Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) is an ideal immunoassay method for large-scale screenings to detect mycotoxin contaminants. However, the matrix effect of complicated samples has always been challenging when performing immunoassays, as it leads to false-positive or negative results. In this study, convenient QuEChERS technology combined with optimizing the dilution solvent was ingeniously used to eliminate interference from the sample matrix to greatly improve the detection accuracy, and reliable ic-ELISAs for the two official tolerance levels of 60 and 500 μg/kg were developed to screen zearalenone (ZEN) in edible and medical coix seeds without any further correction. Then, the 122 batches of coix seeds were determined, and the positive rate was up to 97.54%. The contaminated distribution was further analyzed, and risk assessment was subsequently performed for its edible and medical purposes. The findings indicated that consumption of coix seeds with higher ZEN contamination levels may cause adverse health effects for both medical and edible consumption in the adult population; even under the condition of average contamination level, ZEN from coix seeds was the more prominent contributor to the total risk compared to other sources when used as food; thus, effective prevention and control should be an essential topic in the future. Mycotoxins are secondary metabolites produced by fungal species such as Aspergillus, Alternaria, Claviceps, Penicillium and Fusarium. Of the known mycotoxins, zearalenone (ZEN), which is primarily generated by Fusarium molds, is regarded as a serious problem worldwide, as it can lead to reproductive and fertility disorders by competing with 17β-estradiol for estrogen receptor binding [Coix seed, known as Job’s tears, belongs to the grass family Poaceae and is a minor grain that is extensively cultivated in East and Southeast Asia  . Coix seIndirect competitive enzyme-linked immunosorbent assay (ic-ELISA) is an ideal immunoassay method for large-scale screening to detect mycotoxin contaminants ,12,13. HQuEChERS  is an extraction and clean-up technique that was initially developed to determine pesticide residue by gas chromatography with mass spectrometry (GC–MS/MS)  and thenIn this study, a reliable ic-ELISA method for ZEN in coix seeds was developed. A combination of an extremely simple QuEChERS pretreatment strategy and the optimization of the dilution solvent can effectively eliminate the matrix effect in real sample analyses. The proposed method was devised for both official tolerance levels of ZEN in coix seeds by adjusting the dilution parameters, the method was applied to investigate the contamination of ZEN in 122 batch coix seeds, and the results were further validated by LC–MS/MS. Moreover, health risk assessment was performed to help clarify the present situation of ZEN contamination and the potential health risks of coix seeds.3COONa), anhydrous magnesium sulfate (MgSO4) and other chemical reagents were of analytical grade and were from Guangzhou Chemical Reagent Factory .The mycotoxin standards ZEN, α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) were purchased from Pribolab (Singapore). ZEN monoclonal antibody and ZEN-BSA were purchased from Shenzhen Anti Biological Technology Co., Ltd. . Goat anti-mouse IgG polyclonal antibody-HRP (IgG-HRP) was purchased from Nanning Blue Light Biotechnology Inc. . Bovine serum albumin (BSA) was obtained from Phygene Biotechnology Co., Ltd. . The 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent set was purchased from Bosf. . Methanol and acetonitrile were provided by Macklin . Sodium acetate (CHOne hundred and twenty-two batches of coix seeds (including 22 batches of powdered coix seed) were collected from supermarkets and drugstores in China. The samples (except powdered coix seed) were ground through a high-speed disintegrator  and passed through a 24-mesh sieve. The coix seed sample powder (1.0 g) was added to 5 mL of 80% acetonitrile. The mixture was vortexed for 3 min and then vigorously stirred with 1.0 g anhydrous magnesium sulfate and 0.25 g sodium acetate for 1 min . Then, t2O2 substrate solution ) was added for 10 min. Finally, the reaction was terminated by hydrochloric acid, and the absorbance was determined at 450 nm with a spark multimode microplate reader.The 96-well microplates were coated (100 μL per well) with ZEN-BSA in sodium carbonate coating buffer at 4 °C overnight. Then, the plates were washed three times with PBST (PBS containing 0.05% Tween 20) and blocked at 37 °C for 2 h. Each plate was washed two times with PBST. ZEN standard solution or diluted sample extract (50 μL per well) was added simultaneously along with anti-ZEN antibody (50 μL per well) at 37 °C for 1 h. Then, the plates were washed three times with PBST. IgG diluted with PBS  was added to the plates. After incubating for 1 h at 37 °C, the plates were washed four times, and 100 μL per well of the TMB/HThe method was based on that described by Wu  with modThe risk assessment is discussed separately in this work because coix seed is used for both edible and medical purposes.m refers to the concentration of ZEN (μg/kg), K represents coix seed consumption and is expressed in kilograms per day, and bw is the average weight of adults (70 kg).The estimated daily exposure (EDE) was determined using mycotoxin contents in coix seeds and food consumption data for the adult population (Equation (1) ).(1)EDEThe risk related to ZEN exposure from daily consumption of coix seeds was evaluated by calculating the ratio (% TDI) between the estimated daily exposure (EDE) and the tolerable daily intake (TDI) (Equation (2)).As food, the daily consumption of coix seeds was set to 0.0252 kg according to Zhu’s survey of coix seed consumption in Shanghai, China . Hence, The %TDI value < 100% is assumed to be within the acceptable range, equal to or exceeding %TDI may concern potential non-carcinogenic effects. Note, however, that the risk will increase as %TDI increases ,29.50) of the calibration curve, which ensures that the measurements are more accurate when the contamination levels of ZEN are near the MRLs in real samples. Therefore, the optimization and subsequent validation of the developed ic-ELISA were carried out separately for these two MRLs.Considering the two MRLs (60 μg/kg or 500 μg/kg) of ZEN in coix seeds, we tried to adjust the different sample dilution processes to make the concentration of MRLs around the inhibitory concentration 50%  and sodium acetate (CH3COONa) was added, and the sample was vortexed in situ for 1 min. After centrifugation, ZEN could be transferred into the organic phase, while some polar components of the matrix remained in the aqueous layer. It is worth mentioning that this QuEChERS procedure could better reduce the matrix effect both in the absorbance and inhibition rate compared to the direct dilution procedure, especially for the dilution of MRL 60 μg/kg  (The developed method achieved different sensitivities to satisfy different detection requirements. The ICectively . The spe * 100%) . BecauseThe limits of detection (LOD) and quantification (LOQ), defined as the analyte concentration necessary for obtaining a 10% and 20% inhibition for ELISA , were 6.Accuracy was determined at three concentration levels by spiking blank samples of coix seeds that were determined by LC–MS/MS to 30, 60, and 120 μg/kg for MRL 60 μg/kg and 250, 500, and 1000 μg/kg for MRL 500 μg/kg. The results are shown in One hundred and twenty-two batches of coix seeds were determined by the developed ic-ELISA. The contamination levels of ZEN in the samples can be seen in According to the higher occurrence rate and contamination levels of ZEN in coix seeds, an exposure assessment to ZEN for both medical and edible consumption of coix seeds is necessary. The exposure assessment was performed by a deterministic approach. Three scenarios were considered, including the minimum, average, and maximum concentrations of ZEN in coix seeds .As food, the calculated %TDI value of 733.64 for ZEN in scenario III was observed to far exceed the safety reference value of 100%. As a medicinal herb, the EDE value was about one-third of the food’s, but the calculated %TDI value of 215.72 was still much higher than 100%. The above observations indicated that consumption of coix seeds with higher ZEN contamination levels might cause adverse health effects for both edible and medicinal use. This finding was consistent with previous studies ,28. For As mentioned in In this work, a reliable ic-ELISA for the detection of ZEN with two different MRLs in coix seeds was developed without any correction. It is worth noting that we proposed a strategy to eliminate the matrix effect from coix seed samples that was problematic for previous immunoassay-based rapid detection. It was found that using simple QuEChERS technology as sample pretreatment could reduce the matrix effect both on absorbance and sensitivity. In addition, optimizing the dilution solvent could especially improve interference on sensitivity when the dilution ratio was relatively low, but when the dilution ratio was higher, this improvement was mainly observed for the absorbance. The 122 batches of coix seeds were determined, and a better correlation was obtained compared with that of ultrahigh-performance LC–MS/MS. The contamination distribution was further analyzed, and the results indicated that the occurrence of ZEN in coix seeds is a universal problem in various districts in China. Moreover, based on the edible and medical aims, the dietary exposure to ZEN for adults was assessed separately. The findings indicated concern about intake of the ZEN through consuming coix seeds with higher contamination levels. When used as food, the intake of ZEN through consuming coix seeds with an average concentration was within the acceptable risk range but identified as a significant contributor to the total risk compared to other sources. There was a regional difference in health risk associated with dietary exposure to ZEN from coix seeds. Still, the effect of different regions on the risk assessment is limited by a lack of data on regional consumption, which is worthy of further investigation.


