Escherichia coli and most bacteria, dimers are resolved to monomers by site-specific recombination, a process performed by two Chromosomally Encoded tyrosine Recombinases (XerC and XerD). XerCD recombinases act at a 28 bp recombination site dif, which is located at the replication terminus region of the chromosome. The septal protein FtsK controls the initiation of the dimer resolution reaction, so that recombination occurs at the right time (immediately prior to cell division) and at the right place (cell division septum). XerCD and FtsK have been detected in nearly all sequenced eubacterial genomes including Proteobacteria, Archaea, and Firmicutes. However, in Streptococci and Lactococci, an alternative system has been found, composed of a single recombinase (XerS) genetically linked to an atypical 31 bp recombination site (difSL). A similar recombination system has also been found in 𝜀-proteobacteria such as Campylobacter and Helicobacter, where a single recombinase (XerH) acts at a resolution site called difH. Most Archaea contain a recombinase called XerA that acts on a highly conserved 28 bp sequence dif, which appears to act independently of FtsK. Additionally, several mobile elements have been found to exploit the dif/Xer system to integrate their genomes into the host chromosome in Vibrio cholerae, Neisseria gonorrhoeae, and Enterobacter cloacae. This review highlights the versatility of dif/Xer recombinase systems in prokaryotes and summarizes our current understanding of homologs of dif/Xer machineries.The separation and segregation of newly replicated bacterial chromosomes can be constrained by the formation of circular chromosome dimers caused by crossing over during homologous recombination events. In  It initially involves (1) recognition of the DNA lesion by the complexes RecBCD or RecFOR, depending on the type of DNA lesion; (2) formation of 3′-ssDNA overhangs processed by the exo and endonuclease activity of the Rec proteins, and subsequent coating by RecA; (3) strand invasion of the 3-terminal ssDNA into the homologous duplex DNA molecule and search of the complementary strand; (4) formation of a D-loop intermediate, transformation into a branched intermediate and Holliday junction (HJ) formation and (5) completion of the recombination process by resolution of the HJ, catalyzed by the systems RuvABC or RecG  (dif (Deletion-Induced Filamentation).Because circular chromosomes do not have “ends,” they are vulnerable to concatenation during formation of an odd number of crossover events. Thus, swapping DNA flanking regions tangles the sister chromatids and forms larger ring chromosomes that compromise cellular division (igure 1) . To ensuE. coli, homologous recombination repair is processed by two predominant recombinational pathways; The RecBCD pathway associated with DSB repair, replication fork collapse, replication fork reversal and replication fork arrest, and the RecF pathway, which is mostly involved in the repair of SSG, and under certain conditions, can also repair DSBs . All known site-specific recombinases are classified into two unrelated families, tyrosine–type or serine-type recombinases (Tyr or Ser) based on the amino acid residue that forms a covalent linkage between the protein and a phosphate at the DNA cleavage site  were shown to act on a 28 bp DNA sequence (dif site), located in the ter region. The synaptic XerCD/dif complex consists of two XerC and two XerD subunits respectively bound to two dif sites . Limited structural information of some tyrosine recombinases have revealed a conserved catalytic domain fold   intermediate, subsequent isomerization forms a XerC-HJ intermediate that is rapidly resolved to recombinant DNA by XerC, see below and Figures 3A (The chromosome dimer resolution (CDR) process and heritable stability were originally elucidated in E. coli , 2004, wain fold , facilitain fold  consistiain fold , XerA  . This prgure 2B) . In the gures 3A .dif concerns how the system is controlled to ensure a proper CDR into monomers in the right place and at the right time without promoting the reverse reaction, which would generate dimers from monomers. It is understood that Xer-mediated recombination mostly depends on an active HR system because it is the major process that provides concatenated chromosomes. However, catenation problems caused by replication may require Xer-recombination system as well , where oppositely oriented KOPS (FtsKOrienting Polar Sequences) converge and guide FtsK DNA translocation toward the dif locus (Filamentous Temperature-Sensitive cell division protein K), a large division septum-associated DNA translocase, which coordinates chromosome segregation and cell division when chromosome organization has been affected  (E. coli TOE44 (AB2497 ftsK44) mutant cells and their ability to form long chains of