The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies. Adipose tissue (AT) has long been known for its filling ability, and autologous fat transfer, also called lipofilling, is performed daily to correct soft tissue defects ,2. AT isFrom a regulatory point of view, autologous SVF is considered an advanced therapy medicinal product (ATMP) according to the European Directive No. 1394/2007, as it is prepared using enzymatic digestion  and not intended to be used for the same essential function in the recipient and the donor (non-homologous use). Thus, since it is considered a drug product, SVF manufacturing must strictly follow good manufacturing practices (GMP) from its development to the markering authorization. In others words, appropriate measures and arrangements must be applied to safeguard the quality and the security of the product for their intended use. It included trained personnel, qualified premises and equipment, appropriate documentation, quality of production operations, quality of starting and raw materials, validated quality control and analytical methods, and a qualified person for the batch release. Common steps of SVF manufacturing include AT harvesting and washing, enzymatic digestion, centrifugation, and SVF pellet isolation and washing . HoweverIn this context, we aimed to develop an effective GMP-compliant and cost-optimized method to isolate therapeutic SVF. This study details the developpement of the standardized method, the validation of critical production parameters, and finally provides in vitro and in vivo comparability data on the manually isolated SVF with reference to SVF obtained from the most commonly used automated method.AT was obtained as excedent surgical residue from healthy donors undergoing liposuction for aesthetic purposes in the department of plastic surgery, La Conception University Hospital, Marseille, France. The study was conducted in accordance with the declaration of Helsinki and all subjects provided informed consent for scientific use of the surgical residues through a survey that was validated by the regulatory affairs department of AP-HM hospitals. AT samples were anonymized before transmission to the cell therapy unit.oume, Lipoplasty Products, Sunrise, FL, USA), a 12-gauge 12-hole multiperforated cannula, through a closed circuit, preventing contamination of the harvested product. Infiltration was performed using a mixture of xylocaine 10 mg/mL and adrenaline 0.05 mg/mL diluted in a saline solution . The mean volume of harvested AT was 187.74 mL ± 68.28 mL. The AT was packaged in a medical device . Once harvesting was complete, the bag was immediately transported to the cell therapy unit.AT harvesting was performed by experienced surgeons from the plastic surgery department of La Conception university hospital in an operating room under general anesthesia after a standardized skin aseptic preparation using a Khouri cannula  was transferred aseptically into a 250-mL Puregraft bag  using 60-mL syringes . AT was washed three times, with a volume of lactate Ringer’s solution  equivalent to half of the total Puregraft volume, preheated at 37 °C, using the Puregraft device, allowing the drainage of residual wetting/tumescent solution, red blood cells, and other debris. After discarding the waste bag of the Puregraft device, the sample was then weighed and diluted with RL 1:1 (v/v) at 37 °C (A class). The diluted AT was transiently incubated in a 37 °C incubator  for 15 min before the addition of collagenase enzyme  directly into the Puregraft device (injection performed under laminar airflow), at concentrations of 0.10 or 0.25 U/mL for 15, 30, or 45 min at 37 °C under constant agitation using an orbital shaker. Collagenase was previously sampled and validated for free germs. Then, the cell suspension was transferred aseptically into a new medical device  to allow for cellular concentration by centrifugation (5 min at 400 g)  and filtration using a cell strainer with porosity of 200 µm  to avoid any remaining debris. The filtration and washes to remove residual enzymes were performed with 2:1 (v/v) RL or saline solution (Baxter) supplemented with 5% of human serum albumin  (NaCl 5% HAS). All steps after digestion were aseptically performed in a closed circuit established with subsequent transfers of the cell suspension between transfer bags through sterile connections created using a sterile tubing welder . Finally, the active substance was concentrated and resuspended with 10 to 15 mL of RL or NaCl 5% HSA. First, under laminar airflow, the harvested AT contained in a MACO bag ; the Celution protocol, used here as the reference protocol, is described in several investigational medical product dossiers for clinical trial authorizations . Various biological characteristics of both SVF final products were comparatively assessed and classified as SVF specifications defined for batch release (viability), critical process parameters (recovery yield), and critical quality attributes  according to the International Conference for Harmonization . The extraction method for the Celution protocol is provided below.Once the LG protocol was developed and validated, six GMP preparations of SVF were performed and retrospectively compared to batches of SVF (n = 33) previously manufactured using the Celution device (Cytori Therapeutics) according to the manufacturer’s instructions. The collected lipoaspirate was washed with RL (Baxter) and enzymatically digested with Celase, a GMP cocktail of enzymes provided with consumables . The cells were concentrated, washed, aseptically recovered, and resuspended in RL.SVF was obtained using the Celution 800/CRS automated processing system  in duplicate. Recovery yield was calculated as the number of total VNCs obtained divided by the initial volume of AT, which was measured after the removal of infiltration liquid.The VNCs concentration and cell viability percentage were determined using a NucleoCounter NC-100 (Chermo Fisher Scientific) plates in triplicate in proliferation medium . Then, the colonies were grown for 12 to 14 days, depending on the growth rate of the cells, with medium changes 4 days after seeding, and then every 2 days. At the end of the assay, the culture dishes were rinsed twice with phosphate-buffered saline , fixed with abolute ethanol , and stained with Giemsa reagent  for 20 min before two washes with distilled water. Cell colonies were counted under phase-contrast microscopy . All three culture dishes were counted, and the average and standard deviation were calculated to generate the final frequency percentage value, which was expressed as the colony-forming efficiency  divided by the number of cells seeded × 100.The clonogenic potential of MSCs present in the SVF was assessed by CFU-F assay as follows. First, 500 VNCs were seeded into six-well , phycoerythrin (PE), phycoerythrin-Texas Red-X (ECD), and phycoerythrin-Cyanin 5.1 (PC5) (references listed in 4Cl for 10 min before the cells were centrifuged and resuspended in PBS Ca++/Mg++-free medium (Life Technologies). Then, NucBlue (Thermo Fisher Scientific), which allows the discrimination of viable and dead cells, was added for 5 min prior to flow cytometry analysis on a NAVIOS instrument . Data files were analyzed using Kaluza software (Beckman Coulter) with a multiparameter gating strategy. Briefly, CD45− regenerative cells were discriminated as CD34−CD146+ pericytes and transitional cells, CD34+CD146+ endothelial cells (ECs), and CD34+CD146−CD90+ MSCs. The CD45+ population includes all hematopoietic cells .Characterization of the SVF cell subpopulations was performed by flow cytometry. Aliquots of 5 × 10bcam, Cambridge, UK) according to the manufacturer’s instructions and analyzed on an optical density reader .Washing solutions obtained after all SVF washing steps were collected and sampled to measure the enzyme residual quantity using a collagenase activity assay colorimetric kit  uses a computer-controlled incubation/detection system. The media used contained proprietary factors designed to inactivate a wide variety of antibacterial and antifungal agents. Bact/alert culture bottles were incubated at 37 °C and 5% CO2 for a total of 10 days, and automated readings were taken every 10 min . The detection of organisms resulted in an audible alarm and automatic recording of time of detection.SVF  was sampled in Bact/alert culture bottles . The Bact/alert method  coated with 10 μL of growth factor-reduced Matrigel  previously polymerized for 30 min; then, they were maintained in endothelial cell growth medium  at 37 °C and 5% CO2. Capillary-like structures were recorded during 96 h using a Leica DMI8 video-imaging inverted microscope equipped with an Incubator I8 (Leica Microsystems) at 5× magnification; images were captured using the Leica Application Suite X software . The network, length of tubes, and nodes were quantified using ImageJ software  with the Angiogenesis Analyzer plug-in http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&lang=en). Each experiment was performed in triplicate.SVF samples produced for the intra-donor comparative analysis (n = 2) were loaded at a density of 20,000 cells per well in a μ-slide angiogenesis system  and seeded in non-adherent round-bottom 96-well plates , leading to the formation of spheroids with a defined cell number. After 72 h, spheroids were collected and embedded in collagen gel . The spheroid-containing gel was rapidly transferred into pre-warmed Lab-Tek II slides (Thermo Fisher Scientific) and allowed to polymerize (30 min); then, 100 μL of EGM2-MV medium was added on the top of the gel. Following 24 h of culture in EGM2-MV medium, spheroids were fixed for 30 min in 4% paraformaldehyde at room temperature. After washing and permeabilization with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA), for 2 h at 4 °C, spheroids were immunolabeled overnight at 4 °C with phalloidin coupled with Alexa-647 (Thermo Fisher Scientific) (1/100), and nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (1/5000) diluted in PBS containing 1% BSA. After washing, we then captured fluorescent optical image stacks along the z-axis at 20× magnification using two lasers in sequential mode under a Leica DMI8 microscope (Leica Microsystems). The Las X software (Leica Microsystems) was used during all image acquisition procedures. Image processing prior to image measurements was performed using Huygens Essential Deconvolution software  using up to 40 iterations of the classical maximum likelihood estimation algorithm, with a theoretical point spread function and automatic background correction. Then, the images were analyzed using the Sprout Analysis plug-in developed by Eglinger et al. [SVF samples produced for the intra-donor comparative analysis (n = 2) were suspended in culture medium containing 0.2% (wt/vol) carboxymethylcellulose  with a square hole at each center (3 cm per side). This implant was folded and inserted into the incision surrounding the flap to prevent rapid cutaneous revascularization and healing of the edges of the flap. To improve animal welfare conditions, general analgesia by buprenorphine was administered daily for 1 week postoperatively. Four 6-mm punch excisional wounds were made in the dorsal skin of each mouse, according to Sullivan. Silicone donut-shaped splints fashioned from a 0.5-mm thick sterile silicone sheet  were fixed to the skin surrounding the wounds