Most studies report that the common position of cervical spinal manipulation (CSM) for treating symptomatic cervical disc herniation (CDH) is lateral bending to the herniated side. However, the rationality of lateral bending position on performing CSM for CDH is still unclear. The purpose of this study is to investigate the biomechanical effects of lateral bending position on performing CSM for CDH. A finite element (FE) model of CDH (herniated on the left side) was generated in C5-6 segment based on the normal FE model. The FE model performed CSM in left lateral bending position, neutral position, and right lateral bending position, respectively. Cervical disc displacement, annulus fiber stress, and facet joint stress were observed during the simulation of CSM. The cervical disc displacement on herniated side moved forward during CSM, and the maximum forward displacements were 0.23, 0.36, and 0.45 mm in left lateral bending position, neutral position, and right lateral bending position, respectively. As the same trend of cervical disc displacement, the annulus fiber stresses on herniated side from small to large were 7.40, 16.39, and 22.75 MPa in left lateral bending position, neutral position, and right lateral bending position, respectively. However, the maximum facet stresses at left superior cartilage of C6 in left lateral bending position, neutral position, and right lateral bending position were 6.88, 3.60, and 0.12 MPa, respectively. Compared with neutral position and right lateral bending position, though the forward displacement of cervical disc on herniated side was smaller in left lateral bending position, the annulus fiber stress on herniated side was declined by sharing load on the left facet joint. The results suggested that lateral bending to the herniated side on performing CSM tends to protect the cervical disc on herniated side. Future clinical studies are needed to verify that. Cervical disc herniation (CDH) is a common cause of cervical radiculopathy which occurs in approximately 85.4 of every 100000 persons . SymptomCervical spinal manipulation (CSM) is one of the important conservative treatments for symptomatic CDH –5. StudiThe purpose of the present study is to compare the biochemical effects of different lateral bending positions on performing CSM through three-dimensional finite element analysis, so as to evaluate the rationality of lateral bending to the herniated side on performing CSM for CDH.Following the methods of Zhong Jun Mo et al. 2014 [The slice images were imported into medical image processing software  to reconstruct the vertebrae geometry volume of C3–7. The geometry of other structures , which were difficult to separate from the CT images, was modeled using the solid modeling software, SolidWorks 2014 .Finite element modeling software  was used to build and mesh the cervical spine components. The vertebrae were made up of a solid volume (cancellous bone) and a layer of shell  with a thickness of 0.4 mm , 15. TheThe assigned material properties  selectedSimilar to simulation methods reported by Hussain et al. , the FE The Simulation and Loading of CSM in C5–6 with CDHThe inferior endplate of C6 was fixed.The C5 vertebra, along with the entire model, was rotated 2°, 0, -2° around y-axis to simulate the left lateral bending, neutral, and right lateral bending positions, respectively.In the left lateral bending, neutral, and right lateral bending positions, the cervical FE model was rotated 2° to the right side (the opposite side of CDH) around z-axis  to simulate rotation to the right side, respectively.The cervical FE model continued to rotate a further 0.5° to the right side within 0.15 seconds so as to simulate the high-velocity, low-amplitude CSM.In this research, we used the material properties of moderately degenerated disc to investigate the biomechanical effects of lateral bending position on performing CSM for CDH. The mechanical loading steps in sequence to simulate CSM were as follows :The infeCervical disc displacement, annulus fiber stress (von Mises stress) and facet joint stress (von Mises stress) were observed during the simulation of CSM.The range of motion (ROM) of each functional spinal unit in the FE model of C3-7 is shown in When performing CSM, the left posterolateral (the herniated side) cervical disc moved left, forward, and up in three positions: left lateral bending, neutral, and right lateral bending . The maxWhen performing CSM, the maximum annulus fiber stresses were 22.52, 17.61, and 23.60 MPa in left lateral bending position, neutral position, and right lateral bending position, respectively. The distribution of annulus fibers stress in left lateral bending position was concentrated on the right posterolateral (the heathy side) of cervical disc. However, the distributions of annulus fibers stress in neutral position and right lateral bending position were both concentrated on the left posterolateral (the herniated side) of cervical disc . And theWhen performing CSM, the maximum facet stresses at left superior cartilage of C6 in left lateral bending position, neutral position, and right lateral bending position were 6.88, 3.60, and 0.12 MPa, respectively .Cervical disc herniation is most common at the C5-6 level , 29. PatInterestingly, we discovered that the cervical disc on herniated side moved forward during CSM in three positions: left lateral bending, neutral, and right lateral bending. The maximum forward displacements were 0.23, 0.36, and 0.45 mm in left lateral bending position, neutral position, and right lateral bending position, respectively. It reconfirmed that a small forward displacement was generated on the left side (herniated side) of cervical disk during CSM with rotation to the right side  . And it As the same trend of cervical disc displacement, the annulus fiber stress on herniated side from small to large was 7.40, 16.39, and 22.75 MPa in left lateral bending position, neutral position, and right lateral bending position, respectively. The distribution of annulus fibers stress in left lateral bending position was concentrated on the right posterolateral  of cervical disc. However, the distributions of annulus fibers stress in neutral position and right lateral bending position were both concentrated on the left posterolateral (the herniated side) of cervical disc. It suggested that the cervical disc on herniated side may be damaged with high annulus fibers stress during CSM . CompareThe facet joint stress showed the opposite trend with the cervical disc displacement and annulus fiber stress. The maximum facet stresses at left superior cartilage of C6 were 6.88, 3.60, and 0.12 MPa in left lateral bending position, neutral position, and right lateral bending position during CSM. As we known, facet joints play a role in mechanical function which can contribute to spinal stability and load sharing between spinal columns , 39. TheCompared with neutral position and right lateral bending position, though the forward displacement of cervical disc on herniated side was smaller in left lateral bending position, the annulus fiber stress on herniated side was declined by sharing load on the left facet joint. In summary, lateral bending to the herniated side on performing CSM for treating CDH tends to protect the cervical disc on herniated side during such manipulation. Future clinical studies are needed to verify the biomechanical effects of lateral bending position on performing CSM for treating CDH.The present study has certain shortcomings. First, the current results were based on the FE model of a healthy female who did not have cervical disc herniation. The simulation of cervical disc herniation in C5-6 segment was made by the model adjustment, but other parameters and geometry did not change from the normal FE model. The interpretation of the results should be cautious because the results were drawn from the adjusted model. Second, the loading conditions were highly idealized and could not represent the complicated condition of cervical spinal manipulation. Therefore, it should be kept in mind that the present results were driven by these assumptions. However, the results of FE analysis represent trends rather than precise values because of the necessary simplifications and assumptions concerning the geometry, material properties of the different tissues, contact behavior, and applied loads . Third, Compared with neutral position and right lateral bending position, though the forward displacement of cervical disc on herniated side was smaller in left lateral bending position, the annulus fiber stress on herniated side was declined by sharing load on the left facet joint. In summary, lateral bending to the herniated side on performing CSM for treating CDH tends to protect the cervical disc on herniated side. Future clinical studies are needed to verify that.


Primary immunodeficiencies (PIDs) are a group of over 300 hereditary, heterogeneous, and mainly rare disorders that affect the immune system. Various aspects of immune system and PID proteins and genes have been investigated and facilitate systems biological studies of effects of PIDs on B cell physiology and response. We reconstructed a B cell network model based on data for the core B cell receptor activation and response processes and performed semi-quantitative dynamic simulations for normal and B cell PID failure modes. The results for several knockout simulations correspond to previously reported molecular studies and reveal novel mechanisms for PIDs. The simulations for CD21, CD40, LYN, MS4A1, ORAI1, PLCG2, PTPRC, and STIM1 indicated profound changes to major transcription factor signaling and to the network. Significant effects were observed also in the BCL10, BLNK, BTK, loss-of-function CARD11, IKKB, MALT1, and NEMO, simulations whereas only minor effects were detected for PIDs that are caused by constitutively active proteins . This study revealed the underlying dynamics of PID diseases, confirms previous observations, and identifies novel candidates for PID diagnostics and therapy. The human immunome covers the entirety of genes and proteins that are essential for innate and adaptive immunity . Many ofvia pro- and pre-cell stages to immature and mature B cells and finally to plasma or memory cells. The B cell subtypes play different and sometimes overlapping roles in immune response by virtue of their differing master regulator TFs (B cells produce antibodies. The membrane-bound antibody component of the B cell receptor (BCR) recognizes foreign antigens and triggers a cascade of signal transduction events that lead to the activation as well as nuclear transport of specific transcription factors (TFs) . In the ator TFs  and surfator TFs –7.Primary immunodeficiencies (PIDs) are intrinsic diseases of the immune system, mostly rare and typically with heterogeneous phenotypes. Already over 300 PIDs have been described. Disease-causing variants have been collected to IDbases  and someThe reconstruction of cellular networks in systems biology facilitates simulations where diseases are modeled as perturbations or alterations , 12. Sinin silico. The simulations reproduced known trends due to variations in PIDs in essential signal transduction pathways during B cell development from pre- to mature B cell . Another signal  through the costimulatory receptor, response , 25. Theresponse –28. The response .We identified paths from signals 1 and 2 to the major response TFs including ATF2, CREB1, ELK1, ETS1, FOXO1, JUN, MEF2C, MTOR, NFKB1, and NFAT. For this purpose, we converted the Boolean network into an interaction network  to downstream TFs. Proteins whose Boolean update equations are along most of the loops were considered essential. In