First Draft Genome Assembly of Root-Lesion Nematode Pratylenchus scribneri Generated Using Long-Read Sequencing.

Root-lesion nematodes (genus Pratylenchus) belong to a diverse group of plant-parasitic nematodes (PPN) with a worldwide distribution. Despite being an economically important PPN group of more than 100 species, genome information related to Pratylenchus genus is scarcely available. Here, we report the draft genome assembly of Pratylenchus scribneri generated on the PacBio Sequel IIe System using the ultra-low DNA input HiFi sequencing workflow. The final assembly created using 500 nematodes consisted of 276 decontaminated contigs, with an average contig N50 of 1.72 Mb and an assembled draft genome size of 227.24 Mb consisting of 51,146 predicted protein sequences. The benchmarking universal single-copy ortholog (BUSCO) analysis with 3131 nematode BUSCO groups indicated that 65.4% of the BUSCOs were complete, whereas 24.0%, 41.4%, and 1.8% were single-copy, duplicated, and fragmented, respectively, and 32.8% were missing. The outputs from GenomeScope2 and Smudgeplots converged towards a diploid genome for P. scribneri. The data provided here will facilitate future studies on host plant-nematode interactions and crop protection at the molecular level.

Three-dimensional, label-free cell viability measurements in tissue engineering scaffolds using optical coherence tomography.

In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.

Recent updates on the biological basis of heterogeneity in bone marrow stromal cells/skeletal stem cells.

Based on studies over the last several decades, the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering. However, the markers for osteogenic precursors; i.e., bone marrowderived skeletal stem cells (SSCs), in association with other cells of the marrow stroma (bone marrow stromal cells, BMSCs) and their heterogeneous nature both in vivo and in vitro remain to be clarified. This review aims to highlight: i) the importance of distinguishing BMSCs/SSCs from other "mesenchymal stem/stromal cells", and ii) factors that are responsible for their heterogeneity, and how these factors impact on the differentiation potential of SSCs towards bone. The prospective role of SSC enrichment, their expansion and its impact on SSC phenotype is explored. Emphasis has also been given to emerging single cell RNA sequencing approaches in scrutinizing the unique population of SSCs within the BMSC population, along with their committed progeny. Understanding the factors involved in heterogeneity may help researchers to improvise their strategies to isolate, characterize and adopt best culture practices and source identification to develop standard operating protocols for developing reproducible stem cells grafts. However, more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations.

Comparison of intrathecal bupivacaine-fentanyl and bupivacaine-butorphanol combinations for joint replacement surgeries.

Background and Aims: The objective of the study was to compare duration of analgesia of fentanyl versus butorphanol as adjuvants to bupivacaine in spinal anesthesia. Material and Methods: A prospective, randomized, double-blinded study conducted in 80 patients of 18-75 years age group and American Society of Anesthesiologists Grades I and II undergoing joint replacement surgeries. A total of 40 patients in each Group A and Group B received 0.5% bupivacaine 3 ml with 25 mcg fentanyl and 25 mcg butorphanol respectively, in a total volume of 3.5 ml made with saline. Duration of analgesia, number of rescue analgesia, sensory, and motor block characteristics were compared between the two groups. Statistical analysis was done using t test and Chi-square test with SPSS 19.0 software. Results: Mean duration of analgesia was found more in Group B in comparison to Group A (P < 0.05). A number of doses of analgesic required postoperatively were more in Group A compared to Group B (P < 0.001). Time required for onset of sensory and motor block was comparable in both the groups. However, two segment regression of sensory block was slower in Group B compared to Group A (P < 0.05). Conclusion: We conclude that addition of butorphanol 25 µg as an adjuvant to 0.5% hyperbaric bupivacaine provided prolonged duration of analgesia compared to 25 mg fentanyl.

Early Detection and Temporal Dynamics of Pratylenchus scribneri Infection in Potato Roots Determined Using Quantitative PCR and Root Staining.