with immediate-bonding adhesive and 5-0 nylon sutures , according to Galiano [All experimental procedures were approved by the Region #14 Animal Ethics Committee (reference no. 2015102110259745). A mouse model of cutaneous ischemia was created using 8-week-old female nude Foxn1 Galiano . This cu2 were injected at the wound edges of the four punch excisional wounds (0.4 mL of SVF for each wound). Wound closure was measured weekly for 28 days using standardized digital photographs  and analyzed with ImageJ 1.52p. Mice were randomly separated into three groups: control group, LG group, and Celution group. Each group received subcutaneous injections of SVF obtained from the same donor with the LG or Celution protocol or a control RL injection. Two million VNCs/cmraphPad Software, La Jolla, CA, USA). Quantitative variables are reported as the mean ± standard deviation (SD) or the median 25–75 th percentile according to the normality of their distributions; minima and maxima are also provided. Continuous variables were compared using Student’s t-test or the nonparametric Mann–Whitney t-test. One-way analysis of variance (ANOVA) was employed to compare more than two groups. For assessment of wound closure in the mouse model, data are reported for each group as the average percentage of wound healing ± SD from the four wounds in each mouse. Two-way ANOVA was used for the in vivo wound healing follow-up. A p-value < 0.05 was considered to indicate a statistically significant difference.Statistical analyses were performed using Graph Pad Prism 5  was obtained under conditions of 0.25 U/mL and 45 min. Flow cytometry analyses of the percentage of CD45Altogether, the 0.25 U/mL and 45 min condition was adopted, as it allows SVF to comply with the usual viability specification with acceptable yields for VNCs and the proportion of regenerative cells.p = 0.01)  and a variation coefficient of 139.27%. Conversely, the yield was more repeatable with the NaCl 5% HSA washing solution, with a median value of 4.84 × 105 VNCs/mL AT (2.79–11.93 × 105 VNCs/mL AT) and a variation coefficient of 89.9%. Proportions of MSCs  or NaCl 5% HSA (n = 5). The measured cell viability of the finished product was statistically increased in the NaCl 5% HSA group compared to the RL group (median 92.50% (90.10–94.90%) vs. 78.90% (72.10–82.50%),  = 0.01) A. The am = 0.01) B. Howeve of MSCs C and ECs of MSCs D identifThus, NaCl 5% HSA was chosen as the main excipient for filtering and washing of the isolated SVF due to the high viability it confers to the cell product and its ability to maintain its viability (data not shown).Four independent SVF batches (A–D) were produced using the parameters validated above. After each centrifugation/washing step, the washing supernatants were retained to quantify collagenase concentrations. Analysis of the kinetics of substrate degradation by collagenase over 180 min indicated that collagenase was undetectable when at least two washing steps were applied A. LinearCollectively, these results identify production parameters that allowed us to optimally design the LG protocol and comparatively evaluate it with reference to the Celution-based method. p < 0.01). Similarly, the LG protocol generated a greater number of VNCs/mL AT, with a median value of 4.20 × 105 (3.35–10.58 × 105 VNCs/mL AT) vs. 2.52 × 105 (1.50–3.18 × 105 VNCs/mL AT) in the Celution group (p < 0.01). The clonogenic potential of MSCs, assessed via CFU-F assay, did not statistically differ between the two processes (p = 0.43). The distribution of various cell subpopulations of SVF from the LG protocol was similar to that of SVF from the reference protocol, as the respective proportions of MSCs, ECs, pericytes, and leukocytes did not statistically differ between the two protocols. The results are summarized in The viability of nucleated cells manufactured with the LG protocol (94.15% (89.83–93.03%)) was significantly higher compared to that acquired from the Celution device (87.30% (84.60–90%), 5 VNCs/mL AT (3.74–11.45 × 105 VNCs/mL AT) vs. 3.39 × 105 VNCs/mL AT (2.25–4.54 × 105 VNCs/mL AT) with the Celution device  versus 91.70% (82.95–92.70%) for the reference protocol A. The LGn device B. The cln device C. The ceSamples of SVF (n = 3) produced using the LG protocol were submitted to sterility testing; the results established that no microorganisms were present.5 µm (0.94–2.06 × 105 µm) vs. 77.50 (72.00–83.00) and 1.31 × 105 µm (1.27–1.34 × 105 µm), respectively)  and 1.50 × 10ctively) A,B. The on group E. The nuon group F. The nuon group G. Finallon group H. These p > 0.05) and 25.70% (± 19.35%) for the Celution group (p > 0.05) vs. 12.99% (±13.39%) for the placebo group. The same conclusion was observed at D14, with an average wound healing of 79.98% (± 26.50%) and 87.96% (±21.71%) for mice who received SVF from the LG (p < 0.05) or Celution protocol (p < 0.001) vs. 61.08% (± 33.67%) for placebo. At D21, the percentages of wound healing were 95.56% (± 8.20%), 96.80% (± 9.41%), and 91.16% (± 24.49%) for the control, Celution, and LG groups, respectively; no statistically significant difference was observed. At D28, all wounds were healed.SVF produced for the intra-donor comparative analysis was tested for its wound-healing ability  in a mouse model of subcutaneous ischemia inspired by the Galiano model . Three gIn this study, we developed and validated a protocol, based on a classical enzymatic digestion using collagenase , that wa− SVF cells are also crucial for pro-healing and angiogenesis, as ECs and pericytes make direct contributions to vessel formation and maturation [We defined each step of the process by evaluating changes in manufacturing parameters that are critical both for regulatory requirements and potential effectiveness of the final medicinal product. Viability of the nucleated cells recovered after AT enzymatic digestion was chosen to be the only product specification, as that should confirm the quality and ensure the potential efficacy of the final product according to ICH guidelines. The yield of VNCs per mL AT was defined as a critical process parameter as it is the most representative of isolation protocol efficiency. In addition, it may have an impact on the product quality and should be controlled according to ICH guidelines. The applicability of SVF isolation methods is sometimes hampered by the poor availability of large volumes of AT as the starting biological material in patients with a low body mass index such as those presenting with vascular and/or inflammatory chronic disease. The use of a recommended AT volume of ≈100 mL in the Celution procedure can be viewed as a potential limitation. Therefore, optimization of the SVF yield is critical, making monitoring of the digestion temperature one of the most crucial parameters to obtain an effective digestion. Manual methods for SVF isolation allow digestion to be directly run in a 37 °C thermostatic chamber. Finally, the cell subset distribution and clonogenic potential of MSCs are considered critical quality attributes according to ICH guidelines. Thus, although the mechanism of action of the SVF is not fully established, and no study to date has reported a link between the cell subset distribution of SVF and its clinical benefit in patients, it has been established that the MSC counterpart plays a prominent role through its paracrine activities and/or multipotent differentiation ability . MSCs frturation ,33 and pturation ,35. For Based on these parameters, the selected protocol consists of an AT digestion step with collagenase at 0.25 U/mL AT for 45 min. The best cell viability was achieved by using the highest concentration and the longest incubation time for collagenase. Consistently, as reported by Lee and colleagues, SVF viability was shown to decrease with increasing treatment duration, but this effect is significant only for a duration equal to or greater than 60 min . The folIn the second part of our work, we first retrospectively compared the final product manufactured with the LG protocol to a reference process based on use of the Celution automated device, which was the first commercially available device for SVF production following the discovery of multipotent stem cells in AT by Zuk and her team . InteresMore importantly, the robust intra-donor comparison performed on five batches from the same AT harvest and discriminated according to the LG or Celution-based process allowed us to attest the comparability of the two methods. The viability, yield of VNCs per mL AT, CFU-F assay, and cell subset distribution were statistically similar between both cell suspensions. One trend was a higher percentage of leukocytes within SVF obtained with the LG protocol. This could be explained by the automated initial washing step of the AT provided by the Celution device. This automated washing takes into account the level of blood contamination of the initial AT and is supposed to be more effective, as rinsing and washing steps are repeated until obtaining non-bloody AT. In the LG protocol, the initial washing step is defined by three rounds of washing with RL in 1:1 proportion with AT, leading to an acceptable visual aspect of the AT before digestion. No further development was performed on this initial step, as collagenase activity is driven by required electrolytes present in RL.To our knowledge, our study is the first to implement in vitro angiogenesis testing to analyze the comparability of SVF manufacturing methods. The expected added value is to provide functional testing that can be evaluated in the future as a potency assay. Such testing is still not defined and remains challenging in the context of the use of SVF as an experimental ATMP product. Our results showed that the sprouting ability as well as the capacity to self-organize into capillary-like structures in a Matrigel matrix in vivo was not different between the two SVF production methods. This observation is in line with the fact that the main cell subsets supporting SVF angiogenic activity were similarly represented in SVF from both processes. This also suggests that the functional paracrine interactions between SVF cells during capillary formation are equivalent regardless of the SVF manufacturing process. Consistently, both SVF products tested in an innovative wound model of cutaneous excision and ischemia in nude mice were able to accelerate wound closure. The pro-healing activity did not significantly differ between SVF obtained from the LG or Celution protocols.In the few last years, a substantial number of automated and semi-automated devices have been introduced or are currently in development, with a total of 17 devices reported in the review of Oberbauer and colleagues . A largeIn conclusion, this study reports for the first time the development of a GMP-compliant process for manual SVF isolation. This process showed satisfactory performance criteria and allows the manufacturing of clinical-grade SVF with angiogenic and pro-healing potency in vivo comparable to that obtained with an automated device previously used in clinical trials. Although future studies are required to evaluate the SVF obtained using this validated protocol in clinical settings, our study offers an easy to implement, cost-effective, and standardized technical option that is part of a global strategy aimed at promoting SVF-based cell therapy and favoring patients’ access to medical innovations.


