Specific Damage Research Articles

Evaluation of the vital viability and their application in fungal spores’ disinfection with flow cytometry

More attention was focused on fungi contamination in drinking water. Most researches about the inactivation of fungal spores has been conducted on disinfection efficiency and the leakage of intracellular substances. However, the specific structural damage of fungal spores treated by different disinfectants is poorly studied. In this study, the viability assessment methods of esterase activities and intracellular reactive oxygen species (ROS) were optimized, and the effects of chlorine-based disinfectants on fungal spores were evaluated by flow cytometry (FCM) and plating. The optimal staining conditions for esterase activity detection were as follows: fungal spores (106 cells/mL) were stained with 10 μM carboxyfluorescein diacetate and 50 mM ethylene diamine tetraacetic acid at 33 °C for 10 min (in dark). The optimal staining conditions for intracellular ROS detection were as follows: dihydroethidium (the final concentration of 2 μg/mL) was added into fungal suspensions (106 cells/mL), and then samples were incubated at 35 °C for 20 min (in dark). The cell culturability, membrane integrity, esterase activities, and intracellular ROS were examined to reveal the structural damage of fungal spores and underlying inactivation mechanisms. Disinfectants would cause the loss of the cell viability via five main steps: altered the morphology of fungal spores; increased the intracellular ROS levels; decreased the culturability, esterase activities and membrane integrity, thus leading to the irreversible death. It is appropriate to assess the effects of disinfectants on fungal spores and investigate their inactivation mechanisms using FCM.

Myeloid cells in sensing of tissue damage.

Myeloid cells are components of the innate immune system that represent the first line of defense. Tissue damage, associated with pathological conditions such as infection, cancer or autoimmunity, leads to the exposure of the intracellular content to the extracellular environment. Myeloid cells detect ligands exposed or released by dead cells through specific receptors that signal for a diversity of responses. Inflammatory responses triggered by myeloid cells after sensing tissue injury can contribute to resolution of the damage. The signaling response following dead-cell sensing by myeloid cells can contribute either to an inflammatory or a regulatory response. We review herein some representative examples of how myeloid cells react to the recognition of cell death during specific tissue damage contexts. A deep understanding of the cellular and molecular mechanisms underlying these processes would allow to improve therapeutical interventions in pathologies associated with tissue damage.

Repurposing of niclosamide as a STAT3 inhibitor to enhance the anticancer effect of chemotherapeutic drugs in treating colorectal cancer

AimsColorectal cancer (CRC) is the third most common cancer worldwide. Mutation of various cell signaling molecules or aberrant activation of signaling pathways leads to poor response to chemotherapy in CRC. Signal transducer and activator of transcription protein 3 (STAT3) is an important signaling molecule, which plays crucial roles in regulating cell survival and growth. In this study, the potentitation of chemotherapy by putative STAT3 inhibitors for treating CRC was investigated. Main methodsA few putative STAT3 inhibitors were investigated. Niclosamide, originally indicated for the treatment of tapeworm infection, was chosen for further investigation in five CRC cell lines (HCT116, HT29, HCC2998, LoVo and SW480). Western blot analysis was used to evaluate the expression of STAT3/phospho-STAT3 and its downstream targets. Sulforhodamine B assay was used to evaluate the cytotoxicity of drug combinations. Flow cytometric assays were used to investigate the apoptotic and cell cycle effect. Key findingsNiclosamide was found to inhibit expression and activation of STAT3 in a concentration- and time-dependent manner, thereby downregulating STAT3 downstream targets including survivin and cyclin-D1 to induce apoptosis and cell cycle arrest. When combined with niclosamide or specific STAT3 inhibitor (C188-9), the cytotoxicity and DNA damage response from SN38 (the active metabolite from irinotecan) were significantly enhanced. The sequential exposure of SN38 followed by niclosamide was found to be the most potent treatment sequence for the drug combination. SignificanceNiclosamide represents a promising candidate for repurposing to potentiate the anticancer activity of chemotherapeutic drugs.