total, we found 6,542 such loops of which the longest spans 26 nodes and the shortest 2 nodes. The mean length of the loops is 15 nodes. All the loops included PID proteins , and loss-of-function CARD11, CD19, CD21, CD40, CD81, IKKB, KRAS, LYN, MALT1, MS4A1, NEMO, NFKB1, NFKBIA, ORAI1, PI3K, PLCG2, PTPRC, STIM1, and WIPF1 deficiencies. Although no case of LYN deficiency has been reported, LYN was studied to represent PIDs connected to the Src-family kinases (SFKs). Thus, BLK was represented by LYN in the network model. To the best of our knowledge, BLK is the only SFK involved in B cell PIDs . The knoNone of the major TF signaling pathways were disrupted in the CARD11, KRAS, and PI3K overexpression PID attractors, and CD19, CD81, and WIPF1 knockout attractors Figure . SeveralThe simulation results agree with experimental evidence. We have chosen examples from the different parts of the network. CD40, variants which can lead to hyper-IgM syndrome (HIGM3) , 50 due NEMO is an inhibitor of κB kinase gamma and regulates NF-κB by phosphorylating IκB leading to degradation of it and subsequent activation of NF-κB . Thus, tPrimary immunodeficiencies differ greatly in severity from very mild to life-threatening conditions. Severe combined immunodeficiency (SCID) is associated with high vulnerability to infectious diseases and can be lethal . AccordiCombined immunodeficiencies (CIDs) are less severe compared with SCIDs and have variable clinical phenotypes. NEMO, GOF NFKBIA, ORAI1, STIM1, and WIPF1 constitute CIDs. Most of these proteins are components of calcium signaling, and impair, as expected, the NF-κB and NFAT pathways, both of which are activated by calcium signaling.PIK3CB gene that codes for the catalytic subunit of the PI3K heterodimeric complex are associated with a mild PID , and less profound, but noticeable effects, in the intermediate and late events .GAB1, GRB2, LAT2, MAP2K1, MAP3K7, MAPK14, MAPK3, MAPK8, PIK3AP1, PRKCB, RAF1, and SYK have been designated as candidate PID genes  equivalent of the Boolean update functions, where Odefy  transforiB, the Boolean discrete update function. Since molecular interactions are not switch-like in behavior, the Hill function,HillCube  as follon (the Hill coefficient) represents the cooperativity between the protein interactions, while parameter k represents the value at which the activation is half-maximal.The parameter iB, the HillCube functions were normalized to the unit interval to give the normalized HillCube (To obtain perfect homologs of the Boolean update functions HillCube  as follon = 3, k = 0.5, and τ = 1. Except for the nodes in Table n represents the Hill exponent of the Hill function and is used for converting the discrete Boolean update functions that take value 0 or 1 into their continuous BooleCube equivalents that have values . It captures the influence that the nodes of the same Boolean equation have on each other. k is a variable to control the continuous relaxation of the Boolean step function. It represents the value at half-maximal activation of a protein. τ is a decay parameter; for each signaling molecule, the higher its value, the slower the decay of the molecule.The Odefy toolbox was used to simulate the qualitative dynamics of the network model . We usedThe parameters in Table Analysis of PID effects was performed for each protein encoded by a PID gene using the normalized HillCube simulations. For each perturbation, the node was converted to an input before assigning a state, either off or on, depending on the PID. This state was maintained until the simulation transitioned into the attractor. The parameter values used in the wild-type simulations were maintained for all the PID-perturbed simulations. The result of the simulation is the perturbed PID attractor.Primary immunodeficiency proteins expressed in B cells were retrieved from the IDbases , 44, theGT designed and developed the systems biology approach, interpreted data, performed data processing, interpreted the results, and wrote the manuscript. MV designed the research question, interpreted data, corrected the manuscript, and supervised the work. GT and MV have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmo salar), ranging from 4,500 to 6,000 tonnes. All of these farms used treatments with high doses of antibiotics. Thus, 15 healthy fish were selected and euthanised in order to isolate the bacteria resistant to the antibiotics oxytetracycline and florfenicol from the gut microbiota. In total, 47 bacterial isolates resistant to florfenicol and 44 resistant to oxytetracycline were isolated, among which isolates with Minimum Inhibitory Concentrations (MIC) exceeding 2048 μg/mL for florfenicol and 1024 μg/mL for oxytetracycline were found. In addition, another six different antibiotics were tested in order to demonstrate the multiresistance phenomenon. In this regard, six isolates of 91 showed elevated resistance values for the eight tested antibiotics, including florfenicol and oxytetracycline, were found. These bacteria were called “super-resistant” bacteria. This phenotypic resistance was verified at a genotypic level since most isolates showed antibiotic resistance genes (ARGs) to florfenicol and oxytetracycline. Specifically, 77% of antibiotic resistant bacteria showed at least one gene resistant to florfenicol and 89% showed at least one gene resistant to oxytetracycline. In the present study, it was demonstrated that the high use of the antibiotics florfenicol and oxytetracycline has, as a consequence, the selection of multiresistant bacteria in the gut microbiota of farmed fish of the Salmo salar species at the seawater stage. Also, the phenotypic resistance of these bacteria can be correlated with the presence of antibiotic resistance genes.The high use of antibiotics for the treatment of bacterial diseases is one of the main problems in the mass production of animal protein. Salmon farming in Chile is a clear example of the above statement, where more than 5,500 tonnes of antibiotics have been used over the last 10 years. This has caused a great impact both at the production level and on the environment; however, there are still few works in relation to it. In order to demonstrate the impact of the high use of antibiotics on fish gut microbiota, we have selected four salmon farms presenting a similar amount of fish of the Atlantic salmon species ( The phenomenon of antibiotic resistance is, according to the General Assembly of the United Nations, a priority topic for human development, being on par with global warming . The numConsidering the current and projected situation, antibiotic resistance must be addressed not only from perspectives of human health, but also with considerations to veterinary health and the impact that the use and liberation of antibiotic substances may have on the environment . To thisPiscirickettsia salmonis, which is the causative agent of salmonid rickettsial syndrome (SRS) [Salmo salar), coho salmon (Oncorhynchus kisutch), and rainbow trout (O. mykiss) [P. salmonis is the primary pathogen in the Chilean salmon farming, none of the 40 commercially available vaccines are sufficiently effective at protecting against disease [The Chilean salmon industry is constantly affected by bacterial, parasitic, fungal, and viral infections that cause a series of diseases, many of which can result in the death of millions of fish and, consequently, significant production losses . Withoutme (SRS) . This ba mykiss) . Althoug mykiss) , Canada  mykiss) ,18, Scot mykiss) , and Aus mykiss) , this ba mykiss) ,20. Alth disease –24. ConsAccording to a report by the National Fisheries Service (Sernapesca), the salmon industry used more than 5,500 tons of antibiotics between 2007 and 2017, with each ton of produced salmon receiving, on average, 500 g of antibiotics . AntibioAntibiotics in the aquaculture industry are administered through medicated feed, immersion baths, or, in extreme circumstances, through an intramuscular or intraperitoneal injection ,27. MediThe primary aim of this study was to characterize the antibiotic-resistant bacteria present in the intestinal microbiota of farmed Atlantic salmon treated with high antibiotic doses. Four salmon farms were sampled, and different antibiotic-resistant bacteria were recorded at each. Specific genetic elements involved in resistance to florfenicol and oxytetracycline were identified, as were integron elements. This paper establishes a clear relationship between the use of antibiotics, the presence of resistant bacteria and the high prevalence of antibiotic resistance genes in the intestinal-microbiota system from the Atlantic Salmon. Additionally, which could be an important source for the dispersion and liberation of ARGs into the environment, with potential impacts for human health.Salmo salar species in a similar period of the productive cycle; (ii) similar mass of fish (between 4500 and 6000 tons); (iii) all the farms had more than one outbreak of SRS in the productive cycle; and (iv) the four farms had more than one treatment with medicated food at the time of sampling.  were assessed. The farms were chosen for four fundamental characteristics: (i) all the farms contain fish of the ampling. .i.e., no clinical signs of SRS) Atlantic salmon were randomly selected from each salmon farm. Fecal matter was obtained by applying perianal stimulation to the fish. Prior to fecal extraction procedure fish were anesthetized with benzocaine (25 μg/mL). The collected fecal samples from three individuals were pooled, stored in a 5 mL saline solution (0.85% NaCl), and kept on ice until laboratory analyses 24 h later. To collect the intestine samples, the animals were euthanized by immersion in a solution of 50 mg/L of benzocaine. The intestines of three fish were also pooled. This study was carried out in accordance with law 20,380 regarding animal welfare, as set out by the Chilean Health Ministry in the use of wild or protected animal species in biomedical research and approved by the National Fisheries Service (SERNAPESCA) and the Pontificia Universidad Católica de Valparaíso Bioethical Committee.In order to obtain the largest number of bacterial isolates, two sources were used: (i) fecal matter; and (ii) the intestines. For this, 15 apparently healthy (−4 and 10−5 dilutions (100 μL) were seeded in sextuplet on tryptic soy agar plates (TSA) . Plates were incubated at 25°C, 30°C, and 37°C for 48 h and at 15°C for 72 h, the aim of which being to assess the widest temperature range possible. TSA medium and incubation temperature have been commonly used to isolate bacteria from fish microbiota [To obtain the bacterial isolates, homogenates were obtained from the pooled fecal matter and intestines. A dilution series was applied, where 10crobiota –33. To pColonies grown in TSA medium were classified according to standard patterns: shape, color, texture and shape of the colony-border. Each of the differentiated colonies was seeded in 96-well plates in TSB medium and grown at the temperature at which it was isolated, regardless of whether it came from intestine or fecal material. In order to ensure the purity of the isolates, each of the colonies grown in TSB medium were Gram-characterized and the mixed cultures or cultures contaminated with yeasts were discarded.2) with the Müller-Hinton medium  and increasing concentrations of florfenicol and oxytetracycline , using a 96 pin replicator (Boekel 140500). As a control was used the strain E. coli K12. Once the MIC of each isolate was calculated, those isolated showing a MIC ≥ 128 μg/mL for florfenicol and ≥ 64 μg/mL for oxytetracycline were considered to be resistant bacteria, according to EUCAST clinical standard [The MICs for all the