The root-lesion nematode, Pratylenchus scribneri, is a migratory endo-parasitic nematode that impacts potato production on a large scale. Effective management of this nematode requires an understanding of its population dynamics alongside early detection. Typically, the nematode population estimates are made from infested soil; however, considering the endo-migratory lifestyle of this nematode, it also is crucial to determine the nematode population residing inside the host roots. In this study, a SYBR green-based quantitative real-time PCR (qPCR) assay was developed for detection and quantification of P. scribneri in potato roots. The assay used a previously reported primer pair (ITS-2F/ITS-2R), and it proved to be specific and sensitive, detecting as low as 1/128th equivalents of a P. scribneri individual per 0.2 g of potato roots. The robustness of the assay was reflected in high correlation observed between the P. scribneri densities determined microscopically and the densities detected by qPCR in artificially inoculated (R2 = 0.93) and naturally infected (R2 = 0.73) root samples. A time-course experiment conducted in the greenhouse using qPCR detected P. scribneri in potato roots as early as 5 days after planting. The results correlated well with the microscopic observations (R2 = 0.80) and were complemented further with root staining. Additionally, three P. scribneri reproduction peaks were observed during one 3-month growth cycle of potato. Overall, the assay developed in this study is specific to P. scribneri in DNA extracts of root tissue and allows early detection and understandings of reproduction timings of this important nematode of potato.

Quantitative Craniofacial Analysis and Generation of Human Induced Pluripotent Stem Cells for Muenke Syndrome: A Case Report.

In this case report, we focus on Muenke syndrome (MS), a disease caused by the p.Pro250Arg variant in fibroblast growth factor receptor 3 (FGFR3) and characterized by uni- or bilateral coronal suture synostosis, macrocephaly without craniosynostosis, dysmorphic craniofacial features, and dental malocclusion. The clinical findings of MS are further complicated by variable expression of phenotypic traits and incomplete penetrance. As such, unraveling the mechanisms behind MS will require a comprehensive and systematic way of phenotyping patients to precisely identify the impact of the mutation variant on craniofacial development. To establish this framework, we quantitatively delineated the craniofacial phenotype of an individual with MS and compared this to his unaffected parents using three-dimensional cephalometric analysis of cone beam computed tomography scans and geometric morphometric analysis, in addition to an extensive clinical evaluation. Secondly, given the utility of human induced pluripotent stem cells (hiPSCs) as a patient-specific investigative tool, we also generated the first hiPSCs derived from a family trio, the proband and his unaffected parents as controls, with detailed characterization of all cell lines. This report provides a starting point for evaluating the mechanistic underpinning of the craniofacial development in MS with the goal of linking specific clinical manifestations to molecular insights gained from hiPSC-based disease modeling.

Bioactive three-dimensional silk composite in vitro tumoroid model for high throughput screening of anticancer drugs.

HYPOTHESIS: Modeling three-dimensional (3D) in vitro culture systems recapitulating spatiotemporal characteristics of native tumor-mass has shown tremendous potential as a pre-clinical tool for drug screening. However, their applications in clinical settings are still limited due to inappropriate recapitulation of tumor topography, culture instability, and poor durability of niche support. EXPERIMENTS: Here, we have fabricated a bio-active silk composite scaffold assimilating tunable silk from Bombyx mori and - arginine-glycine-aspartate (RGD) rich silk from Antheraea assama to provide a better 3D-matrix for breast (MCF 7) and liver (HepG2) tumoroids. Cellular mechanisms underlying physiological adaptations in 3D constructs and subsequent drug responses were compared with conventional monolayer and multicellular spheroid culture. FINDINGS: Silk composite matrix assists prolonged growth and high metabolic activity (Cytochrome P450 reductase) in breast and liver 3D-tumoroids. Enhanced stemness expression (Cell surface adhesion receptor; CD44, Aldehyde dehydrogenase 1) and epithelial-mesenchymal-transition markers (E-cadherin, Vimentin) at transcript and protein levels demonstrate that bio-active matrix-assisted 3D environment augmenting metastatic potential in tumoroids. Together, enhanced secretion of Transforming growth factor ß (TGFß), anchorage-independency, and colony-forming potential of cells in the 3D-tumoroids further corroborates the aggressive behavior of cells. Moreover, the multilayered 3D-tumoroids exhibit decreased sensitivity to some known anticancer drugs (Doxorubicin and Paclitaxel). In conclusion, the bio-active silk composite matrix offers an advantage in developing robust and sustainable 3D tumoroids for a high-throughput drug screening platform.

Tissue Engineering Measurands.

A metrological perspective for thinking about the characterization of tissue engineered medical products (TEMPs) may help improve communication between researchers. During the development lifecycle of a TEMP, many product properties are measured over the long path to a product release. The selection of each measurement is designed to establish that the product is safe and efficacious (i.e., successful). However, there is often miscommunication during discussions of product characterization. The miscommunication stems from inherent assumptions that are made about the measurements. A "measurand chart" can help clarify these assumptions to enable a more coherent discussion of the value of each measurement. A measurand is defined as "the quantity or property intended to be measured". Tissue engineering measurands are discussed in terms of three case studies including "cell viability in a scaffold", "potency", and "biocompatibility". Topics including a measurement model, defining tissue engineering measurands and definitional uncertainty, are discussed to further refine thinking about tissue engineering measurands. Awareness of these concepts while discussing product characterization can enhance communication and strategic thinking so that the resulting plan is clear and purposeful.