Novel Hallux Valgus Cadaveric Model: Role of Ligament Damage and Tendon Pull in Recreating the Deformity

Category:Bunion; Midfoot/ForefootIntroduction/Purpose:Hallux valgus (HV) is of uncertain origin, which explains the plethora of treatment options available. In the last few years it has been shown that it is not a one plane deformity, but a multiplanar one. It is the authors belief that Hallux Valgus originates from instabilities located at medial ray joints. To be able to further understand this frequent pathology, a cadaveric model should be developed. Through specific and limited medial column ligament damage, a model was developed trying to obtain similar deformities to the ones observed in real life, i.e. first ray varus and pronation.Methods:7 fresh frozen lower leg specimens were used without evident foot and ankle pathology. Tibio-talar and talo-navicular joints were fixed. Luminous markers were attached to the medial and middle foot ray bones, including talus. A constant tibial axial load of 25 kg was used. Cyclic tibial rotation (to simulate gait phases) and 5 kg pull on the extensor and flexor hallucis tendons (EHL and FHL) were applied to every specimen. Limited and sequential medial and middle column ligamentous sectioning were performed, including the naviculocuneiform, intercuneiform, metatarsocuneiform, metatarsophalangeal medial capsule and intermetatarsal ligaments. After each ligament damage, 3D bony alignment was registered using high speed high definition infrared cameras.Results:After ligament sectioning, an increase in intermetatarsal angle was observed of 2.5 degrees (range 0.8-5) (p=0.001), with 1 degree (0-3) (p=0.02) of further increase when Hallucis tendons pull was applied. Intercuneiform angle increased 2.3 degrees (0- 6) (p=0.01), with no change with tendons pull. Regarding bone rotation, the metatarsal pronated in relation to the navicular (metatarso-navicular angle) 6.3 degrees (3-20) (p=0.015) and the cuneiform pronated in relation to the navicular (naviculo- cuneiform angle) 3.3 degrees (0.5-6) (p=0.005). At the metatarso-cuneiform joint, a non-significant rotation was identified (1 degree (p=0.39). Hallucis tendons pull did not significantly affect any rotation angle. An evident hallux valgus deformity could be created, which further deformed with hallucis tendons pull.Conclusion:After sequential medial and middle ray ligament sectioning, a progressive and simultaneous increase in medial column varus and pronation were observed. Hallucis tendons pull, increased the medial column varus, but appeared not to play a role in bone rotation. Bone rotation occurs along the medial column joints, not isolated to the tarsometatarsal joint. Progressive medial ray ligamentous sectioning was able to recreate a Hallux valgus deformity, including medial ray varus and pronation.

