14, more data about mitf nucleotide and amino acid alignments and protein structure is presented in Supplementary information online) and contribute to the transformation of a new chromatophore subtype that is involved in red spot formation. It is hypothesised that erythrophores and/or surrounding cells in red spots partially preserve the expression profile of the precursor/progenitor pigment cell  or that chromatophore precursors are present in red spots, generating subtype 2 erythrophores. An additional change in the expression of one of the duplicated gene copies might trigger the emergence of a new cell subtype (e.g. an erythrophore). Nevertheless, the surrounding cells, which were analysed together with chromatophores in differently pigmented skin regions, probably also play an important role in chromatophore positioning and pigment patterning. The specific expression profile and interactions of the surrounding cells may also contribute greatly to the transformation/differentiation state of a cell type or the emergence of new cell type/subtype.The expression profile of paralogue genes is shown to be only partially differentiated in differently pigmented brown trout skin regions. However, there is no clear distinction between the expressions of individual paralogues in differently pigmented skin regions, with the exception that multiple paralogues are overexpressed in red spots compared with other skin regions. These results still suggest that duplicate gene copies at least partially adopt new functions differentiation in vitro and its dependence on interactions with other cells (i.e. subtype 2 erythrophores with melanophores) could be a promising approach to determine the appropriateness of classifying cells into types or subtypes.The present study has opened a number of hypotheses regarding erythrophore origin and differentiation into two types, subtypes, or states in brown trout and has also provided an important basis for further analyses. Defining cell types and states and tracing their developmental origin requires single-cell assays45.Ten adult brown trout individuals (aged > 2 years) were collected from Bled fish farm, Bled, Slovenia. Their average body length was approximately 30 cm. All fish were first-generation offspring reared in the fish farm from wild-caught parents originating from Malešnica stream, Slovenia. Individuals had been fed with Biomar INICIO Plus (Denmark) as a starter feed and Biomar EFICO Enviro 920 during adulthood. Prior to skin sample collection, the fish were sedated in anaesthetic Tricaine-S  and killed by a blow to the neck. Small pieces of skin from the lateral part of the trunk of the body Fig.  were obt4. Briefly, the samples of two red and two black spots were stained en bloc with 2% uranyl acetate in sterile distilled water for 2 h, dehydrated in a graded series of ethanol solutions, and embedded in Epon 812 resin . Semi-thin (1 µm) and ultra-thin (65 nm) sections were cut using an Ultracut UCT microtome . Semi-thin sections were contrasted with toluidine blue and examined under a light microscope to determine the position of the pigment cells. The centres of the black and red spots were cut into ultra-thin sections, which were then contrasted with 2.5% of filtered uranyl acetate in sterile distilled water and lead citrate solution prepared according to the protocol described in46 and examined under a CM100 transmission electron microscope  equipped with a CCD camera  for cell ultrastructure analysis. Altogether we analysed two independent red and two independent black spots, from which we altogether took 49 images. Twenty-seven images were taken for red spot, 16 and 11 from each red spot sample, respectively. Similarly, 22 images were taken for black spot, 16 and 6 from each black spot, respectively.The skin samples of two brown trout were dissected and observed directly under a light microscope . Small pieces of skin from the lateral part of the trunk of the body Fig.  were obtCLEM combines the strengths of light microscopy and electron microscopy, enabling complementary analysis of cellular events or structures within the same sample. This method was used to further describe the two erythrophore types in brown trout skin.47 with slight modifications. Briefly, pieces of skin were treated with trypsin solution  in phosphate-buffered saline (PBS)) for 80 min at 20 °C. After washing three times with PBS, the samples were treated with a collagenase solution , 0.1 mg/mL DNase I , 100 μL/mL foetal bovine serum , 1.2 mg/mL bovine serum albumin in PBS) for 80 min at the agitation speed of 1000 rpm  at room temperature. The cell suspension was then filtered through a 25 μm pore size mesh and centrifuged at 40 g at room temperature. To separate the pigment cells from other cell types, the cell suspension was further centrifuged over a Percoll gradient . The cell pellet was then resuspended in medium  and plated on a MatTek Petri dish  with a gridded glass bottom coated with Geltrex substrate (Thermo Fisher Scientific). This grid helps locate the exact same cell, which was primarily observed under a light microscope, under an electron microscope. The isolated cells from the black and red spots were cultured separately under ambient light (fluorescent neon lamp) at 20 °C overnight. Only the red-coloured cells from the black and red spots (i.e. from both MatTek Petri dishes) were selected for further analyses. The cells were first observed under a light microscope, after which they were immediately submerged in a mixture of 3% paraformaldehyde and 3% glutaraldehyde in 0.1 M cacodylate buffer fixative overnight at 4 °C. Following immersion in 2% osmium tetroxide in sterile double distilled water for 2 h at room temperature, they were stained with 2% uranyl acetate in sterile double distilled water for 2 h, dehydrated in an increasing gradient series of ethanol solutions, and finally embedded in Epon 812 resin . Semi-thin (1 μm) and ultra-thin (65 nm) sections were prepared using an Ultracut UCT microtome . The semi-thin sections were contrasted with toluidine blue and examined under a light microscope to find the position of a specific pigment cell. Then the regions appropriate for ultra-thin sections were determined. To enhance the contrast of ultra-thin sections, 2.5% of filtered uranyl acetate in sterile distilled water and lead citrate solution prepared according to the protocol described in46 were used. The ultrastructure of specific cells was examined with a CM100 transmission electron microscope  equipped with a CCD camera .Pigment cells were isolated separately from the red spots that mostly contain type 2 erythrophores and from the black spots that mostly contain melanophores and type 1 erythrophores. We followed the protocol described in9) were selected. We used the following selection criteria: i) genes are upregulated (≥ six-fold) in red spots compared with other skin regions, ii) genes are classified into gene groups with a known role in pigmentation , iii) genes are involved in cell–cell communication or ion channels (e.g. kcnk3s and cldn24), or iv) genes are involved in extracellular matrix organization (e.g. acana and coch). According to our previous results9, genes in these groups may contribute to cell–cell communication between chromatophores or with surrounding cells and to the migration and positioning of the cells within tissues. qPCR was performed using SYBR Green PCR Master Mix  and a Viia7 Real-Time PCR System . All primers for the selected candidate genes . Two internal reference genes previously determined to exhibit stable expression in brown trout skin (see9) were used: rps20 and pgk1. The expression of the candidate genes was analysed with eight biological replicates, each performed in triplicate. A no-template-control was used to check for potential external contamination. Standard curve analysis was performed for the reference and target genes to assess amplification efficiency, which was comparable between genes. The conditions for all reactions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. At the end of each run, a melting curve analysis was performed to confirm a unique amplicon reaction. The differential gene expression results were calculated using the Pfaffl method49, normalised to the geometric mean of the above-mentioned reference genes and with the use of amplicon-specific efficiency. The Student’s t-test was performed to assess the statistical significance of the differential expression between samples, and results with p < 0.05 were considered statistically significant.Small