bacterial isolates were determined following the agar double-dilution protocol established by the Clinical and Laboratory Standards Institute,35. Briestandard . Finallyi.e., FB and OB) were measured by estimating MICs to treatment with chloramphenicol (CHL), tetracycline (TET), ciprofloxacin (CYP), erythromycin (ERY), ampicillin (AMP), and kanamycin (KAN). The previously described methodology for establishing MICs was applied, using increasing concentrations of each antibiotic . Resistance levels was defined by the EUCAST values for any antibiotic in E. coli K12: AMP ≥ 64 μg/mL, CHL ≥ 32 μg/mL, CIP ≥ 4 μg/mL, ERY ≥ 16 μg/mL, KAN 16 μg/mL and TET 32 μg/mL [The cross-resistance potentials of isolates from both banks (32 μg/mL 5'-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). The amplification conditions were as follows: initial denaturalization at 95°C for 3 min, followed by 35 cycles at 95°C for 45 s, 55°C for 30 s, and 72°C for 1 min, with final extension at 72°C for 5 min. The obtained sequences were analyzed using the BLAST tool [To identify the resistant isolates, 16S rRNA gene sequencing analysis was used for taxonomic classification. Genomic DNA extracts were obtained with Chelex-100  according to manufacturer instructions. PCR amplification of the 16S gene was carried out using the universal primers 27F  were submitted to the GenBanK database . For flofloR ,39, fexAoR [fexA ,41, and exA [cfr . For oxynd tet35 . The priStatistical analyses were performed using SPSS 22.0 for Windows . The prevalence of antibiotic resistance bacteria and antibiotic resistance genes was compared among isolates from different origins or taxonomic groups using the chi-square test at a significance level of 0.05.More than 12,000 colonies were obtained in the TSA plates without antibiotics from the four farms sampled. Of these colonies, 28% was obtained at 15°C, while 63% of the isolates came from the growth at 25°C. Finally only 9% of the colonies came from the growth at 30°C. In none of the fish farms were bacteria found that could grow at 37°C. After the classification of the colonies according their pattern of size, shape, color, texture and the shape of the border, and after discarding the mixed cultures or those contaminated with yeast, the number of colonies was considerably reduced: 2,628 coming from the intestine and 2,390 colonies from the feces, showing no differences between this two different sources. The individual analysis per farm showed that 1,248 colonies were obtained from the farm I; while 1,035 are from farm II; 1,472 from farm III; and 1263 from farm IV. The amount and diversity of the patters of size, shape, texture, color and shape of the border from the colonies were similar for the four farms.The MICs to florfenicol or oxytetracycline were determined using microdilutions in Müller-Hinton agar, adding increasing concentrations  of each antibiotic for all isolates of the bacterial bank. Once MICs were determined, those isolates evidencing resistance to ≥64 μg/mL for oxytetracycline or ≥128 μg/mL for florfenicol were classified as resistant. A total of 47 (23 from feces and 24 from intestine) florfenicol-resistant and 44 (21 from feces and 23 from intestine) oxytetracycline-resistant isolates were classified   was calculated, and for the florfenicol resistance samples this rate was estimated in 9.4x10i.e., MIC ≥64 μg/mL for oxytetracycline or ≥128 μg/mL for florfenicol), MIC values were determined against the following antibiotics: chloramphenicol, tetracycline, erythromycin, ampicillin, ciprofloxacin, and kanamycin. Among the FB resistant isolates, 4.3% showed resistance phenotype to the eight assessed antibiotics, and 57.3% showed resistance to at least four antibiotics , thus facilitating expression . These e or fexA . Regardient fexA . These rtet genes included tetA, tetB, tetE, tetH, tetL, tet34, and tet 35, all of which were previously described in sediments associated with salmon farms [tetM was also included considering prior reports of being a widely dispersed and prevalent resistance mechanism against tetracyclines [tet genes, the more prevalent of which was, not surprisingly, tetM (72.7%). Following in prevalence was tetL (68.1%). Among this group of bacterial isolates, 4.5% of the bacterial isolates carried six tet genes, 9,1% five and 15.9% four. In other words, 29.5% of oxytetracycline-resistant bacteria carried at least four different resistance genes to this antibiotic  [i.e., not in the field) and found a notable enrichment of Aeromonas salmonicida, a bacterial pathogen. In the present study, only one Aeromonas isolate was found in the OB group, thus corroborating the possible selection of this bacterium under high oxytetracycline concentrations.Various studies have extensively characterized the gut-microbiota composition of Atlantic salmon, reporting total dominance by the Proteobacteria phylum. More specific still, resented ,60. Thes. (2008)  exposed P. salmonis infection by the end of the 80s, oxytetracycline and quinolones have been used to tackle the infection. Also, oxytetracycline is the most widely used antibiotic during freshwater rearing, with florfenicol use nearly null during this stage [i.e., the constant florfenicol treatments may have selected and maintained only those bacteria with the most efficient mechanisms against this antibiotic. This is the perfect scenario for Pseudomonas members, the genomes of which are equipped with multiple resistance elements that make these bacteria competitive in environments saturated with antibiotics [A fundamental finding of the present study is that oxytetracycline-resistant isolates were found to be more diverse than those resistant to florfenicol. This could have direct implications on treatments and the strategies applied to the use of these antibiotics. Since the early report of is stage . Consequis stage . This cois stage . In contibiotics ,63.Pseudomonas, the present study detected most of the resistance genes included in assessments, and the majority of these genes, excepting tetM, were drug efflux pumps. These findings corroborate that these genetic elements are a principal trait of Pseudomonas bacteria [Pseudomonas, it was unsurprising that the majority of the obtained florfenicol-resistant isolates were of this genus. Regarding oxytetracycline-resistant isolates, the detected genera, as with Pseudomonas, are highly ubiquitous in different systems. For example, Hafnia alvei has been isolated from the intestinal microbiota of various organisms, including pigs, cows, and fish [H. alvei is an ideal resistant-isolate candidate in any system. These traits can also be found in species of Shewanella [Serratia [Carnobacterium [Brochotrhix [Kluyvera [Aeromonas molluscorum [Psychrobacter nivimaris [In addition to the well-known antibiotic resistance capacity of bacteria . Consideand fish ,66. Thisand fish , meaningewanella ,69, SerrSerratia ,71, Carnacterium , Brochotchotrhix ,74, and Kluyvera ,76. All luscorum ,78 and Pivimaris  are excltet genes found for the FB and OB could be explained by the high frequency at which these genes localize in mobile genetic elements, such as plasmids or transposons [tetA, tetB, tetE, tetL, tetH, tet34, and tet35 have been recorded in the sediments and water column of Chilean salmon farms [tetM, this globally distributed gene presents a clinically confirmed resistance mechanism to tetracyclines [Tetracyclines are commonly used in veterinary medicine around the world, and, specifically, oxytetracycline is the most widely used antibiotic during the freshwater stage of salmon growth in Chile . Origin nsposons . The genon farms ,80. An icyclines ,82. One cyclines ,84; thistet genes, the presence of floR, a drug efflux pump of the major facilitator superfamily (MFS), has previously been reported in bacteria collected from marine sediments associated with salmon farming in Chile [tet and qnr genes, the latter of which are implicated in resistance to quinolones [floR in the gut microbiota of salmon. Furthermore, this investigation is the first to find and report on fexA, an efflux pump of the major facilitator superfamily also, in association with Chilean salmon farming, with no previously published study having detected this gene in relation to the Chilean aquaculture industry. This fenicol-resistance gene was first described in the bacterium Staphylococcus lentus [Staphylococcus genus [Staphylococcus genus, fexA has been described in members of the Enterococcus genus, specifically in association with industrially farmed terrestrial animals [Staphylococcus or Enterococcus bacteria were found among any of the obtained resistant isolates, but the presented findings do support the ability of fexA to disperse across a wide variety of both Gram-positive and–negative bacteria.As with the in Chile . This geinolones . Howevers lentus , and hasus genus ,51,86–88 animals ,90. No SfloR, but, by contrast, 52,2% did present fexA. As mentioned, the most widely used antibiotic used in Chilean salmon farming is oxytetracycline, and there is a nearly null use of florfenicol in the freshwater stage. Prior reports have found fexA in plasmids containing a great number of resistance genes to other antibiotics [fexA has almost exclusively been isolated from industrially farmed terrestrial animals. This is another compelling case for ARGs primarily originating from human activities and not directly from salmon farming.One curious outcome of the conducted study was that none of the oxytetracycline-resistant bacteria presented ibiotics . TherefoBetaproteobacteria native to soils, freshwater, and saltwater. Genetic cassettes are incorporated alongside elements that are highly varied and much more diverse than ARGs [qnrA and qnrB), trimethoprims (drfA12), and aminoglycosides (aad2) have recently been described in the Chilean farming system, which is in addition to some clinical Escherichia coli also carrying these plasmid-transferred integrons [et al. (2018) [In contrast to findings from sediments related to intense salmon farming in Chile , the prehan ARGs . Integrohan ARGs . Integrontegrons . This ap. (2018) .Another important aspect to consider in this work is the Antibiotic Resistance Rate (ARR). Overall, it was found that less than 1% of the total bacterial isolates were resistant to oxytetracycline or florfenicol (0.94% for florfenicol and 0.87% for oxytetracycline). Although there are few works in which this rate is addressed at an environmental level ,98, valuThe information collected in the present study clearly indicate that using large amounts of antibiotics to treat industrially farmed animals has the consequence of selecting for multiresistant bacteria in the intestinal microbiota, which then have undeniable advantages when liberated into the environment through the feces. While the obtained results are somewhat biased towards “culturable” bacteria, metagenomics data are currently being analyzed, the results of which will reveal all of the bacteria that did not grow in a TSA medium. This information will provide a complete picture for the behaviors of the bacterial populations interacting in this complex system. Meanwhile, the currently reported data do show that the intestinal microbiota, as a system “semi-isolated” from the environment yet in direct contact with applied antibiotics, is the perfect place for interactions to occur between bacteria from different environments, including from the human clinical context. Therefore, the gut microbiota of farmed salmon could also serve as the perfect reservoir for ARGs, the dispersion of which would be fundamentally related to feces. The release of feces with resistant bacteria and ARGs is creating an environment constantly rich in