VIsualizing myocardial injury from elective cardioversion

Introduction Despite everyday use of electrical interventions in cardiovascular care, the extent and type of concomitant myocardial injury is not fully understood. Current literature disagrees about the question whether and how cardioversion or defibrillation damage the myocardium, especially when serologic markers are used. Such markers are not always cardiac-specific, nor diagnostic for type and region of myocardial injury. Cardiovascular magnetic resonance (CMR) allows for the visualization of specific damage in the myocardium and can aid in diagnostic accuracy. In particular, parametric mapping techniques can localize edema and fibrosis. We aimed to investigate whether the acute and long-term impact of electrical cardioversion on myocardial structure and function are detectable using CMR imaging. Methods Patients scheduled for elective cardioversion were enrolled to undergo three CMR exams: on the morning prior to cardioversion to assess pre-existing injury; two to five hours after cardioversion to assess the acute response; and six to ten weeks later to investigate chronic injury. The CMR exam studied left ventricular (LV) function, T2 mapping to measure edema, extracellular volume (ECV) to measure diffuse fibrosis, and quantified both degree of injury and proportion (%) of myocardial area affected in the imaged slices. Data are mean±SD. Results Eight patients completed the study, requiring 1-2 shocks (totalling 120-300 J biphasic energy) to achieve sinus rhythm. LV ejection fraction increased after cardioversion from 47±13% to 55±15% (p=0.020), and was 52±16% at the third exam (p=0.199). Even prior to intervention, some patients showed edema (baseline T2>40ms) afflicting 49±23% of their LV myocardium. Area affected by edema expanded to 72±18% after cardioversion (p=0.002) and returned to 54±24% by the third exam. T2 signal rose from baseline (40.4±1.8ms) after cardioversion acutely to 44.1±5.2ms (p=0.028) and normalized until the late exam (40.8±3.1ms, Figure). The degree of ECV increased from 28.0±0.8% at baseline to 29.3±2.5% at 6-10 weeks after cardioversion (p=0.016). Myocardial area affected by diffuse fibrosis (ECV>30%) was 28.3±9.4% at baseline and 38.8±18.9% late after cardioversion (p=0.018). Pathologic T2 increases (indicative of edema) were not observed in all patients, but individuals with higher baseline ECV also experienced greater T2 increase after cardioversion (r=0.840, p=0.036). Discussion Patients with atrial fibrillation scheduled for elective cardioversion may exhibit pre-existing myocardial edema at CMR. Cardioversion improves left ventricular systolic function, but also aggravates myocardial edema acutely within hours, and possibly adds to diffuse fibrosis during several weeks thereafter. Such sequelae of cardioversion were observed mainly in patients with a greater burden of pre-existing myocardial injury. More data is needed to corroborate these preliminary findings and to study whether this type of myocardial injury predicts worse outcome.

IMMUNE MECHANISMS FOR THE DEVELOPMENT OF HEPATORENAL SYNDROME IN LIVER CIRRHOSIS

Hepatorenal syndrome is a life-threatening complication of liver cirrhosis. 90% of patients with liver cirrhosis die within 2 months since the onset of hepatorenal syndrome development. For many years the hypothesis of the hemodynamic mechanism of hepatorenal syndrome development was accepted as the only true one, according to this hypothesis compensatory systemic vasodilation in response to portal hypertension causes renal ischemia and the development of functional specific acute kidney damage, the so-called “hepatorenal syndrome – acute kidney damage”. In recent years some works were published that substantiate the role of a systemic inflammatory reaction in the development of hepatorenal syndrome; this inflammatory reaction being associated with activation of innate immunity cells in response to a bacterial infection, including that to the microflora of the intestine which is adjacent to the liver. Data has been obtained which indicated that Toll-like receptors (TLRs), in particular TLR4 and TLR9 are involved in the development of hepatorenal syndrome.

Influence of Crack Width in Alternating Tension–Compression Regimes on Crack-Bridging Behaviour and Degradation of PVA Microfibres Embedded in Cement-Based Matrix

The use of high-performance polymeric microfibres in enhancing the ductility of cementitious composites is widespread. A vivid example is the application of strain-hardening cement-based composites (SHCCs) in the construction industry. However, there are a few challenges which need to be addressed with respect to material design. For instance, the ductility of SHCC diminishes under alternating tension–compression loading, where the fibres lose their crack-bridging capacity due to specific damage mechanisms. The damage development and its influence on crack-bridging capacity have been studied in previous works by the authors. The paper at hand focuses on the influence of crack width on the crack-bridging capacity of polymeric microfibres in conjunction with the number of cycles in an alternating tension–compression regime with different cyclic compressive force levels. It shows that bridging capacity can be markedly influenced by crack width: an increase in crack width leads to more severe damage to the fibres and thus to lower crack-bridging capacity. Then, after analysing the specimens by means of electron microscopy, a hypothesis is presented to address the effect of crack width on damage development. Finally, a simple approach is proposed for estimating the influence of different parameters on fibre degradation.

The Synthetic Myeloperoxidase Inhibitor AZD3241 Ameliorates Dextran Sodium Sulfate Stimulated Experimental Colitis.