Datasets were downloaded from the Gene Expression Omnibus online database. Differential expression analysis was first performed using the blood exosome dataset GSE39833 from colorectal cancer and normal humans to identify differentially expressed (DE) miRNAs, and then, transcriptional enrichment analysis was performed to identify important enriched genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed by FunRich software. Using the muscle atrophy sample GSE34111, the DE mRNAs in the muscle atrophy sample were analyzed, a regulatory network map was established based on miRNA‒mRNA regulatory mechanisms, further GO and KEGG enrichment analyses were performed for the DE genes in muscle atrophy via Cytoscape’s ClueGO plug-in, and the network pharmacology pharmacophore analysis method was used to analyze the celastrol therapeutic targets, taking intersections to find the therapeutic targets of celastrol, using the artificial intelligence AlphaFold2 to predict the protein structures of the key targets, and finally using molecular docking to verify whether celastrol and the target proteins can be successfully docked. A total of 82 DE miRNAs were obtained, and the top 10 enriched target genes were identified. The enrichment of the 82 miRNAs showed a close correlation with muscle atrophy, and 332 DE mRNAs were found by differential expression analysis in muscle atrophy samples, among which 44 mRNA genes were involved in miRNA‒mRNA networks. The DE genes in muscle atrophy were enriched for 30 signaling pathways, and 228 target genes were annotated after pharmacophore target analysis. The NR1D2 gene, the target of treatment, was found by taking intersections, the protein structure of this target was predicted by AlphaFold2, and the structure was successfully docked and validated using molecular docking. In our present study, colorectal cancer likely enters the muscle from blood exosomes and regulates skeletal muscle atrophy through miRNA‒mRNA regulatory network mechanisms, and celastrol treats muscle through NR1D2 in the miRNA‒mRNA regulatory network.Muscle atrophy due to colorectal cancer severely reduces the quality of life and survival time of patients. However, the underlying causative mechanisms and therapeutic agents are not well understood. The aim of this study was to screen and identify the microRNA (miRNA)–mRNA regulatory network and therapeutic targets of celastrol in colorectal cancer causing muscle atrophy  Cachexia is a severe atrophy syndrome ; in manyMany factors produced by the body and cancer cells, including inflammatory cytokines, contribute to the cachexia of tumors . The recChinese medicine has been in clinical practice in China for thousands of years . NumerouIn this study, blood exosome data from colorectal cancer were used to analyze differentially expressed (DE) miRNAs, and then, DE mRNAs were analyzed in muscle atrophy samples to model the miRNA‒mRNA regulatory network and to analyze the potential network targets of celastrol for the treatment of colorectal cancer-related muscle atrophy based on network pharmacology using AlphaFold2 and molecular docking for molecular dynamics validation. The overall goal was to provide new insights into the potential miRNA‒mRNA regulatory mechanisms of colorectal cancer muscle atrophy and the targets of celastrol therapy and explore the role of the AI AlphaFold2 in drug development.First, the keywords “colorectal cancer” and “muscular atrophy” were retrieved from the Gene Expression Omnibus (GEO) database , and thep < 0.05 were used as screening conditions to identify significant DE miRNAs.The samples were grouped according to the two groups of the GSE39833 dataset and analyzed by the “limma” package  in R , TFs enriched with DE miRNAs were determined, and the top 10 significant TFs were graphically displayed. Gene Ontology (GO) terms were annotated and mapped by FunRich software, including biological processes, cell composition, and molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified.p < 0.05. DE mRNAs with significant expression were screened out.In the GSE39833 dataset, we selected samples from six healthy controls and 12 patients with muscle atrophy in the R software package “limma” for differential expression analysis, with the filtering conditions |log2 FC (fold change)| > 0.5 and The miRNA target genes were predicted within three databases: miRTarBase, miRDB, and TargetScan. Subsequently, we constructed miRNA‒mRNA gene regulatory networks for the screened DE miRNAs with DE mRNAs based on miRNA‒mRNA regulatory mechanisms. Cytoscape (version 3.9.0) was used to visualize miRNA‒mRNA gene regulatory networks.Homo sapiens” was selected for GO enrichment analysis, including the biological process, cell composition, and molecular function, as well as KEGG signaling pathway enrichment analysis, and the map was drawn.DE mRNAs were imported into Cytoscape’s ClueGO plug-in , and “Hohttp://www.lilab-ecust.cn/pharmmapper/) database was used to select the top 600 ranked targets for pharmacophore target prediction. The names of the analyzed target genes were annotated in the UniProt (https://www.uniprot.org/) database by selecting the species “Homo sapiens,” and the network was constructed in Cytoscape based on the drug–target relationships.PharmMapper is an online platform for pharmacophore matching and potential identification of targets. The PharmMapper , and downloaded a PDB format file of the corresponding NR1D2 protein structure, a display map of the protein structure, the protein sequence map used, and the predicted confidence map.AlphaFold2 is an artificial intelligence program that predicts protein structure online. We selected the target gene The 2D structure of the small-molecule ligand celastrol was downloaded from the PubChem database. NR1D2 was selected as the protein receptor, and the PDB file predicted by the AI AlphaFold2 was used. AutoDockTools was used to read the protein receptor file, which was converted into the PDBQT format after hydrotreating ion modification. This structure was then converted to a 2D structure to draw active pockets. Finally, AutoDock Vina was used for molecular docking, and the model with the lowest free energy was selected for visualization.p < 0.05 was considered statistically significant.R (4.1.0) was used for bioinformatics analysis, and the R package was used for statistical analysis. p < 0.05, a total of 82 DE miRNAs were obtained in the colorectal cancer group compared to the healthy group  were graphically displayed   enrichmep < 0.05 , ascorbate and aldarate metabolism, drug metabolism, TGF-beta signaling pathway, tyrosine metabolism, sphingolipid metabolism, fatty acid degradation, pyruvate metabolism, TNF signaling pathway, relaxin signaling pathway, and fluid shear stress and atherosclerosis. The citrate cycle (TCA cycle), pyruvate metabolism, and fatty acid degradation have also been predicted to be important signaling pathways in muscle atrophy in spinal cord injury . The TGFNR1D2 for drug treatment. In GeneCards, NR1D2 is presented as a transcriptional repressor that coordinates circadian rhythms and metabolic pathways in a hemoglobin-dependent manner. NR1D2 is a component of complex transcriptional machinery that controls circadian rhythms and forms a key pathway in the biological clock by directly repressing the expression of the core clock components ARNTL/BMAL1 and CLOCK. It also regulates genes involved in metabolic functions, including lipid metabolism and inflammatory responses. In the previous literature, NR1D2 was shown to regulate mitochondrial function by regulating the circadian rhythm, and impaired mitochondrial function leads to Duchenne muscular dystrophy. Many important signaling pathways in the KEGG enrichment of DE mRNAs in muscle atrophy are also associated with hypo, including lipid metabolism and inflammatory responses.The therapeutic targets of celastrol were analyzed by pharmacophore target analysis of network pharmacology, and 228 genes were successfully annotated and then intersected with mRNAs of the miRNA‒mRNA regulatory network to find the disease target gene We then used the AI AlphaFold2 on the protein structure of NR1D2 to predict the human protein structure of the gene, successfully molecularly docking it to celastrol.AlphaFold2 is a revolutionary change in the field of biology, demonstrating the power of AI, which is changing the way drugs are developed, greatly accelerating the development of new drugs, and helping millions of patients around the world who are unable to develop drugs because of unknown protein structures. AlphaFold2 will be a powerful AI weapon in the fight against various diseases. We offer a new strategy for drug development by combining bioinformatics, network pharmacology, and artificial intelligence.In this study, we identified a potential miRNA‒mRNA regulatory network of colorectal cancer regulating muscle atrophy through exosomes in a bioinformatics approach. There are 22 miRNAs and 44 mRNAs in this network. Molecular docking validation by the AI AlphaFold2 predicted the structure of the NR1D2 protein and revealed the molecular target of celastrol for the treatment of colorectal muscular atrophy. AlphaFold2 will help develop potential therapeutic drugs for more diseases.