resistance elements, creating an ideal situation for genetic exchanges to occur between different bacterial populations, whether in the water column or in marine sediments. These populations are precisely where fish pathogens can be found, thus notably increasing the risk that these bacteria will acquire resistance elements. If this occurs, no effective treatments will exist for the control of these pathogens. Fortunately, a number of aquaculture producers are beginning to understand the gravity of the situation. Measures aimed at decreasing antibiotic usage are increasingly being adopted, with expectations that by 2020, there will be at least a 50% decrease in the use of these substances.Salmo salar species, showing high resistance levels to antibiotics oxytetracycline and florfenicol, were characterised. The feces, represent important vehicles of dispersion of resistant bacteria and ARGs. The analyses showed that the high use of these antibiotics selects bacteria that are multiresistant to a wide range of antibiotics. In turn, these bacteria show a high prevalence of antibiotic resistance genes, thus, verifying the phenotypic resistance with the mechanism present in the bacteria to cope with these substances. Furthermore, almost 100% of these (antibiotic) resistant isolates showed class 1 integrons, dangerous elements involved in the resistance phenomenon due to their capacity of dispersion.In this study, bacterial isolates from fish gut microbiota of the S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Cortex Dictamni is a commonly-used traditional Chinese herbal medicine for the treatment of skin inflammation, tinea, and eczema. Recently, some studies reported that Cortex Dictamni might induce liver injury, suggesting more attention to its safety. The current study was designed to investigate subchronic toxicity of Cortex Dictamni aqueous extract (CDAE) and ethanol extract (CDEE) in mice and the potential hepatotoxicity mechanisms in vitro. Firstly, CDAE or CDEE groups were administrated with varying dosages  in mice for 28 days in subchronic toxicity studies. General clinical signs and biochemical parameters were examined, and morphological analyses were conducted. Secondly, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS and chose major components for further study. In order to determine the toxic components, we investigated the cytotoxicity of extracts and chosen components using CCK-8 assay in HepG2 cells. Furthermore, we explored the possible hepatotoxicity mechanisms of Cortex Dictamni using a high content analysis (HCA). The results showed that no significant differences of general clinical signs were observed in mice. Aspartate alanine aminotransferase (ALT) and aminotransferase (AST) were significantly increased in the high-dose CDAE and CDEE groups compared to the control group. Meanwhile, the absolute and relative liver weights and liver/brain ratio were significantly elevated, and histological examination of liver demonstrated cellular enlargement or nuclear shrinkage. In UPLC analysis, we compared the chemical constituents between CDAE and CDEE, and chose dictamnine, obakunone, and fraxinellone for hepatotoxicity evaluation in the in vitro studies. In the CCK-8 assay, CDAE, CDEE, dictamnine, obakunone, and fraxinellone decreased the cell viability in a dose-dependent manner after treatment for 48 h. Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of reactive oxygen species were shown to increase, meanwhile, mitochondrial membrane potential was also changed in HepG2 cells following 48 h of compounds treatment using HCA. Our studies suggested that CDAE and CDEE have potential hepatotoxicity, and that the alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component, whose potential hepatotoxicity mechanism may be associated with cell apoptosis. Moreover, this study could provide valuable data for clinical drug safety research of Cortex Dictamni and a good example for safety study of other Chinese herbal medicines. Drug-induced liver injury (DILI) often results in the termination of drug development at the clinical stage or withdrawal of a drug from the market . CompareDictamnus dasycarpus Turcz, which has about six species and is widely spread in Europe and Asia. There are two species in China, Dictamnus dasycarpus Turcz and Dictamnus angustifolius G. Don, which are mainly distributed in Xinjiang, Henan, and northeast China [Cortex Dictamni is a commonly-used TCM herbal medicine that originates from the dried root bark of st China . Accordist China ,8. Thougst China , Xiaoyinst China , Keyin pst China , Vitiligst China , and Zhist China . In the st China . Furtherst China , Huang rst China . We found no reports of toxicity on Cortex Dictamni in ancient TCM literature, but pharmacological studies have shown that Cortex Dictamni contains alkaloids, saponins, and other chemical compounds which may increase its toxicity risk . HoweverNo significant differences in body weight and food consumption were observed between the drug-treated groups and the control group, as shown in No drug-related deaths and toxicity signs  were observed in the drug-treated groups during the administration period.p < 0.05). Meanwhile, significant increases in ALB and TP were observed in male mice treated with CDEE at a dose of 9.2 g/kg . In the groups with 9.2 g/kg dose CDAE and CDEE, TG significantly increased . In female mice with the 9.2 g/kg CDEE, ALT and AST significantly increased . ALB, GLB and TP increased in the CDAE and CDEE-treated group with 4.6 g/kg and 9.2 g/kg dose compared to the control group .p < 0.05, p < 0.01). Meanwhile, the absolute liver weight, relative liver weight, and liver/brain weight ratio in the high-dose CDAE and CDEE groups were significantly elevated in female mice  compared to the control group.Observations of hepatic tissue sections showed that the high-dose CDAE and CDEE groups had nuclear shrinkage, suggesting apoptosis. Cellular enlargement was also observed in these groups .The results of the subchronic toxicity test in mice showed that both CDAE and CDEE had potential hepatotoxicity and that CDEE may be more toxic than CDAE. It is speculated that the active components of Cortex Dictamni inducing the toxicity would be enriched in alcohol extract. Thus, the ingredients differences between CDEE and CDAE were investigated in order to find out the active components of Cortex Dictamni for potential toxicity using HPLC.p < 0.05). Among them, dictamnine, obakunone, fraxinellone, and limonin, which have the most remarkable difference, were further studied.Chemical constituents in CDAE and CDEE were systematically analysed using the HPLC-Orbitrap MS. After the original mass spectrometry data were processed by SEIVE 2.1, a total of 5000 common components were obtained from the original mass spectrometry data of CDAE and CDEE. After normalisation and standardisation, the data were imported into SIMCA-P 13.0 software for multidimensional statistical analysis. As shown in The absolute content of dictamnine, obakunone, fraxinellone, and limonin in CDAE and CDEE was further determined in p < 0.5, p < 0.01). Meanwhile, the data showed that CDEE was more toxic than CDAE. Furthermore, exposure of HepG2 cells to dictamnine, obakunone, and fraxinellone at the highest dose significantly decreased the viability . It was found that dictamnine had stronger toxicity than other two constituents. The three compounds were used for further studies of hepatotoxicity mechanisms.In order to determine the toxic constituents in Cortex Dictamni, we used the CCK-8 assay to estimate the potential cytotoxic effect of compounds. The results in 50 values of extracts and compounds of these parameters were shown in To further explore the mechanisms of hepatotoxicity of these components, the multiparameter cytotoxicity assay endpoints were evaluated in HepG2 cells following 48 h of compounds exposure using HCA . As showDILI is a major concern during drug development, and the evaluation of potential hepatotoxicity of drugs plays a key role in drug development and use. The toxicity of TCM herbs is an important concern of TCM practice as the premise and guarantee TCM safety.Cortex Dictamni was recorded for the first time in “Shen Nong’s Herbal Classic”, which told little about its toxicity. However, in recent years, the toxicity of some formulations containing Cortex Dictamni have been frequently reported, such as Qingdai pills, Keyin pills, Vitiligo capsules, and Zhixue capsules. Furthermore, there have been studies related to the toxicity of Cortex Dictamni. Jang reported four cases of Cortex Dictamni decoction-induced toxic jaundice hepatitis . McRae rIn the Chinese pharmacopoeia, the recommended highest daily dose of Cortex Dictamni was 10 g (0.17 g/kg for a 60 kg human). However, the clinical application of Cortex Dictamni complex prescription has been raised; the commonly used daily dosage is 15 g or 30 g, sometimes it could even reach the dosage of 60 g. In this study, Cortex Dictamni, in quantities of 2.3, 4.6, and 9.2 g/kg/day were given to mice to estimate the human equivalent doses of 15, 30, 60 g/day, respectively. Ultimately, these research results could provide new data on the clinical safety of Cortex Dictamni and help guide rational drug use in clinical applications. Furthermore, we found that previous studies have mainly involved the toxicity of the aqueous extract of Cortex Dictamni, but the ethanol extract is often used in clinic, such as Zhixue capsules. In our previous studies, we have found that the main reason for the hepatotoxicity of Zhixue capsules was related to the ethanol extraction process, and Cortex Dictamni was the main drug factor. Thus, in this study, we compared the two different extracts of Cortex Dictamni. We used two different dimensions of extracts and doses of Cortex Dictamni to evaluate the safety of the drug, combining the gold standard of histopathology with the indexes of organ and blood biochemistry to evaluate the toxicity of Cortex Dictamni comprehensively. This is of important clinical significance as it provides a base for drug safety. Firstly, we evaluated the toxicity of Cortex Dictamni using animal models. Changes in general behaviours and body weight have previously been used as important indicators in toxicity evaluations ,25. In tThe relative organ weight is regarded as an indicator of toxic effect . An incrThe observations above demonstrated that there was hepatotoxicity in mice treated with 9.2 g/kg of CDAE or CDEE, which is equivalent to 60 g/kg in humans; this dosage is also used in some diseases. Besides, in the present study, the period of drug administration is equivalent to one week of clinical medication, but the actual course of drug usage of Cortex Dictamni is longer than one week. As far as we know, the toxicity of drugs is closely related to the dosage and duration of medication. Thus, the results suggested us that ethanol extraction process could increase the toxicity of Cortex Dictamni, and more close observation to the clinical use of Cortex Dictamni shall be paid in the future.Secondly, in order to find out the possible toxic material basis of Cortex Dictamni, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS. The results revealed that the main components of CDAE and CDEE were dictamnine, obakunone, fraxinellone, and limonin, and the contents of dictamnine, obakunone, fraxinellone, and limonin in CDEE were 2.27, 5.28, 2.15, and 1.33 times of CDAE, respectively. Thus, the possible toxic ingredients maybe enriched in CDEE, which could be the reason for having higher toxicity. Besides, limonin is often used as food additive. Therefore, dictamnine, obakunone, and fraxinellone were determined as the candidates of toxic compounds contained in the extracts of Cortex Dictamni. In recent years, some studies have reported that animal models cannot adequately predict human liver toxicity, possibly due to differences in the immune system, the metabolic capacities, and genetic sensitivities between animals and humans . Thus, aThe CCK-8 assay is a useful technology for estimating the potential cytotoxicity in vitro for further research of DILI screening . The resIn order to obtain more information about the mechanisms of hepatotoxicity, HCA was chosen, since it may have more endpoints and is considerably more reliable ,35. TechIn our present study, we studied five parameters: the cell number, nuclear intensity, mitochondria membrane potential, cell membrane permeability, and concentration of reactive oxygen species. These five indicators reflect the basic mechanisms of cell apoptosis and death in different angles of hepatotoxicity. The cell number can be used to evaluate the degree of cell injury, which is related to cell death. Nuclear intensity mainly evaluates the occurrence of nuclear shrinkage and cell apoptosis. Cell membrane permeability is mainly used to reflect the integrity of cells, whose increase indicates the disruption of ionic gradients or mitochondrial dysfunction . Oxidati50 values obtained from the HCA assay indicated that dictamnine, obakunone, and fraxinellone were all cytotoxic to hepatocytes. We found that dictamnine may have stronger toxicity when comparing the IC50 values in HCA.Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of ROS were shown to increase, meanwhile, mitochondrial membrane potential also changed in HepG2 cells following 48 h of compounds exposure using HCA, suggesting that hepatotoxicity induced by Cortex Dictamni may lead to cell apoptosis. The results of the cell viability assay and the ICNowadays, experts on clinical issues have proposed causes of the toxicity of Chinese herbal medicine, such as the lack of theoretical guidance of Chinese medicine, the ignorance of the compatibility of traditional Chinese medicine, and improper use of dosages. The pharmacological and toxicological effects of toxic ingredients have also become fundamental measures to control the toxicity risks of Chinese herbal medicines. Cortex Dictamni mainly contains alkaloids, lactones, and sesquiterpene glycosides. Recent studies have reported that limonin, dictamnine, obacunone, and fraxinellone are the four main constituents of Cortex Dictamni extract ,43 that In conclusion, the results demonstrated that CDAE and CDEE have potential hepatotoxicity, and alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component. Moreover, the potential mechanism of hepatotoxicity may be associated with changes in the cell number, nuclear intensity, mitochondria membrane potential, cell membrane permeability, and concentration of reactive oxygen species, which may induce cell apoptosis. Systematic research on common adverse drug reactions in clinical practice has always been an important issue in the field of traditional Chinese medicine. Our study provides applicable strategies for the safety research of many other herbal medicines. Furthermore, in future research, we seek to find more evidence for the appearance of clinical adverse reactions of Cortex Dictamni and to study the intrinsic mechanisms of toxicity, which may safeguard clinical usage of traditional Chinese herbal medicine based on the scientific research data.Organic solvents and reagents were all of HPLC grade. Acetonitrile was purchased from Merck  and dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich . Deionised water was prepared in a Milli-Q laboratory water purification system . Dictamnine, obakunone, and fraxinellone were purchased from Yuanye Bio-Technology Co. Ltd. . HepG2 cells were purchased from China Infrastructure of Cell Line Resources . The ToxInsight DILI cartridge was acquired from Thermo Fisher Scientific . Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were purchased from Gibco . Penicillin, streptomycin, cyclosporine A, and aspirin were obtained from Sigma Aldrich . The cell counting kit (CCK-8) was from Dojindo . Liquid paraffin and neutral formalin were purchased from Beijing Chemical Works .Dictamnus dasycarpus Turcz. (Rutaceae), was purchased from Beijing Yan Bei decoction Chinese Herbal Pieces Co., Ltd.  and identified by Prof Xiangri Li .Cortex Dictamni (Bai-Xian-Pi) from the root bark of w/w) and 8.2% (w/w). The dried residue was stored at 4 °C and was resuspended in distilled water for use. For preparation of the aqueous and ethanol extracts, 10 kg of Cortex Dictamni decoction pieces were boiled with 80 L of distilled water or 85% ethanol under reflux for 2 h, twice. The decoction was filtered and then the filtrates were combined and concentrated using a rotary evaporator and were lyophilised under reduced pressure. The yields of these extract were approximately 31.7% (The aqueous and ethanol extracts of the Cortex Dictamni (15 g) were boiled in 120 mL of deionised water or 85% ethanol under reflux for 2 h, twice. The filtrates were combined and concentrated under vacuum and then filtered with a 0.45-μm membrane filter before use. A few milligrams of dictamnine, obakunone, fraxinellone, and limonin were dissolved in 10 mL of methanol. 1 mL of these compound solutions was mixed into a 10 mL volumetric flask, and the mixturefiltered with a 0.45-μm membrane filter before use. m/z 50 to 1000 Da. The resolving power was 30,000 for the full scans and 15,000 for the MS2 scans. The spray and capillary voltages were set to 4.0 V and 25.0 V, respectively; the tube lens was set to 110 V; and the source temperature was set to 300 °C. Nitrogen (purity > 99.99%) was used as both the sheath gas (30 units) and auxiliary gas (10 units).UPLC analysis was performed on a Thermo Scientific Dionex UltiMate 3000 UHPLC system  equipped with an autosampler, a vacuum degasser unit, a quaternary pump, a diode array detector (DAD), and a column compartment. Samples were separated on a ZORBAX SB-C18 column  at 35 °C. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), and the elution gradient was set as follows: 5–53% B (0–20 min), 53–68% B (20–40 min), 68–80% B (40–42 min), 80–90% B (42–47 min), 90–95% B (47–47.1 min), and 95% B (47.1–52 min). The flow rate was set at 1.0 mL/min, and the injection volume was 10 µL. ESI-MS analyses were carried out on a Thermo Fisher Scientific LTQ-Orbitrap XL hybrid mass spectrometer  equipped with an electrospray ionization interface and an HESI-II source. Samples were analysed in negative ion mode, and full-scan mass spectra were acquired in a mass range of High-resolution MS data processing was carried out using the QualBrowser of Xcalibur 2.1 (Thermo Fisher Scientific), including EIC, calculation of elemental compositions using potential metabolite ions with 5-ppm mass tolerance and isotope pattern simulation. By comparison to data regarding reference substances and by referring to the relevant literature, the components of each extract were identified, and the structure and composition of these components were determined.Adult male and female ICR mice (18–22 g) were obtained from the Beijing SPF Animal Technology Company . The mice were housed in groups of matched weights in certain humidity (40–70%) and temperature (20–25 °C) controlled room and were housed in a 12-h light/dark cycle environment during the tests. Food and water were available ad libitum. The study was approved by the experimental animal ethics subcommittee of Beijing University of Chinese Medicine (BUCM), the ethical review number is BUCM-4-2017122501-4037.The protocol was in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. In total, 140 mice were used in the study. The tests were performed according to the Guideline on Repeated Dose Toxicity . The ICR mice were weighed and randomly divided into seven groups (one control group and six treated groups), with 10 males and 10 females in each group. The control group was orally administered distilled water and the treated groups were administrated CDAE or CDEE, respectively, at doses of 2.3, 4.6, or 9.2 g/kg/day, which were set as the low-, medium-, and high-dose groups, respectively. The mice were administered with the same volume of 10 mL/kg/day based on their body weight during the 28-day treatment period. Clinical observations, food consumption, body weights, biochemical parameters, organ weights, and the histopathology of mice were examined at the end of the 28-day treatment period.The general clinical signs of the mice, such as general activity, behaviour, appearance, hair lustre, and urine, were observed after daily administration. Body weight and food consumption were measured weekly.The blood samples were collected from the mice ocular vein. Serum was obtained by centrifugation of whole blood at 3500 r/min for 10 min. The parameters of the biochemistry assays: alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), direct bilirubin (DBIL), total bilirubin (TBIL), albumin (ALB), globulin (GLB), total protein (TP), high density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC), triglyceride (TG), and cholesterol (CHO).At the end of the administration, necropsies were performed on all animals. The organ weights of the liver and brain were measured. The ratio of each organ weight to the terminal body weight in grams per 100 g body weight (relative organ weight) was calculated, respectively. The livers were fixed in 10% neutral buffer formalin and embedded with paraffin. Subsequently, samples were sliced into 3 µm thickness using a rotary microtome. Then the sections were stained with haematoxylin–eosin (HE) After staining, the slides were observed and examined using an optical microscope at 300× magnification . v/v) of FBS and 1% antibiotics  at 37 °C in a humidified incubator containing atmospheric air and 5% CO2. The cells were subcultured when they were 80–90% confluent.Cells were cultured in DMEM medium supplemented with 10% . Percentage (%) =  × 100%. The concentrations of CDAE were 5, 2.5, 1.25, 0.63, and 0.31 mg/mL, which were diluted in the culture medium, while the CDEE stock solution was prepared in DMSO and was serially diluted in the culture medium for different concentrations . Dictamnine, obakunone, and fraxinellone were also prepared in DMSO. Dictamnine was serially diluted in the culture medium for different concentrations , while obakunone and fraxinellone were serially diluted in the culture medium for different concentrations . The final DMSO concentration in the culture medium never exceeded 0.5% (v/v).Cells in the logarithmic growth phase were selected for the experiment. The viability of HepG2 cells after treatment with CDAE and CDEE was measured using CCK-8 assay. Exponentially-growing HepG2 cells were seeded into 96-well plates at a density of 5 × 105 cells/mL in a final volume of 20 µL medium for 24 h. Then, the cells were treated with varying doses of CDAE or CDEE. After 48 h incubation, the cells were applied to a fluorescent dye cocktail, which contained SYTOX Green Nucleic Acid Stain for Cell Membrane Permeability, Cell ROX Deep Red Reagent for ROS, TMRM for MMP, and Hoechst 33342 to determine the cell number or nuclear intensity. The excitation and emission wavelengths were 346 nm and 460 nm, respectively. The signal of the cell membrane permeability dye was acquired with 504 nm excitation and 523 nm emission wavelengths. For the ROS signal collection, the excitation and emission wavelengths used were 644 nm and 655 nm, respectively. The mitochondrial signal (MMP) was detected at 540 nm excitation and 580 nm emission wavelengths. The images were observed and examined using 10 × objective. The