A. protothecoides heretofore. Temperature was a critical factor affecting growth and lipid metabolism of A. protothecoides. It also remained largely unknown whether these TFs would respond to temperature stress and be involved in controlling lipid metabolism process.Both APETALA2/Ethylene Responsive Factor (AP2/ERF) superfamily and R2R3-MYB family were from one of the largest diverse families of transcription factors (TFs) in plants, and played important roles in plant development and responses to various stresses. However, no systematic analysis of these TFs had been conducted in the green algae Hereby, a total of six AP2 TFs, six ERF TFs and six R2R3-MYB TFs were identified and their expression profiles were also analyzed under low-temperature (LT) and high-temperature (HT) stresses. Meanwhile, differential adjustments of lipid pathways were triggered, with enhanced triacylglycerol accumulation. A co-expression network was built between these 18 TFs and 32 lipid-metabolism-related genes, suggesting intrinsic associations between TFs and the regulatory mechanism of lipid metabolism.This study represented an important first step towards identifying functions and roles of AP2 superfamily and R2R3-MYB family in lipid adjustments and response to temperature stress. These findings would facilitate the biotechnological development in microalgae-based biofuel production and the better understanding of photosynthetic organisms’ adaptive mechanism to temperature stress. AP2/ERF superfamily was a large family of plant transcription factors, containing one or two conserved AP2 DNA-binding domains (60–70 conserved amino acid sequences) . The AP2Chlamydomonas reinhardtii . The inoculum cultures were incubated at 28 ℃ under 50 μmol m−2 s−1 PAR in the dark/light cycles of 12-h Light/12-h Dark on an orbital shaker at 140 rpm for 48 h to reach the midlog phase (×108 cells/mL) as of seeds. Then, they were inoculated at the ratio of 10% (v/v) into a 500-mL Erlenmeyer flask with 200 mL medium with orbital shaking at 140 rpm under different temperature conditions (10 ℃/28 ℃/32 ℃). Cells were cultivated without light illumination and any external supply of CO2. The experiments were repeated at least three times.escribed , includiCells were collected at 96 h in 10-mL tubes, rapidly harvested by centrifugation, frozen in liquid nitrogen and stored at − 80 °C until lipid extraction. The lipid inactivation and extraction steps were performed twice and lipid extracts were pooled and dried. All LC–MS analyses were carried out on an Exion UPLC coupled to QTRAP 6500 Plus . Further details of lipid extraction and LC–MS analyses were described in the Additional file P < 0.05).Data statistical analyses were performed using the SPSS 21 software. Data were presented as mean values ± SDs. For the statistical analyses of lipidomic data, non-parametric comparisons were performed by applying Kruskal–Wallis test  and DGDG (B) in A. protothecoides grown for 96 h under low and high temperature stress. Figure S8. Heat map of differential expression of genes encoding lipases and genes involved in lipid transport under low and high temperature stress. Figure S9. Phylogenetic relationships of lipid acyltransferases from A. protothecoides, other green algae and plant.Additional file 2: Table S1. The RNA-seq data for the putative AP2/ERF and R2R3-MYB transcriptional factors of A. protothecoides. Table S2. The relative transcript levels of AP2/ERF and R2R3-MYB genes in response to low and high temperature stress in A. protothecoides grown for 168 h. Table S3. The detailed information of lipid species identified in this study. Table S4. The contents of the glycerolipid molecular species in A. protothecoides grown for 96 h under low, normal and high temperature conditions. Table S5. The RNA-seq data for the putative genes in glycerolipid metabolism of A. protothecoides grown for 96 h under low and high temperature stress. Table S6. Subcellular localization predictions for candidate proteins involved in glycerolipid metabolism in A. protothecoides. Table S7. The relative transcript levels of 32 individual genes associated with lipid metabolism in response to low and high temperature stress in A. protothecoides grown for 168 h. Table S8. Primers sequences used for RT-qPCR analysis.Additional file 3. Additional methods.


Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.To further obtain insights into the  Rhipicephalus microplus is a one-host tick, which preferentially feeds on bovines, and it is considered the most harmful cattle parasite2. The economic losses associated with R. microplus parasitism are due to direct effects of the tick itself, which causes skin injuries and long-standing blood loss, leading to anemia and reduction of both weight gain and milk production, or are produced indirectly via transmission of tick-borne pathogens such as Babesia spp. and Anaplasma marginale3. In spite of its huge impact on the economy, current tick control strategies still rely mostly on the use of chemical acaricides, even though selection of resistant tick populations to major used acaricides has been confirmed5. This is recognized as a worldwide drawback to successful tick control. Immunization of cattle against R. microplus and other ticks has been recognized as an alternative approach against chemical control strategy6. Thus, a deeper understanding of tick physiology is needed as a means to find molecular targets that can be useful in the development of novel tick control methods.The cattle tick 8. The genomes of Ixodes persulcatus, Ixodes scapularis, Haemaphysalis longicornis, Dermacentor silvarum, Hyalomma asiaticum, Rhipicephalus sanguineus, and Rhipicephalus microplus have been sequenced and now are helpful resources for studying tick physiology and biology11. The genome of the cattle tick R. microplus was estimated to be 7.1 Gbp in length and consists of approximately 70% repetitive DNA12. Recently, a cattle tick genome draft was reported13 using a hybrid sequencing and assembly approach to capture the repetitive fractions of the genome. According to this study, the R. microplus genome assembly is composed of 51.4% repetitive sequences, containing 38,827 putative R. microplus gene loci, of which 24,758 are protein coding genes13. In spite of the large number of loci and of the significant advance provided by this genome assembly, based on the proportions of highly conserved single copy genes found in all arthropods, the degree of completeness of this assemblage was estimated to be about 41%, still leaving a vast open field for gene discovery in this organism. Recently, a remarkable publication reported the genome assembly of six tick species, including that of R. microplus11. A BUSCO analysis of the predicted proteome of this tick indicated a 51.8% of complete and single BUSCOs, an improvement over the assembly of Barrero et al.13.Transcriptomic analyses have contributed with valuable information regarding the physiology of several parasites, including ticks13, transcriptomes17, and proteomes of ticks19. Based on these datasets, many different experiments have been carried out that elucidated tick-host20 or tick-pathogen interactions19, and can support the development of new control strategies15. Taken together, these reports highlighted the complexity of tick physiology mechanisms comprising both known and novel proteins participating in multiple cellular pathways. Recently, one study in Ixodes ricinus transcriptome shows that the genes involved in cuticle formation, chitin metabolism, and blood digestion enzymes are more related to fed stages. The basic energy metabolism pathway genes are more actively expressed during unfed stages, egg development, and embryogenesis21. In another recently published study, an integrated R. microplus transcriptome and proteome analyses of larvae and salivary glands of nymphs, males and females feeding on susceptible and resistant bovine hosts showed that the expression of genes involved in the host-parasite interaction are associated with host immune activation profile22.Currently, there are several studies describing the genomeR. microplus transcriptome, we used RNAseq to carry out a study of tissue differential gene expression: (i) embryos from 1, 3, 5, 7, 9, 11, 13 day-old eggs; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v and vi) digestive cells from partially and fully engorged females. Reads from each library were mapped back into assembled transcriptome, giving us a view of gene expression in different tissues. Taken altogether, this study offers additional transcriptomic data for this important tick species. The data illustrate the dynamic gene expression changes during the parasitic phase and the embryo development of R. microplus and reveals sets of genes that have a highly tissue-specific expression profile, pointing to pathways that may perform essential roles in the physiology of these tissues. These findings could be useful for future studies of tick physiology, contributing to the development of new tick control methods, as well as new biotechnological and pharmacological applications.Here, aiming to obtain a further insight into Rhipicephalus microplus, we used RNA-seq to carry out a transcriptome study:(i) embryo from 1, 3, 5, 7, 9, 11, 13-day-old eggs (EMB); (ii) ovaries (OV) from partially and fully engorged females; (iii) salivary glands (SG) from partially engorged females; (iv) fat bodies (FB) from partially and fully engorged females; and (v) digestive cells from partially engorged females, and (vi) fully engorged females . We obtained > 500 million raw reads, and after removal of Illumina primers and trimming of low quality bases, we obtained > 190 million high quality reads  were obtained  using BLASTP. Over 9198 (48%), 10,308 (54%), and 13,692 (72%) of protein-coding sequences in the transcriptome had at least one significant BLASTP hit when using E-values < 1e–10, < 1e–5, or < 1, respectively , in order to avoid inclusion of poorly expressed contigs, totaling 16,240 CDS. The data presented a characteristic transcriptional pattern for each tissue, revealing a clear tissue-specific clustering of overexpressed genes in all samples  to highlight that some alterations are introduced in this storage lipoprotein after endocytosis. Vt is a major source of raw materials that support embryo growth. Proteases involved in Vt digestion in R. microplus have been described33. Interestingly, these enzyme transcripts are not abundant in ovaries, but are highly expressed in gut cells and fat body  transcripts , which were highly expressed in fat body and in gut digestive cellsdy Table –S8. This25. Other examples are the tick heme-binding aspartic proteinase (THAP—Rm-14882) and Boophilus yolk-pro-cathepsin (BYC—Rm-849). From all the cathepsin-B coding transcripts with a role in oogenesis, only one (Rm-23945) is overexpressed in the ovary when compared with other tissues, suggesting a role in Vt degradation. A distinct pattern is observed for the Vg receptor (VgR—Rm-80856), since its transcript was expressed mostly in the ovary. The VgR is responsible for Vg uptake; the main reserve protein stored in oocytes for embryo development35.Transcripts coding for proteases involved in Vt digestion are overexpressed, such as the vitellogenin-degrading cysteine endopeptidase (VTDCE—Rm-15854), which was not significantly found in the ovary, and was overexpressed in DIG-F and FB Table . The VTD36. However, a sequence coding to a transcript that is highly homologous