Chronic inflammatory bowel disease (IBD) is a condition with multifactorial pathophysiology. To date, there is no permanent cure and the disease is primarily managed by immunosuppressive drugs; long-term use promotes serious side effects including increased risk malignancies. The current study aimed to target neutrophil-myeloperoxidase, a key contributor to the pathogenesis of IBD, through the use of AZD3241that inhibits extracellular myeloperoxidase. Experimental colitis was induced in C57BL/6 male mice by 2% dextran sodium sulfate in drinking water ad libitum over 9 days. Mice received either normal drinking water and peanut butter (control), 2% DSS in drinking water and peanut butter or 2% DSS in drinking water and AZD3241 (30 mg/kg) dispersed in peanut butter daily for 9 days. Administered AZD3241 attenuated body weight loss (10% p<0.05) and improved clinical score (9 fold p<0.05; a score comprising the time-dependent assessment of stool consistency and extent of rectal bleeding), loss of colonic crypts (p<0.001), preserved surface epithelium (p<0.001) and enhanced expression of the transcription factor Nrf-2 (regulator of antioxidants) and enhanced expression of the downstream antioxidant response element haeoxygenase-1 (HO-1) in the colon tissue. Also, the concentration of fecal hemoglobin and the myeloperoxidase specific oxidative damage biomarker 3-chlorotyrosine in the colon were significantly decreased in the presence of AZD3241. This latter result was consistent with AZD3241 inhibiting MPO activity in vitro. Overall, AZD3241 ameliorated the course and severity of experimental colitis through ameliorating MPO derived tissue damage and could be considered a potential therapeutic option, subject to further validation in chronic IBD models.

Structural Aspects of DNA Repair and Recombination in Crop Improvement.

The adverse effects of global climate change combined with an exponentially increasing human population have put substantial constraints on agriculture, accelerating efforts towards ensuring food security for a sustainable future. Conventional plant breeding and modern technologies have led to the creation of plants with better traits and higher productivity. Most crop improvement approaches (conventional breeding, genome modification, and gene editing) primarily rely on DNA repair and recombination (DRR). Studying plant DRR can provide insights into designing new strategies or improvising the present techniques for crop improvement. Even though plants have evolved specialized DRR mechanisms compared to other eukaryotes, most of our insights about plant-DRRs remain rooted in studies conducted in animals. DRR mechanisms in plants include direct repair, nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), non-homologous end joining (NHEJ) and homologous recombination (HR). Although each DRR pathway acts on specific DNA damage, there is crosstalk between these. Considering the importance of DRR pathways as a tool in crop improvement, this review focuses on a general description of each DRR pathway, emphasizing on the structural aspects of key DRR proteins. The review highlights the gaps in our understanding and the importance of studying plant DRR in the context of crop improvement.

AMPK Activity Contributes to G2 Arrest and DNA Damage Decrease via p53/p21 Pathways in Oxidatively Damaged Mouse Zygotes.

In zygotes, the capacity of G2/M checkpoint and DNA repair mechanisms to respond to DNA damage varies depending on different external stressors. In our previous studies, we found that mild oxidative stress induced a G2/M phase delay in mouse zygotes fertilized in vitro, due to the activation of the spindle assembly checkpoint. However, it is unclear whether the G2/M phase delay involves G2 arrest, triggered by activation of the G2/M checkpoint, and whether AMPK, a highly conserved cellular energy sensor, is involved in G2 arrest and DNA damage repair in mouse zygotes. Here, we found that mouse zygotes treated with 0.03 mM H2O2 at 7 h post-insemination (G1 phase), went into G2 arrest in the first cleavage. Furthermore, phosphorylated H2AX, a specific DNA damage and repair marker, can be detected since the early S phase. We also observed that oxidative stress induced phosphorylation and activation of AMPK. Oxidative stress-activated AMPK first localized in the cytoplasm of the mouse zygotes in the late G1 phase and then translocated to the nucleus from the early S phase. Overall, most of the activated AMPK accumulated in the nuclei of mouse zygotes arrested in the G2 phase. Inhibition of AMPK activity with Compound C and SBI-0206965 abolished oxidative stress-induced G2 arrest, increased the activity of CDK1, and decreased the induction of cell cycle regulatory proteins p53 and p21. Moreover, bypassing G2 arrest after AMPK inhibition aggravated oxidative stress-induced DNA damage at M phase, increased the apoptotic rate of blastocysts, and reduced the formation rate of 4-cell embryos and blastocysts. Our results suggest the G2/M checkpoint and DNA repair mechanisms are operative in coping with mild oxidative stress-induced DNA damage. Further, AMPK activation plays a vital role in the regulation of the oxidative stress-induced G2 arrest through the inhibition of CDK1 activity via p53/p21 pathways, thereby facilitating the repair of DNA damage and the development and survival of oxidative stress-damaged embryos. Our study provides insights into the molecular mechanisms underlying oxidative-stress induced embryonic developmental arrest, which is crucial for the development of novel strategies to ensure viable embryo generation.