concentrations of CDAE and CDEE were the same as that of CCK-8 assay. The positive control compounds were cyclosporin A and ketoconazole, and the negative control compound was aspirin. DMSO (0.5%) was used as Vehicle control.In this assay, we used five parameters to determine the drug-induced liver toxicity: cell number, nuclear intensity, MMP, cell membrane permeability, and concentration of ROS. The exponentially-growing HepG2 cells were seeded into 384-well plates at a density of 1 × 10For the cell number, % vehicle control = (cell number compound/cell number vehicle) × 100%For the nuclear intensity, mitochondria membrane potential, reactive oxygen species, and cell membrane permeability, % vehicle control = (read compound/read vehicle) × 100%50 of the cell number and mitochondria membrane potential, the % vehicle control was plotted against the concentration of test compounds and the data was fitted to a sigmoidal dose–response curve with a variable slope using GraphPad Prism 5.0. The IC50 of the compound was calculated from the curve as Y = Bottom + (Top − Bottom)/(1 + 10^((LogIC50 − X) × HillSlope)) Max Inhibition = (100% − % vehicle) at the concentration that showed the highest inhibition effect. To determine the IC50 of the nuclear intensity, reactive oxygen species, and cell membrane permeability, the % vehicle control was plotted against the concentration of the test compounds, and the data was fitted to a sigmoidal dose–response curve with a variable slope using GraphPad Prism 5.0 . The EC50 of the compound was calculated from the curve as Y = Bottom + (Top − Bottom)/(1 + 10^((LogEC50 − X) × HillSlope)) Max Induction = % vehicle at the concentration that showed the highest induction effect.To determine the ECp < 0.05) were considered significant.The statistical analysis was performed using GraphPad Prism software . The variance in data for body weight, serum biochemistry, and organ weights (both absolute and relative weights) was checked for homogeneity by Bartlett’s procedure. The data were assessed by one-way analysis of variance (ANOVA), for the data with equal variances, followed by Dunnett’s tests. In contrast, the data that demonstrated unequal variance were analysed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons tests. Probability values lower than 5% (


Philornis downsi Dodge and Aitken (Diptera: Muscidae) is an avian parasitic fly that has invaded the Galapagos archipelago and exerts an onerous burden on populations of endemic land birds. As part of an ongoing effort to develop tools for the integrated management of this fly, our objective was to determine its long- and short-range responses to bacterial and fungal cues associated with adult P. downsi. We hypothesized that the bacterial and fungal communities would elicit attraction at distance through volatiles, and appetitive responses upon contact. Accordingly, we amplified bacteria from guts of adult field-caught flies and from bird feces, and yeasts from fermenting papaya juice (a known attractant of P. downsi), on selective growth media, and assayed the response of flies to these microbes or their exudates. In the field, we baited traps with bacteria or yeast and monitored adult fly attraction. In the laboratory, we used the proboscis extension response (PER) to determine the sensitivity of males and females to tarsal contact with bacteria or yeast. Long range trapping efforts yielded two female flies over 112 trap-nights (attracted by bacteria from bird feces and from the gut of adult flies). In the laboratory, tarsal contact with stimuli from gut bacteria elicited significantly more responses than did yeast stimuli. We discuss the significance of these findings in context with other studies in the field and identify targets for future work. Philornis downsi Dodge and Aitken, 1968 (Diptera: Muscidae) is an avian nest parasitic fly that invaded the Galapagos archipelago in the latter half of the twentieth century [ century ,3,4. Fem century . P. downsi was first observed in 1997 [Camarhynchus pauper) has lost >50% of its population on Floreana island [Camarhynchus parvulus) are declining as levels of parasite infestation continue to rise [Camarhynchus heliobates) is literally on the verge of extinction, due to the combined effects of predation, habitat loss, and Philornis parasitism [The broad host range of these flies, lack of competitors and natural enemies, and their high dispersal ability and adaptability to harsh environments have all contributed to their successful invasion . This su in 1997 , nearly  in 1997 . Further in 1997 ,3. Thus, to rise . On the P. downsi in the Galapagos Islands [P. downsi, as exemplified by the extreme difficulty of rearing this fly in captivity [Due to the protected status of the Galapagos Islands, a control approach based on indiscriminate application of insecticides is out of the question. Accordingly, an international consortium of researchers, coordinated by the Charles Darwin Foundation (CDF) and the Directorate of the Galapagos National Park in Puerto Ayora, Santa Cruz, is seeking to develop and implement strategies and tools for the management of  Islands . In the  Islands , applica Islands ,12, and  Islands . The lon Islands ), implemaptivity  and our Complementary to these approaches are methods to manipulate the microbiome of the target insect. The relationship between insects and microorganisms has received much attention in the past two decades, and hardly needs an introduction, as the contributions of symbionts to host nutrition, environmental adaptation, immunity, and ultimately, fitness, are quite well known . In theoP. downsi were attracted to traps containing active bakers’ yeast (Saccharomyces cerevisiae) and in particular to acetic acid and the ethanol produced by the yeast. Currently, the standard method of capturing P. downsi in the field is with McPhail traps baited with fermenting papaya juice [P. downsi/trap/day. This method provides an index for the population, but does not reflect the true population size, as there is no direct correlation between number of larvae in nests and flies caught in traps during long-term monitoring [In flies (Diptera), gut and environmental bacteria have been found in many instances to provide cues that are important in foraging for food ,24,25,26ya juice . This atnitoring . FurtherP. downsi management such as improving diets for mass rearing for SIT and for developing effective attractants for use in traps. Recently, we characterized the bacterial microbiome of P. downsi [P. downsi will elicit attraction at distance through volatiles, and appetitive responses upon contact. Accordingly, we amplified bacteria from guts of adult field-caught individuals, and from bird feces , and yeast from fermenting papaya juice, on selective growth media, and assayed the response of flies to these microbes or their exudates. In the field, we baited traps with bacteria or yeast and monitored adult fly attraction. In the laboratory, we used the proboscis extension response (PER) , we ) e.g., ,39) to d to dP. dP. downsi is commonly collected. Traps were deployed overnight on 8, 9, 10, 14, and 15 February and 7, 8, and 9 March 2018, and on 11, 13, and 15 February 2019, for a total of 112 trap-nights , and inoculated them on plates with a selective growth medium; either Luria–Bertani (LB), tomato (TA), or brain heart (BH) agar (BD Difco). Twenty-four hours following the inoculation, the growth plates, with the bacterial colonies, were positioned (uncovered) as bait in the traps. In addition to bacteria from flies, we also cultured bacteria from fresh feces of a medium ground-finch (Geospiza fortis) on LB plates and baited traps in a similar manner. To assay attraction to yeast volatiles, a sample from fermenting papaya juice (routinely used for baiting traps) was inoculated on yeast-extract potato dextrose  agar plates supplemented with chloramphenicol (50 µg/mL) for suppression of bacteria. Bacterial and yeast communities developing on media plates were subsequently identified, respectively, by sequencing the 16S rRNA and ITS genes (see following section).To assay attraction to bacterial volatiles, we dissected the gut of 4–6 freshly caught female and male https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/). Resulting reads were merged, and quality filtered using Mothur v.1.42.1 [https://www.mg-rast.org/mgmain.html?mgpage=project&project=mgp91882.Genomic DNA was isolated from the bacterial and fungal consortia harvested from the different media plates we used (LB and TA for bacteria and YPD for yeasts). DNeasy Blood and Tissue Kit  was used for the isolation of DNA with sample-specific pre-treatment according to the manufacturer’s instructions. From the isolated bacterial DNA, the V4 region of 16S rRNA gene was PCR amplified with eubacterial primers 515F and 806R ,43,44. Fv.1.42.1 . After rv.1.42.1 . For ITSv.1.42.1 . The seqCordia lutea, Boraginaceae) and water ad lib. On the day of the experiment they were 6–9 days old.The PER experiment was done on two populations of flies. The first were adults that emerged from pupae that were collected from nests after fledglings had left the nest or died. The nests were brought back to the laboratory and pupae removed from the nest material. Following emergence, flies were held in sex specific cages (dimensions: 45 × 45 × 45 cm) and provided with ripe berries of Muyuyo (The second group were adult flies caught in McPhail traps baited with fermented papaya juice that were placed around the Charles Darwin Research Station (CDRS) in Puerto Ayora , and brought back to the laboratory between 26 January 2019 and 2 February 2019. These flies were at least 14–21 days old on the day of the experiment (15 February 2019). Following capture, they were held in cages as described above, and fed on a mixture of pulped papaya (34.2%), protein powder (2.6%), egg powder (3%), milk powder (2.6%), sugar (7.9%), and water (50%). Four hours prior to the experiment, flies were attached with liquid silicon to the base of a disposable pipette tip and allowed to rest in a humidified environment a. not responding to the positive control  were excluded from the analysis.The assay itself consisted of presenting the tarsi of the flies with a series of stimuli and recording the response of the proboscis. The stimuli were presented as droplets in the following order: Water , phosphate buffered saline , bacteria or yeast suspensions in PBS (test stimulus), and 20% sucrose solution (positive control) b. Apart The results of the PER experiment were analyzed using a generalized linear model (GLM) with binomial distribution and log link function . Microbiome data were relativized according to the sum of read abundance in each sample. Statistical analysis was performed with the multiresponse permutation procedure (MRPP) using Bray–Curtis distances.The community composition of culture-dependent bacteria and yeasts, which were used as baits in traps and as stimuli in the PER experiment is shown in p = 0.23 after Bonferroni correction). These communities were dominated by Morganella (Providencia (17.8% ± 7.4%), Enterobacter (17.8% ± 12%), and Acinetobacter (25.3% ± 22.4%). Notably, this community structure differed from the bacteria acquired on agar plates inoculated with finch feces . In fecal samples Bacillus (57.9% ± 20.11%), Pantoea (16.09% ± 22.7%), and Enterococcus (11.7% ± 16.6%) were dominant on LB, while either Enterobacter (46.9% ± 64.2%) or Pantoea (45.7% ± 64.6%) dominated the TA plates.The gut microbial communities developing on LB and TA plates did not differ significantly .The yeast communities used in the trapping and PER experiment were dominated by species of P. downsi females . This model also