to a juvenile hormone acid methyltransferase is overexpressed in the ovary (Rm-49336).Transcripts encoding for proteins involved in nuclear regulation and export from the nucleus such as cell division control protein, chromatin assembly and modification-related proteins, enzymes involved in DNA synthesis and repair, and histone-related proteins represent about 18% of total RPKM for the ovary. Similarly, proteins involved in signal transduction correspond to about 6% of total RPKM Table , includiThe analysis of expression of the detoxification genes Fig.  in all o37, a role that could well be assigned to these tick homologs.The presence of a robust flow of antioxidant potential calls for an equally high source of reactive oxygen species. The fact that two transcripts coding for Duox type NADPH oxidases also have a predominant expression in ovaries make these proteins a candidate for this role. Interestingly, this enzyme has been ascribed a role as the source of hydrogen peroxide for chorion protein cross-linking in an insect39. In ticks, the ovary is the residence of intracellular bacteria symbionts that are essential to tick development42. However, Guizzo et al.43 recently reported a quantitative analysis of the R. microplus microbial community revealing that, different from most animals, the tick gut harbors a very modest (if any) resident microbiota, in contrast to the ovary which has a large population of a Coxiella mutualist symbiont, that increase following the blood meal, paralleling the course of ovarian growth43. Interesting, Amblyomma sculptum and Amblyomma aureolatum have distinct transcriptional profiles of gut44 and microbiota composition45 that can be related to susceptibility to Rickettsia rickettsii.Duox enzymes also have been ascribed a central role in the control of gut microbial symbionts in several insect speciesR. microplus microbial population in the ovary might also involve production of reactive oxygen species, making it essential for a panel of antioxidants to maintain intracellular redox homeostasis. This last hypothesis is in accordance with the identification of seventeen transcripts showing similarity to the antimicrobial peptide microplusin , aurora kinases, polo-like kinases, and checkpoint kinases  are the most important protein kinases related to cell division. Figure R. microplus and I. persulcatus ticks53. Yet regarding control of cell fate and development, nucleoredoxin, a member of the thioredoxin family known to regulate the Wnt/beta-catenin signaling pathway in a redox-dependent way, also showed a high expression level in the ovary54.Research on insect reproduction has characterized many physiological and molecular properties of ovary and egg developmentComparative analysis of embryo allowed the identification of 347 transcripts that are five times overexpressed in embryos than in other analyzed samples Table . Interes57. In insects, these protective cellular layers prevent the loss of water and secrete defensins against bacterial infections; these protective layers are absent in tick embryonic development. These secreted proteins may have a role in immune protection during the long embryogenesis of R. microplus, which takes about 21–22 days in a potentially harmful environment 58.Among these 347 embryo-specific transcripts, those encoding transposons, lipocalins, glycine-rich proteins, and secreted proteins are the most abundant. These secreted proteins are homologous to genes previously described as defensins or genes involved in immune response. Some of these secreted molecules specifically expressed during embryogenesis could be trans-peptides involved in immune response during embryogenesis. Such a finding would be important for embryo survival, since chelicerates such as ticks do not display an extraembryonic enveloping protective layer such as the serosa, which is involved in embryonic immune response in insect egg. Conversely, protective layers have independently evolved in other chelicerates such as scorpionsR. microplus saliva is a complex mixture rich in bioactive compounds that modulate host hemostasis and immunological defenses to allow tick feeding activity60. In recent decades, cumulative information from transcriptomic and proteomic analyses of salivary glands and tick saliva of several tick species have provided insights into the immunological interactions at the tick–host interface. In addition to facilitating blood-feeding, the antihemostatic and immunomodulatory activities of tick saliva may also support survival and establishment of hemopathogens in the host61. R. microplus larvae attach to its host and then begin to feed and molt in nymphs before molting into immature adults, a process that takes around 12 days59. After mating, adult partially engorged females take larger blood meals to complete the maturation within 21–22 days and detach62. In the current study, SG were dissected from adult females feed on the host during 17 and 22 days. Therefore, data shown here combine part of the slow feeding phase and of the final rapid feeding phase. Consistent with reports that other tick species change salivary composition during feeding67, data in this study reveal that 320 transcripts are overexpressed in SG when compared to other tissues, including contigs encoding metalloproteases, proteinase inhibitors, and lipocalins olytic activities68. In a related study, RNAi silencing of metalloproteases from I. ricinus impaired blood meal feeding and egg laying70. In R. microplus, vaccination with recombinant metalloprotease 4 (rBrRm‐MP4) significantly decreases the number of ticks that complete the life cycle on the host and egg hatching, providing an overall protection of 60% against tick infestation65.Tick metalloproteases have been suggested as participating in platelet disaggregation and blood coagulation by fibrinolytic and gelatinase activities, thereby facilitating blood feeding71. In ticks, some protease inhibitors have several Kunitz domains in tandem, and they are classified as monolaris, bilaris, trilaris, and so on, according the number of Kunitz domains. In R. microplus, 11 Kunitz protease inhibitors are overexpressed in the salivary glands, including nine monolaris and two bilaris  signature domains have been reported in blood-feeding mosquitoes and tick sialomes76. In R. microplus, TIL family members include peptides closely related to tick elastase inhibitors, which also have antimicrobial activity77, with a total of three contigs overexpressed in SG , and cystatin were overexpressed in SG Table . Most Kuis Table . SeveralSG Table .78. Contigs matching the tick histamine-binding domain were identified being overexpressed in SG of R. microplus us Table . The hig80. After erythrocyte lysis, blood proteins are taken up by endocytosis and digested in acidic vesicles by lysosomal hydrolases81. To address this singularity of the biology of ticks, we prepared cDNA libraries from DIG that were isolated from (1) partially engorged adult females (DIG-P) that were still engaged in the process blood feeding and were in the “big sip” stage, and (2) fully engorged females (DIG-F) that had already completed blood meal and have detached from the host. To our knowledge, this is the first transcriptomic analysis of this cell type, as other previous studies on gene expression in tick gut have used whole organ extracts83.A unique feature of tick physiology that differs from hematophagous insects is that blood digestion is entirely intracellular, occurring inside a differentiated cell lineage: the so-called digest cells (DIG)Dermacentor variabilis gut84 and accounts alone for close to 20% of the total digestive secretome RPKM of R. microplus  were found in the DIGs libraries. Noticeably, although several inhibitors of cysteine proteinase (cystatins) were identified, these were nearly ten times less expressed than inhibitors of serine proteases. These inhibitors can be related to the inhibition of mammalian proteases involved in blood coagulation and complement cascades87, acting as a regulator of blood digestion in gut88 and modulation of the tick immune proteolytic cascades, that have been ascribed relevant roles in the interaction with the microbiota and with pathogens89. However, it is also possible to speculate that part of these inhibitors might be negative regulators of hemoglobin degradation, as the proteolytic activity involves the release of large amounts of heme, a pro-oxidant molecule90. Keeping the pace of hemoglobin degradation with heme detoxification should be of paramount importance for this cell. Regarding this topic, it is relevant to observe that in DIGs a significant expression of three transcripts coding for an aspartic proteinase, whose activity was shown to be inhibited by heme , was previously described as related to oogenesis28. In this study, THAP was found almost exclusively in the gut (97% of the RPKM); a result that makes it very likely that THAP may also play a relevant role in dietary hemoglobin degradation. However, we also identified the presence of two other contigs of probable paralogous enzymes  was highly transcribed in DIGs, similar to 97.Analysis of transcripts coding for classical antioxidant enzymes showed a common core of antioxidant potential sustained by glutathione synthesis , thioredoxin, and two transcripts coding for thioredoxin reductases, a phospholipid-dependent glutathione peroxidase, and the mitochondrial Mn-SOD that are expressed in all tissues. DIG libraries, however, showed particularly high expression levels of specific isoforms for the cytosolic Cu, Zn SOD, and a thioredoxin peroxidase (2-cys family), plus a selenium glutathione peroxidase and catalase, highlighting a focus on hydrogen peroxide detoxification by this cell type. A result that is in line with previous experimental data reporting the severe effect of chemical inhibition of catalase on blood fed ticks98. This hypothesis is consistent with some sulfotransferases  decreased expression in digestive cells after blood meal, making it likely that those enzymes are acting as players in extracellular matrix production that might support the growth of cells  is a sulfate donor to the biosynthesis of sulfated polysaccharides, major components of extracellular matrix of the tick gut101, where ABC-like transporters were implicated on heme transport in digestive cells of R. microplus. In addition, although this transporter has considerable expression levels in gut, it is even more intensively expressed in the ovary, which raises the interesting possibility that these transporters may be involved in heme transport to the growing oocyte, where vitellin is a major heme-storage protein.A major feature of tick gut DIGs is the presence of an organelle dedicated to heme detoxification, the hemosome. A heme transporting pathway directs heme derived from hemoglobin degradation in the acidic digestive vacuoles to the cytosol and then to the hemosome. Several ABC transporters have high expression levels in DIGs and therefore are likely candidates to function as heme transporters, including Rm-73791 that is almost exclusively expressed in DIGs. This is in accordance with previous biochemical data102.Among typical immune effectors, only defensins seem to be highly expressed in the DIG. However, transcripts receiving an immune label were mainly related to bacterial killing by phagocytosis . This is consistent with the digestive function of these cells being exerted by means of digestion of blood proteins in a lysosomal acid