cells due to a single substitution of one amino acid in the N-terminal domain (FtsKN), by then, FtsK was thought to participate in septum formation as a peptidoglycan-modifying enzyme. Then, ftsK1::cat minB double mutants of E. coli minicells. Finally, in N) is responsible for attachment of the protein to the membrane by four transmembrane segments and interaction with other proteins of the division septum such as FtsZ , usually referred as the motor of the FtsK, is comprised of three separated subdomains called α, β and γ. Structural studies of the translocation module FtsKαβ of Pseudomonas aeruginosa demonstrated that it assembles as a hexameric ring around double-stranded DNA forming a central channel of 30 Å in diameter, where double stranded DNA (dsDNA) passes through . KOPS are over-represented on the leading strand of replication where their concentration gradually increases as dif is reached; indeed, more than 90% of KOPS sequences nearby dif are located on the leading strand   . FtsK wa as FtsZ . The linresidues . Its len Vibrios  or ∼125 udomonas . Experimresidues . Followi through . Later sg FtsKαβ . The Ftsg strand ; giving g strand  with a fg strand . The secsolution . How Fts of FtsK . It is l of FtsK . Once a  restart . FtsK ha restart . It has  restart . Howevero 67 A°)  followedE. coli, and other bacteria. Current estimates have identified more than 1300 tyrosine recombinases where many of them are associated with other host proteins to regulate their activity, directionality, or processivity , a non-codifying region of 280 bp where two additional proteins act with XerCD to catalyze SSR reactions: the arginine repressor (ArgR)    ,b, and aed XerA) . SSR in  product . The cerreaction . The two of ArgR . This isnd XerD) . In cer,bination . Additio protein . Rcd binis pause .B. subtilis, two homologs of XerC and XerD called CodV and RipX perform dimer resolution at a 28 bp dif site (Bsdif) close to the terminus region . The Bsdif region is comprised of two 11 bp half-sites with imperfect dyad symmetry where CodV and RipX bind simultaneously and a 6 bp central region where DNA exchange occurs. Both CodV and RipX share a 37 and 44% identity with the XerC and XerD respectively, and 39% between them  and the soluble SftA protein (Septum-associated FtsK-like Translocase of DNA). Both translocases harbor AAA+-ATPase and C-terminal domains with 56% of sequence similarity between them. SftA exhibits 50% identity with respect to the E. coli FtsKγ domain whereas SpoIIIE exhibits a 50% of similarity to the FtsKαβ subdomain and 42% of similarity to the FtsKγ subdomain of E. coli (SpoIIIE Recognition Sequences) which are similar to the E. coli KOPS sequences. The SRS motifs are mostly located on the leading strand (up to 85%), and direct translocation toward the Bsdif site . However, unlike FtsK that activates XerCD recombination reactions, neither SftA nor SpoIIIE directly activate CodV or RipX recombinases. In this case, SftA and SpoIIIE affect the CodV/RipX reaction by proper positioning of the  to date .Vibrio cholerae, as well as 10% of sequenced bacteria to date, possess a very distinct property among bacteria; it harbors more than one chromosome  whereas some small-sized chromosomes still require Xer/dif recombination machinery as demonstrated by some Rickettsiales (genome ranging from 0.85 to 1.52 Mb in size) (romosome . One ancromosome . Harboriin size) .V C and XerDV C with 53 and 68% of amino acid similarity to E. coli XerC and XerD, respectively (dif-like sequences are present in both chromosomes (dif1 and dif2) located near GC skew shift-points  in a similar way to that found in E. coli. Once the dif sites are brought together nearby, FtsKV C activates XerDV C, which is positioned to cleave the bottom strand, and perform the first strand cleavage. Then XerCV C cleaves the top strand and performs the second strand cleavage; these reactions are carried out on both chromosomes at their respective dif sites   separate earlier than terI and that this early separation keeps terII sites at midcell by the macro domain MatP/matS organization system. This restriction during concatenation induces several collisions at midcell between terII sites, increasing the number of recombinational events and the likelihood of dimer resolution. It also ensures that ter sites of bacterial chromosomes remain exclusively in mid-cell to be processed by FtsK.Homologs of XerC/XerD and FtsK have been characterized on chrI, referred as XerCectively . Whereast-points . Interesposition . Dimer rgure 3E) . Additio complex . An addi complex . This caV. cholerae, the causative agent of the potentially fatal human disease cholera, XerCV C and XerDV C are hijacked by some vibriophages to integrate their genomes into the chromosome. They are usually referred to as IMEX (Integrative Mobile Elements