pieces of differentially pigmented skin (i.e. black and red spots and background colour regions) were obtained with biopsy punches and immediately immersed in liquid nitrogen. The pieces of skin were then homogenised with Percellys 24 homogenizer (Bertin corp.) using zirconium oxide beads (Bertin corp). Total RNA was isolated using the RNeasy Plus Universal Mini Kit according to manufacturer’s instructions and subsequently treated with RNase-free DNase I (Thermo Fisher Scientific). Only RNA with good purity, i.e. with A260/280 values of ~ 2.0, checked with a NanoVue spectrophotometer , was used for cDNA synthesis, which was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Twelve DEGs determined with transcriptome analyses SRP157513575139 we50 was used to conduct enrichment analysis for the tested DEGs. The Search Tool for the Retrieval of Interacting Genes (STRING) database version 11.0 10 was used to validate and examine the network of associations among all the genes confirmed to be upregulated in the red spots. Since the STRING database does not include data for brown trout or any other Salmonidae taxon, putative homologues of candidate genes in D. rerio were analysed.gProfilerTranscripts were obtained from transcriptome data, available as PRJNA485234, and blasted against brown trout genome assembly  using ENSEMBL . The original primers, designed based on transcript sequences, were also blasted against brown trout genome assembly. The top hits were recorded, as well as the paralogue gene copies in the brown trout genome stemming from two duplication events (Ss4R and FSGD).48 with an amplicon size of 90–300 bp and a melting temperature of 60 °C  or indels) to enable paralogue-specific amplification.Paralogue-specific primers were designed based on sequences from the brown trout genome assembly using Primer-BLAST°C Table . SequencThe study was conducted according to the guidelines of the Animal Protection Act , and approved by the Ethics Committee of Ministry of Agriculture and Environment, Slovenia, under decision letter number U34401–60/2013/4.Supplementary Information.


Capreolus capreolus) roadkills occurring in the period 2002–2006 in southeastern Austria. Using a comprehensive dataset, consisting of 11.771 data points, we examined the influence of different time units , illumination categories  and lunar phases on deer-vehicle accidents by performing linear and generalized additive models. Thereby, we identified peak accident periods within the analyzed time units. Highest frequencies of deer-vehicle accidents occurred in November, May and October, on Fridays, and during nights. Relationships between lunar phases and roe deer-vehicle accidents were analysed, providing evidence for high frequencies of deer-vehicle accidents during full moon phases. We suggest that deer-vehicle accidents are dependent both on human activity in traffic and wildlife activity, which is in turn affected by phenology, intra- and interspecific competition, climatic and astronomical events. Our results highlight, that short-term mitigation measures (e.g. traffic controls and speed limits) can be highly effective to reduce deer-vehicle accidents, but should be flexibly adapted to specific temporal periods.Wildlife-related accidents, especially deer-vehicle accidents, pose a serious problem for road safety and animal protection in many countries. Knowledge of spatial and temporal patterns of deer-vehicle accidents is inevitable for accident analysis and mitigation efforts with temporal deer-vehicle accident data being much more difficult to obtain in sufficient data quality. We described the temporal patterns of roe deer ( Capreolus capreolus) is the cervid species being involved in the highest numbers of DVAs in Europe [Roadkill, and especially deer-vehicle accidents (DVAs), have received considerable attention in recent years –6. In adn Europe , 16–19. Spatial distribution of DVAs and the effects of associated factors have been addressed in many roadkill studies , 20–22. Thus, while some studies focused on coarse temporal patterns (e.g. throughout seasons or months), fine scaled temporal analyses of DVAs . The comprehensive dataset, consisting of 11,771 data points, allowed investigating temporal patterns of roe deer-vehicle accidents to an unprecedented temporal degree. According to season, month, day of the week, time of the day (illumination) and lunar phase we evaluated numbers and frequencies of accidents and we discuss influencing factors and predictors as well as the resulting possibilities of roadkill prevention methods. We assumed that high frequencies of DVA are associated with i) rutting period of roe deer in late summer, lasting from the end of July until the beginning of August , 39, ii)2 is consistent with the mean value of all Austrian provinces, whereas the spatial portion of traffic area reaches 3,8%, which is the second largest value within Austria [We analyzed the temporal patterns of roe deer roadkills in Burgenland, the easternmost , and—with an area of 3,961.8 square kilometers and a human population density of 72.5 residents per square kilometer —least po Austria . The dai Austria .The landscape of our study area is characterized by gradients from alpine mountainous patches to steppe lowlands of Eastern Europe with illyrian (submediterranean) climate in the South and continental to pannonic climate in the northern districts of the province. Mean annual temperature in Burgenland is 11.4°C and average annual rainfall is below 700 mm . A great2 represent 93% of the country´s territory [De jure, hunting in Austria and consequently in all its provinces is inseparably linked with land ownership, and the system is thus based on individual hunting grounds . For roeerritory . Roe deeerritory .Roadkill data was provided by district authorities (police data) and the Hunting Association of Burgenland. The province consists of seven districts, with police reports being collected at the responsible district authority, respectively. The so called „incident reports”are archived for three years and cover files on police operations from crime procedures to status reports, therefore stringent data protection is inevitable. Files on accidents with animals have to comprise information on date, local time and location  of the event, as well as the species involved. In total, approximately 15,000 police-reports of wild animal-vehicle interactions, occurring in the period 2002–2006, were gathered. Data on other species than roe deer or reports with vague or fragmentary information were discarded from analysis, yielding 11,771 remaining specific roadkill records for roe deer for the study period. Carcass removal is not a standard practice in the Road Administrations responsibilities but is incumbent on the respective hunting party. Data from the Hunting Association covers information of roe deer numbers in the hunting bag, killed in traffic or by other mortality reasons per year and district respectively. It serves as comparative value to identify report rates to police.All statistical analyses were carried out using R version 3.6.1 . We used2) was calculated. To test whether frequency of accidents varied throughout the week, we ran a post hoc Tukey´s HSD test and plotted results  of roe deer roadkill were analyzed by performing linear models (R package ‘MASS’ ), which ultcomp’ ).To evaluate seasonal patterns, accident dates were classified according to the beginning and ending of the astronomical seasons with spring lasting from 20./21.3. - 20.6. (summer solstice), summer from 21.6. - 21./22.9., autumn from 22./23.9. - 20./21.12. (winter solstice) and winter from 21./22.12. - 19./20.3. of the years under investigation, respectively .Prevailing illumination at accident date and the specific time of day were determined using internet-based calculators  for sunrDifferences in temporal patterns of roe deer roadkill throughout the year (day of year) and at different days of the moon cycle were analyzed by performing generalized additive models with integrated smoothness estimation (R package ‘mgcv’ ). A cyclNote that if no horizontal line fits within the 95% confidence bands this corresponds to a formal test for nonlinear association between explanatory and dependent variable with significance level p < 0.05 .Because the sample size is very large, a normal distribution for the residuals was used. For large numbers count models like a poisson or quasipoisson model converge to the normal distribution due to the law of large numbers .The effective day of the moon cycle at accident date