Multiple primary malignant neoplasms (MPMNs) are defined as multiple tumors with different pathogenic origins. MPMNs are rare, but the morbidity rate is on the rise. With the development of anti-tumor treatments, such as targeted therapy and immunotherapy, the overall survival of cancer patients has been significantly prolonged, leading to an increased number of patients with MPMNs. A crucial aspect of MPMNs management is deciding how to schedule further treatments according to individual tumor risk. This process involves a multidisciplinary physician team to ensure favorable outcomes. Herein we report a 60-year-old male who developed four different malignancies, including esophageal squamous cell carcinoma, upper urinary tract urothelial carcinoma, mediastinal small cell lung cancer, and left lung squamous cell carcinoma over 20 years and received appropriate treatment of each cancer with long survival. Accor2Between 1998 and 2020, a case report described a patient with four primary tumors including ESCC, UTUC, SCLC, and LUSC with separate pathologic features and explored sustainable and reasonable management of multiple primary tumors, who had a long overall survival .In 1998, a 42-year-old male was admitted to the Thoracic Surgery Department of the General Hospital of Jinan Military Region due to food obstruction. A barium meal fluoroscopy showed that the lumen of the middle part of his esophagus was narrow with a mucosal injury; the length of the lesion was approximately 7.5 cm. A transthoracic radical resection of the esophageal tumor and an esophagogastric thoracic anastomosis were performed. Postoperative pathologic evaluation revealed a grade II ESCC with infiltration into the adventitia. The resection margins were negative in the upper and lower tangent lines, and 0 of 6 peripheral lymph nodes had tumor metastases . The pat2 qw × 8 cycles) intravesical instillation chemotherapy was administered for 1 year. Additional instillations were administered (20 mg/m2 qm × 10 cycles). Periodic re-examinations were performed after the infusion chemotherapy was completed.In July 2011, he visited the hospital for painless hematuria. An enhanced computed tomography (CT) of urinary system showed that the left renal pelvis was spherical and dilated, with a long diameter of approximately 7.8 cm. A soft tissue density was noted in the lower end of the walking area . He then2 d1-3) and cisplatin (40 mg/m2 d1-2q21d) for six cycles with concurrent intensity modulated radiotherapy (60 Gy/30F). The therapeutic evaluation was a partial response according to the RECIST 1.1 standard treatment effect evaluation after treatment. Regular follow-up evaluations were performed after treatment.In June 2019, a chest CT revealed multiple mediastinal lymphadenopathy . Given tIn February 2020, he sought evaluation for blood in the sputum in our department. A enhanced chest CT showed that there was an irregular tissue mass in the lingual section of the left upper lobe near the hilum . BronchoInformed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.3MPMNs defined as the occurrence of two or more primary malignant tumors have increased malignant behavior and a worse prognosis in comparison with a single primary tumor –8. Si etIt has been reported that most patients with four primary tumors have breast and upper gastrointestinal tumors –13. SquaAs a result of advances in cancer research and treatment, the overall survival of patients after diagnosis continues to lengthen. Due to the growth in the number of cancer survivors, the long-term side effects of chemotherapy and/or radiotherapy, the increase in diagnostic sensitivity, and the continued influence of genetic and behavioral risk factors, the risk of MPMNs is also increasing. The specific mechanism underlying MPMNs has not been established. The root causes of primary cancers may include genetic, environmental, and treatment-related factors . The sen


Treatment with 30 and 40 μg mL−1 of TCPP induced marginally significant micronuclei (MN) frequencies as well as cytotoxic effects. Freshwater microalgae species treated with TCPP  showed different growth rates over time. All the tested microalgae species were adversely affected after exposure to TCPP during the first 24 h. However, differences among the microalgae species’ sensitivities were observed. In the case of the freshwater species, the most sensitive was found to be Chlorococcum sp. The marine algal species Dunaliella tertiolecta and Tisochrysis lutea were significantly affected after exposure to TCPP. The effects of TCPP on Aliivibrio fischeri that were observed can classify this flame retardant as a “harmful” compound. Our results suggest a potential risk to aquatic organisms and humans from the wide utilization of TCPP and its consequent release into the environment. These results highlight that further research should be conducted to investigate the effects of TCPP individually and in combination with other organophosphorus flame retardants in various organisms. In addition, the concern induced by TCPP points out that measures to control the introduction of TCPP into the environment should be taken.Tris(chloropropyl) phosphate (TCPP) is a characteristic and widely used organophosphorus flame retardant. TCPP is comprised of four isomers and the most abundant is tris(1-chloro-2-propyl) phosphate. TCPP can be released into the environment, with potential impacts on living organisms and humans due to its extensive industrial use. Aiming to assess the potential risks of TCPP on human health and the environment, its toxic and genotoxic effects—using organisms from different trophic levels, i.e., bacteria, green microalgae, and human cells—were investigated. TCPP exposure at nominal concentrations of 10, 20, 30 and 40 μg mL Organophosphates esters (OPEs) are a large class of compounds widely used in industry as flame retardants and plasticizers for a variety of products and applications, i.e., electronics, building materials, vehicles, furniture, car seats, plastics, and fabrics. The widespread and frequent use of OPEs poses a risk to human health, given that they have been detected and quantified in various tissues in the human body ,2,3,4.One of the most-used organophosphates, used as a flame retardant, is Tris(chloropropyl) phosphate (TCPP). Commercial TCPP is a mixture of isomers, and its composition may vary. Tris(1-chloro-2-propyl) phosphate is the most used and most frequently detectable isomer in commercial products. Additional isomers that are frequently used include bis(2-chloro-1-methylethyl)-2-chloropropyl phosphate, bis(2-chloropropyl) 2-chloroisopropyl phosphate and tris(2-chloropropyl) phosphate  5]..5].TCPP is used in the form of a flexible and a rigid polyurethane foam in furniture and construction materials, respectively, as well as in other products such as textiles, paints, coatings, adhesives and electronics. TCPP can be used as a substitute for various brominated as well as other chlorinated flame retardants because of their toxicity concerns . The totThe regulatory status of TCPP in relation to the potential risk of the use of TCPP from 2015 until today can be described as follows: TCPP is listed on the Toxic Substances Control Act (TSCA) Inventory and is under investigation by the U.S. Environmental Protection Agency (EPA) for its impact on consumers, the general population and aquatic organisms. Currently, no regulations restrict the production or use of TCPP in the United States . In EuroTCPP can be released into the environment and has potential adverse effects on living organisms and humans due to its extensive industrial use in manufacturing and numerous consumer products . The maiA recent review reported that TCPP is an environmentally abundant OPE and has been detected at elevated concentrations in indoor and outdoor air, wastewater and surface water in several countries around the world . Rauert Human exposure to TCPP can occur through inhalation, oral and/or dermal contact or when working with consumer products containing TCPP. The presence of TCPP in indoor as well as outdoor environments increases its potential effects on human health because of its intensive use as a consumer product. The oral and dermal exposure of children to items containing TCPP  poses a potential health and safety issue that should be always under investigation and control by the regulatory authorities ,13,18. TTo our knowledge, there are limited genotoxicity studies on TCPP ,22, and Taking into consideration the detectable levels of TCPP in the environment worldwide, this compound can cause toxic and genotoxic as well as cytotoxic effects in various organisms. Therefore, there is an imperative need for combined genotoxicological studies on the effects of TCPP in organisms from different trophic levels to clearly determine the risks to human health and the environment.Aliivibrio fischeri) and green microalgae  as well as human cells (lymphocytes).Aquatic pollution is an important environmental issue that faces humanity ,24,25. TAliivibrio fischeri bioassay, using the marine bacterium Aliivibrio fischeri (formerly known as Vibrio fischeri), is a widely used short-term toxicity test. The application of this method is based on the bioluminescent properties of the used bacteria; reductions in the bioluminescence of a bacterium when exposed to chemicals reflects the toxic effect of the tested substances. The Aliivibrio fischeri bioassay is used as a standard (eco)toxicological bioassay [The bioassay ,28.The use of freshwater and/or marine microalgae has found extensive applications and constitutes an efficient experimental tool for environment contamination assessments and for aquatic ecosystems. For the assessment of TCPP’s toxic effects on freshwater and marine microalgae, the guidelines produced by the Organization for Economic Cooperation and Development (OECD) were followed ,30.in vitro micronucleus assay for the detection of micronuclei (MN) has been extensively applied and proposed as a well-established tool for genotoxicity screening due to its simplicity, rapidity and sensitivity [Over the last few decades, the implementation of the sitivity ,32. MN fsitivity ,33. In tScenedesmus rubescens (strain SAG 5.95) and Chlorococcum sp. (strain SAG 22.83) were purchased from the bank SAG collection of the Gottingen University (Germany). Dunaliella tertiolecta (strain CCAP 19/6B) as well as Tisochrysis lutea  were purchased from the Scottish Marine Institute, Oban, Argyll, Scotland. BG-11 medium (Cyanobacteria BG-11 Freshwater Solution) and F/2 medium (Guillard’s (F/2) Marine Water Enrichment Solution) were purchased from Sigma-Aldrich. Aliivibrio fischeri bacteria (Microtox® Acute Reagent) and Microtox® Reconstitution Solution were supplied from Modern Water. Sodium chloride, phenol and ZnSO4·7H2O were obtained from Sigma-Aldrich. For the cell