Exploiting Xer), and the best known ones are VGJϕ (Vibrio Guillermo Javier filamentous phage), TLCϕ (Toxic Linked Cryptic), and CTXϕ (Cholera Toxin Phage). CTXϕ is a lysogenic [(+)ssDNA] filamentous bacteriophage that encodes the A-B type enterotoxin CT in V. cholerae (attP), a dif-like site that serves to classify the three different groups of IMEX,  (attP sites (attP1 and attP2) separated by 90 nt on the ssDNA sequence   . Althouggure 3F) . The twoexcision . Althougormation . Once threaction . Interessmission . In the smission .V C catalytic reaction at the dif1 site, but unlike CTXϕ, it only harbors one dif-like attachment site (V GJattPϕ) of 29 bp that allows its integration into the chromosome as a dsDNA. The attP central region contains four different nucleotides close to the XerD binding side with respect to the central region of the dif1 site . The lack of homology at the XerDV C central region side prevents XerDV C participation in the catalytic reaction. Once integrated, prophage VGJϕ acquires two attP sites (attPL and attPR), equally functional for the XerCV C excision reaction, in contrast to CTXϕ, where Xer recombinases can process VGJϕ excision from the host genome . The prophage form of TLCϕ is almost always linked to CTXϕ integration confirming the regular synergistic interactions found in most IMEX. Paradoxically, despite the lack of homology between the XerD binding sites of dif1 and TLCattPϕ, TLCϕ integration/excision is mediated by XerDV C and then completed by XerCV C resembling dimer resolution in bacteria, but independently of FtsK participation  genes  and a divergent XerD-binding site. GGI insertion into the Neisseria genome follows a CDR-like process where FtsK activates XerD to perform the first strand cleavage between Ngdif and GGIdif followed by isomerisation of the synaptic complex and activation of XerC to perform the second strand cleavage, creating a GGI integrated form with two active Xer binding sites. Interestingly, the GGI synapse has given important clues about how IMEX might remain integrated in the host genome despite the presence of dif sites. in vitro that a trimeric form of the E. coli FtsK protein (t-FtsKαβγEc) was unable to activate XerCD recombination at one of the two dif sites (the GGIdif site), in fact, the XerCD/GGIdif complex was unable to stop t-FtsKαβγEC translocation. As a consequence, the XerCD complex is dissociated from GGIdif and the excision process is inhibited.IMEX are recombination platforms that permit bacteria to evolve and adapt through the acquisition and reordering of relevant genes. They have strengthened bacterial evolution, playing an important role in the rise of multidrug resistance, gene transfer mechanisms and virulence factors among clinically relevant bacteria . Besides species  and the ludwigii . GGI is S) genes . GGI carE. ludwigii (ECAA-01) that carries the NMC-Abla gene that encodes for a serine carbapenemase. It was first characterized by E. ludwigii. The results indicated the presence of a new 29-kb region with lower GC content when compared to the bacterial genome, indicating a possible gene transfer acquisition  that acts on an atypical 31 bp recombination site called difSL in the presence of dimers. Unlike E. coli, the xerS gene is found immediately adjacent to the recombination site difSL acting as a single module. The difSL site differs from most dif sites because of its large central region of 11 bp as opposed to the normally found 6–8 bp in all other dif regions   versus the left part of the sequence (TTTCCGAAAA). This additional spacing suggests XerS/difSL may be biased to favor binding in one-half site over the other. It was later confirmed by difSL than the right-half site, and a preference for initiating the recombination reaction on the bottom strand of the difSL site. These results indicate that, although the difSL site is relatively symmetric and XerS is a single tyrosine recombinase, there is a bias for where the proteins initially bind to difSL and where they initiate the strand cleavage reaction. Thus, the left-bound monomer could activate the right-bound monomer by bending the DNA or changing the conformation of the second monomer which could explain the preferential cleavage and exchange of the bottom strand. This behavior resembles what XerC displays with weak binding but stronger strand exchange when compared to XerD  involved in XerD interaction  . Thus, d to XerD . This inerfamily . The C-teraction . On the bacteria . This wor motifs . XerS alculative .Helicobacter sp and Campylobacter sp led to the discovery of another type of single recombinase called XerH that acts on a recombination site called difH in a FtsK-dependent manner. It was shown to be involved in chromosome segregation and possibly dimer resolution in Helicobacter pylori  possess a system analogous to the