tested the effects individually and revealed that there was a significant difference in the reaction to the stimulus (bacteria or yeast) , while the source of the flies , and their sex , were not significant in eliciting a different response to bacteria or yeast.The PER experimental results were more conclusive . Bacteri2 = 6.6, p = 0.035, n= 77). While there were still no differences between the responses of males or females , the response to the stimuli differed significantly , with more flies responding to stimuli from bacteria compared to yeasts. On excluding the source of the flies (which showed no differences) from the analysis, the picture becomes even stronger , amines, indole, putrecine, methyl-amine, and fermented papaya juice. The latter proved to be the strongest attractant, and is currently used in all monitoring efforts [The development of a selective and efficient trap for programs . In ligh efforts . Recentl efforts  identifiGeospiza fortis). The feces we used were from a fresh deposit made by an individual frequenting the cafeteria at CDRS. In our culture-dependent samples, community structure in feces was significantly less diverse than the communities found in flies. A recent study by Knutie et al. [Interestingly, one of the females we caught was in a trap baited with bacteria from the feces of a medium ground-finch  from the two populations we assayed showed a significantly higher response to the bacterial cues. Thus, although flies responded to volatiles from active yeasts and fermented papaya juice , suspendd nectar ,3,5, as d nectar . ClearlyP. downsi to microbial volatiles and tactile cues. We found no evidence for long-range attraction to microbial volatiles, yet recognize that this may be due to a flaw in our trap design. Conversely, tethered flies exhibited a feeding response to bacterial cues with alacrity, significantly more so than to fungal cues. Future work will continue to investigate the importance of both bacteria and fungi in the nutritional ecology of this invasive fly.Our study examined responses of


It is well recognized that type 2 diabetes mellitus (T2DM) is an age-related metabolic disease, emerging gradually as a major global health burden that has gained public attention. Meanwhile, increasing attention is paid to the crucial role of gut microbiota in the pathogenesis and therapeutic mechanisms of metabolic disorders, especially T2DM. In this study, we used C57 BL/KS db/db male mice as a T2DM murine model. We found that the β-diversity and relative abundances of gut bacteria were obviously altered in db/db mice, associated with a significant increase in Verrucomicrobia at six levels  and family S24-7 and a significant decrease in Bacteroidaceae at family, genus, and species levels, as well as Prevotellaceae at family and genus levels. Furthermore, fecal bacteria from db/db and m/m mice transplanted into pseudo-germ-free mice showed a significant change in the metabolic parameters, including the body weight, fasting blood glucose, fluid and food intake, and alterations in the composition of the gut microbiota. Taken together, these findings suggest that abnormalities in the composition of the gut microbiota might contribute to the development of T2DM and that potential therapeutic strategies improving gut microbiota might provide beneficial effects for individuals with T2DM and age-related glucose intolerance. Aging is highly correlated with a decline in the metabolic rate and glucose intolerance, which might trigger the potential onset of diabetes mellitus . DiabeteRecently, an increasing number of studies have focused on the early diagnosis and therapeutic mechanisms of T2DM since T2DM is a chronic disease in the elderly population with an intricate pathogenesis characterized by different levels of genetic susceptibility and environmental risk factors –11. Inde14 microbes that play a critical role in energy homeostasis, metabolic signaling, and the immune system [Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia [Bacteroidetes and Proteobacteria, were found to be increased in patients with T2DM [Bacteroides and lower levels of Firmicutes and Prevotella compared with healthy individuals [Bacteroidetes/Firmicutes ratio as well as the Bacteroides/Prevotella ratio was observed to be highly correlated with plasma glucose concentration [The human gut, which is a complex ecosystem, consists of more than 10e system , 18. It microbia . Interesith T2DM , and theividuals , 22, whentration . There iIn the present study, we adopted C57 BL/KS db/db male mice as a T2DM murine model, and we determined alterations in the gut microbiota using 16S rRNA sequencing. Furthermore, we examined the effects of fecal bacteria transplantation from db/db and m/m mice on pseudo-germ-free mice, assessing the metabolic parameters, including the body weight, fasting blood glucose, and fluid and food intake, and we detected the gut microbiota compositions of the host.It is well acknowledged that db/db mice are a rodent model that is genetically diabetic owing to missense mutations of leptin receptors . In the We used 16S rRNA gene sequencing to compare the alterations in the relative abundances of gut microbiota in db/db and m/m mice. The results showed that a total of 17 gut bacteria differed between fecal samples from db/db and m/m mice at six phylogenetic levels  –2Q. The Heat maps of the gut microbiota composition at the phylum, class, order, family, genus, and species levels between db/db and m/m mice are shown in As shown in After fecal bacteria transplantation in the pseudo-germ-free mice, we analyzed the profiles of gut microbiota among the CONT, vehicle, m/m, and db/db groups –5G. The Bacteroidetes, class Bacteroidia, class Deltaproteobacteria, order Bacteroidales, order Desulfovibrionales, family Desulfovibrionaceae, family S24-7, and species Unclassified  were significantly altered among the CONT, vehicle, m/m, and db/db groups –6AR. Antassified , and 6ARHeat maps of the composition of fecal microbiota transplants in pseudo-germ-free mice at the phylum, class, order, family, genus, and species levels in the CONT, vehicle, m/m, and db/db groups are shown in Aging represents a predisposing factor for abnormalities in glucose metabolism and regulation . It has It has been demonstrated in various studies that obese and overweight individuals typically exhibit the onset of systemic low-grade inflammation  and thatVerrucomicrobia at six levels  and a significant decrease in Bacteroidaceae at the family, genus, and species levels, as well as Prevotellaceae at the family and genus levels. Several lines of evidence suggest that Gram-negative bacteria, including Bacteroidetes and Proteobacteria, could secrete LPS endotoxins, finally leading to metabolic endotoxemia and increased levels of Bacteroidetes and Proteobacteria in patients with T2DM [Bacteroidaceae at the family, genus, and species levels and Prevotellaceae at the family and genus levels were decreased in db/db mice, which is inconsistent with the findings of previous studies. This discrepancy might be attributable both to the differential profiles in gut microbiota between humans and rodents and to the differential dietary intake, although detailed studies on the role of Bacteroidaceae and Prevotellaceae are required [16S rRNA gene sequencing has become an available approach for exploring the differential composition of gut microbiota –37. In tith T2DM . Interesrequired .Verrucomicrobia as a beneficial bacterium widely distributed in the healthy human gut has the potential to control the inflammation process [Verrucomicrobia are indicative of an unstable microbial community or gut dysbiosis [Verrucomicrobia was shown to be decreased in individuals with T2DM [Verrucomicrobia at six phylogenetic levels in our study, implying that there is likely a positive feedback response to regulate the onset of inflammation. Altogether, an abnormal composition of the gut microbiota might directly, at least partially, contribute to low-grade inflammation, finally acting as a predisposing factor for the progression of obesity and T2DM.It has been identified that  process  and thatysbiosis . Furtherith T2DM . IntriguBacteroidetes, class Bacteroidia, and order Bacteroidales were increased in the pseudo-germ-free mice after db/db fecal bacteria transplantation. These findings appear to be inconsistent with the results of gut microbiota in db/db mice. The physiological functions of gut microbiota are extremely complicated, and the interactions among the bacteria are fully determined. These complicated factors, combined with the use of antibiotics, make the changes in the gut microbiota more confusing. Although the specific mechanism of this difference is still unclear, these findings support the likelihood that alterations in the gut microbiota could ameliorate or aggravate the development of T2DM.Pseudo-germ-free mice induced by large doses of antibiotics were commonly used in fecal microbiota transplants in our previous studies and in other studies , 37, 42.Overall, the findings described above could support the possibility that potential therapeutic strategies improving gut microbiota might exert beneficial effects for type 2 diabetes mellitus. Actually, increasing evidence has suggested that microbiota transplantation generated from animals or even humans into a germ-free environment may ameliorate or aggravate metabolic abnormalities. Intriguingly, a study on gut microbiota transplant from twins discordant for obesity into mice  has provThere are several limitations in the study. First, a large number of mice are needed to diminish the discrepancy among the groups. Second, we did not adopt db/db mice at different age stages, especially the elderly. Third, clinical studies are required to figure out clinical and therapeutic implications, which would provide alternative strategies in the treatment of T2DM.In conclusion, the findings of this study strongly suggest that an abnormal composition of gut microbiota contributes to the pathogenesis and progression of T2DM. In addition to lifestyle changes and antidiabetic medication prescriptions , 48, regn = 6), C57BL/KS male m/m mice , and C57BL/6J male mice  were purchased from Beijing Vital River Laboratory Animal Technology . All mice were housed under standardized conditions on a 12 h light/dark cycle, with ad libitum access to food and water. The laboratory conditions were maintained with a consistent temperature of 22°C ± 2°C and relative humidity of 60% ± 5%. The mice were allowed to acclimate for a week before the experiments commenced. All experimental protocols and animal handling procedures were conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health . This study was approved by the Experimental Animal Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology .Eight-week-old C57 BL/KS db/db male mice  to Beijing Genomics Institute  before performing a 16S rRNA analysis. These procedures were completed within one day; this protocol was similar to the one reported in previous studies by our group –38, 49. P < 0.05 was considered to be statistically significant. Comparisons among groups were analyzed using one-way or two-way analysis of variance, followed by post hoc Tukey’s test and Student’s t-test using GraphPad Prism 7.0 . Other analyzes, including data normality and statistical significance of relationships among independent variables of nonnormal distributions, were performed using Kolmogorov–Smirnov test, Mann–Whitney U test, Fisher’s exact test, and SPSS software version 21.0 . PCA and PLS-DA were performed to visualize the similarities and the discriminations in db/db and m/m mice.Data are shown as the mean ± standard error of the mean (SEM).