cultures, all the chemicals/reagents reported in our previous work were used [TCPP (CAS Number:13674-84-5) as a mixture of isomers was purchased from Sigma-Aldrich. Ultrapure water was used for the preparation of the solutions. The microalgae species ere used . Nylon SScenedesmus rubescens (strain SAG 5.95) and Chlorococcum sp. (strain SAG 22.83), as well as the saltwater species Dunaliella tertiolecta (CCAP19/6B) and Tisochrysis lutea (T. ISO strain CCAP 927), according to the OECD 201 protocol [4 cells mL−1) in the logarithmic growth phase . All the cultures were incubated under sterile conditions and continuous illumination (4300 lux). Five different TCPP nominal concentrations  were tested in duplicate cultures for 72 h under continuous stirring. The cell numbers were determined using a Neubauer hemocytometer and the algae growth (μ) and inhibition (% I) rates were determined at 24, 48 and 72 h of incubation.Algal bioassays were conducted using the freshwater species protocol . BG-11 mAliivibrio fischeri Bioluminescence Inhibition bioassay using a Microtox Model 500 Toxicity Analyzer from Azur Environmental. The aqueous samples of TCPP were tested in four dilutions in 2% NaCl and incubated for 5 and 15 min. MicrotoxOmni Windows Software was employed for the calculation of the bioluminescence inhibition (%) of each sample and EC50 upon 5 and 15 min. Positive (Phenol and ZnSO4·7H2O) and negative controls (2% NaCl) were also tested, employing the same procedure.The ecotoxicity of TCPP was also assessed by the The cytokinesis-block micronucleus (CBMN) assay was performed according to the established protocol of our laboratory  which is50 values , Probit analysis (p < 0.05) was performed using log-transformed values (IBM SPSS 25 Inc. software package). Levene’s test of equality of error variances was used to check the homogeneity of variance, whereas significant differences in the algae number (mL−1 × 104) were assessed by post-hoc multiple comparison tests . Using Microtox assay the bioluminescence inhibition (%) of each sample, as well as the effective concentration of the TCPP that reduced the bioluminescence by 50% (EC50), were calculated using MicrotoxOmni Windows Software. Regarding the CBMN assay, the G-test was used for MN data analysis, whereas the CBPI data set was analyzed using the Chi-square (×2) test.For the determination of ICThe study was approved by the Research Ethics Committee of the University of Patras (Ref. No. 7682/6 June 2021). Participants used as blood donors signed informed consent forms to say that they were not exposed to radiation, drug treatments or any viral infection in the recent past.Anthropogenic chemicals with extensive use in a plethora of applications—such as TCPP—tend to accumulate in ecosystems, posing a risk to living organisms. The undesired effects range from potential toxicological alterations of micro-organisms to genotoxicological damages in higher organisms, including humans. Species from different trophic levels have been used in many studies as bioindicators to evaluate the potential toxicological effects of several environmental contaminants ,30. The −1—to identify its potential risk of inducing genotoxic effects in cultured human lymphocytes. As observed, there are no statistically significant differences between control and TCPP-treated cultures at the lowest tested nominal concentrations of 10 and 20 μg mL−1. Statistically significant differences in MN frequencies in comparison with the control were recorded at TCPP nominal concentrations of 30 and 40 μg mL−1  at the highest examined concentrations of 30 and 40 μg mL−1 . No mutagenic effects were observed for TCPP in eight Salmonella strains using concentrations of up to 1 mM in the presence and absence of S9. On the other hand, a recent study reported that TCPP induced genotoxic and apoptotic effects in human umbilical vein endothelial cells (HUVECs) and may affect the human vascular system [−1) induced a marginally genotoxic effect and a significant cytotoxic effect under the specific experimental conditions. Our findings agree with the results of the aforementioned studies focused on the genotoxicological activities of TCPP in different cell lines and organisms.In a study performed by Föllmann and Wober (2006) , the genr system . In a rer system , no evid−1, as stated in a very recent review where the global distribution of OPEs in aquatic systems was analyzed. Moreover, carcinogenic risks were not reported for OPEs and potable water in the examined countries and the averages are above the limit for only one country (China) [−1) are below or comparable to the maximum average detected concentration of TCPP in China. Our findings and the available literature data suggest a potential risk for the utilization of TCPP, taking into consideration the observed in vitro genotoxicological effects in human lymphocytes as well as in various human cell lines.The maximum average concentration of TCPP worldwide was detected in Chinese water resources, at around 67 μg mL (China) . The conThe continuous release of OPEs into aquatic systems and their potential toxic effects on living organisms have caused a growing concern about their impact on the environment. Algae, crustaceans and fish are representative aquatic organisms that have been used as bioindicators for the environmental risk assessment of pollutants ,47, inclScenedesmus rubescens treated with 1, 10, 20 and 50 μg L−1 of TCPP for 24 h showed significantly lower growth rates compared to control cultures (−1). In contrast, lower effects on growth rates were observed after the exposure of Scenedesmus rubescens for 72 h to TCPP, as confirmed by the increase in IC50 values over time (Chlorococcum sp. (−1). At 48 h, significantly lower growth rates and higher inhibition (%I) of algae growth were also recorded as compared to the control—specifically at the highest nominal concentrations (20 and 50 μg L−1). Finally, inhibition rates after algae exposure for 72 h revealed that TCPP still inhibited the growth rate of Chlorococcum sp., while the %I stayed higher at the highest tested concentrations (20 and 50 μg L−1). IC50 values .Fresh-water algal species treated with TCPP showed different growth rates over time, as depicted in cultures a. The saver time . In the ccum sp. b, a tota0 values  were in Dunaliella tertiolecta cultures (−1) showed high growth inhibition at the 24 and 48 h at all tested nominal concentrations, with IC50 values of 0.002 and 0.114 mg L−1 respectively. After 72 h of treatment, the inhibition rate was still high—especially for the nominal concentrations of 10, 20 and 50 μg L−1—and the IC50 value was 1.596 mg L−1. Similarly, lower growth rates and algal density were observed for Tisochrysis lutea (−1) over time (24–48–72 h), showing a dose–response relationship. IC50 values . Growth inhibition in microalgae reflects the toxic effects of different pollutants on microalgae such as TCPP, affecting their physiological function [50 value for the species Scenedesmus subspicatus was 45 mg L−1 after exposure to TCPP for 72 h, which is consistent with our findings for the Scenedesmus rubescens. According to the United Nations, compounds are classified based on ecotoxicity values as follows [(i)50 ≤ 1 mg L−1;highly toxic: EC(IC)(ii)−1 < EC(IC)50 ≤ 10 mg L−1;toxic: 1 mg L(iii)−1 < EC (IC)50 ≤ 100 mg L−1.harmful to aquatic organisms: 10 mg LHowever, differences among the algal species’ sensitivities were observed at higher exposure times—especially at 72 h. The differences in their IC species . In the function . Niu et function  mentione follows :(i)highl50 values calculated in the present study  was also calculated  production, oxidative stress and apoptosis [Ciona intestinalis [Based on the results, TCPP can be classified as a “harmful” compound to ia magna . Similarpoptosis . Moreovestinalis .−1) inducing genotoxic and cytotoxic effects in cultured human lymphocytes was evaluated using a CBMN assay. Only the highest nominal concentrations (30 and 40 μg mL−1) induced marginally significant MN frequencies as well as cytotoxic effects. The effects of TCPP  in freshwater and marine algal species were also evaluated. Reduced growth rates for all tested microalgae species were observed after exposure for 24 h, reaching a 100% inhibition of the growth rate in some cases. Among the freshwater species, Chlorococcum sp. was found to be the most sensitive. Both tested marine microalgae species  were significantly affected after exposure to TCPP. The effect of the tested flame retardant was also estimated against the bacteria Aliivibrio fischeri. According to the categories established by the United Nations, TCPP can be considered as highly toxic to Chlorococcum sp., toxic to Dunaliella tertiolecta and harmful to Scenedesmus rubescens, Tisochrysis lutea and Aliivibrio fischeri. These findings suggest that the wide utilization of TCPP and its consequent release into the environment could have adverse effects on aquatic organisms and humans.In the present study, the implementation of a battery of toxicity bioassays in aquatic micro-organisms in combination with the application of an in vitro genotoxicity assay in human cells was performed to evaluate the potential effects of TCPP in the environment and humans. The potential risk of TCPP (10, 20, 30 and 40 μg mL