was determined using a internet-based calculator . Time anAccording to hunting statistics, numbers of roe deer losses due to traffic did not vary much within the study period with an overall mean value of 3,163 accidents per year . OfficiaThroughout the year, high variation in number and frequency of DVAs occurred. Day of year had a significant effect on DVA . DVA frequencies were highest in the months November (mean ± SD = 11.0 ± 1.0%), May (10.7 ± 1.2%) and October . The lowest frequencies of DVA were recorded in February (5.9 ± 0.4%) and January (6.5 ± 0.9%).Seasonal distribution of DVAs showed highest numbers in autumn (mean ± SD = 30.1 ± 1.8%) followed by spring (27.8 ± 2.3%), summer (23.0 ± 1.4%) and winter (19.1 ± 1.5%). Frequency of DVA was significantly lower in summer than in spring, but increased after summer and was significantly higher in autumn than in summer, before it decreased again in winter (2 = 0.669).There were no significant differences in frequency of DVA between days from Monday until Thursday . However, frequency of DVA peaked on Friday (16.2 ± 0.7%), but decreased again on Saturdays (14.8 ± 1.0%) before it reached the lowest value on Sunday . More detailed analyses of the effect of illumination on frequency of DVA showed that highest number of DVA occurred during nights after dusk  and daylight  and lowest numbers of DVA occurred during nights before dawn , dusk  and dawn .Frequency of DVA during the night (mean ± SD = 52.4 ± 1.2%) was significantly higher than during daylight (25.4 ± 1.3%) and and twilight . The additive model with integrated cyclic cubic regression smooth spline for day of moon cycle explained 22.6% of deviance . FrequenOur study confirmed temporal patterns of DVA frequencies throughout the different time units analyzed. For the first time, analyses were performed based on a fast amount of data collected within five years, thereby allowing definitive conclusions and proposing relevante measures to alleviate the occurrence of accidents, not being hampered by small sample sizes. None of the analyzed time units showed a uniformly distributed occurrence of accidents, but featured distinct patterns. Peak accident periods occurred during nights and full moon phases. However, in contrast to our expectations, highest numbers of DVA were found after rutting season in autumn (October and November) and on Fridays. Our findings highlight that a variety of decisive factors may influence the number and characteristics of DVAs within the given temporal units, e.g. activity of wildlife species and humans in traffic.Our study showed that number of DVAs did not vary much within the study period lasting from 2002 to 2006. However, numbers of roe deer accidents officially reported by police authorities were lower than numbers reported by hunting associations. Reporting rates to police or insurance agencies depend on the quantum of damage, the driver´s insurance coverage and the drivers fear of implications and consequences of a report . It is thus not surprising that report rates found in literature range from 60% , 59–63 tIn contrast, roadkill data collected by hunting authorities are commonly based on carcass removal numbers per year and offer thereby not much information about temporal patterns of DVAs. Hunters also tend to collect roadkill data only within their hunting grounds; and mainly from low level roads , 67. TheAlces alces) [Odocoileus virginianus) [Cervus elaphus) [Cervus nippon; [Dama dama; [The rut of deer species, accompanied by increasing overall deer activity and movement, is often reported to account for highest roadkill numbers  , white-tinianus) , red deeelaphus) ). Therefelaphus) , 39. In elaphus) . Studies nippon; ), fallowma dama; ) or red ma dama; , where tma dama; –73. The Season, being a time unit of low temporal resolution within a year, is frequently used in roadkill research , 74, 75.Subsequent monthly analyses showed that temporal roe deer roadkill patterns are characterized by a pronounced peak in November (11.0%), the month with highest DVA value, and October (10.6%) with the second highest DVA value  of the year . Our preAs often stated in literature, the phenology of roe deer constitutes the most relevant driver of temporal roadkill patterns , 78. We For the distribution of roe deer accidents per day of the week, no deer-specific biological, ecological or behavioral driving factors are known and we therefore assume traffic values to be the main drivers. Austria-wide mobility surveys have shown that 83% of persons are mobile on working days, while only 77% and 66% are mobile on Saturdays and Sundays, respectively . Thus, wRoe deer exhibits a number of diurnal activity-bouts depending on season, habitat and disturbance level . With adBased on the analysis of prevailing light conditions at accident times there is clear evidence for highest DVA numbers during the dark hours of the day (52.4%). It also shows the importance of twilight hours as this period of time, occurring twice a day, represents only 40 minutes before sunrise and after sunset, respectively, but accounts for 22.3% of DVAs during 24 hours. The more detailed temporal analysis of roadkills into five graduations of light conditions  demonstrates substantial differences of previously cumulated day segments . During The diurnal distribution of DVAs has strong implications for the application of mitigation methods. In Austria , 88 and Data on effects of lunar cycles on roadkill is scarce to almost non-existent in specific literature. Higher probability of moose-train accidents  and highIn theory, activity rhythms of roe deer seem to be primarily responsible for the temporal distribution of road-kills , 78. ButAs already stated, knowledge of detailed temporal roadkill patterns is important from a scientific point of view to extract and assess roadkill influencing factors but also to enable stakeholders and practitioners to apply optimal mitigation methods to reduce DVA numbers. Addressing the presumed high impact of agriculture on DVA patterns, measures from hunters, farmers and road authorities to improve habitats such as maintaining cover and food supply after harvest may help to avoid or reduce negative effects. However, the location of such compensation areas might determine their effectivity as they are frequently placed adjacent to traffic infrastructure which aggravates the underlying problem. To aid recognition, signage of hunting or harvesting activities alongside potentially affected roads can certainly be of advantage. As humans and animals show some kind of habituation effect when warnings or signals are presented too excessively over a long period of time it may be essential to focus methods in terms of space and time.Hazards in traffic resulting from increased animal activities are made public by road safety boards and motor clubs in Austria on a regular basis, which should be intensified during the most dangerous months to further raise drivers´ awareness. Permanent signage from road authorities, warning of possibly crossing game animals is frequently deployed in abundance due to legal reasons and is consequently mostly ignored by drivers. Intensified information campaigns as well as permanent or digital signage and warnings in navigation devices, activated only during peak accident times could help remedy this situation. In the future, further developments of the automotive industry such as advanced driver assistance and break assistant systems could also help to early detect crossing animals and avoid collisions.Therefore, we strongly recommend more studies analyzing temporal patterns of roe deer roadkill numbers with high-resolution temporal data (time of accident) allowing for an extraction of detailed driving factors for roadkill apart from species specific phenology. Further research is needed to address the phenomenon of apparently increased nocturnal activity and the related DVAs of roe deer during full moon. To raise road safety for both humans as well as animals, precise knowledge of spatial as well as temporal roadkill patterns is needed as it enables researchers, hunters and road administrations to adequately place mitigation measures in space and time to achieve the best possible result in lowering DVAs at roadkill black spots.S1 FigSince the synodic month repeats on average every 29.53 days, exceptionally low frequencies of DVA were found on moon day 30. Therefore, moon day 30 was removed before performing analyses.(TIFF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 File(TXT)Click here for additional data file.


Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic subtype of cancer. Because of its rarity and heterogeneity, the management of these patients is challenging. Here, we present the case of a rapidly progressive MBC with mesenchymal differentiation in a 37-year-old female, treated with trimodal therapy consisting of neoadjuvant chemotherapy with paclitaxel and carboplatin, followed by dose-dense cyclophosphamide and doxorubicin (ddAC), modified radical left mastectomy, and adjuvant radiotherapy. Despite the need to anticipate the surgery after the first cycle of ddAC, because of a life-treating adverse event, there was a pathologic complete response. Nevertheless, 6.2 months after completing adjuvant radiotherapy, the patient had a recurrence on the central nervous system (CNS) (two lesions), which was managed with excisional biopsy and stereotactic body radiation therapy. The patient also started “complementary” chemotherapy with capecitabine. Still, 18 months after being diagnosed, she died due to CNS disease progression. Non-epithelial tumors of the breast are rare. Metaplastic breast carcinoma (MBC) comprises less than 1% of all invasive breast cancer tumors and is associated with poor prognosis . BecauseA fit and well 37-year-old female with no relevant medical and family history was referred to the medical oncology department after being diagnosed with a locally advanced MBC with mesenchymal differentiation. She had detected a rapidly growing lump in her left breast four months earlier, just two months after giving birth. On physical examination, there was a hard, painless mass located on the upper outer quadrant of the left breast, measuring approximately 100 mm in diameter and presenting an exophytic friable component measuring 40 × 40 × 20 mm Figure . The ipsAt the time of referral to medical oncology, the patient had already undergone several investigation tests. Four months earlier, she was observed by her primary physician because of a small painful lump on her left breast. The first ultrasound examination revealed a hypoechoic solid lesion measuring 20 mm in diameter located on the upper outer quadrant of the left breast, suggestive of fibroadenoma. However, as the lump continued to grow, two months later, a second ultrasound examination was performed, which confirmed that the lesion had doubled in size (46 mm) and became more heterogeneous; moreover, it was now evident that it was adenomegaly measuring 17 mm in diameter in the ipsilateral axilla. Biopsy of both lesions led to the diagnosis of MBC with mesenchymal differentiation (carcinosarcoma). Immunohistochemical staining was performed on paraffin-embedded blocks of tumor tissue, and the carcinomatous epithelial cells displayed a positive reaction for cytokeratin , vimentin, and Ki-67 (superior to 30% of cells) and a negative reaction for ER, PR, and HER2neu; the mesenchymal cells exhibited a positive reaction for p63 , vimentin, and CD10  and normal CEA (0.5 ng/mL) with normal hemogram and renal function. The genetic test did not detect any known pathogenic variant on BRCA1, BRCA2, PALB2, ATM, and CHEK2 genes.2 and carboplatin AUC 6 every three weeks (four cycles), followed by dose-dense cyclophosphamide 600 mg/m² and doxorubicin 60 mg/m² (ddAC) every two weeks (four cycles with eight days of prophylactic filgrastim). Prior to the beginning of treatment, an echocardiogram was obtained, and her ejection fraction was estimated to be 62% with normal left ventricle wall motion. Tumor response was closely monitored during the entire treatment. There was a rapid clinical response (shrinking and softening) since the first cycle ), the patient was proposed for neoadjuvant chemotherapy with weekly paclitaxel 80 mg/me Figure -1E.After the first cycle of dose-dense cyclophosphamide and doxorubicin, the patient was diagnosed with pancytopenia  complicated with febrile neutropenia grade 4. At the time, she only referred a mild and generalized abdominal pain, and on physical examination, she was prostrated and hypotensive, and no focus of infection was identified. The patient was admitted to home hospitalization and treated with piperacillin/tazobactam for seven days and filgrastim for three days, with full recovery. In the multidisciplinary group meeting, it was decided to suspend chemotherapy and schedule surgery. Less than four weeks after the last chemotherapy administration , the patient was submitted to a modified radical left mastectomy. Pathology examination showed a complete pathologic response ypT0N0, with response signs in one of the 22 removed lymph nodes. Less than eight weeks after surgery, she underwent adjuvant radiotherapy to the chest wall, supraclavicular/infraclavicular regions, and internal mammary chain (50 Gy in 25 daily fractions delivered over five weeks).Thirteen months after diagnosis, the patient was admitted to the emergency department due to an episode of a generalized tonic-clonic seizure. Physical examination revealed stable vital signs and no signs of head trauma or neurologic deficits. The cerebral CT revealed an intra-axial lesion measuring 25 × 18 mm located on the right frontal lobe, suggestive of metastasis. An MRI was later performed and confirmed the presence of the suspicious lesion measuring 22 × 25 × 20 mm on the right frontal lobe and revealed a second lesion measuring 9 × 10 × 7.5 mm on the left temporal lobe near the skull base ; surgical site on right frontal lobe: 27 Gy in three fractions for 0.5 weeks (VMAT)). Pathology examination was consistent with breast cancer metastasis.2 twice per day on days 1 to 14 every three weeks. However, after three cycles of treatment, with good clinical and hematological tolerability and no neurologic sequels, the patient was admitted to the emergency department with intense headaches. The cerebral CT scan revealed an increase of the lesion located on the frontal lobe (32 × 30 mm), with more vasogenic edema and associated hydrocephaly. The patient was admitted to an intermediate care unit and initiated high-dose corticotherapy. Nevertheless, the patient died that same night, 18 months after being diagnosed with metaplastic breast cancer.After completing the radiotherapy, the patient started “complementary” chemotherapy with capecitabine 1250 mg/mMBC is a rare and heterogeneous histologic type of breast cancer that exhibits the transformation of part or all of its glandular carcinomatous component into a non-glandular or metaplastic component . The WorAs MBC was not officially recognized as a distinct pathologic diagnosis until 2000, knowledge about treatment patterns and outcomes is limited. Recent studies reported that metaplastic breast carcinomas are associated with poor prognosis compared with other histologic subtypes -7. The mIn recent years, neoadjuvant chemotherapy has been increasingly used as part of the multidisciplinary management of breast cancer. This treatment strategy allows the evaluation of tumor response to specific chemotherapy regimens, and complete pathologic response after neoadjuvant chemotherapy has been shown to be an independent predictor for survival compared with cases that fail to achieve such response ,11. TherOur clinical case demonstrates the excellent response to neoadjuvant chemotherapy (mainly to paclitaxel and carboplatin) of an aggressive locally advanced MBC with mesenchymal differentiation. The inclusion of platinum agents as neoadjuvant chemotherapy for triple-negative breast cancer is controversial, and we decided to incorporate this compound due to the urgent need for local tumor control . NeverthRegarding subsequent treatment strategy, in case of progression, due to the limitations in the medical management of MBC, molecular profiling has been initiated to elucidate targeted therapies. Recently, small studies identified frequent mutations in PIK3CA (42%-48%) ,15 and PThere is a lack of evidence on the management of locally advanced MBC. As suggested by international guidelines, the treatment plans for MBC according to the hormone receptor and HER2 classification are effective. This report highlights the aggressive subtype of MBC with a poor prognosis even in patients with a complete pathological response. Nevertheless, the fast and complete response to neoadjuvant treatment support this strategy.