classical XerCD system. Additionally, unlike other tyrosine Xer recombinases, XerH activity appears to be affected by a second Xer recombinase called XerT in H. pylori (the TnPZ transposon associated recombinase) since under XerT deletion, difH recombination levels increased (Helicobacter difH comprises two highly conserved imperfect inverted binding sites of 11 and 10 bp (AGTTATGAAAA and AAAAGTTTGA) in the left and right sides respectively, separated by a 6–10 bp central region  (dif appears to be determinant in the order of binding and cleavage reactions). The left half site preference is due to stronger interaction between XerH and difH left site ΔG = -21.3 kcal/mol compared to the right half site ΔG = -15.4 kcal/mol). The extra nucleotide thymine (T4) in the outer region of the left half site confers a specific hydrogen bond between the left arm and the lysine (K290) of XerH that favors stronger protein–DNA interaction with the other three outermost nucleotides, DNA bending and specific positioning of the nucleophilic tyrosine. Surprisingly, XerH assembly on difH does not induce strong DNA bending alone and it seems to require FtsK to generate the required conformational rearrangements to favor XerH DNA exchange (difH of C. jenuni (campdifH) demonstrated that XerH binding to either the left or right site of campdifH resulted in similar affinities compared to the full campdifH site possibly due to the similarity between the outer sequences of both arms. Additionally, XerH binding to campdifH appears to be less efficient than XerS which suggests that it is less cooperative than XerS/difSL system. Additionally, these results contradict XerH binding affinities observed in H. pylori by in vivo between two campdifH sites located on the same plasmid; it is also suggested that XerH might be involved in decatenation processes because of the apparent absence of Topo IV proteins in H. pylori  .Studies in r pylori . XerH (3r pylori . Anotherpression . Interesncreased . Recent gure 3J)  (Unpubligure 3J) . Two subexchange . Results. pylori . Interesdif-like site located in the replication terminus region  in a FtsK-independent manner that acts on a s region . XerA sheference .Archaea Short Polarized Sequences) that are skewed toward dif sites, showing a similar triangle-shaped diagram observed in Bacteria of skew inversion at dif sites  called ASPS  and stochastic optical reconstruction microscopy (STORM), plus the use of other techniques as Förster (Fluorecence) resonance energy transfer (FRET), tethered fluorophore motion (TFM), single-molecule Flourescence resonance energy transfer (smFRET), among others. They are providing a powerful blueprint for investigators studying short- and long-range changes in DNA, DNA/protein, and protein/protein interactions. Researchers in SSR systems and protein–protein interactions might be the most direct beneficiaries of these techniques, especially when it is becoming urgent to further understand IMEX insertion and its subsequent influence in antibiotic resistance and bacterial virulence.Much information has been gained on site specific recombinases and dimer resolution. This review has highlighted the complexity of All the authors contributed equally to the work by providing: Substantial contributions to the conception or design of the work; the acquisition, analysis, and interpretation of data for the work; Drafting the work or revising it critically for important intellectual concepts; Final approval of the version to be published; and Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


A 57-year-old African American female with a history of ischemic cardiomyopathy and a recent stroke with no residual deficits presented with apraxia and confusion. Non-contrast CT scan of the head revealed multiple embolic strokes in both cerebral hemispheres. Transthoracic echocardiography raised the suspicion for increased trabecular meshwork in the left ventricle. Cardiac MRI confirmed the findings of isolated left ventricular non-compaction (LVNC) syndrome. A contrast-enhanced transesophageal echocardiogram demonstrated the characteristic features of this unusual disease with the additional demonstration of contrast filling the trabecular meshwork. Interestingly multiple transthoracic echocardiograms in the past had failed to identify myocardial non-compaction. The patient was started on warfarin for prophylactic anticoagulation and an implantable defibrillator was placed to lower the risk of sudden death. LVNC is a rare type of genetic cardiomyopathy characterized by excessively prominent trabeculations and deep inter-trabecular recesses in the ventricle wall. Non-compaction remains frequently overlooked even by experienced echocardiographers. Failure to diagnosis may lead to insufficient treatment since it is often associated with a risk of