Generation of human induced pluripotent stem cell line (NIDCRi001-A) from a Muenke syndrome patient with an FGFR3 p.Pro250Arg mutation.

Muenke syndrome is the leading genetic cause of craniosynostosis and results in a variety of disabling clinical phenotypes. To model the disease and study the pathogenic mechanisms, a human induced pluripotent stem cell (hiPSC) line was generated from a patient diagnosed with Muenke syndrome. Successful reprogramming was validated by morphological features, karyotyping, loss of reprogramming factors, expression of pluripotency markers, mutation analysis and teratoma formation.

Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Comparative study of hemodynamic effects of intrathecal bupivacaine with butorphanol in cardiac and non-cardiac patients.

BACKGROUND AND AIMS: The synergism between intrathecal opioids and low dose local anesthetics makes it possible to achieve reliable spinal anesthesia (SA) with minimal hypotension. The study objective was to compare the hemodynamic effects of reduced dose of 0.5% intrathecal bupivacaine (2mL) with 25 µg butorphanol in cardiac vs non-cardiac patients. MATERIAL AND METHODS: We included sixty patients aged 30-80 years, undergoing infraumbilical surgeries in the study and compared thirty cardiac patients with mild to moderate reduction in left ventricular ejection fraction (LVEF) on 2D echocardiography (Group C) with 30 non-cardiac patients (Group NC) for similar types of surgery. Both the groups received 0.5% bupivacaine 2.0 ml with 25 µg butorphanol. RESULTS: The spinal block characteristics were similar in both groups (P > 0.05). The blood pressure of the patients in the two groups was comparable till 80 min P > 0.05 after which Group NC had significant increase in blood pressure compared to Group C upto 95 min (P < 0.05). Similarly, heart rate was comparable until 90 min (P > 0.05) after which Group NC had significant increase in heart rate versus Group C upto 100 min (P < 0.05). Eight patients in group C and five patients in group NC showed hypotension. Bradycardia was seen in 4 patients in group C in comparison to only one patient in group NC. CONCLUSION: We can safely consider spinal anesthesia with 10 mg bupivacaine and 25µg butorphanol in cardiac patients with mild to moderately reduced ejection fraction presenting for infraumbilical non-cardiac surgeries with the advantage of intraoperative hemodynamic stability and adequate postoperative analgesia.

Phytochemicals based chemopreventive and chemotherapeutic strategies and modern technologies to overcome limitations for better clinical applications.

Naturally occurring phytochemicals or plant derivatives are now being explored extensively for their health's benefits and medicinal uses. The therapeutic effect of phytochemicals has been reported in several pathophysiological settings such as inflammatory disorders, metabolic disorders, liver dysfunction, neurodegenerative disorders, and nephropathies. However, the most warranted therapeutic effects of phytochemicals were mapped to their anticancerous and chemopreventive action. Moreover, combining phytochemicals with standard chemotherapy has shown promising results in cancer therapy with minimal side effects and better efficacy. Many phytochemicals, like curcumin, resveratrol, and epigallocatechin-3-gallate, have been extensively investigated for their chemopreventive as well as chemotherapeutic effects. However, poor bioavailability, low solubility, hydrophobicity, and obscure target specificity restrict their therapeutic applications in the clinic. There has been a continually increasing interest to formulate nanoformulations of phytochemicals by using various nanocarriers, such as liposomes, micelles, nanoemulsions, and nanoparticles, to improve their bioavailability and target specificity, thereby maximizing the therapeutic potential. In the present review, we have summarized chemopreventive as well as chemotherapeutic action of some common phytochemicals and their major limitations in clinical application. Also, we have given an overview of strategies that can improve the efficacy of phytochemicals for their chemotherapeutic value in clinical settings.

Marine Drugs: A Hidden Wealth and a New Epoch for Cancer Management.