In the second trial, SPM increased the yield to 7.0 and 6.4 tha−1 during main and off seasons, respectively, indicating that it was significantly higher than control and the average yield reported by KADA. The yield of SPM treatments improved by 25 and 33% with an increment of farmer’s income at main and off seasons, respectively. Stomatal width was significantly higher than control at 11.89 µm. In conclusion, irrespective of the tested granaries and rice variety, spermine mediated plots displayed increment in grain yield.Rice which belongs to the grass family is vulnerable to water stress. As water resources get limited, the productivity of rice is affected especially in granaries located at drought prone areas. It would be even worse in granaries located in drought prone areas such as KADA that receives the lowest rainfall in Malaysia. Spermine (SPM), a polyamine compound that is found ubiquitiosly in plants is involved in adaptation of biotic and abiotic stresses. The effect of SPM on growth,grain filling and yield of rice at three main granaries namely, IADA BLS, MADA and KADA representing unlimited water, limited water and water stress conditions respectively, were tested during the main season. Additinally, the growth enhancer was also tested during off season at KADA. Spermine increased plant height, number of tillers per hill and chlorophyll content in all three granaries. Application of SPM improved yield by 38, 29 and 20% in MADA, KADA and IADA BLS, respectively. Harvest index showed 2.6, 6 and 16% increases at IADA BLS, KADA and MADA, respectively in SPM treated plants as compared to untreated. Except for KADA which showed a reduction in yield at 2.54 tha Oryza sativa L.), the staple food of half the human population is a cereal crop that is susceptible to soil water deficit, which causes huge yield losses in many Asian countries2. Plants adapt to drought stress or water shortage by altering physiological, biochemical and molecular processes4. Severe stress such as drought leads to significant difference in actual and potential rice yield. Tuong and Bouman5 predicted that by 2025, 15–20 million hectares of irrigated terrain will endure water shortage resulting in water stress that can affect the development and yield of rice7 especially if it occurs during grain filling. As a result, the farmers income and local stockpiles of rice will decrease and in fact becomes worsened in areas where rice cultivation is an annual event. When drought prevails, there are no immediate solutions that can be offered to the farmers. As a consequence, the farmers suffer from less profit and or total losses to their farming venture.Rice .The irrigation system in major granary areas in Malaysia depends mostly on rainwater. During the main-season (August-February), paddy is mainly grown using rainfed system whereas in off-season (March to July), planting normally depends on irrigation system which might lead to water stress if it is insufficient. The major drawback of rice breeding program, a long-term approach, is the inability to increase productivity under normal growing conditions. Undoubtedly, managing rice under limited water availability is highly desired to ensure crop survival while enhancing yield in general for the given situation. Foliar application of growth enhancer such as polyamines that can be economical, feasible, easy to apply and readily available to the farmers could be a possible solution for rice growing in water stress environment11.Several measures are available to mitigate the impact of drought on rice which include improved irrigation facilities in all granaries, breeding programme and simple agronomic manipulation14. The three major forms of PAs, namely, diamine putrescine (PUT), triamine spermidine (SPD), and tetraamine spermine (SPM) mainly exist in a free, soluble conjugated or insoluble bound form. PAs play pivotal role and are central to seed germination, organogenesis, embryogenesis, budding, floral development, leaf and fruit senescence, fruit development and ripening, and plant stress responses17. However, the prominent accumulation of PAs takes place under abiotic stress such as salinity, drought, chilling, heat, hypoxia, ozone, UV-B and UV-C, heavy metal toxicity, mechanical wounding and herbicide treatment16. Prior research agree that the exogenous application of the many forms of PA at variable concentrations has conferred the plants with enhanced tolerance to various stresses 18 besides regulating stomatal responses by reducing their aperture and prompting its closure under water deficit conditions as a vital drought tolerant mechanism21.Low molecular weight aliphatic polyamines (PAs) that are present in plant cells are fundamental ubiquitous compounds for they guard and control diverse cellular functions and processes in response to biotic and abiotic stresses22. It was also involved in different cellular processes ranging from growth promotion and cell division to inhibition of ethylene production and senescence22. Meanwhile, Yamaguchi et al.23 found that an Arabidopsis mutant plant, which cannot produce SPM, is hypersensitive to drought and that this phenotype was cured by SPM pretreatment.Spermine, also a phytohormone, improved plants subjected to water deficit by increased dry matter yield and net photosynthesis that was associated with the maintenance of leaf water status,membrane properties and improved water use efficiency in rice26. Therefore, the main objective of the study is to validate the efficacy of spermine to different variabilities  that prevailed during the growth cycle of rice plants at different locations, planting seasons and prevailing climate under field conditions.At present, the roles of spermine on local rice varieties in relation to grain filling and yield of rice under various water regimes are not widely examined. Furthermore, earlier findings were mainly reported from the studies under normal conditions onlyEffects of spermine on growth and yield of rice at three main granary areas  during main planting season .Effects of spermine on yield of rice during the main and the off planting seasons at Kemubu Agricultural Development Authority (KADA), Kelantan .To establish the bio-environmental based performance of the growth enhancer, two experiments were conducted in different granary areas and planting seasons. The experimental trials were carried out as follows:27 and chemical analysis, namely total nitrogen, available phosphorus and available potassium according to methods of Kjeldahl, Bray and Kurtz28 and Metson29, respectively. The field trial experiment was conducted in different rice granary areas from