thromboembolism, life-threatening arrhythmias and sudden death. Furthermore, because of the familial association described with ventricular non-compaction, screening of first relatives with echocardiography is recommended. Left ventricular non-compaction (LVNC) is a rare type of genetic cardiomyopathy. It has been associated with significant risk of developing arrhythmias, thromboembolic complications, severe systolic dysfunction and sudden cardiac death . EchocarA 57-year-old female was brought to the hospital because of new onset of confusion and apraxia. The patient had a past medical history significant for nonischemic cardiomyopathy for 2 years. The patient had a stroke several weeks prior during an admission to another facility. At that time, work-up for the etiology of the stroke was negative except for cardiomyopathy with an ejection fraction of 30%. The patient did not have any residual focal motor neurologic deficits, although she was cognitively impaired. Medications at discharge included aspirin, carvedilol, lisinopril and atorvastatin.On admission, the patient was hemodynamically stable without any distress. Her speech was incoherent and tangential. She had difficulty identifying objects and could not use eating utensils appropriately. Pupils were equal and reactive without any nystagmus or gaze disturbance. No other neurologic deficits were present. On cardiac auscultation, first and second heart sounds were heard normally without any murmur. Lungs were clear. Carotid and other peripheral pulses were palpable and symmetric. Trace bilateral ankle edema was present.The patient had a normal complete blood count, electrolytes, liver function and thyroid function tests. The cardiac BNP value was 340 pg/mL. Electrocardiogram revealed normal sinus rhythm with T wave inversions in the lateral leads. A chest X-ray was negative for active cardiopulmonary disease. Non-contrast CT scan of the head showed areas of low attenuation in the right posterior frontal and parietal regions consistent with subacute infarctions which were outlined with high attenuation representing hemorrhagic conversion. Subsequent MRI was consistent with these findings with additional tiny acute infarcts in the parietal lobes .A transthoracic echocardiogram for an embolic source showed severe reduction of the left ventricular systolic function with ejection fraction of 10-15%. On close observation, there was a suspicious appearance of prominent trabeculations in the left ventricle . CardiacIsolated LVNC is an uncommon finding and the true prevalence of it remains unknown. It was first described by Chin et al in 1990 . LVNC isTo date, the arrest in the embryonic endomyocardial morphogenesis is believed to be the mechanism underlying the cardiomyopathy. This is supported by genetic factors as well as from murine data, in which mice with mutations of these genes developed LVNC , 8. ThisLVNC is a genetically heterogenous disorder, with both X linked as well as autosomal dominant inheritance described. Because of the possible genetic influence, there has been emphasis on the screening of all first-degree family members of the affected individuals. Mutations in the G4.5 gene that encodes the tazaffin family of proteins result in infantile X linked cardiomyopathies, including Barth syndrome with dilated cardiomyopathy and LVNC in a pediatric patients -11. ThesThe four major clinical manifestations of non-compaction that have been described to date include heart failure, arrhythmias, embolic events and sudden death , 5, 14. Arrhythmias that are associated with LVNC include ventricular tachyarrhythmias 47%), atrial fibrillation 25%), paroxysmal supraventricular tachycardia, complete heart block and sudden cardiac death (accounted for more than 50% of deaths) [5%, parox7%, atriaSystemic embolic events including stroke as seen in our patient can be associated with LVNC because deep inter-trabecular recesses with slow and sluggish blood flow with stasis increase the risk of thrombus formation.The diagnosis of LVNC is primarily based on 2D echocardiographic findings. Chin et al first proposed diagnostic criteria for non-compaction syndrome in 1990, followed then by Jenni and Stollberger , 18. TabKohli et al studied all three criteria for identification of LVNC in HF and control groups . There wCardiac MRI has been recently utilized as a sensitive tool for the diagnosis of LVNC. It provides a good correlation with transthoracic echocardiography for the localization and extent of non-compaction and is useful in cases with poor echocardiographic image quality. Petersen et al proposed that NC/C > 2.3 in diastole accurately diagnosed LVNC .Recently contrast echocardiography has been utilized as an extension of the existing routine echocardiographic examination. It is a low-cost, real-time, non-invasive technique which shows