compartment and can be related to very low bacterial levels observed in the gut, including during blood feeding103, carbohydrate metabolism in this organism deserves some discussion concerning the correlation with this very rich protein diet. Two hexokinase coding transcripts were found in the six libraries analyzed. The isoform represented by Rm-54671, is almost 30 times more expressed than Rm-19014, but it is from six to seventy times less expressed in digestive cells than in any other tissue , UDPG-pyrophosphorylase (Rm-37883), UTP-glucose-1-phosphate uridyltransferase (Rm-26034), glycogen synthase (Rm-18733), and glycogen phosphorylase (Rm-26011) are observed in all tissues. The enzymes involved in glycogen accumulation are poorly expressed in digestive cells of fully engorged females. The transcript coding for hexokinase, the first enzyme of the glycolytic pathway, has a very low expression in digestive cells of both partially and fully engorged females; we could not find a transcript coding for phosphofructokinase-1. In contrast, the glucose 6-phosphate-phosphatase transcript was highly expressed, especially in digestive cells of partially engorged females. The high abundance of this transcript in digestive cells led us to speculate that most of the glucose in these cells might not come from the blood meal but from gluconeogenesis, and later being exported to hemolymph and other tissues. The following six enzymes catalyze reactions that are reversible in cellular conditions; the direction of these reactions thus being governed by the law of mass action, and therefore participating both in glycolysis and gluconeogenesis: fructose-bisphosphate aldolase (Rm-1568), triosephosphate isomerase (Rm-4663), glyceraldehyde 3-phosphate dehydrogenase (Rm-10107), 3-phosphoglycerate kinase (Rm-26739), phosphoglycerate mutase (Rm-62495), and enolase (Rm-19039) were found. Their expression levels, although varying among organs, are higher than those of transcripts coding for enzymes that are participating only in glycolysis but not in gluconeogenesis Table .R. microplus , were expressed in all organs tested. Nevertheless, their expression is higher in salivary glands. Moreover, ORFs of all enzymes necessary for β-oxidation were found, showing that conversion of fatty acids into ketone bodies occurs in us Table .Enzymes coding for the pentose phosphate pathway are all present in all tissues but are particularly highly expressed in the ovary. This could account for the high demand for NADPH reducing power by the glutathione/thioredoxin dependent antioxidant mechanisms described above, as well as by the very pronounced concentration of the lipid biosynthesis in the ovary .R. microplus revealed 210 CDS classified in the immune system category -related Lipid-recognition)] domain containing proteins , and GILT 108. GILT are the only molecules capable of keeping essential SH residues of cysteine proteinases reduced at acid pH, therefore preserving their activity inside the endosomal pathway109. As mentioned above, the R. microplus gut is inhabited by a very minute microbial population, probably reflecting the operation of a powerful microbe killing control, such as the production of hemocidins111. Data presented here suggest that the endosomal/lysosomal pathway also play a central role in the gut immunity of ticks  are missing in ticks, as well as in plesiomorphic orders of insects (Hemiptera) and crustaceans. However, it was observed that IMD pathway is functional in I. scapularis115 and in R. microplus116 , although little is known about its activation and effectors. Reports on functional characterization of components of tick immune signaling pathways are also scarce. It has been suggested that the JAK/STAT pathway plays a role in the control of infection by both Anaplasma phagocytophilum118 and Borrelia burgdorferi119 in I. scapularis. Moreover, it was reported that the host IFNγ upregulates a tick GTPase through the JAK/STAT pathway, limiting B. burgdorferi proliferation120. The IMD pathway was also enrolled in protection of ticks against infection. The knockdown of the transcription factor Relish increased the susceptibility of R. microplus to Anaplasma marginale116 and of I. scapularis to A. phagocytophilum115. Interestingly, it was shown that the activation of IMD pathway does not occur through recognition of bacterial peptidoglycans by PGRPs (peptidoglycan-recognition proteins) as in insects, but through recognition of bacterial lipids115.Different from the vastly available information on the immune signaling pathways in insects, very little is known about this subject in ticks. Sequences encoding components of immune signaling pathways were identified in the genome of 114. This result reinforces the constitutive expression of signaling pathway components in R. microplus, as previously suggested114.In the current study, we identified five CDS of Toll-like receptors and two CDS of the cytokine spaetzle, which are components of Toll pathway Table . Other c121. Among the AMPs described in ticks, defensins are a prominent group. These small peptides are synthesized as preprodefensins and are present in several species of both soft and hard ticks122. In R. microplus, one defensin was found in hemocytes47. Importantly, the knockdown of varisin, a defensin from the tick D. variabilis, reduced in 50% the activity of hemolymph against the Gram-positive bacterium Micrococcus luteus123. On the other hand, silencing of this same gene intriguingly reduced the infection of the tick-borne pathogen A. marginale124. Also, defensins were reported to be differentially expressed upon infection with Rickettsia montanensis125. In addition to hemolymph, expression of defensins was also reported to occur in other tick organs, for example the gut and salivary glands. Nineteen defensin CDS were here identified in the R. microplus transcriptome. Among them, one CDS exhibited high transcript levels in digestive cells and another in salivary glands  and cysteine (cathepsin L-type) peptidases. Notably, transcripts of both cathepsins D- and L-types were detected in the current study  and in synganglion of Amblyomma hebraeum129. It was shown that microplusin has the property of chelating copper, inhibiting the respiration of both bacteria and fungi131. Twenty-one microplusin CDS were detected in the present study . This study was conducted considering ethic and methodological aspects in agreement with the International and National Directives and Norms by the Animal Experimentation Ethics Committee of the Universidade Federal do Rio Grande do Sul (UFRGS). The protocol was approved by the Comissão de Ética no Uso de Animais (CEUA) – UFRGS (project 14403).Rhipicephalus microplus ticks (Porto Alegre strain) free of Babesia sp. and Anaplasma sp. were maintained on Hereford bovines acquired from a naturally tick-free area . The bovines were housed in individual thick-proof pens on slatted floors and infested with 20,000 10-day-old R. microplus larvae per animal59. Partially engorged female (PEF) ticks were manually removed from cattle, while fully engorged female (FEF) ticks were obtained after spontaneous detachment from the host. After collection, ticks were washed with 70% ethanol and had the dorsal surface dissected with a scalpel blade.81. Essentially, ticks were dissected in Petri dish containing sterile PBS, guts were isolated, opened gently with tweezers, and cells were detached from the gut wall manually with tweezers. Subsequently, cells were carefully collected using a 1-mL pipette tip, washed three times in sterile PBS and placed in a 12-well culture plate. In order to reduce the amount of debris and gut soluble contents, cells were washed with a gentle flow of culture medium (L-15 Leibowitz’s medium supplemented with 150 mM NaCl) produced by a 1-mL pipette tip. During the isolation of the digestive cells, the gut was dissected and kept in an ice-cold medium. The procedure can be observed by the movie file included as Digestive cells were isolated as previously describedTissues (as described below) were separated with fine-tipped forceps and washed in ice-cold phosphate buffered saline pH 7.2 (PBS). For egg collection, FEF ticks were collected and incubated in Petry dishes at 28 °C and 85% relative humidity until oviposition. For embryos, eggs were kept during twelve days in disposable tubes under the same conditions as described above. Total RNA was extracted from the following tissues: (i) embryos ; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat bodies from partially and fully engorged females; (v and vi) digestive cells from partially and fully engorged females, using TRIZOL reagent according to the manufacturers’ instructions . A total amount of 10 µg RNA was used as input material for the library preparation. Sequencing libraries were generated using the IlluminaTruSeqTM RNA Sample Preparation Kit  following the manufacturer’s recommendations.14. Illumina adaptor sequences and low-quality bases were removed from the raw reads. The quality-filtered sequence reads of each sample were pooled to generate a single transcriptome assembly of the R. microplus. Reads were assembled with Abyss 1.3.3 software (https://www.bcgsc.ca/resources/software/abyss)133 with various k values . Because the Abyss software tends to miss highly expressed contigs, we have also run the Trinity 2.0 assembler (https://trinityrnaseq.github.io/)134 on the raw data. The resulting assemblies were joined by an iterative BLAST and CAP3 assembler14.Bioinformatic analyses were performed as described previously14, based on similarities to known proteins, or by obtaining coding sequences from the larger open reading frame (ORF) of the contigs containing a signal peptide identified by version 3.0 of the SignalP software 135. A non-redundant set of the coding sequences and their protein sequences were mapped into a hyperlinked excel spreadsheet. Signal peptide, transmembrane domains, furin cleavage sites, and glycosylation sites were determined with software from the Center for Biological Sequence Analysis (https://www.cbs.dtu.dk/services/). The automated annotation of the proteins was based on matches to various databases, including Gene Ontology (https://geneontology.org/)136, Pfam (https://pfam.xfam.org/)78, Swissprot (https://www.ebi.ac.uk/uniprot/), KOG (https://mycocosm.jgi.doe.gov/help/kogbrowser.jsf)137, SMART (https://smart.embl-heidelberg.de/)138, Refseq-invertebrates, and sequences containing Acari [organism] protein sequences obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The manual annotation was performed as detailed in previous article14.Coding sequences (CDS) were extracted using an automated pipelineTo estimate the transcripts abundance, reads were mapped back into the CDS using BLASTn with a word size of 25 (-W 25 switch). The matches were used if they had less than two gaps and if their scores were equal to the best score. The resulting BLAST file was used to compile the number of reads each CDS received from each library, and to count the number of hits at each base of the CDS, allowing for the determination of the average CDS coverage, percent linear coverage, as well as maximum and minimum coverage. Mapping of the reads and RPKM values were included in an Excel spreadsheet.13 were used as reference to assess the assembly quality in terms of gene completeness using BUSCO v. 4.1.3 (https://busco.ezlab.org/)23.Finally, tick genomesSupplementary Table S1.Supplementary Information.Supplementary Video 1.