November 2013 to April 2014. The locations of the study were at the rice fields of Kampung Hutan Buloh, Peringat in the Kemubu Agricultural Development Authority (KADA), Kelantan , Kampung Padang Teluk Mukim Jeram, Tunjang, Jitra, in the Muda Agricultural Development Authority (MADA), Kedah  and Parit 4, Sungai Burung, Sekinchan, in the Integrated Agriculture Development Area Barat Laut Selangor (IADA BLS), Selangor . The properties of soil collected from the upper 20 cm at each field were analyzed. These three locations were selected on the basis of medium, low-medium and high rice productivity  were prepared and separated by a 0.5 m boundary (approximately 16 hills per meter squared) in each of the granary areas  and spermine (SPM) were arranged in a Randomized Complete Block Design (RCBD) with four replications. Four plots for each experiment with 16 mThe seed variety, method of plant establishment and different cultivation practices in each of the granary areas were as follows:−1 (360 kgha−1), 0.28 kgplot−1 (175 kgha−1) and 0.28 kgplot−1 at 15, 50 and 70 DAS; urea was top-dressed at rate of 0.16 kgplot−1 (100 kgha−1) at 35 DAS. The field was kept flooded from transplanting until 15 days before maturity when the field was drained except at KADA trial plots that experienced water deficit during the planting season.The soil in the experimental plot was fertilized with NPK compound fertilizer at the rate of 0.58 kgplot10. Each of the treatments was added with 1% Tween 20 (Sigma-Aldrich) as a surfactant. These solutions were stored at 5 °C before the time of application. Foliar application of SPM was carried out at 35 and 55 days after transplanting by spraying the plants uniformly to the point of run-off (approximately 100 mL m−2) using electric knackpack sprayer  for plot size 4 × 4 m2. Each plant in the pots was sprayed until all leaves were wet. The treatments were applied between 9.00 to 11.00 am on a clear sky day.The control treatment was prepared by adding distilled water and 1% of Tween 20 (Sigma-Aldrich). Stock solution of SPM  was prepared by dissolving 1 g of SPM in 100 ml of water. It was then diluted with distilled water to reach a final concentration of 70 µM of SPM according to the method described by Farooq et al.30 where measurement was made from the plant base to the tip of the highest leaf blade. Tiller number per hill was counted by fully expended tiller. Four hills per pot were taken randomly and measured at 45 and 65 days after sowing (DAS). Chlorophyll content of the leaves was measured by the indirect method using Portable Minolta SPAD 502 Plus  chlorophyll meter. The third fully expended leaf from top was chosen for data measurements at 45 and 65 DAS. Three replicates were taken for each treatment.Plant height was measured at 45 and 65 DAS according to methods described by YoshidaThe rainfall data as shown in the Fig. 2 for yield components determination. For biomass determination, the plants were separated into straw and panicles and then dried at 70 °C to for dry weight determination. The panicles were hand-threshed and filled spikelets were separated from unfilled spikelets manually. Filled and unfilled grains were counted and weighed to determine three yield components 30.The plants were harvested at maturity within a quadrat of 1 mAll data were statistically analysed using SAS software . Two-sample T-test was used to test significant differences between treatments at each granary. Additionally, a one-way ANOVA was done on yield components of the three granaries for comparison. The differences among treatments were determined using the least significant difference (LSD) test at 0.05 probability level.−1) as a direct consequence of low rainfall (< 200 mm) as recorded by Meteorogical Department Malaysia. The properties of soil collected from the upper 20 cm at each field were analyzed. The soil properties were analyzed for physical properties  according to methods by Gee and Bauder27 and chemical analysis, namely total nitrogen, available phosphorus and available potassium according to methods of Kjeldahl, Bray and Kurtz28 and Metson29, respectively.Second field trial experiment was conducted at the National Key Economic Areas (NKEA) 11, Tegayong, KADA, Pasir Puteh Province, Kelantan  with farmers participatory research during main-season and off-season in 2014/2015 (September–December and February-May). This granary area was selected due to lower yield . Treatments were arranged in a Randomized Complete Block Design (RCBD) with three replications. The results were analysed using Statistical Analysis System  by T-test at P ≤ 0.05.−1 (360 kgha−1), 71 kgacre−1 (175 kgha−1) and 71 kgacre−1 at 15, 50 and 70 DAS; urea was top-dressed at rate of 41 kgacre−1 (100 kgha−1) at 35 DAS.Sixty kilogram of MR219 rice seeds per acre were used in both seasons for direct seeding. Field was thoroughly prepared and leveled before seeding. The farmer’s plot sizes were 0.5, 1.0 and 1.5 acres which were used as replication with farmer’s participatory research. The soil in the experimental plot was fertilized with NPK compound fertilizer at the rate of 146 kgacreFoliar application of SPM was carried out at 35 and 55 days after transplanting. The SPM spray was applied until run-off with an equivalent volume of 56 L/acre by using 14 L mist blower sprayer  in each of the plots.Figure −1) with farm-gate price . Moreover, the Malaysian government gives an additional incentive for every 1 metric tonne increase in rice yield compared with usual yield per hectare31. To determine the economic feasibility of SPM foliar spray, benefit cost analysis and net present revenue were carried out. Benefit–cost ratio (BCR) was calculated using the method of Nwaobiala and Adesope32 whereas, net present value (NPV) was determined as described by Ingabire et al.33.:r = discount rate of rice field for per season (t). If NR or NPV > 0, then the total revenue is greater than the total cost, if NR or NPV = 0, than the total revenue is equal to the total cost, and if the NR or NPV < 0, than the total revenue is less than the total cost. In this study, NR and NPV are measured in Malaysian Ringgit (RM) and is based on one hectare. If BCR > 1, then the total revenue is greater than the total cost, if BCR = 1, than the gross revenue is equal to the total cost, and if the BCR < 1, than the gross revenue is less than the total cost.After 80% grain maturity, all plants were harvested whereby yield was determined with three replications for each treatment at the end of the experiment (120 DAS). The yield was separately harvested by a harvester machine, filled in separate lorries and weighed in the rice mills . The revenue from rice yield was calculated by multiplying rice yield  was used to view the samples at high vacuum acceleration voltage of 15 kV with a working distance of 20 mm.This study on rice comply with relevant institutional, national, and international guidelines and legislation.Permission was obtained from Malaysian Agricultural Research and Development Institute (MARDI) to use the rice seeds for this research.