BACKGROUND: Malignant tumors are the leading cause of death in humans. Due to the tedious efforts and investigations made in the field of marine drug discovery, there is now a scientific bridge between marine and pharmaceutical sciences. However, currently only few marine drugs have been lined towards anticancer direction yet many more to are be established in future as well. METHOD: This review gives an overview of present status of marine natural products MNPs both at the level of research and clinical stages. The authors haved summarized the detail information of diverse marine organisms that were reportedto be active or potentially active in cancer treatment in the last two decades. Interstingly, marine organisms are abundant producer of plenty of structurally incomparable bioactive metabolites that have unusual mode of actions and diverse biosynthetic pathways. RESULTS: This review summarizes the associated anticancer properties of different classes of marine natural compounds based on their structural diversity, biological activity, and the molecular mechanisms of action. Emphasis has also be given to recent advances in clinical development of marine agents used in clinical trials. CONCLUSIONS: The present review is summarising the various sources of marine chemicals and their exploration of anticancerous potential. There is justified hope for the discovery and development of new anticancer agents from the marine environment.

Intricatinol synergistically enhances the anticancerous activity of cisplatin in human A549 cells via p38 MAPK/p53 signalling.

Platinum containing drugs are widely used to treat advanced lung carcinomas. However, their clinical success is still limited due to severe side effects, and drug resistance. Alternative approaches are warranted to augment efficacy of platinum based chemotherapeutic drugs with minimal side effects. Intricatinol (INT), a homoisoflavonoid, has been shown to possess anti-tubercular, antioxidant, hypoglycaemic, and hypolipidemic activity. However, its anticancer activity largely remains unknown. In the present study, we have evaluated anticancer potential of INT alone or in combination with cisplatin (CIS) in non-small cell lung carcinoma (A549) cells. Treatment with INT alone reduced the viability of A549 cells in a dose-dependent manner. Interestingly, the combination of low doses of INT and CIS exerted a synergistic effect and induced apoptosis as evident by DNA fragmentation and Annexin V positive cells. Enhanced Bax:Bcl-2 ratio, loss of Δψm, cytochrome c release, cleavage of caspase 3 and PARP1 strongly corroborated our findings. Further, increased expression of p53, p38 MAPK and their phosphorylated counterparts, loss of clonogenicity and reduced migration potential were also recorded with INT + CIS treatment. Most interestingly, INT could not induce any significant cell death in primary mouse embryonic fibroblasts (MEFs). Moreover, no additive or synergistic effect was noted with INT + CIS in MEFs under similar treatment conditions. In conclusion, INT has a selective anticancer potential and could synergize cytotoxicity of CIS. Therefore, the combination of INT and CIS may serve as an effective anticancer strategy for the treatment of non-small cell lung carcinoma.

Necroptosis: Modules and molecular switches with therapeutic implications.

Among the various programmed cell death (PCD) pathways, "Necroptosis" has gained much importance as a novel paradigm of cell death. This pathway has emerged as a backup mechanism when physiologically conserved PCD (apoptosis) is non-functional either genetically or pathogenically. The expanding spectrum of necroptosis from physiological development to diverse patho-physiological disorders, including xenobiotics-mediated toxicity has now grabbed the attention worldwide. The efficient role of necroptosis regulators in disease development and management are under constant examination. In fact, few regulators (e.g. MLKL) have already paved their way towards clinical trials and others are in queue. In this review, emphasis has been paid to the various contributing factors and molecular switches that can regulate necroptosis. Here we linked the overview of current knowledge of this enigmatic signaling with magnitude of therapeutics that may underpin the opportunities for novel therapeutic approaches to suppress the pathogenesis of necroptosis-driven disorders.

Enhanced chemoprevention by the combined treatment of pterostilbene and lupeol in B[a]P-induced mouse skin tumorigenesis.

The present study is aimed to evaluate the inhibitory effect of the combination of two phytochemicals; pterostilbeneand lupeol (generally obtained from blue berries, grapes, white cabbage, green pepper, olive and mangoes) on mouse skin tumorigenesis. We hypothesized that the concomitant topical treatment of selected phytochemicals would lead to improved impediment of skin cancer. Swiss albino mice (n = 25) received a topical dose of Benzo[a]pyrene (B[a]P, 5 µg/animal) with pre/post application of pterostilbene (16 µM/0.2 ml acetone/animal) and/or lupeol (500 µM/0.2 ml acetone/animal) for 32 weeks. Results showed that pterostilbene and/or lupeol treatment resulted in a significant delay in onset of tumorigenesis. However, a more promising effect on tumor suppression was noted with the combination of both the phytochemicals. A significant reduction in average tumor volume, cumulative number of tumors and tumor multiplicity was recorded in combination treated group. The histopathological analysis illustrated the marked suppression in epidermal hyperplasia and necrotic cells in combination treated groups. Our study suggests that the combination of pterostilbene and lupeol was more effective in prevention of skin cancer as compared to either of the phytochemical alone. Therefore, the combined treatment of phytochemicals has better potential to prevent skin carcinogenesis.