A patient’s survival may depend on several known and unknown factors and it may also vary spatially across a region. Socioeconomic status, accessibility to healthcare and other environmental factors are likely to contribute to survival rates. The aim of the study was to model the spatial variation in survival for colorectal cancer patients in Malaysia, accounting for individual and socioeconomic risk factors. We conducted a retrospective study of 4412 colorectal cancer  patients diagnosed from 2008 to 2013 to model survival in CRC patients. We used the data recorded in the database of the Malaysian National Cancer Patient Registry-Colorectal Cancer (NCPR-CRC). Spatial location was assigned based on the patients’ central district location, which involves 144 administrative districts of Malaysia. We fitted a parametric proportional hazards model in which the spatially correlated frailties were modelled by a log-Gaussian stochastic process to analyse the spatially referenced survival data, which is also known as a spatial survival model. After controlling for individual and area level characteristics, our findings indicate wide spatial variation in colorectal cancer survival across Malaysia. Better healthcare provision and higher socioeconomic index in the districts where patients live decreased the risk of death from colorectal cancer, but these associations were not statistically significant. Reliable measurement of environmental factors is needed to provide good insight into the effects of potential risk factors for the disease. For example, a better metric is needed to measure socioeconomic status and accessibility to healthcare in the country. The findings provide new information that might be of use to the Ministry of Health in identifying populations with an increased risk of poor survival, and for planning and providing cancer control services.  Cancer is a major health burden across the world, with over 14.1 million new cancer cases worldwide in 2012. Of these, around 1.35 million cases (9.6%) are new cases of colorectal cancer. The number of colorectal cancer cases is expected to increase by 80% by 2035, climbing to approximately 2.4 million new colorectal cancer cases and contributing to 1.3 million deaths worldwide [A number of studies have been carried out to investigate and model the spatial variation in survival for various types of cancer. For example, the spatial model of spatial variation in leukaemia survival in north west England , the spaSpatial variation in the survival of colorectal cancer patients has been observed in several studies conducted in developed countries. A study observed disparities in survival across New Jersey among more than 25,000 people diagnosed with colorectal cancer between 1996 and 2003. The lowest survival rates were found mostly in economically deprived areas while those in affluent areas had longer survival times; lack of healthcare accessibility is assumed to be one of the key predictive factors here .Existing research has quantified the geographical variation in survival using a discrete-time multilevel logistic survival analysis for colorectal cancer patients . This reIn Spain , space-tMaterial deprivation and geographical accessibility to healthcare were found to influence survival in colorectal cancer in a study involving cases from three cancer registries in France and one cancer registry in England . This stThere have been several previous studies that have examined the spatial distribution of colorectal cancer incidence in areas of Malaysia, and which have described variation in colorectal cancer incidence, but none have investigated the spatial variation in survival from this disease ,15,16. TOur aim is to model the spatial variation in survival for colorectal cancer patients in Malaysia while accounting for individual and socioeconomic risk factors. We also aim to investigate how individual and socioeconomic factors might affect survival from colorectal cancer, adjusting for spatial location.Identifying the factors that influence the difference in survival rates across the region may help public health authorities to better plan healthcare delivery, and thus eventually reduce disparities in colorectal cancer survival in Malaysia.There are two national cancer registries in Malaysia: the National Cancer Registry (NCR) and the National Cancer Patient Registry (NCPR) . Both arThe NCR captures the data on diagnoses from all regions in Malaysia. Diagnoses are reported to a state registry and from there to the National Registry. However, reporting cases to the state registries from hospitals is voluntary and therefore it is not always completed. However, the NCR is not passive; it conducts active case finding and routine checks. Assessment of the completeness of registration in the NCR is difficult because it is not clear how many of the 165 main hospitals in Malaysia are sending records to the registry, or how accurately diagnoses are recorded even when they are sent .The NCPR collects data on registrations of cancer from participating sites. These participating sites include 34 hospitals that diagnose and treat cancer patients in Malaysia. The objectives of the NCPR are to describe the natural history of cancers and to determine the effectiveness of treatments, to monitor safety, and to evaluate access to treatments. The NCPR collects data on four cancers: colorectal cancer, blood cancers, breast cancer and nasopharyngeal cancers. The NCPR records diagnoses and collects clinical data on risk factors, treatments and patient outcomes. This makes the NCPR data useful for research into the effects of treatments and survival from cancers . This stOur data consisted of 4501 patients with histologically verified primary colorectal cancer diagnosed between 2008 and 2013 . After excluding patients without Malaysian citizenship, patients with negative age and negative survival time, there were 4412 subjects’ data available for analysis.There are many instances in this dataset where information was missing or incomplete, but these are recorded inconsistently with a variety of indicators such as “missing”, “not applicable”, “not available”, “unknown” or “NA”. These do not always match up with the categories defined for each variable in the data. We therefore decided to combine the various missing data as ”unknown” or ”not applicable” if these categories were stated as such in the patients’ form, or otherwise just as ”missing”. We also obtained advice from our data provider about the uncertain category recorded in the database to justify our decision regarding data categorization. For example, even though the variable ”tumour differentiation” has categories of ”not applicable”, ”not available” and ”missing”, most of the data in these categories did not tally with the data definition. Our data provider clarified this, and assured us that all data in those categories of this variable were actually just ”missing”.We collected exposure data on ethnicity as Malay and non-Malay. All others were coded as other because of the small numbers. Smoking status was categorised as: non-smoker, former smoker, active smoker and missing status. Education level was classified as “Nil”, “Primary”, “Secondary”, “Tertiary” or “missing”.Clinical data was recorded as three categories: yes, no and missing. However, cancer stage was recategorised to “Stage I”, “Stage II’, “Stage III”, “Not staged”, and missing. Since the “site of distant metastases” contains many categories, each of which only contained a small number of data points, we decided to combine all the metastases regardless of their specific site. Irrespective of where and when it has been detected, the “presence of distant metastases” was therefore reclassified into yes, no and missing. ”Tumour differentiation” was recorded as “well”, “moderate”, “poor” and “missing”.The treatment modalities were categorised into four types of treatment received by the patients. They are patients who underwent surgery alone, patients who underwent surgery followed by chemotherapy and/or radiotherapy, patients who underwent chemotherapy and/or radiotherapy and patients who got other alternative treatments or palliative care. Patients without information of the treatment received were recorded as an unknown group.The specific cause of death provided in the data was not verified and therefore could not be deemed reliable, so, we decided to perform the analysis on all-cause mortality. The data records whether each patient is dead or alive at the end of the study period; we relabelled each patient’s status as either dead or censored. The censored group are patients who were alive until the end of the study period as recorded in the database. We obtained ethical clearance from the Ministry of Health Medical Research Ethical Committee (MREC), Malaysia and the Faculty of Health and Medicine Research Ethics Committee (FHMREC), Lancaster University (Ref no: NMRR-15-311-24656(IIR).The spatial data involved at the district level analysis includes all districts in Peninsular and East Malaysia and was based on polygonal data. The analysis for Peninsular Malaysia and East Malaysia was done separately as they are physically separated. We therefore separated the shapefile of Malaysia into Peninsular Malaysia and East Malaysia.In order to conduct analysis at the district level, each participant was assigned to their correct district based on the town variable recorded in the data. Each of the 520 unique names of towns seen in the data belong to one of the 144 administrative districts in Malaysia, of which 87 districts are in Peninsular Malaysia and 57 districts are in East Malaysia.We included a measure of socioeconomic status  and the On the other hand, we created a proxy-measure for accessibility of healthcare using the estimated number of hospitals per unit area (the density), calculated by the “density.ppp” function from the “spatstat” package in R software. Then, we included this density as one of the parameters in the spatial survival model. To model the correlation in space, we used an exponential correlation function, using the distance between the centroids of regions to determine the correlation.ho is the baseline hazard function, t is the observed time for the i th individual, Xi is a vector of covariate values for the i th individual, ψ = h are covariate effects, the parameter of the baseline hazard and the parameter of the covariance function of a spatially latent Gaussian field Y, respectively. Yi is the value of the field at the location of individual i.We used the spatial survival model to analyse our spatially referenced survival data. We created our spatial survival model using the “spatsurv” package . The spaβ, ω, η and Y. We compared plots of prior and posterior