Diabetes Mellitus (DM) is a significant public health issue worldwide due to the associated comorbidities. Recent studies have demonstrated a strong relationship between blood glucose levels and serum ferritin levels in patients with type 2 DM. The aim of the study was to investigate the association between Ferritin Levels and Inflammatory Markers on HbA1c in the Type 2 Diabetes Mellitus Patients.This single-center, cross-sectional, controlled study included patients who were admitted to the Endocrine and Metabolic Disorders outpatient clinics of the Private Kütahya Hospital in the province of Kutahya in the Western Turkey. The study included a total of 172 patients, 84 of whom had type 2 DM and 88 without diabetes and constituted the control group. A total of 190 patients with DM were admitted to the Adult Endocrine and Metabolic Diseases Outpatient Clinics of the hospital between July 1, 2018 and September 1, 2018, and among these, the study was conducted on 172 volunteer patients who met the study inclusion criteria and who did not have any missing data. The HbA1c levels, serum ferritin, hemoglobin (Hb), insulin, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), C-Reactive Protein (CRP), lipid profiles, and uric acid levels were compared between the groups.The serum ferritin levels of the patients with type 2 DM significantly increased with increasing HbA1c levels (p<0.01). A strong positive correlation was found between serum ferritin levels and HbA1c and fasting blood glucose (FBG) levels (p<0.01).Our study results show a significant relationship between HbA1c levels and serum ferritin and CRP levels, suggesting that serum ferritin and CRP levels can be used as a routine screening tool for the early diagnosis of DM. However, further large-scale, prospective studies are needed to confirm these findings. Diabetes Mellitus (DM) is one of the most common endocrine disorders around the world, affecting about 366 million people in 2011, although this number is expected to rise to 552 million by the year of 2030.. The studies conducted by Kunutsor et al. also supported these findings.Recent studies into the early recognition of type 2 DM have identified the presence of a significant relationship between serum ferritin levels and Fasting Blood Glucose (FBG) levels in patients with the disease, and these findings have shifted focus on the inflammatory markers of patients with type 2 DM. Normally, serum ferritin levels reflect the status of iron reserves in healthy individuals, and several studies have found increased ferritin levels in association with such diabetic complications as retinopathy, nephropathy, and vascular dysfunction in patients with DM and with elevated FBG.In practice, HbA1c reflects the mean blood glucose levels over the past three months, with an increased HbA1c value This single-center, cross-sectional, study included patients who attended the Endocrine and Metabolic Disorders outpatient clinics of the Private Kütahya Hospital in the province of Kutahya in the Western Turkey. The study protocol was approved by the Clinical Trials Ethics Committee of Afyonkarahisar Health Sciences University, Faculty of Medicine . A written informed consent was obtained from each participant. The study was conducted in accordance with the princibles of the Declaration of Helsinki.The study included a total of 172 patients, 84 of whom had type 2 DM and 88 without diabetes and constituted the control group. A total of 190 patients with DM were admitted to the Adult Endocrine and Metabolic Diseases Outpatient Clinics of the hospital between July 1, 2018 and September 1, 2018, and among these, the study was conducted on 172 volunteer patients who met the study inclusion criteria and who did not have any missing data. The sociodemographic data were obtained using a face-to-face interview technique. The duration of diabetes in the participating patients with current therapies  and comorbidities were evaluated. The serum ferritin levels of the participating patients were carefully reviewed and females with a hemoglobin (Hb) level of ≤12 mg/dl and males with a hemoglobin level of ≤13 mg/dl, those who received therapy for anemia in the last two months, those who donated blood in the last four months, pregnant women, patients with liver or coronary heart disease, those with infections, those receiving any type of radiotherapy or chemotherapy, patients with kidney disease and those with a hematological disease which could affect ferritin levels were excluded from the study. The control group comprised healthy individuals who did not have DM and who did not meet the aforementioned exclusion criteria.2), and classified as follows: <18.5 underweight, ≥18.5–24.9 normal weight, 25–29.9≤ overweight, and ≥29.9 obese.White Blood Cell (WBC) count, platelet (PLT), ferritin, vitamin B12 (Vit- B12), CRP, insulin, HbA1c, creatinine, serum alanine aminotransferase (ALT), FBG, cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), calcium and uric acid (UA) levels in the blood samples collected from the participants following 10 to 12 h of fasting were retrieved from the hospital automation system. The analyses were conducted using the Sysmex XN-1000™ Hematology AnalyzerDCCT) were considered in the classification. Accordingly, a HbA1c value of 4.0–6.0% was considered normalThe heights of the participants were measured with a stadiometer (cm), weight was measured with a weighing scale (kg), and blood pressure was measured with a mercury sphygmomanometer by a single operator. The results were recorded. The patients’ Body Mass Index (BMI) values were calculated using the formula of weight (kg) / height . Continuous quantitative variables were expressed in number, mean and Standard Deviation (SD); while qualitative or score variables were expressed in number, median, or 25A total of 172 patients  were followed during the study. The mean duration of diabetes was 10.69±7.17 (range: 5.00 to 15.00) years in the diabetes group. Of the total patients, 65.5% with DM were taking oral antidiabetics, 15.5% were receiving insulin, and 19% were receiving insulin combined with an oral antidiabetic. The patients being followed were divided into two groups, being the diabetes group and the non-diabetic group, and the baseline characteristics of the groups are shown in 12, serum CRP, HOMA-IR, FBG, TG, and UA values of the patients with type 2 DM were significantly higher than the control group. Furthermore, insulin values were significantly higher in the diabetes group than the control group. When the HbA1c % values of the diabetic patients were classified in the present study, the serum ferritin, serum CRP, FBG, and TG values increased significantly and insulin values decreased significantly with increasing HbA1c % values.In the present study, the mean HbA1c value was 9.09±2.73% among the patients with DM, and 5.02±0.59% in the control group. The mean HbA1c % value was significantly higher in the diabetes group than in the non-diabetic group, indicating a significant difference between the groups. Furthermore, an increase was observed in the HbA1c values with prolonged duration of diabetes, indicating that the compliance of patients with therapy decreased and glycemic control worsened with prolonged duration of DM. Apart from the HbA1c % and serum ferritin levels, the BMI, PLT, WBC, Vit B12, CRP, HbA1c, HOMA-IR, FBG, TG and UA values of the patients with type 2 DM were significantly higher than those of the non-diabetic patients, and the insulin levels of non-diabetic patients were significantly higher than those of the patients with type 2 DM . In a cohort study conducted by Cugy et al.Furthermore, a study conducted by Wolide et al.There are also studies comparing two sexes and reporting different opinions and results, although the present study included general population, and also reported no results in this regard. In a study by Dekker et al.In a cross-sectional study including 8,235 participants in 2009 , the diabetic patients were found to have higher serum ferritin levels, and a significant relationship was found with the HbA1c and HOMA-IR values..8% diabeThe results of the present study are consistent with many other studies, and suggest a significant relationship between serum ferritin and glycaemia. However, further large-scale, long-term, prospective studies in different regions of the world are required to gain a better understanding of the role of serum ferritin levels in the prevention of diabetes.SN: The whole article was prepared by me conceived, designed, data collection, manuscript writing designed, statistical analysis, editing, review and final approval of manuscript.


Juvenile Parkinson’s disease (JPD) is a rare movement disorder that presents before the age of 21 years. Kufor-Rekab syndrome (KRS) is one of the distinct types of JPD caused by the ATP13A2 mutation and inherited as an autosomal recessive. The pathogenesis of KRS is related to an interrelated metabolism of ATP13A2 with Mn+2 and Zn+2, bioenergetics of mitochondria, autophagy lysosomal dysfunction, and synuclein metabolism. Clinically, KRS has a variable phenotype and may present with pyramidal or extrapyramidal symptoms and cognitive impairment. Early diagnosis of KRS is important as most of these patients are levodopa-responsive and genetic counseling and screening is important for the whole family. We present a case of a 16-year-old boy who presented with tremors and walking difficulty. His physical examination showed an expressionless face, decrease in eye blink frequency, and slow vertical saccadic eye movements. His movements were slow. All laboratory investigations were normal, except the genetic study, which led to the diagnosis of KRS. Parkinson's disease (PD) is a movement disorder caused by a degeneration in the basal ganglia. It has four cardinal symptoms consisting of tremor, rigidity, bradykinesia, and postural instability. The incidence of PD increases with age, with an incidence of 0.5 per 100,000 in the < 40-year age group compared to 13.4 per 100,000 in the overall age group . Several genetic mutations have been identified as a cause of JPD. Kufor-Rekab syndrome (KRS) is one of the distinct types of JPD that is caused by the ATP13A2 mutation with an autosomal recessive pattern of inheritance. It is one of the rare diseases with less than 50 cases reported so far [A 16-year-old adolescent boy presented in our neurology clinic with a history of tremors in the left hand for eight months, followed by difficulty in walking for three months. The tremors were only present during rest without any change in frequency. Similarly, no exaggerating or initiating factors were identified. For the last two years, the patient has experienced increased restlessness, sweating, and nervousness, especially during social gatherings. The patient was born to a consanguineous marriage and has no significant past medical or family history. The patient was prescribed propranolol but no improvement was seen. The patient had also gone under cognitive-behavioral therapy without any beneficial outcome.On clinical examination, the Glasgow Coma Scale (GCS) was 15/15 . His vitals were a blood pressure of 120/70, heart rate of 80 beats per minute, respiratory rate at 18 breaths per minute, and afebrile. He had an expressionless face with a decrease in eye blink frequency. Tremors were noticeable in his left hand while resting on the table. Cranial nerve examination showed slow vertical saccadic eye movements. His vision and funduscopic examination were normal. Slit-lamp examination showed no Kayser‐Fleischer ring. Limb examination showed a Grade 2 rigidity ) in the left upper and lower limbs with 5/5 power and +2 deep tendon reflexes (DTR). Slowness was observed during hand movement, finger tapping, and toe-tapping. During walking, the patient had difficulty maintaining his balance with swaying on the left side. The patient also had difficulty in writing and letters became progressively smaller. Multiple investigations were carried out that involved a complete blood profile, comprehensive metabolic panel, serum iron, copper, ceruloplasmin, and 24-hour urine copper levels, which appeared to be normal. Magnetic resonance imaging (MRI) of the brain and spinal cord, along with visual evoked potentials, were also normal. On the basis of his history, physical examination , and age of onset of symptoms, a clinical diagnosis of juvenile parkinsonism (JPD) was considered. Molecular screening of the ATP13A2 gene on the deoxyribonucleic acid (DNA) extracted was performed. Sequence analysis revealed a homozygous deletion on exon 22 that resulted in the replacement of serine with threonine, followed by a stop codon. This confirmed the diagnosis of autosomal recessive juvenile parkinsonism. The patient's parents consented for the testing of a gene mutation which showed a heterozygous mutation of ATP13A2 gene, confirming an autosomal recessive pattern of disease.The patient was started on a low dose of carbidopa/levodopa, resulting in a gradual improvement in tremor and walking. After four months of follow-up, the patient experienced dyskinetic movements of tongue and foot due to a peak concentration of dose. We lowered the dose of levodopa and started the patient on pramipexole (the dopamine agonist). He was followed for a year after that and his symptoms were stable but a little difficulty in writing still persisted.KRS is an autosomal recessive, juvenile-onset, levodopa-responsive parkinsonism that is characterized by the onset of extrapyramidal, pyramidal, and cognitive dysfunction. It was first identified in 1994 when five cases of a Jordanian family were reported living in Kufr Rakeb, hence gaining the name of the disease . Clinically, KRS manifests as parkinsonian signs of tremor, bradykinesia, rigidity, and postural instability. In addition, slow saccadic eye movement, supranuclear gaze palsy, ataxia, partial or total paralysis of the legs, spasticity, dystonia, dyskinesia, and hyperreflexia. Neurodegeneration involving multiple organs is evidenced by the presence of pyramidal signs and cognitive impairment. Many patients first present with learning difficulties and intellectual disability, and it is the most common non-motor symptom. Other non-motor symptoms include anxiety, hyposmia or anosmia, panic attacks, and auditory and visual hallucinations . KRS can be confused with many diseases, such as dopa-responsive dystonia (DRD), Wilson’s disease, a juvenile form of Huntington’s disease, Parkinson-plus syndromes, neuroacanthocytosis, and several toxic and metabolic causes of PD. The few cases that have been reported in the literature have been delayed in diagnosis due to the variability of presentation . Jha et The mainstay of treatment is the symptomatic treatment of PD, with the aim of maximizing functional recovery and minimizing medication-related side effects. Levodopa has shown an important effect in the resolution of symptoms but long-term use and high doses can cause dyskinesia and motor fluctuations . FurtherOur case highlights the importance of diagnosing juvenile Parkinson's disease with such a rare genetic cause. KRS may present with phenotype variability, which may delay the diagnosis. However, young patients who have any kind of pyramidal and extrapyramidal symptoms must be screened for ATP13A2. Since most of these patients respond to levodopa, early diagnosis can then prevent long-term sequelae in these patients.