−1 and 306.4 mg kg−1 at IADA BLS and the lowest were 10.6 mg kg−1 (MADA) and 67 mg kg−1 (KADA), respectively. In term of textural class, KADA and MADA were classified as clay loam soil while IADA BLS as silty clay soil.The physico-chemical properties of the soil in the different granaries are listed in Table Spermine treated rice consistently had significantly higher values in growth measurement parameters compared to control plants Table , except Plant height and tiller number per hill at both DAT were significantly (P ≤ 0.05) higher in SPM treatments as compared to control. In addition, chlorophyll content of SPM showed an increase of 8% at 65 DAT as compared to control. A slight difference in plant height and tiller number per hill in both treatments at 45 and 65 DAT were observed Table . It mighHighly significant differences (P ≤ 0.05) were observed in plant height, tiller number per hill and chlorophyll content at 45 and 65 DAT in SPM treated plants compared to control (Table Table −1) in both treatments compared to the average yield reported by KADA, 2013 (4.0 tha−1). Harvest index showed 6% increment in SPM treated plants as compared to control at 0.50.Yield components showed significant differences between control and SPM treated plants at KADA, Kelantan Table . Interes−1 compared to average yield of 6.3 tha−1 in year 2013 as reported by DOA. Harvest index in SPM and control treatments were 1.16 and 1.19 respectively.Table All the yield components, namely grain number, 1000 grain weight, grain filling percentage, biomass and yield showed significant interaction between treatment and granary Table . Both faThousand grain weight, grain filling percentage, grain number, biomass and yield were very highly significant Table . Spermin−1 during main and off seasons, respectively, indicating that the yield was significantly higher than control (5.3 and 4.3 tha−1) and the average yield reported by KADA (2015) with 4.8 tha−1. The yield of SPM treatments improved by 25 and 33% with an increment of RM 1612 ha−1 and RM 2129 ha−1 in farmer’s income at main and off seasons, respectively.The rice yield production in control and SPM treatments during both seasons were presented in Fig. −1) as compared to the national  average yield (4.8 t ha−1) in both seasons. For every RM 1 spent in adopting the SPM treatments, RM 1.60 and RM 1.62 were gained in return during main and off season, respectively. In terms of net present value, the SPM treatments more economically viable compared to control with additional income of RM 1621.71 ha−1 and RM 2128.98 ha−1 during main and off season, respectively.Table Stomatal density was found to be higher in control plants at 43 per area though not significantly higher than SPM treatment at 39 Table . Althoug34. The reproductive stage of rice plant is critical and the most sensitive to water shortage which can lead to sterility of the reproductive organs which will then result in empty grains35 and hence can reduce grain yield by 35–75% in different cultivars of rice, which display wide genotypic differences in susceptibility to water stress during this period37.Abiotic stress such as drought is a multifaceted stress condition that inhibits crop growth and causes serious crop yield limitations37 while puddling in wetland rice cultivation needs 30% of the total water consumption38.Although transplanting requires less seed, it is highly labour intensive and requires longer time to maturity due to transplanting stress39. Puddling which is done to create a hard pan below the plough zone had led to high losses of water. As water crisis becomes severe, this method of planting will give rise to high labour cost, thus reducing profit. Although transplanting could inprove yield as seedlings with well-developed rootlets are used, it has to be done at the optimum transplanting time, otherwise weak tillering will lead to reduced yield 40. Hence, in Malaysia, direct seeding method has gained much attention for its low water demand. In this method, seeds or pre-germinated seeds are directly sown in the field and as such transplanting shock is avoided. Three methods are employed in direct seeding, namely dry-seeding, wet-seeding and water-seeding41. The proportion of direct seeded land under rice cultivation is believed to be highest in Peninsular Malaysia amongst all the Asian countries.In this study, both methods of rice plant establishment, transplanting and direct seeding were used. Generally, rice planting is dependant on about 27% of total fresh water withdrawal42. Plants reach maturity stage earlier for they need not reproduce rootlets as in the case of transplanted rice. Nevertheless, direct seeding has its drawbacks too. There is greater competition between weed and rice due to same age. Hence, farmers incur extra cost to control the notorious weeds. In addition, plants tend to lodge more due to poor and shallow root anchorage as seeds are planted at shallow depth42. Furthermore, high plant density in direct seeded rice results in excessive stem elongation and thin stems43. Poor and uneven establishment and inadequate weed control are the major reasons for lower unfilled or and partially filled spikelets45. Other major problems with direct-seeded rice include difficulties in controlling snails and quality deterioration resulting from harvest that may occur during the rainy season. Seed priming is often employed to improve the germination performance and seedling establishment of direct-seeded rice 46. Besides, this technique could to improve speed and uniformity of seed emergence, elevate seedling vigor, and yield of cereal crops47. In a nutshell, both methods have their fair share of benefits and down-falls, thus it is at the discretion of the farmers to decide on the method of rice cultivation in addition to the planting material, herein rice variety.Advantages of direct seeded rice are reduced labour requirement, as much as 50% depending on environment, increased water use efficiency and shorter crop rotation as a result of reduced stress49. In the present study, SPM, a form of PA was found to have a profound effect in improving growth and yield of rice under differing water availability. Three granaries, namely, KADA, MADA and IADA BLS were selected on the basis of low, medium and high rice productivity, respectively  had the lowest total nitrogen content and available potassium content compared to other granaries. Additional nutrients in the soil enhanced plant growth, which indicates higher translocation of assimilates and partitioning of carbon reserved in stem or leaves and grains55.Polyamines are endogenous plant growth regulators that mediate many plant physiological processes and the manipulation of endogenous PA metabolism or external application of PA could affect the resistance of crops to drought stressely Fig.  as indicely Fig. . In the −1, it could reach a maximum yield of 10.7 ton ha−156. The maturity period was 105–111 days; the shortest life cycle compared to previously released varieties and was fairly resistant towards blast and bacterial leaf blight 57. As for MR263, it was released a year later to ensure the rice granaries had back up varieties with good agronomic traits in case there was a disease breakout and to complement the existing pool of rice varieties. It was also a high yielding variety with an average yield of 7.5–9 ton ha−1 with a shorter maturation date (97–104 days)58. MR263 was initially developed to be planted in moderately fertile soils and organic rice fields. To put things in perspective, any rice variety will succumb to pest and disease attack if planted for more than 20 consecutive seasons. In 2015, a total of 4,033 ha was infected by rice blast which led to 50–70% of yield losses59. Present day farmers are more open to a change in cultural agronomic practices, hence are more accommodative to try new