Thirty Wistar rats were randomly divided into six groups of five animals apiece. Group one served as the negative control and received distilled water while the study lasted. Animals in groups 2–4 received 100 mg/kg/day AlCl3 throughout the study. Animals in groups 3 and 4 were also administered 100 mg/kg/day gallic acid and 100 mg/kg/day hesperidin, respectively. Groups 5 and 6 were treated with 100 mg/kg/day gallic acid only and 100 mg/kg/day hesperidin only, respectively. Treatments were administered orally via gavage for 28 days with distilled water as the vehicle. Animals were sacrificed after which levels of potassium, calcium, magnesium, phosphate, chloride, and bicarbonate ions were evaluated in the serum, while activities of Na+/K+ and Ca2+/Mg2+ ATPases were determined in kidney homogenate. Results showed that AlCl3 significantly (p < 0.05) inhibited activities of Na+/K+ and Ca2+/Mg2+ ATPases in addition to increasing serum levels of potassium, calcium, phosphate, and chloride, with concomitant decrease in serum levels of magnesium and bicarbonate. However, coadministration of AlCl3 with either gallic acid or hesperidin ameliorated all the disruptions caused by AlCl3. It could be concluded that gallic acid and hesperidin could be relevant in managing electrolyte imbalances and acidosis occasioned by kidney dysfunction.Nephrotoxicity is usually characterized by inefficiency of the kidney, thereby causing disruptions to electrolyte balance and blood acidity. This study aimed to evaluate the effect of hesperidin and gallic acid on serum electrolytes and ion pumps in Wistar rats subjected to aluminum chloride (AlCl Nephrotoxicity is a condition in which detoxification and excretion functions of the kidney are impaired as a result of damage or destruction to the kidney by toxicants or drugs. Nephrotoxicity is usually implicated in the aetiology of acute kidney injury (AKI) . Acute kIn the course of daily interaction with the environment, humans and animals get exposed to various chemicals and heavy metals, which have the tendency to bioaccumulate in body tissues. Among the metallic elements in the Earth crust, aluminum has the third highest occurrence . ExposurMaintenance of electrolyte levels within normal ranges is important for proper functioning of organs as well as several metabolic processes. The kidneys play an important role in maintaining electrolyte homeostasis. Thus, diseases and dysfunctions affecting the kidneys disturb their regulatory functions, thereby leading to imbalances in electrolyte level, with attendant life-threatening implications .3-induced nephrotoxicity in Wistar rats.Hesperidin belongs to the flavanone class of flavonoids. Its antioxidant and anti-inflammatory effects in acute renal damage were earlier reported . Gallic +/K+ ATPase and Ca2+/Mg2+ ATPase were purchased from MyBiosource, Inc, San Diego, USA. All other chemicals and reagents used were of analytical grade.Hesperidin and gallic acid were obtained from SantaCruz Biotechnology Inc, Heidelberg Germany. Kits for evaluating serum levels of potassium, calcium, and magnesium were obtained from Atlas Medical, Blankenfelde-Mahlow, Berlin, Germany. Kits for estimating serum levels of phosphate, chloride, and bicarbonate were purchased from Teco Diagnostics, Anaheim, USA. ELISA kits for evaluating Nan = 5). Group 1 was the control and was administered distilled water only throughout the study. Animals in groups 2–4 were administered 100 mg/kg AlCl3 while the study lasted. Animals in groups 3 and 4 also received 100 mg/kg gallic acid and 100 mg/kg hesperidin, respectively, in addition to AlCl3. Groups 5 and 6 animals received 100 mg/kg gallic acid only and 100 mg/kg hesperidin only, respectively. All treatments were orally administered. Experimental animals were allowed access to food and water without restriction throughout the acclimatization and study periods. Animals were treated for 28 days. Doses of AlCl3, gallic acid, and hesperidin used in this study were as reported in previous studies [Thirty male rats of Wistar strain (180–200 g) were obtained from the animal research facility of Afe Babalola University, Ado-Ekiti, Nigeria. They were allowed to acclimatize for 10 days under standard conditions before commencement of the study. The experimental animals were randomly distributed into six groups . The homogenates were centrifuged at 3000 rpm for 10 min at 4°C after which the supernatant was used for evaluation of activities of Namal Care . Ethical+/K+ ATPase and Ca2+/Mg2+ were evaluated using ELISA according to kit manufacturers' instructions.Serum levels of potassium, sodium, calcium, magnesium, phosphate, chloride, and bicarbonate were estimated spectrophotometrically by following instructions provided by kit manufacturers. Activities of Nap < 0.05.Data were expressed as mean ± standard deviation. Data was analyzed with one-way ANOVA using using Graphpad prism 5 software. Mean comparison was done with the Tukey test. The level of statistical significance was held at 3 only to rats occasioned a significant decrease in renal Na+/K+ ATPase activity while gallic acid and hesperidin prevented such decrease when they were coadministered with AlCl3. As presented in 3 with either gallic acid or hesperidin to experimental animals significantly (p < 0.05) increased Ca2+/Mg2+ ATPase activity, when compared with administration of AlCl3 only. In Figures 33 only to rats caused a significantly (p < 0.05) higher serum potassium, calcium, phosphate, and chloride levels, respectively. However, coadministration of gallic and hesperidin with AlCl3 prevented such an increase in all the electrolytes. Figures 3 significantly (p < 0.05) lowered serum levels of magnesium and bicarbonate, respectively. This decrease was however prevented in rats administered with either gallic acid or hesperidin, in addition to AlCl3. Results from our study indicated that administration of gallic acid only and hesperidin only did not have adverse effects on the biochemical indices evaluated in this study.In vivo and in vitro studies have established that aluminum is a prooxidant, whose basis of toxicity is oxidative stress and apoptosis, characterized by degeneration of the renal-tubular cells to [Nephrotoxicity is one of the commonest kidney disorders. It can be caused by several therapeutic and nontherapeutic chemicals that have toxic effects on various anatomical components of the kidney. These toxic effects eventually culminate in degeneration of morphology and function of the kidney . Nephrotcells to , 23.+/K+ ATPase and Ca2+/Mg2+ ATPase are enzymes found in all living organisms. They are involved in generating and maintaining ion gradients across biological membranes. Na+/K+ ATPase is highly expressed in the kidney, with the distal convoluted tubules having up to 50 million pumps per cell. The ion gradient generated by the enzyme is utilized by the kidney to filter the blood, maintain pH, and regulate levels of electrolytes in the blood [2+/Mg2+ ATPase is expressed in the basolateral membranes of the kidney where it contributes to the intracellular calcium concentration essential for kidney function and also contributes to maintenance of Ca2+ and Mg2+ homeostasis [+/K+ ATPase can be inhibited by aluminum in the kidney and liver, both in vivo and in vitro [3 only significantly inhibited the activity of Na+/K+ ATPase and Ca2+/Mg2+ ATPase in the kidney of Wistar rats. However, when gallic acid and hesperidin were coadministered with AlCl3, inhibition of the enzymes was prevented. This observation is an indication that both gallic acid and hesperidin might be relevant in maintaining electrochemical gradients across membranes and thereby ameliorate imbalances in concentration of the electrolytes specific to these enzymes, thereby improving aluminum chloride-induced nephrotoxicity.Electrolyte homeostasis is essential for proper functioning of numerous biological processes in the body. Consequently, dysfunctions of the kidney disrupt the electrolyte balance and may lead to life-threatening health conditions . Na+/K+ he blood . Ca2+/Mgeostasis . Chemicaeostasis , 27. Morin vitro , 28. In +/K+ ATPase in the kidney [3 caused hyperkalemia. However, coadministration of hesperidin and gallic acid reversed the high serum level observed in the group which administered AlCl3 only. This suggests that both gallic acid and hesperidin might be relevant in preventing cardiovascular events associated with impaired kidney function as hyperkalemia is an important link between both health conditions [About 98% of potassium, which is the most abundant intracellular cation, is sequestered in the intracellular fluid. The electrochemical gradient of potassium is maintained by the action of Nae kidney . Hyperkae kidney . In the nditions .3 caused a significant increase in serum calcium levels, a situation that was prevented with coadministration of AlCl3 with either gallic acid or hesperidin. It should however be noted that hypercalcemia arising from renal dysfunctions constitutes only a small fraction of all hypercalcemia cases [Calcium ion plays an important role in the regulation of several physiological processes including muscle contraction, secretory mechanisms, and excitation of neurons . One of ia cases , 33.3 only. This is in contrast to the observation in rats coadministered AlCl3 with either gallic acid or hesperidin. Hyperphosphatemia increases the risk of mortality due to cardiovascular events in CKD patients mainly due to calcification of the vascular system [The kidneys play an important role in phosphorus homeostasis, and hence, renal dysfunctions can significantly disrupt phosphorus homeostasis. It was reported that the risk of death increases by 20% for every 1 mg/dl increase in serum phosphorus among CKD patients . Even thr system . Finding3 was observed to significantly lower the serum magnesium level in Wistar rats. Nevertheless, coadministration of AlCl3 with gallic acid and hesperidin to Wistar rats significantly increased the serum magnesium level, when compared with the AlCl3 only group. This observation implies that the ability of gallic acid and hesperidin to prevent hypomagnesemia could be due to improvement of tubular reabsorption of magnesium.Magnesium is one of the most abundant intracellular divalent cations. It serves various essential roles in the body such as DNA synthesis, oxidative phosphorylation, cardiovascular tone, cofactor for enzymes, bone formation, and neuromuscular excitability . Mainten3 induced hyperchloremia in rats. However, gallic acid and hesperidin significantly lowered serum chloride levels when coadministered AlCl3. Our result suggests that gallic acid and hesperidin might have ameliorated the damaging effect of AlCl3 on the glomerular filtration rate in experimental animals.Chloride is a critical driver of several biological processes such as rennin secretion, renal sodium handling, blood pressure, and tubuloglomerular feedback . A previ3−, in addition to synthesizing adequate HCO3− in order to neutralize the intrinsic acid load [3−, while the thick ascending limb of the loop of Henle reabsorbs the balance [3− eventually leads to a reduction of serum bicarbonate concentration which ultimately contributes to metabolic acidosis [3 prompted metabolic acidosis by significantly lowering serum bicarbonate levels. Nonetheless, coadministration of gallic acid and hesperidin prevented metabolic acidosis possibly by preventing reduced renal mass and maintaining tubular reabsorption function of the kidneys.In order to maintain regular acid-base balance, renal tubules reabsorb filtered HCOcid load . The pro balance . Failureacidosis . Moreoveacidosis . Both acacidosis . In the This study corroborates the disruptive effect of aluminum on electrolyte homeostasis due to its nephrotoxic effects on renal functions. Apart from its effect on the serum levels of individual electrolytes, aluminum was also found to disrupt the activities of two enzymes involved in generation and maintenance of electrochemical gradients across membranes. Coadministration of gallic acid and hesperidin however prevented these biochemical alterations, an observation that further underlines the relevance of plant-derived bioactive compounds in health promotion. This study did not also discover a significant difference in the ability of gallic acid and hesperidin as far as maintenance of serum electrolyte levels was concerned.