"Repair Me if You Can": Membrane Damage, Response, and Control from the Viral Perspective.

Cells are constantly challenged by pathogens (bacteria, virus, and fungi), and protein aggregates or chemicals, which can provoke membrane damage at the plasma membrane or within the endo-lysosomal compartments. Detection of endo-lysosomal rupture depends on a family of sugar-binding lectins, known as galectins, which sense the abnormal exposure of glycans to the cytoplasm upon membrane damage. Galectins in conjunction with other factors orchestrate specific membrane damage responses such as the recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to either repair damaged membranes or the activation of autophagy to remove membrane remnants. If not controlled, membrane damage causes the release of harmful components including protons, reactive oxygen species, or cathepsins that will elicit inflammation. In this review, we provide an overview of current knowledge on membrane damage and cellular responses. In particular, we focus on the endo-lysosomal damage triggered by non-enveloped viruses (such as adenovirus) and discuss viral strategies to control the cellular membrane damage response. Finally, we debate the link between autophagy and inflammation in this context and discuss the possibility that virus induced autophagy upon entry limits inflammation.

Cardiovascular complications of COVID-19

The objective : based on publications to provide up-to-date information on the frequency, basic variants and predictive significance of cardiovascular complications of the coronavirus infection caused by SARS-CoV-2. Results : the article identifies variants of cardiovascular disorders in COVID-19 caused by various comorbidities in particular hypertension, specific virus-associated myocardial damage, and side effects of medications. It analyzes the role of angiotensin-converting enzyme 2 type II receptors during infection with the coronavirus and potential changes in the renin–angiotensin–aldosterone system including in patients receiving angiotensin-converting enzyme inhibitors or blockers of angiotensin receptors. The data on the frequency and prognostic role of arrhythmias, virus-associated myocardial damage and heart failure, the risk of ischemia, and myocardial infarction are presented. The adverse cardiovascular events of drugs of different groups prescribed for treatment of COVID-19 and possible drug interactions are described. The current recommendations for cardiotonic and cardioprotective therapy in patients with cardiovascular complications are analyzed. Conclusion . The SARS-Cov-2 virus has a pronounced cardiac tropic effect which requires maximum cardiac alertness in the treatment of patients in this category, timely use of electrocardiography, echocardiography, control of biomarkers of myocardial damage and tension, as well as a reasonable use of cardiotonic and cardioprotective drugs.