to check that our data were sufficient to allow identifiability of the parameters in our model. The model has been fitted using three different distributions for the baseline hazards: Weibull, Exponential and B-spline. The models were compared using the Watanabe-Akaike information criterion (waic) value.In the Bayesian model, a prior density for the parameter of interest was inputted and the data modified the prior by using the likelihood to arrive at the posterior.The spatsurv package uses a Markov chain Monte Carlo (MCMC) algorithm to perform Bayesian inference for the parametric proportional hazards model. The idea is to use MCMC to draw samples from the posterior and estimate the parameters in our model. The spatsurv package implements the Markov chain Monte Carlo (MCMC) inferential algorithms because although they are typically slower than approximate methods, such as those based on the Laplace approximation for example, they deliver an unbiased, joint inference for all model parameters and are relatively easily extensible to wider model classes with additional hierarchies . We lookWe plotted the posterior baseline and cumulative hazard for each model, as well as the spatial covariance function and correlation against distance, for Peninsular Malaysia and East Malaysia. The plots of the posterior spatial correlation function show how similar the hazard is across space, and how fast that similarity decays. A correlation plot with a fast drop  shows that there is little dependence between the hazard and distance. On the other hand, if the correlation plot has a slow drop, this shows that there is strong spatial dependence in the hazard or the risk of death from colorectal cancer is highly associated with place. It means that even though there may be a large distance between places, the correlation in their hazard is high. The interpretation of Ø is that for distances over Ø apart, there is little dependence on space.We also mapped the probability that the covariate-adjusted relative risk exceeds certain thresholds. These plots represent the risk over space that is not accounted for by the covariates in our model. All analysis were carried out using R software.We produced two spatial survival models, one for Peninsular (West) Malaysia and one for East Malaysia. After controlling for spatial location and socioeconomic factors, cancer staging still plays an important role in determining the risk of death from colorectal cancer in Malaysia. Patients diagnosed at Stage IV had six times ) and seven times ) higher risk of death from colorectal cancer in West and East Malaysia, respectively, than patients diagnosed at Stage I. Each year of increase in age led to a slight increase ) in the relative risk of death. Other factors that significantly affect survival in colorectal cancer patients in Malaysia were ethnicity, tumour differentiation and the presence of distant metastases. We included two parameters to represent the socioeconomic distinctions in the population; the density of the hospitals in each district and the middle-class household item index (socioeconomic index). However, the socioeconomic index was only available for Peninsular Malaysia. Both of these variables showed an association with a decrease in the risk of death as the value of the parameters increases, however neither result was significant.The table shows the hazard of death from colorectal cancer for the variables chosen in the spatial survival model. It is represented by the median HR and 95% credible interval.The spatial correlation function plot  shows hoIn this study, we investigated the survival model of colorectal cancer in Malaysia, and incorporated geographical location. Our findings show that there is spatial variation in survival prognoses, or the hazard of death, for colorectal cancer in Malaysia even after adjusting for individual-level and area-level covariates. Cancer staging, tumour differentiation and the presence of distant metastases have a significant effect on survival for colorectal cancer patients. However, we also found that Malays had a significant 26% higher risk of dying from colorectal cancer than non-Malays. An increase in age slightly increased the risk of death from colorectal cancer.We found that a high socioeconomic index in an area did not significantly affect the risk of death from colorectal cancer. Our findings were similar to those of  who founIt is possible that there is a positive association between our variable “education”, an individual-level variable, and our socioeconomic index, which is an area-level factor measure with education as one of its domains. We think that these two measures are not likely to be well correlated for the following reasons. The socioeconomic index includes a measure, at an area level, of the proportion of the population with tertiary education in a district (area level). Education is one of five domains by which the socioeconomic measure is comprised. We realize that education might drive socioeconomic status, but it is not the sole driver of socioeconomic status. The widely used UK Index of Multiple Deprivation (IMD) is comprised of seven domains, one of which is based on education, and the index measures small areas across England, called Lower-Layer Super Output Areas (LSOAs) .The education variable in the model represents the individual education level of the patients in this study, which is classified into five categories, nil, primary, secondary, tertiary and missing status. Thus, we think that the education and the SE index variable in the model represent different things. To see if this is likely, we checked if there was any correlation between the two variables but it was not significant with a correlation coefficient of ρ = 0.01.We noticed that there were patients that had changed their address after diagnosis, but we decided not to take the distance of the patients’ addresses to the hospital as our measure of accessibility to healthcare. Address at diagnosis is important as an “exposure” or proxy for unmeasured exposures, but we had no record of length of residence at the address at diagnosis, which leaves open the likelihood of misclassifying cases by exposure to place. For example, people move house or job for many reasons, but sometimes for health reasons. They may, for example, move nearer a hospital when ill, or away from an exposure when concerned and there is evidence that population movement can lead to misclassification in epidemiological studies, such as when a birth address is used in studies of birth defects . InsteadThe relationship between survival and distance to the treatment hospital is not clear cut: it is not necessarily the closest hospital to patients that they will choose to go to seek treatment and for this reason, we used the smoothed density as a covariate in our model. We expected the coefficient of this covariate to be positive, since, in places where there is a greater concentration of hospitals, patients tend to get diagnosed quicker as early symptoms are recognised and acted upon. We found that greater accessibility to healthcare decreased the risk of death from colorectal cancer but the effect was not significant in our study, which contradicts a previous study reporting that lack of access to healthcare was significantly associated with being diagnosed at a more advanced stage of colorectal cancer , which wThree areas were found to have a high risk of death . They were Jeli district in the North-East, Kinta district in the North-West and Melaka Tengah located in the West of Peninsular Malaysia. In East Malaysia, Sabah had better survival compared to Sarawak as shown by the probability of exceedance risk maps. The reasons for these differences could be such things as delay in chemotherapy treatment  and comoWe have a limitation as Malaysia does not have a formal socioeconomic status instrument that is consistent across the population  . CurrentThe spatial analysis method used here has several strengths. First it allows us to combine data from the individual level with data from aggregated levels and map our findings. It also allows us to estimate and map the effect of unobserved environmental confounders through the use of a spatially correlated random effect terms. We applied MCMC for our analysis as it delivers full joint inference for all model parameters. Some limitations to be considered is that this analysis assumes a particular model form and correlation structure for the spatial variation and that it can be difficult in practice to identify the parameters of the spatial process. Attaining good convergence and mixing of MCMC can be difficult without access to bespoke software but again this is not an issue for us. Despite these limitations, this is the first study to examine the variation in survival for colorectal cancer in Malaysia. Having controlled for the potential individual and area-level factors, we found there is variation in the survival or risk of death from colorectal cancer in the population.Our findings indicate there is wide spatial variation in colorectal cancer survival across Malaysia, after controlling for individual and area-level characteristics. Better healthcare provision and higher socioeconomic index in the districts where patients live decrease the risk of death from colorectal cancer, although the associations were not statistically significant in this study. To obtain reliable SES data from individuals would require a range of questions to be answered as part of the initial data entry form. This might seem an unnatural thing to try to elicit routinely as part of a colorectal cancer diagnosis by medical practitioners. Similar comments apply to our chosen measure of healthcare accessibility; again, this information would be better obtained from each patient.In the future, the following suggestions may improve the quality of data and its resulting analysis. Reliable measurements of environmental factors are needed to provide good insight into the effects of potential risk factors for the disease. For example, a better metric is needed to measure the socioeconomic status and accessibility to healthcare in the country. Ensuring complete, accurate and consistently recorded data in the National Cancer Registry database is vital for reliable analysis and meaningful results. Perhaps ascertainment of active cases across the country and also better training of staff that interact with the database is required in order to minimise the amount of missing data.This study will provide new input for the Ministry of Health to target the population with a high risk of poor survival in providing cancer control services and to enhance health activities that are cancer-related in order to improve survival in the population.