Drosophila melanogaster and Drosophila serrata, to explore the distributions of genetic variance across high-dimensional phenotypic space. Surprisingly, most of the heritable trait covariation was due to few lines (genotypes) with extreme [>3 interquartile ranges (IQR) from the median] values. Intriguingly, while genotypes extreme for a multivariate factor also tended to have a higher proportion of individual traits that were extreme, we also observed genotypes that were extreme for multivariate factors but not for any individual trait. We observed other consistent differences between heritable multivariate factors with outlier lines vs those factors without extreme values, including differences in gene functions. We use these observations to identify further data required to advance our understanding of the evolutionary dynamics and nature of standing genetic variation for quantitative traits.The interaction of evolutionary processes to determine quantitative genetic variation has implications for contemporary and future phenotypic evolution, as well as for our ability to detect causal genetic variants. While theoretical studies have provided robust predictions to discriminate among competing models, empirical assessment of these has been limited. In particular, theory highlights the importance of pleiotropy in resolving observations of selection and mutation, but empirical investigations have typically been limited to few traits. Here, we applied high-dimensional Bayesian Sparse Factor Genetic modeling to gene expression datasets in 2 species,  The maintenance of quantitative genetic variance presents geneticists and evolutionary biologists with a formidable challenge. While models of the evolution of allele frequencies can be relatively simple, evolution of the genetic variance for phenotypic traits also depends on the effects of those alleles . A substFrom a theoretical perspective, the resulting allele frequency spectrum is a key distinguishing feature between models in which selection actively maintains polymorphisms  and models in which selection eliminates variation . Balancing selection mechanisms are predicted to maintain relatively symmetrical allele frequencies at a locus, while under MSB models, genetic variation is determined by rare alleles, where the greater the fitness effect of a locus, the rarer the minor allele at that locus . GenomicDifferent MSB models also make contrasting assumptions about the magnitude of effects of new mutations relative to segregating alleles: Gaussian models assume mutations arise frequently, but have small effects, while House-of-Cards (HoC) models assume rarer, larger effect mutations . GWAS ofn) is much lower than the number of traits measured (p). Indeed, based on genetic load and genetic variance in fitness arguments, n > 200 is predicted to be unlikely  modeling  to internogaster . SeparatD. serrata population in Brisbane, Queensland, Australia, as detailed in D. serrata lines , and a random subset of 3,385 of the 10,096 genetically variable gene expression traits. We analyzed data for males only (as in D. serrata). The previous analysis of these data indicated strong patterns of genetic covariance among expression traits  across traits, then plotted the sorted pooled observed values against the sorted pooled values from the simulated data . The obsG, is modular and of low rank (i.e. sparse). This assumption is consistent with published estimates of G from a range of taxa and traits that suggest both modularity  measures of the 3,385 gene expression traits; X is the 60 × 1 incidence matrix relating observations to the fixed effect of trait means where B is the 1 × 3,385 matrix of trait means; Z is the 60 × 30 incidence matrix relating the 2 replicates per line to their respective line; U is the 30 × 3,385 matrix such that itu is the mean effect of line i on trait t; and E is the 60 × 3,385 matrix of deviations of the replicates from their line mean, such that ijte is the effect of replicate j of line i on trait t.The BSFG analysis is founded in the classical linear mixed model that partitions observed phenotypic variation to genetic and nongenetic sources. Here, the linear mixed model, fit separately to the U and E, are assumed to follow multivariate normal distributions:G) and replicate (R) variance–covariance matrices sum to give P, the phenotypic variance–covariance matrix:The random effects of genotype (line) and replicate (reflecting microenvironment and measurement error), P as:Λ is the 3,385 × k matrix whose columns form the k predicted latent factors underlying the phenotypic variance and Ψ is the diagonal matrix containing the 3,385 trait-specific variances.In the BSFG model, common and specific components of phenotypic (co)variance are estimated by modeling k is free to vary from one iteration of the Gibbs sampler to the next, unless otherwise specified. For computational efficiency, we constrain the model to estimate at most 59 factors; this number corresponds to the maximum number of nonzero eigenvalues in an orthogonal decomposition of a covariance matrix of 60 observations per trait. BSFG analysis is not well suited to estimating the true number of independent latent traits in the data , and is instead primed to provide robust estimates of latent factors with the greatest influence on the total phenotypic variation  and specific (i.e. Ψ) components of P can each be partitioned into genetic and nongenetic components:k × k diagonal matrix of latent factor heritabilities, Ik and Ip are identity matrices of size k and p, respectively. A summary of the outlined parameters can be found in 2ij, as approximately the proportion of phenotypic variance in trait i that can be explained by factor j.The common .To determine statistical significance of the latent factors and their heritabilities, we used the local false sign rate (LFSR) approach with an average error rate of 1%, as described in Other studies investigating the relationship between allelic effect size and frequency have defined effect size quantitatively  is far greater than the number of objects measured per variable (60 samples), random correlations among variables are expected to be common e.g. . To helpAs anticipated, sampling error generated spurious genetic correlations among traits , resultiD. serrata) or 20 (D. melanogaster) times as many large pairwise correlations (i.e. |r| > 0.5) in the observed data than the randomized datasets  D. serrata heritable factors but only 4 of 42 D. melanogaster heritable factors were retained for further analysis based on this criterion  is arbitrary. To aid interpretation of the latent traits, we imposed directionality on each factor by multiplying its trait loadings and latent trait values by the sign of its mean trait loading . This allowed us to investigate whether extreme variants were more likely to be associated with an overall increase or decrease in gene expression across the variance-standardized traits significantly affected by the latent factor.Like an eigenvector, the direction of each factor  were available for 29 of the 30 lines analyzed here. Across these 29 lines, 5 known inversions were segregating, present as 1 or 2 copies across 11 lines (including one line carrying 2 inversions). Two of the 5 inversions, In(2L)t and In(3R)Mo, are associated with variation in the expression levels of hundreds of genes . Of the 3,385 probe sets analyzed, 3,119 were associated with a single FlyBase Gene ID . For D. serrata, we used FlyBase gene IDs corresponding to orthologs in D. melanogaster of the D. serrata transcript-level coding sequence, matched to D. serrata ESTs using BLASTN  using GffRead  or 2,154 (D. serrata) FlyBase Gene IDs. Limiting the background to only the genes that could have been in the gene set of interest reduces the probability of detecting significant enrichment where there is none  factors were statistically supported, collectively explaining 59% of the total phenotypic variance in the 3,385 expression traits, while in D. melanogaster 39% of the phenotypic variance was explained by 47 statistically supported factors. We assessed the effect of these latent traits on the distribution of gene expression phenotypes and, in particular, whether the observed extreme gene expression values (D. serrata (D. melanogaster). That is, latent factors contributed more than trait-specific effects did to extreme values in the data.The BSFG analysis returned evidence consistent with previous analyses of these data, namely that a substantial portion of the variation in expression was shared among traits, not trait specific. In n values  were accn values , and cenn values , top rowD. serrata and D. melanogaster, respectively, 32 and 22 of the latent factors were significantly heritable and met the additional criteria from the randomization analysis  or 94% (D. melanogaster) reduction in extreme genotypic values after model fit. This suggests that the majority of extreme genotypic values may arise via processes affecting multiple traits.In analysis . In D. s to 0.99 , with in to 0.99 . In D. m to 0.99  with bet to 0.99 . We asseD. serrata and 22 D. melanogaster latent factors for which heritability was statistically supported revealed patterns that suggested only some of the heritable factors may contribute to the extreme genotypic values. For 7 (22%) and 11 (50%) of the heritable factors in D. serrata and D. melanogaster, respectively, there were no outlier lines  across the 960 (30 lines × 32 heritable factors) latent trait values  across the 660 (30 lines × 22 heritable factors) latent trait values, with at most a single extreme value for any given line or heritable factor  or 6.7% (D. melanogaster) had at least one outlier line (D. serrata) or 19 (D. melanogaster) IQR from the median ; 127 of these traits were also associated with heritable factors with outlier line(s)  . Similarx-axis in y-axis in x-axis in y-axis in Second, we investigated whether the frequency of outlier trait values for individual lines could be predicted by the magnitude of the latent trait value. We observed a statistically significant but imperfect association . IndividD. serrata) or 20% (D. melanogaster) of the associated individual traits  that were outliers for a heritable factor were outliers for only a small proportion (<10%) of the associated individual traits  allowed us to infer positive (negative) extreme latent trait values to indicate an overall increase (decrease) in variance-standardized gene expression when summed across the traits significantly associated with the factor. In the 25 heritable factors with outliers in D. serrata, 33 of the 40 outlier values deviated above the median  had at least one extreme genotype (line), resulting in higher heritability of these multivariate traits relative to other multivariate traits lacking extreme genotypic values. While extreme latent trait values typically occurred for lines (genotypes) with a higher proportion of individual trait outliers, individual traits could be extreme without resulting in extreme latent trait values for the heritable factor that they were influenced by, and conversely, extreme latent trait values were observed when the heritable factor did not cause extreme values of any individual trait . Thus, tDrosophila populations. First, we presume that extreme trait values are caused by single loci, where for heritable factors these loci have pleiotropic effects. Trans-regulatory factors can influence expression of large numbers of genes . Applying equation (2.13) of μ is the genic mutation rate, predicts that the strength of selection required to maintain q at 1% was ∼0.01–0.1, given μ of 10−5–10−6 (q is in fact lower (higher) than our sample suggests, s must be stronger (weaker) to maintain the allele frequency, assuming q ∼ 1%. Finally, this prediction assumes that the large-effect alleles are recessive; if the observed outlier lines in fact carry alleles with additive or dominant effects, then the resultant increased visibility to selection means that selection 50–100 times weaker could maintain alleles at 1% frequency (Given the presumption of a single (pleiotropic) locus, and the observation that, for most individual and latent traits, only one extreme line was observed, we infer that alleles with (homozygous) major effects were segregating in the base population at a frequency of 0−5–10−6 . If the requency  and 2.15s ∼ 0.01–0.1) detailed above is within the range estimated for new mutations affecting fitness traits, such as viability and fecundity, based on the ratio of mutational to genetic variance  of gene expression traits in the D. serrata lines analyzed here, inferring selection within the same range for 5D multivariate axes of expression (median s = 0.032), but weaker selection on the individual gene expression traits  cause coordinated changes in expression of functionally unrelated genes via horizontal mechanisms. Second, the observed pattern  of heritable factors with outliers could reflect extreme values of pleiotropic effects. The distribution of effects of pleiotropic alleles across multiple traits is largely unknown, but might be expected to include alleles with very weak effects on some trait(s) (Heritable factors with outliers were less likely to be enriched for specific functions and exhibited patterns among lines where expression was elevated for some traits but depressed for others (i.e. unbiased direction of loadings). There are several possible nonexclusive explanations for these observations, which our data cannot further distinguish among. Heritable factors with outliers might capture gene coexpression caused by a mixture of so-called horizontal (e.g. shared chromatin status) and vertical (e.g. shared transcription factors) processes. trait(s) ; the herSaccharomyces cerevisiae found that mutations more frequently increased than decreased expression of 2 of the 10 genes studied, while a third gene exhibited the opposite bias, with more mutations decreasing expression , expression was biased upward; the question of emergent strong bias in the multivariate distribution of phenotypic effects warrants further investigation.Quantitative genetic theories of the maintenance of genetic variance that incorporate indirect (apparent) stabilizing selection assume that mutations have directionally biased effects on fitness itself (decreasing it), but that the pleiotropic effects of those mutations on other traits are unbiased, equally frequently increasing as decreasing trait values . EmpiricResolution of theory predicting the maintenance of genetic variation for quantitative phenotypes depends on better insight into the distributions of (pleiotropic) allelic effects on multiple traits and fitness. Widespread availability of data on many expression traits from the same genotypes provides a particularly powerful system for investigating shared variation among high-dimensional phenotypes, while the intermediary causal nature of expression traits, connecting genotype to more complex traits (iyac122_Supplemental_TablesClick here for additional data file.iyac122_Supplemental_FiguresClick here for additional data file.