THE IMPACT OF DOUBLE-STRANDED SPERM DNA BREAKS ON ICSI OUTCOME

We sought to evaluate specific sperm DNA damage, double-stranded DNA breaks (dsDNA), and its effect on embryo development and implantation. Over a year-long period, a prospective pilot study was carried out on sperm samples to evaluate the proportion of dsDNA on samples screened by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) to assess total DNA fragmentation. Once a correlation was established, we extrapolated dsDNA values retrospectively onto patients who had their ejaculates screened by TUNEL to evaluate clinical outcome. Samples from consenting couples were screened for dsDNA rates by neutral Comet assay using an in-house protocol; 200 spermatozoa were assessed per patient. These samples were also assessed by TUNEL using a commercially available kit, analyzing at least 500 spermatozoa per patient. ICSI was performed in the standard fashion. The pilot study reported an average total DNA fragmentation of 11.3±6% by TUNEL and an average dsDNA of 2.2±3% by neutral Comet. The results showed a linear relationship between the overall SCF and dsDNA rates (R2=0.96). This equation was applied to extrapolate the dsDNA levels from 573 normozoospermic men (volume of 2.6±1 mL, concentration of 42.3±33x106/mL, 43.1±10% motility, and 4.2±1% normal morphology) with an average SCF of 14.2±8%. Therefore, on the basis of this preliminary test, we established a dsDNA threshold of 3%. A total of 417 couples (maternal age, 37.0±4 yrs; paternal age, 38.6±5 yrs) underwent 777 ICSI cycles and presented with dsDNA levels of 1.8±0.6%. These cycles had a 73.0% fertilization rate, a 12.7% (123/966) implantation rate, and a 23.2% (102/440) clinical pregnancy rate (CPR), of which 15 were lost (14.7%), leaving a 19.7% (87/440) ongoing/delivery rate. There were 155 couples with dsDNA levels of 4.2±1%. These couples underwent 268 ICSI cycles with a comparable maternal age of 37.3±5 years, but with older male partners at 41.3±8 years of age (P<0.001). These cycles had a comparable fertilization rate of 71.0%, implantation rate of 11.4% (53/464), and clinical pregnancy rate of 24.3% (44/181). However, these couples were much more likely to lose their pregnancy at 29.5% (13/44; P = 0.03), leaving an ongoing/delivery rate of 17.1% (31/181). These findings provide further evidence that dsDNA has an important role in the success of a pregnancy generated by ICSI. dsDNA damage has been linked to aneuploidy and consequent pregnancy loss. The use of an assay in a laboratory setting to screen exclusively for dsDNA would help to identify paternally linked aneuploidy that traditional screening would only report as total SCF.

Evaluation of structural safety reduction due to water penetration into a major structural crack in a large concrete project

Structural damage to an arch dam is often of major concern and must be evaluated for probable rehabilitation to ensure safe, regular, normal operation. This evaluation is crucial to prevent any catastrophic or failure consequences for the life time of the dam. If specific major damage such as a large crack occurs to the dam body, the assessments will be necessary to determine the current level of safety and predict the resistance of the structure to various future loading such as earthquakes, etc. This study investigates the behavior of an arch dam cracked due to water pressure. Safety factors (SFs), of shear and compressive tractions were calculated at the surfaces of the contraction joints and the cracks. The results indicated that for cracking with an extension depth of half the thickness of the dam body, for both cases of penetration and non-penetration of water load into the cracks, SFs only slightly reduces. However, in case of increasing the depth of crack extension into the entire thickness of the dam body, the friction angle of the cracked surface is crucial; however, if it reduces, the normal loading SFs of stresses and joints tractions reduce significantly.

An Evaluation of Different Parameters to Screen Ornamental Shrubs for Salt Spray Tolerance.

In the context of seaside landscaping, one of the greatest challenges for practitioners and scientists is to select suitable species that are able to tolerate salt spray. This is the key aspect for a wide number of potentially suitable species. The objectives of this study were (1) to identify plant traits associated with species tolerance to salt stress and (2) to evaluate the responses of different shrub species to salt spray. For this purpose, a study was conducted to determine the effects of salt spray on twenty-four ornamental shrubs using rapid and easy-to-use methodology. The species were subjected twice a week to nebulization treatment with simulated seawater solution for 60 days. Every 20 days, net photosynthesis, stomatal conductance, transpiration rate, and chlorophyll a fluorescence were determined. Furthermore, dry biomass of the different organographic portions, leaf number, leaf area, Specific Leaf Area, chlorophyll content, and leaf damage were determined at the end of the experiment. The species exposed to seawater solution showed different physiological and morphological responses. Based on the above indices, these ornamental shrubs were screened and categorized as tolerant, moderately tolerant, or susceptible. The results suggest that Convolvulus, Ceratonia, and Ligustrum are more tolerant to salt spray than numerous other genotypes; L. langmaniae, Cascabela, and L. frutescens, conversely, are more sensitive. Among the plant traits, the morphological parameters thoroughly characterized the effects of the salt spray, but they were destructive, with the only exception being the leaf damage percentage. This last non-destructive parameter is interesting considering the aesthetic value that ornamental plants must have. The physiological parameters, and in particular photosynthesis activity, can instead be used as a non-destructive screening method to select species suitable for ornamental green spaces near the sea.