Emergence of acute kidney injury (AKI) in patients with nephrotic syndrome (NS) requires prompt diagnosis and differentiation between acute tubular necrosis (ATN) and proliferative glomerulonephritis. We studied the potential use of commercial urinary biomarkers' tests in the diagnosis of AKI in patients with NS.A cross sectional estimate of urinary concentrations of KIM-1 and NGAL was performed in 40 patients with NS: 9 with proliferative glomerulopathy, being 4 with AKI and 31 without proliferative glomerulopathy, being 15 with AKI. AKI was defined using the KDIGO criteria.The mean age was 35 ± 16 years. The main diagnoses were focal and segmental glomerulosclerosis , membranous glomerulopathy , minimal change disease , lupus nephritis , and proliferative glomerulonephritis . Patients with ATN had higher levels of urinary KIM-1 (P = 0.0157) and NGAL (P = 0.023) than patients without ATN. The urinary concentrations of KIM-1 (P= 0.009) and NGAL (P= 0.002) were higher in patients with AKI than in patients without AKI. Urinary NGAL and KIM-1 levels were significantly higher in patients with ATN without proliferative glomerulonephritis than in patients with proliferative glomerulonephritis .Neutrophil gelatinase associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) estimates correlated with histological signs of ATN and were able to discriminate patients with AKI even in conditions of NS. Furthermore, urinary levels of NGAL and KIM-1 may be useful in the differential diagnosis of acute tubular necrosis and exudative glomerulonephritis in patients with nephrotic syndrome. ATN usually requires supportive measures, while proliferative lesions can require immunosuppression ,-Currently, the diagnostic criteria for AKI are defined by elevation in serum creatinine levels and a decrease in urinary output, although serum creatinine is a later marker of kidney dysfunction and does not reflect kidney damage in some instances In this work, we studied the use of tests based on the urinary concentrations of KIM-1 and NGAL for the diagnosis of AKI in patients with nephrotic syndrome and examined the association of urinary concentration of these biomarkers with histological lesions consistent with ATN or proliferative glomerular lesions.Patients: This was a cross sectional study in which we reviewed the biopsies and clinical data of patients with nephrotic syndrome subjected to renal biopsies during relapse or initial presentation in the referral hospitals for renal disease in Salvador, Bahia, Brazil, between January 2012 and December 2013. The patients were divided in three groups according to presence of ATN histologically defined and proliferative glomerulonephritis, as follows: control group (CON), patients with neither proliferative glomerulonephritis nor ATN; acute tubular necrosis group (ATN), patients with ATN and without proliferative glomerulonephritis; and proliferative glomerulonephritis group (PGN), patients with proliferative glomerulonephritis or the presence of crescent lesion in renal biopsy, regardless of the presence of ATN. We studied the potential use of urinary estimates of KIM-1 and NGAL in the diagnosis of AKI in patients with nephrotic syndrome and correlated histological lesions of ATN with urinary concentrations of KIM-1 and NGAL.All of the biopsies were examined at the Instituto Gonçalo Moniz - Fiocruz, Salvador, Brazil. Cases were excluded if the histological slides were not available, if interstitial fibrosis was estimated to encompass 30% or more of the renal cortex area, or if there were fewer than 7 glomeruli in the sample.Clinical data: The following data were obtained from biopsy request forms and from the patients' medical records: age, sex, diagnosis of nephrotic syndrome, serum creatinine, albumin, cholesterol, and triglyceride concentrations, 24-hour urine protein excretion rate, and diagnosis of systemic arterial hypertension. Patients were diagnosed with nephrotic syndrome when the urine protein excretion rate was greater than 3.5 g/24 h associated with edema, hypoalbuminemia and dyslipidemia. AKI was defined as a serum creatinine increase of 0.3 mg/dL or 1.5x the baseline value, according to the KDIGO definition of AKI. Baseline creatinine value was defined as the lowest measurement during the previous year. When previous measurements were not available, the lowest creatinine value estimated during hospitalization was used.Histological analysis: All of the renal specimens were obtained by percutaneous biopsies. They were fixed in Bouin's solution, embedded in paraffin, cut in 2-µm slices, and stained with hematoxylin and eosin. A pathologist (WLCS) reviewed all of the slides. The severity of ATN was estimated as the percentage of the renal cortex that contained tubules with evidence of recent necrosis on examination by conventional optical microscopy. The following tubular changes were considered evidence of recent ATN: tubular dilatation with thinning of the tubular epithelium, accompanied by cellular casts and interstitial edema and the presence of morphological markers of epithelial regeneration, such as nuclear hyperchromatism, mitosis and binucleation. As shown previously, even mild ATN is associated with higher risk of renal failure. Hence, we used the extent of tubular necrosis for separating patients into two groups: with ATN, 10% or more of renal cortex presenting necrotic tubules and without ATN, less than 10% of the renal cortex presenting necrotic tubules. The intensity of tubular interstitial fibrosis was also estimated as a percentage of the renal cortex with increased extracellular matrix by visual assessment.KIM-1 and NGAL measurements in the urine: Morning urine samples were collected prior to renal biopsy. They were centrifuged at 2,000 x g at 4°C, and the supernatant was stored in 2-mL aliquots at -80°C. Urinary concentrations of KIM-1 and NGAL were measured by enzyme linked immunosorbent assay , according to the manufacturers' instructions. All the samples were run in duplicate.Statistical analysis: Data are reported as absolute numbers or percentages and are summarized as means, standard deviations, or medians, using lower and upper percentiles when appropriate. The performance of the biomarkers estimate for the diagnosis of AKI was assessed by analyzing a receiver operating characteristic (ROC) curve. Comparisons between groups were performed using the t-test or the Mann-Whitney (nonparametric) test when applicable. Differences involving proportions were analyzed using the chi-square test. The results were considered statistically significant if P < 0.05. Data were analyzed using Prism software, version 5.01 , and Stata IC software , version 11.Ethics approval: The study was conducted in accordance with the resolution No. 196/96  and the resolution No. 039/2011 (http://conselho.saude.gov.br/web_comissoes/conep/carta_circular/Uso_de_dados_de_prontuarios_para_fins_de_Pesquisa.pdf) of the National Health Council, and the procedures were approved by the Ethics Committee for Research Involving Human Subjects of Instituto Gonçalo Moniz, Fiocruz, Protocol No. 188/09 and No. 380/12.In accordance with these resolutions, written informed consent was obtained from all patients involved in the study. Patient records were only reviewed after approval by the Ethics Committee and authorization by the Hospital Clinical Board as required by the resolution No. 039/2011. All the informed consents are maintained in the records of the Laboratório de Patologia Estrutural e Molecular - LAPEM, Fiocruz Bahia.Sixty-five patients with nephrotic syndrome subjected to renal biopsies between January 2012 and December 2013 were randomly selected, and clinical data and urine samples were collected prior to renal biopsy. Twenty-five patients were excluded: 20 (30%) had more than 30% cortical fibrosis estimated, and 5 (8%) had fewer than 7 glomeruli in the biopsy. Forty patients were included and the clinical and laboratory data of the three groups are presented in Baseline serum creatinine value was available in 31 patients, of whom 19 (59%) presented with AKI according to KDIGO criteria. In nine patients, the baseline creatinine value was not available. Seven of these nine patients had isolated serum creatinine concentrations in the normal range. When estimated by the isolated serum creatinine concentration on the day of biopsy, 14 (35%) patients presented with renal failure. ATN was associated to renal failure in patients with nephrotic syndrome  and 0.8647 (95% CI= 0.7068-1.0000) respectively. The urinary concentrations of KIM-1 and NGAL were higher in patients with AKI  than in patients without AKI (KIM-1 = 1006 [202-2539] pg/mL), P= 0.009; and NGAL = 23.1 [6.6-182.1] ng/mL, P=0.002). Among the patients with proliferative glomerulonephritis, the KDIGO criteria for AKI were applied in only 6 patients, and 4 of them had AKI. Hence, further comparison between this group and the group of patients with AKI without proliferative glomerulonephritis was not performed  and of NGAL  with the percentage of renal cortex with histological lesions of ATN. Patients with ATN ≥10% had higher concentrations of urinary KIM-1 (4299 [630-9534] pg/mL) and NGAL (126.1 [22.8-1195] ng/mL]), compared to patients without ATN . Urinary concentrations of NGAL and KIM-1 were significantly higher in patients with ATN without proliferative glomerulonephritis than in those with proliferative glomerulonephritis .,In the present study, we found that AKI diagnosed by the KDIGO criteria was frequent in patients with nephrotic syndrome. Appel and colleagues 2007) reported AKI in 24 of 95 adult patients with minimal change disease007 repor,Studies associating the histological patterns of ATN, renal failure, and urinary biomarkers for AKI have been scarce in humans. Most of the knowledge in this field comes from histological studies in experimental animal models, transplanted kidneys, and intensive care patientsMost studies have assessed the performance of urinary NGAL and KIM-1 for predicting the diagnosis and prognosis of AKI in animal models of ischemic and toxic AKI, critical illness, and heart surgery patientsUrinary concentrations of NGAL and KIM-1were higher in patients with ATN associated with non-proliferative glomerulopathy than in patients with proliferative glomerulonephritis. Previous studies have demonstrated induction of NGAL in epithelial cells in inflammatory conditions, and in kidney, there is early expression of NGAL in proximal tubule epithelial cells in ischemic reperfusion injury. In patients with lupus nephritis, urinary NGAL excretion was predictive of the renal disease with better accuracy than Anti-dsDNA, and urinary concentrations of NGAL seem to correlate with disease activity and are elevated in renal flares,Finally, although AKI has been considered an uncommon complication of nephrotic syndrome, recent studies have demonstrated that AKI is, in fact, common in the course of idiopathic nephrotic syndrome in adults with minor glomerular abnormalities. Proteinuria has been associated with impaired antioxidative response, tubular epithelial cell injury, and apoptosis in animal models of tubular necrosisATN is a common cause of renal failure in patients with nephrotic syndrome;Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) estimates were correlated with histological signs of ATN;The commercially available kits for NGAL and KIM-1 measurement were able to distinguish patients with AKI from among patients with nephrotic syndrome without requiring further processing steps;Future studies evaluating urinary concentrations of NGAL and KIM-1 could be used to differentiate ATN alone from proliferative glomerulonephritis;The use of these markers does not rule out the requirement of kidney biopsy for the diagnosis of kidney disease in adult patients with nephrotic syndrome.