Populus tremula L.), goat willow , rowan (Sorbus aucuparia L.), and sycamore (Acer pseudoplatanus L.). Based on the previous work from mature forests, we hypothesize that bark properties of young trees are also species-specific and change along the stem profile. Thus, across the regions of Slovakia, we selected 27 forest stands composed of one of the target broadleaved species with ages up to 12 years. From the selected forests, 600 sample trees were felled and stem bark properties were determined by measuring bark thickness, weighing bark mass after its separation from the stem, and drying to achieve a constant weight. Since the bark originated from trees of varying stem diameters and from different places along the stem , we could create regression models of stem characteristics based on the two mentioned variables. Our results confirmed that bark thickness, thus also specific surface mass, increased with stem diameter and decreased with distance from the stem base. While common aspen had the thickest stem bark (4.5 mm on the stem base of the largest trees) the thinnest bark from the analyzed species was found for sycamore (nearly three times thinner than the bark of aspen). Since all four tree species are very attractive to large wild herbivores as forage, besides other uses, we might consider our bark mass models also in terms of estimating forage potential and quantity of bark mass consumed by the herbivory.Considering the surface of individual tree compartments, it is obvious that the main portion of bark, i.e., the largest area and the greatest bulk mass, is located on the stem. We focused on basic bark properties, specifically thickness, surface area, biomass, and specific surface mass (expressed as dry weight per square unit) on stems of four broadleaved species: common aspen ( Plants, which also include trees growing in forest ecosystems, are composed of a structural complex of organs characterized by their typical forms but mainly by fulfilling specific functions . It is gDBH, i.e., measured 130 cm from the ground level) as a predictor. For instance, Stängle and Dopremann [DBH are suggested as explanatory variables of models predicting bark thickness. The authors pointed out that the interest in accurate bark thickness estimates is driven by the need to obtain not only accurate inside-bark diameters and volumes, but also precise estimates of bark volume. The importance of these estimates increased with the shift in the commercial relevance of bark from a useless residue to a valuable fuel and feedstock for high-value biomaterial [Several authors , cork pmaterial , or for material . Moreovematerial .D0), and showed a decreasing contribution of bark mass to total stem mass as well as to total tree biomass with tree size. Here, we utilized the database which had been used for the preparation of the monograph and constructed more detailed models of bark thickness and biomass respecting not only tree size, but also the position along the vertical stem profile.As most of previous bark-focusing studies and models were motivated by certain economic interests (bark utilization), nearly all authors focused on mature trees. One of a few studies about bark quantity of young trees was published by Pajtík et al. . In theiPopulus tremula L.), goat willow , rowan (Sorbus aucuparia L.), and sycamore (Acer pseudoplatanus L.) have been particularly attractive to large wild herbivores, especially red deer (Cervus elaphus L.). Bark browsing  and the overall impact of large wild herbivory on forest vegetation has been increasing in many European countries because of continuously rising population density of these animals, especially red deer . The. ThePopur  is: aspen, sycamore, rowan, and goat willow . These rIn the next step, we derived species-specific regressions for using Equation (5) to calculate stem radii at different heights on stems. The models were highly significant and explained from 59 to 91.6% of radius variation along the stem .Next, we analyzed bark thickness along the stem profile. Bark thickness varied between approximately 0.5 to 4.5 mm depending on the species and height on stem . There wFurther analyses proved large differences in the surface area of bark between tree species . Note thD0 of 100 mm and 0–50 cm section from the ground). For all tree species, large differences in bark mass were recorded between the lowest section 0–50 cm and all the other sections.Bark mass was obtained from the information on bark thickness and its surface area using Equation (6) . The larD0 and individual stem sections  and upper stem parts exhibited much more contrast for common aspen and goat willow than for rowan and sycamore.Finally, specific surface mass was calculated respecting both sections . The larOur results clearly demonstrate that from the intraspecific point of view, bark thickness varies with stem diameter as well as along the vertical stem profile. In fact, both of these variables are prevailingly related to bark age, i.e., older bark (on older trees and/or bark situated at a lower stem part) is thicker than younger bark (younger trees and/or at higher stem parts). In general, each tree stem contains a layer of dividing cells called the vascular cambium . These cMoreover, large differences in bark thickness occur between the species. The differences between the species with the thinnest bark  and the thickest bark (common aspen) are nearly three-fold. Trees are sometimes categorized as thin-barked and thick-barked , althouBark thickness of all investigated species decreased with height on stems (distance from ground), which is consistent with studies on other species ,36. The DBH, while it measured approximately 2.5 mm for trees with DBH of 10 cm, and doubled in trees with DBH of 20 cm. Larger trees with DBH of 30 cm and more had similar values of bark thickness equal to nearly 9 mm. If we consider bark thickness (2.5 mm) for the lower limit of the stem diameter (10 cm) shown in the paper by Johansson [Sequoia sempervirens (D. Don) Endl. was approximately 8, 14, and 18% thicker than the bark of dominant trees with the same dimensions, respectively. However, the authors did not show exact ages of the individual trees, which were compared and the differences might be related primarily to different tree ages. At the same time, Berill et al. [We could not find any results about bark thickness for the four studied broadleaved tree species in other works, except for common aspen . The papohansson , our resohansson ,37,38,39ohansson ) or a coohansson ). The fiohansson . Anotherohansson . The autl et al.  proved tA positive relationship between tree size and stem bark thickness has been previously interpreted to mean that bark thickness is strongly influenced by its defensive role . AccordiFraxinus excelsior L.). The authors showed that the specific surface mass of bark for ash was between 4.0 and 7.5 g per dm2 for trees with D0 between 20 and 60 mm [Fagus sylvatica and Picea abies . On theOur research can be understood as both a pilot and a tentative one. It can be regarded as a pilot study because previous work that applies modeling of bark properties focused almost exclusively on large trees with a minimum stem diameter of about 10 cm e.g., ,42,46). ,46. 42,4http://gis.nlcsk.org/lgis/, accessed on 10 February 2022). From these stands, we further chose forest stands that grew at moderately fertile sites; therefore, nearly all of them were found on mesotrophic cambisols. Since mesotrophic cambisols [Our sampling covered four broadleaved tree species, namely: common aspen, goat willow, rowan, and sycamore. Firstly, we selected naturally regenerated even-aged forest stands for each target tree species with a maximum age of 12 years. These were from the current national database of forest stands and based on data from Forest Management Plans  during the existence of the selected stand.Field work was conducted each year between 2016 and 2020, always in the second half of the growing season. Each tree species at a specific location was represented by 15–25 individuals. Thus, totally, we sampled between 100 and 200 trees per species . Trees wSampled trees were randomly selected in each stand while avoiding damaged, dying, deformed, or atypically shaped individuals, e.g., ones growing at forest stand edges. We tried to choose individuals of all bio-sociological positions, i.e., dominant, co-dominant, intermediate, and suppressed ones. However, we omitted severely suppressed individuals with the symptoms of initial die-back.D0) and at breast height (DBH) were measured using a digital caliper with ±0.1 mm precision. Then, all branches were cut off by garden scissors and disposed. A precise tree height (±1.0 cm) was measured using a metal tape measure. Afterward, all tree stems were packed in labeled paper bags and transported to our laboratory .The stems were divided into several sections, while the number of sections depended on the tree height (more sections were taken from higher trees). When dividing the tree into sections, we implemented two principles: (1) to have at least 4 sections per tree, and (2) not to exceed the length of 1 m, which was set as the maximum length of a section. Diameters of each section were measured with a digital caliper (precision ±0.1 mm) in two perpendicular directions at its both ends, and in the middle. The measurements were repeated twice: before peeling the bark off  and after peeling the bark (under bark). Bark thickness at a certain position was calculated as a difference between the diameters before and after bark peeling.°C for 120 h to reach the constant weight. Afterward, they were weighed with an accuracy of 0.1 g.At a tree level, we took overall bark  that weH is a tree height (m);D0 is a diameter at stem base (mm);b0, b1, b2 are regression coefficients.The relationship between tree height and stem base diameter was described with the following regression:DBH is a diameter at breast height (mm);D0 is a diameter at stem base (mm);b0, b1 are regression coefficients.The relationship between diameter at breast height and stem base diameter was described with the following linear function:D0 (independent variable). Thus, relationships between D0 and tree height (or diameter DBH) also create the possibility for potential users to implement these tree characteristics for estimating bark characteristics.Values of regression coefficients and statistical characteristics of both relationships are presented in Bark thickness at a certain height above the ground was calculated using a power function with two predictors:bT is bark thickness (mm);D0 is a diameter at stem base (mm);gH is a distance from the ground level (cm);b0, b1, b2 are regression coefficients.d0 was derived from the measured data on bark thickness at specific heights. The heights were not constant for the whole dataset, because the sections were of different lengths. The model of bark thickness was derived only for the bottom part of the stem up to the height of 2.5 m.The model for the calculation of bark thickness based on the height above the ground and the stem base diameter Bark surface was calculated for individual stem sections using the formula for calculating the surface of a truncated cone, omitting the areas of base and top circles:bS is bark surface (cm2);r1 is a radius of the bottom end (cm);r2 is a radius of the top end (cm);sl is the length of the section (cm).r1 and r2 at any height above the ground (gH), we derived a continuous regression model for stem radius with D0 and gH as predictors. This relationship was also derived for a stem part up to 2.5 m above the ground. The formula is:r is a stem radius (mm);D0 is a diameter at stem base (mm);Hg is a distance from the ground level (cm);b0, b1, b2 are regression coefficients.To determine the radii r1 and r2 for a tree with a diameter D0 at predefined heights above the ground . The calculated values of r1 and r2 were then used in Equation (4) to derive the bark surface of sections 0–50, 51–100, 101–150, 151–200, 201–250 cm. Models (3) and (5) were derived in forms of allometric equations in very similar ways as previously published by Marklund [Model (5) allowed us to determine Marklund , LedermaBark mass of individual sections was derived using the equation:bW is bark mass weight (g);bV is bark volume (cm3);bρ is bark density (kg per m3).where bark volume bV of each section was calculated as a difference between the section volume outside bark and the volume inside bark. The section volume (cm3) was determined using the volume equation for a truncated cone:ρ were taken from the only available paper by Pajtík et al. [The species-specific values of bark density k et al. . Bark mak et al. ).Specific surface mass of bark was calculated as follows:SPH is specific surface mass of bark (g per dm2);bW is bark mass (g);bS is bark surface (dm2).This study evaluated vertical changes in bark properties along the stems of four broadleaved tree species in the Western Carpathian Mountains., Slovakia. The results confirmed that bark thickness is species-specific and changes with tree size and height on stem. The thickest bark was found for common aspen. Goat willow, rowan, and sycamore had much thinner bark than aspen . Our results revealed the same order of tree species according to bark thickness as in the case of mature trees published in other previous works. The bark thickness was greatest at the bottom part of trees and decreased with the increasing height on stems, irrespective of species. Hence, biomass studies of young trees should consider the changes along the vertical stem profile. The models derived in this study account for these changes and may be used under similar growing conditions in Central Europe.The revealed substantial differences between bark properties of tree species indicate that using generic values of bark thickness or bark mass may cause significant bias in estimated biomass. Therefore, species-specific and, if possible, also local models that account for tree size should be implemented for tree biomass estimates including stem bark. The new information about bark properties can be considered as another scientific contribution of our work because a substantial part of previous studies did not consider stem bark as a separate tree component but often included it in over-bark stem estimates. Finally, we would like to conclude that findings on the thickness and/or biomass of stem bark should be not considered only as an output for estimating potential residues in the wood processing industry. Such novel knowledge could be applied in studies of stem bark utilization for humankind and as a part of large wild herbivory nutrition.