Saving time maintaining reliability: a new method for quantification of Tetranychus urticae damage in Arabidopsis whole rosettes

BackgroundThe model species Tetranychus urticae produces important plant injury and economic losses in the field. The current accepted method for the quantification of the spider mite damage in Arabidopsis whole rosettes is time consuming and entails a bottleneck for large-scale studies such as mutant screening or quantitative genetic analyses. Here, we describe an improved version of the existing method by designing an automatic protocol. The accuracy, precision, reproducibility and concordance of the new enhanced approach are validated in two Arabidopsis accessions with opposite damage phenotypes. Results are compared to the currently available manual method.ResultsImage acquisition experiments revealed that the automatic settings plus 10 values of brightness and the black background are the optimal conditions for a specific recognition of spider mite damage by software programs. Among the different tested methods, the Ilastik-Fiji tandem based on machine learning was the best procedure able to quantify the damage maintaining the differential range of damage between accessions. In addition, the Ilastik-Fiji tandem method showed the lowest variability within a set of conditions and the highest stability under different lighting or background surroundings. Bland-Altman concordance results pointed out a negative value for Ilastik-Fiji, which implies a minor estimation of the damage when compared to the manual standard method.ConclusionsThe novel approach using Ilastik and Fiji programs entails a great improvement for the quantification of the specific spider mite damage in Arabidopsis whole rosettes. The automation of the proposed method based on interactive machine learning eliminates the subjectivity and inter-rater-variability of the previous manual protocol. Besides, this method offers a robust tool for time saving and to avoid the damage overestimation observed with other methods.

Non-targeted metabolomic profiling of atrazine in Caenorhabditis elegans using UHPLC-QE Orbitrap/MS

The widespread use of the herbicides Atrazine (ATR) has been raised attention due to its ubiquitous occurrence in the environment. As an endocrine disruptor, ATR causes reproductive, immune, nervous system toxicity in biota. In this study, we aimed to investigate metabolic profile characteristics and potential metabolic biomarker that reflects specific damage in toxic effect after ATR exposure. Hence, a metabolomics study was performed to determine the significantly affected metabolites and the reproduction and locomotion of C. elegans were investigated. Mediation analysis was used to evaluate the mediating effect of metabolites on association between ATR exposure and toxic effect. ATR (≥0.04 mg/L) caused the significant dose dependent reduction of brood size and locomotion behavior, however, the body length and width were significantly decreased only in 40 mg/L group. These results suggesting that brood size, head thrashes and body bends are more sensitive indictor to assessment ATR toxicity in C. elegans. Meanwhile, metabolomics analysis revealed that ATR exposure can induce metabolic profiles significant alterations in C. elegans. We found that 9 metabolites significantly increased and 18 metabolites significantly decreased, such as phosphatidylcholine, GMP, CDP-choline, neopterin etc. Those alteration of metabolites were mainly involved in the pathways: glycerophospholipid metabolism, glycolysis/gluconeogenesis, folate biosynthesis, glycine, serine and threoninemetabolism, pyrimidine and purine metabolism. Overall, these changes are signs of possible oxidative stress and ATP synthesis disruption modification. Mediation analysis showed a significant indirect effect of ATR exposure on brood size, via 7,8-dihydroneopterin 2′,3′-cyclic-p, and phosphatidylcholine might mediate association between ATR exposure and body bends, suggesting that 7,8-dihydroneopterin 2′,3′-cyclic-p and phosphatidylcholine might be potentially specificity marker for brood size and body bend respectively. This preliminary analysis investigates metabolic characteristics in C. elegans after ATR exposure, helping to understand the pathways involved in the response to ATR exposure and provide potential biomarkers for the safety evaluation of ATR.