rice varieties when one fails. In our trial, SPM-mediated treatment using MR263 at IADA BLS exhibited the highest yield followed by MR219 at MADA and KADA.In the current study, we opted to replicate in-situ planting conditions and thus used MR219 and MR263. Both varieties were released by Malaysian Agricultural Research and Development Institute (MARDI) which has a paddy and rice unit under its wing and does rice breeding. MR219 that was released in 2001, was the first variety developed using the direct seeding method and was a high yielding variety that used to be planted in about 90% of granaries up till a couple of years back. While the average yield is in the region of 6.5 ton ha60, humus in organic matter could enhance soil water holding capacity and CEC, and aeration through good soil structure.The IADA BLS granary is the most productive granary in Malaysia for it produces an average of 6.28 kg/ha of rice Fig. . Good cu61. As such farmers will need to supplement with an adequate supply of nutrients.Besides, the plant in IADA BLS is grown in silty clay soil compared to MADA and KADA where rice is grown in clay loam soil Table . The dif62 have proven that as WHC of the soil more than doubled, the soil organic matter content increased by five-folds. Correspondingly, clay soil has a higher physical protection of soil organic matter as compared to sandy soil63. As soil texture affected the available water holding capacity and organic matter, clay soil positively impacted yield components by producing more tillers and grain bearing panicles, heavier seeds and higher grain filling against sandy soil.Hudson64, the best technological practices are in IADA BLS followed by MADA and lowest in KADA. Tillage practices, water management, land preparation and seed preparation at IADA BLS is the best among the granaries in the country. This was evident in thousand grain weight, grain number grain filling percentage and yield of untreated plants as these parameters recorded much higher values at IADA BLS. In addition, the soil at KADA had a much lower potassium content compared to MADA which might have caused early leaf senescence leaf wilting, and leaf rolling when temperature is high and humidity is low in addition to unhealthy root system reduction in the uptake of other nutrients.Though both KADA and MADA share the same type of soil, the same can’t be said of its effect on yield components. Having a high percentage of sand particles in the soil will drain the soil off water and nutrients at a faster pace. While it may not be an issue at MADA which gets uninterrupted water supply, KADA which is located in a drought prone area will be unable to compensate for water loss. Lack of water at crucial stages such as at reproductive stage may lead to high percentage of grain sterility. According to Harun et al.The dynamic increase of yield in three different locations with different climatic conditions and soil properties were proven by SPM foliar applications. Based on these results, a second trial was conducted at KADA, Kelantan for it was the most infertile granary as it had the lowest nutrient compositions and has a history of drought related stress as proven by the lowest rainfall received among the three granaries tested.−1. Based on the main season, an increase of 42% will impact the national rice production on average yield and SSL by 5.8 tha−1 and 92%, respectively. Interestingly, the economic viability of adopting SPM foliar spray in rice cultivation was very practical based on increases of NPV by 53 and 67% during main and off season, respectively.The experiment was conducted to evaluate the yield production, cost and income of farmers in both seasons (main and off seasons). In terms of cost, SPM treatments was very economical as if benefited the farmers with better return especially during the off season, a crucial time for farmers to survive. Eventhough the yield production was very low at off season, additional SPM foliar application still improved yield by 67% and an extra income of RM 2129 ha67. Water deficit increased stomatal density on the leaf area whereas stomatal length and width were reduced. Increased stomatal density in a water stress condition is often accompanied by a decrease in the volume of the cells that form the stomatal apparatus which was apparent in this study69. Study of stomatal size and density in various crops growing under water shortage revealed a negative relationship between stomatal density and stomatal size71. In contrast, the size of stomata was higher in SPM treated plants. The size and density of stomata varies according to species and the environment and is controlled by genetic and environmental factors73. As stomata plays a pivotal role in regulating photosynthesis, size and density are important ecophysiological traits controlling plant water use, especially in water-limited environments77.Water stress affects stomatal morphology and the results obtained in this study were in agreement with previous trials79. In a previous study80, it was revealed that the application of SPM and with double spray generated higher stomatal conductance and photosynthesis rate. According to past researches, SPM might stimulate efficient sink activity by the metabolic activity of assimilate uptake and conversion81. At the same time, better dry matter partitioning to generative storage organs compared to meristematic tissues and vegetative storage organs might have occurred with the use growth enhancers.Spermine improved plant height, tiller number and chlorophyll content compared to control treatments due to increased cell division and enlargement of sink size. These results indicated that more translocation of assimilates and partitioning of carbon are reserved in stem or leaves for expansion and elongation of organs83. Studies had shown that SPM enhanced photosynthesis rate, stomatal conductance84 and yield production85. Li et al.86 reported that SPM helped to maintain water balance under drought stress by increasing expression of the Ca2+-dependent AQPs, TrTIP2-1, TrTIP2-2, and TrPIP2-7. In addition, the growth enhancer can regulate several abscisic acid-related genes, which in turn control stomatal closure, stress-response gene expression and osmolyte production87. Meanwhile, Krishnan and Merewitz88 reported that exogenous SPM application increased the concentration of abscisic acid and gibberellic acid in bentgrass under drought stress. Yang et al.89 deduced that the yield maintenance ratio of rice was positively correlated to increasing PAs during water stress. Apparently, the biosynthesis of PA is enhanced during stress in stress-tolerant plants resulting in a two- to threefold increase of endogenous PA levels as opposed to stress intolerant plants 90.It is obvious that the application of SPM on rice plants leads to improved plant tugor and manage to mitigate water stress by adjustment of stomatal opening and also root hydraulic conductivityDouble application of SPM on rice plants is an alternative way to improve plants under water stress conditions through higher grain filling. The yield of SPM foliar sprayed plants were consistently enhanced as compared to control in all granaries. It showed SPM continuosly enhanced grain filling and yield of rice in both planting seasons compared to control. Based on the benefit cost ratio and net present value, SPM treatments was very practical and economical in terms of economic return especially during off season.