Injuries of the acromioclavicular joint (ACJ) occur frequently in young and active people. The best management of acute grade III injuries has been a source of controversy and extensive debate. When surgery is indicated, there is still no gold standard surgical technique for treating acute grade III ACJ injuries.TM Button System, etc. Fourteen studies were selected for the final review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.The methodology of this review was a comprehensive search of PubMed, Medline, Cochrane, and EMBASE databases using various combinations of the keywords “Rockwood,” “type III,” “grade III,” “treatment,” “surgery,” “acromioclavicular joint,” and “dislocation,” since the inception of the databases to December 2020. Surgical techniques were divided into two groups. In group 1 were ACJ fixation techniques using hardware such as the hook plate, Kirschner wires, and wire cerclage; group 2 included coracoclavicular (CC) ligament fixation/reconstruction techniques using double buttons, TightRope®, suture anchors, Endobuttons, the Infinity-LockThis review showed better outcome scores in group 2. Overall, complication rates were higher in group 1 compared to group 2. The results of this review show that CC fixation, using suspensory or loop devices, of Rockwood grade III injuries, has better outcomes and fewer complications than fixation of the ACJ with hardware. Injuries of the acromioclavicular joint (ACJ) are common in young and active people and account for about 9% of all shoulder girdle injuries . The litThe acromioclavicular joint is an amphiarthrodial joint formed at the articulation of the lateral clavicle with the acromion. Essentially attaching the shoulder girdle to the axial skeleton and capable of very complex, previously misunderstood movements , the ACJThe goal of the treatment of an acute ACJ dislocation is to restore the dislocated joint’s normal anatomy and alignment and permit a return to full power without any limitations on the range of movement during activity . RockwooThe best surgical management of grade III injuries is a source of ongoing controversy and debate ,13,14, aThis systematic review aims to compare the outcomes of the different surgical techniques employed, when indicated, in the treatment of grade III ACJ injuries.Methods and materialsEthical approval and patient consent were not required in this review as it is based on previously published studies.Search Strategy and Study SelectionPubMed, Medline, Cochrane, and EMBASE databases were searched since inception following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines Figure  17] by  by 17] bData ExtractionTwo investigators (IO and EA) performed the data extraction using a standardized Excel file . A third reviewer (MA) confirmed the data extracted by the two independent reviewers and reviewed the final study. The following data were extracted from the included studies: first author’s name, year of publication, study design, surgical technique used, the sample size in each group, patient characteristics/demographics, duration of follow‐up, outcome measures, and complications. Surgical procedures were divided broadly into two groups: group 1 included techniques that involve fixation of the ACJ using hardware, e.g., K-wires, wire cerclage, and hook plates, and group 2 comprised procedures requiring repair of the CC ligament by either rigid or non-rigid fixation devices and anatomical or non-anatomical CC ligament reconstruction/augmentation by autogenous or synthetic grafts. Any disagreements between the investigators were resolved by discussion and consensus.Outcome Measures and StatisticsThe outcome measures of interest were the Constant-Murley (CM) score , Taft scResultsThe search query resulted in 306 abstracts, with three more included from cross-referencing selected studies making a total of 309 abstracts. 98 abstracts were excluded due to duplicate records leaving 211 abstracts. After screening titles/abstracts for satisfying inclusion/exclusion criteria, 177 abstracts were further excluded, leaving 34 unique studies for full-text review. Finally, 14 studies that reported outcomes after the surgical management of acute Rockwood grade III acromioclavicular dislocations were included in this systematic review  score was the most frequently reported outcome measure recorded in nine studies Table . The aveŁazarski et al. reported statistically significant improved CM scores in patients randomized to techniques in group 2 compared to the use of hook plates in group 1 (p = 0.04) . Ye et aŁazarski et al. reported Patient-Reported Outcomes Measurement Information System (PROMIS) scores. Patients who had fixation using the Ladermann technique (group 2) had average PROMIS scores of 86.45 (66-90), while those who had fixation using a hook plate and wire cerclage (group2) had average PROMIS scores of 80.25 (61-89) and 89.5 (87-90), respectively. A comparison of the preoperative and postoperative CCD was reported in five studies, with one study reporting only the preoperative CCD. A detailed comparison of the outcomes in the two groups is shown in Table ComplicationsPostoperative loss of reduction/redislocation, pain, infection, CC ligament ossification, and osteoarthritis were the most frequently reported surgery complications. Table DiscussionSeveral factors determine the indication for primary surgical fixation of acute grade III injuries, and they include pre-injury function, post-injury functional deficit, the presence of functional pain, age, sex, hand dominance, and the patient’s preference for operative intervention. The optimal surgical treatment of grade III ACJ dislocations is still controversial, and a topic of debate among surgeons, as the current evidence, is inconclusive. Although different surgical techniques have been described in the literature for the management of ACJ joint dislocations, controversies regarding the timing of surgery, open versus arthroscopic surgery, and the choice of surgical procedure are still prevalent, more so for grade III injuries. The purpose of this study was to review the literature to determine the best surgical treatment for acute grade III acromioclavicular injuries.The surgical repair of an ACJ dislocation can be grouped into techniques that allow either fixation of the ACJ and achievement of fixation between the coracoid and the clavicle or a reconstruction/augmentation of the CC ligament.The goal of ACJ fixation is to stabilize the ACJ and reduce the CCD to allow the healing of ligaments disrupted by injury. ACJ fixation was historically achieved using Kirschner wires or Steinmann pins as described by Taft et al. and Leidel et al. ,31. As sFixation between the clavicle and coracoid can be achieved by either rigid or non-rigid devices. Excellent outcomes have been reported in the use of Bosworth screws to achieve rigid fixation in grade III injuries ,26,36. INon-rigid fixations of the CC ligament have the benefit of eradicating the need for a second surgery for implant removal and include the use of polydioxanone sulfate (PDS), suture anchors, TightRope®, and Ethibond sutures. The TightRope® was first described as a technique for the treatment of AC dislocation in 2007 . DarabosWith recent advancements in surgical instrumentation and arthroscopic techniques, there has been an increase in the use of arthroscopically assisted or all-arthroscopic reconstruction of the coracoclavicular ligament with graft or synthetic materials.This study showed better functional outcomes in patients with a Rockwood grade III ACJ injury managed with CC ligament repair or reconstruction than ACJ fixation with hardware. Pin migration, pain, and osteoarthritis were the most frequently occurring complications in group 1 and are in keeping with previous studies ,45. DespLimitationsThis review has substantial limitations. Although there were two level I studies, most of the included studies were retrospective and level III evidence. Furthermore, several of the studies had shown evident bias in selecting patients, while others did not include all complications. Some studies did not report the outcome measures being assessed. In addition, several studies did not consider or report radiographic outcomes. Finally, a few of the surgical techniques described have since become obsolete, while others such as the use of threaded Kirschner wires for intra-articular ACJ fixation are outrightly contraindicated today.TM Ligament, as opposed to rigid fixation methods, is more effective for the management of acute ACJ dislocations.The results of this study are in line with current biomechanical and clinical studies that show that the reconstruction of the CC ligament using autogenous or synthetic ligaments, such as the TightRope® and Surgilig/LARSDue to the lack of sufficient level I evidence available in the current literature, appropriately powered randomized control studies are required to compare the various CC ligament repair/reconstruction techniques to determine a gold standard for the surgical management of acute grade III ACJ dislocations.