Therapeutic vulnerabilities in the DNA damage response for the treatment of ATRX mutant neuroblastoma.

BackgroundIn neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research.MethodsTo evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF.FindingsIn isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft.InterpretationATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic.FundingThis work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.

Biomimetic CoO@AuPt nanozyme responsive to multiple tumor microenvironmental clues for augmenting chemodynamic therapy

Chemodynamic therapy (CDT), an emerging therapeutic strategy, has been recently exploited for in situ treatment through Fenton or Fenton-like reactions to generate cytotoxic reactive oxygen species (ROS). However, current systems rely significantly on the high local oxygen levels and strongly acidic conditions (pH = 3.0–5.0). Simultaneously, the produced ROS can be rapidly consumed by intracellular glutathione (GSH) in the electron transport chain. Herein, an original and biomimetic CoO@AuPt nanocatalyst was prepared based on the assembly of Au and Pt nanoparticles (NPs) on the surface of hollow CoO nanocapsules. The as-synthesized nanozyme exhibits extremely high stability under physiological conditions, whereas it undergoes spontaneous disintegration in the unique tumor microenvironment (TME). Subsequently, the decomposition products can catalyze a cascade of biochemical reactions to produce abundant ROS without any external stimuli. Thus, the present nanoplatform can increase intracellular ROS levels through continuous supply of H2O2, relief of local hypoxia and depletion of GSH, which result in remarkable and specific tumor damage both in vitro and in vivo. The findings of this study highlight the promising potential of CoO@AuPt nanocatalyst as a TME-responsive CDT nanomagnet for highly efficient tumor therapy.

The Antitrust Regulation on Abuse of Dominant Market Position in the Field of Big Data：From the Perspective of Methodological Improvement

The provision of prohibiting abuse of dominant market position in China’s antitrust law is limited to such types of behavior as “monopoly pricing” and “limited transaction”, which not only narrows the scope of regulation, but also blurs the criteria of regulation. A lot of anti-competitive behaviors in the field of big data are not stipulated by the antitrust law, which may lead to a lack of supervision. Furthermore, the current regulatory methods do not focus on the analysis of big data market behaviors, but use the deductive method from general to individual, tending to assume the type of monopoly behaviors on the basis of law. The double failure of legal provisions and interpretation methods contrasts strongly with the regulatory requirements in the field of big data. To tackle this problem, the traditional regulatory methodology needs to be adjusted in accordance with the characteristics of the big data market from the three aspects: market power identification, abuse behavior screening, and competition damage effect analysis.First of all, we should take the data value chain as the basis, and fully consider the network effect, cross-border competition, dynamic competition and many other characteristics of the big data market, to evaluate the market power. Secondly, with the help of the infringement identification of “exclusionary abuse” and “exploitative abuse”, we may quickly identify and summarize the behavior types of market dominant position abuse in the field of big data. Lastly, we should establish specific identification methods and competitive damage analysis to regulate those anti-competitive behaviors associated with big data such as “restricting interoperability” and “excessive collection and use of data”.As China’s anti-monopoly law is being comprehensively revised and improved, we should take this opportunity to modify the existing provisions and improve the regulatory methodology in view of the development of the digital economy. For one thing, the legislature should summarize special factors according to the characteristics of big data industry in the legal provisions defining relevant markets and evaluating market power. For another thing, China should refer to the legislative model of the EU competition law and determine “exclusionary abuse” and “exploitative abuse” as the basic types of the abuse of dominant market position, so as to expand the scope of application of antitrust laws. In addition, China should issue antitrust law enforcement guidelines, summarize the types of abuses common in the field of big data, and formulate corresponding antitrust law enforcement methods and regulatory measures.