Imaging of Pulmonary Manifestations of Connective Tissue Diseases.

Connective tissue diseases (CTDs) are a heterogeneous group of conditions characterized by circulating autoantibodies and autoimmune-mediated organ damage. Common CTDs with lung manifestations are rheumatoid arthritis, scleroderma or systemic sclerosis, Sjögren syndrome, polymyositis/dermatomyositis, systemic lupus erythematosis, mixed connective tissue disease, and undifferentiated connective tissue disease. The most common histopathologic patterns of CTD-related interstitial lung disease are nonspecific interstitial pneumonia, usual interstitial pneumonia, organizing pneumonia, and lymphoid interstitial pneumonia. Drug treatment of CTDs can cause complications, including opportunistic infection.

Deltamethrin induced RIPK3-mediated caspase-independent non-apoptotic cell death in rat primary hepatocytes.

Deltamethrin (DLM), a synthetic pyrethroid insecticide, is used all over the world for indoor and field pest management. In the present study, we investigated the elicited pathogenesis of DLM-induced hepatotoxicity in rat primary hepatocytes. DLM-induced cell death was accompanied with increased ROS generation, decreased mitochondrial membrane potential and G2/M arrest. Pre-treatment with N-acetyl cysteine/butylated hydroxyanisole/IM54 could partly rescue hepatocytes suggesting that ROS might play a role in DLM-induced toxicity. Interestingly, DLM treatment resulted in a caspase-independent but non-apoptotic cell death. Pre-treatment with pan-caspase inhibitor (ZVAD-FMK) could not rescue hepatocytes. Unaltered caspase-3 activity and absence of cleaved caspase-3 also corroborated our findings. Further, LDH release and Transmission electron microscopy (TEM) analysis demonstrated that DLM incites membrane disintegrity and necrotic damage. Immunochemical staining revealed an increased expression of inflammatory markers (TNFα, NFκB, iNOS, COX-2) following DLM treatment. Moreover, the enhanced RIPK3 expression in DLM treated groups and prominent rescue from cell death by GSK-872 indicated that DLM exposure could induce programmed necrosis in hepatocytes. The present study demonstrates that DLM could induce hepatotoxicity via non-apoptotic mode of cell death.

Evaluation and physiological correlation of plasma proteomic fingerprints for deltamethrin-induced hepatotoxicity in Wistar rats.

AIMS: Uprising reports towards deltamethrin (DLM)-induced toxicity in non-target species including mammals have raised a worldwide concern. Moreover, in the absence of any identified marker, the prediction of DLM elicited early toxic manifestations in non-targets remains elusive. MAIN METHODS: Comprehensive approach of proteome profiling along with conventional toxico-physiological correlation analysis was performed to classify novel protein based markers in the plasma of DLM exposed Wistar rats. Animals were exposed orally to DLM (low dose: 2.56mg/kg b.wt. and high dose: 5.12mg/kg b.wt.) up to seven consecutive days. KEY FINDINGS: The UPLC-MS/MS analysis revealed a dose-dependent dissemination of DLM and its primary metabolite (3-Phenoxy benzoic acid) in rat plasma. Through 2-DE-MS/MS plasma profiling and subsequent verification at the transcriptional level, we found that 6 liver emanated acute phase proteins (Apolipoprotein-AIV, Apolipoprotein E, Haptoglobin, Hemopexin, Vitamin D Binding protein, and Fibrinogen gamma chain) were significantly (p<0.05) modulated in DLM treated groups in a dose-dependent manner. Accordingly, DLM exposure resulted in adverse effects on body growth (body weight & relative organ weight), serum profile, liver function and histology, inflammatory changes (enhanced TNF-ɑ, TGF-ß and IL6 level), and oxidative stress. Moreover, these toxic manifestations were suppressed upon N-acetyl cysteine (NAC) supplementation in DLM treated animals. Thus, DLM-induced inflammatory response and subsequent oxidative injury to liver grounds the altered expression of identified acute phase proteins. SIGNIFICANCE: In conclusion, we proposed these six liver emanated plasma proteins as novel candidate markers to assess the early DLM-induced hepatotoxicity in non-target species with a minimal invasive mean.