high correlation in the diagnosis of LVNC. The added advantage of contrast echocardiography is that it gives a real-time assessment of intracardiac blood flow and depicts contrast filling in the inter-trabecular spaces.We performed a transesophageal echocardiography with contrast to aid in the diagnosis of LVNC. Although transesophageal echocardiography has not been used often for the diagnosis of LVNC, it can be extremely helpful in the diagnosis when transthoracic echocardiography image quality is poor and there is a high suspicion. We suggest that the simultaneous use of contrast echo either transthoracic or transesophageal along with cardiac MRI helps to prove concordance among the findings and highly increases the sensitivity of true diagnosis.There are no proposed guidelines for the management of patients with LVNC. It seems reasonable that for patients who are asymptomatic without any LV dysfunction, follow-up should be echocardiography and Holter monitoring every 2 or 3 years , 22. PatThe concern for ICD implantation in all LVNC patients has been proposed due to high risk of sudden cardiac death. There are no clear cut guidelines for ICD implantation for patients with LVNC. In the present era of knowledge an ICD should be implanted in patients with LVNC presenting with symptomatic ventricular arrhythmias, syncope or severely impaired LV systolic function (left ventricular ejection fraction < 35%) .Finally since LVNC is a genetic cardiomyopathy, family screening with echocardiography is recommended for all first-degree relatives. Hoedemaekers et al in their recent study investigated relatives of 50 unrelated LVNC probands and found familial cardiomyopathy in 64% of families screened .Isolated LVNC cardiomyopathy is a rare form of cardiomyopathy, with many unanswered questions and overall knowledge limited to case reports and case series. It should not be overlooked in patients presenting with nonischemic cardiomyopathy. A multimodality imaging approach may improve the diagnostic accuracy. Contrast echocardiography can significantly improve the yield and detection rates. Functional diagnostic criteria need to be defined apart from the morphologic criteria to better diagnose and determine appropriate management therapy.


Rhamnus species, and it is found to have more potent biological activities than the parent compound emodin. The aim of this study is to characterize the vasodilatory effect of madagascine on vasoconstriction and sphingosylphosphorylcholine induced vasospasm in ex vivo and reveal the potential mechanisms in vitro. The effects of madagascine on vasoconstriction of rat mesenteric resistance arteries (MRAs) induced by K+, methoxamine, and endothelin-1 were, respectively, studied. The cholesterol-enriched porcine coronary vascular smooth muscle (VSM) strips were used to investigate the effects of madagascine on abnormal constriction induced by sphingosylphosphorylcholine (SPC) which has a pivotal role in vasospasm. The vasodilatory effect was induced by madagascine (0.3–100 μM) in isolated rat MRAs and the vasodilatory effect was blocked by NO synthase inhibitor L-NAME and AMPK inhibitor compound C. Madagascine (10 μM) also significantly relaxed the abnormal constriction in porcine VSM induced by SPC and the effect was abolished by compound C. Madagascine significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) in endothelial cells while decreasing the phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in VSM cells. Madagascine-induced vasodilatation was abrogated using small interfering RNA knockdown of AMPK. In summary, madagascine exerted vasodilatation through activating AMPK, leading to the activation of eNOS in endothelium and inhibition of ROCK/MYPT1 in VSM. This study suggests the potential value of madagascine in amelioration of vasospasm related cardiovascular diseases.Madagascine (3-isopentenyloxyemodin) can be chemically synthesized or purified from several  Vascular vasodilatation is beneficial for amelioration of cardiovascular diseases including essential- and renal-parenchymal-disease-related hypertension, vascular remodeling, cardiac infarction, and congestive heart failure . Vascula2+ dependent and Ca2+ independent mechanisms (2+ independent VSM constriction by inhibiting myosin phosphatase via phosphorylation of myosin phosphatase target subunit 1 (MYPT1) (N-deacylation of sphingomyelin which is one of the most abundant lipids in cell membrane (Recent studies suggest AMP-activated protein kinase (AMPK) is a new therapeutic target for vasodilatation. In endothelial cells, AMPK is activated through the phosphorylation by LKB1 and CAMKK . The actchanisms . AMPK ac (MYPT1) . Sphingomembrane . SPC is membrane . SPC-medmembrane . HoweverFigure 1). The trivial name of madagascine derives from the natural source, Harungana madagascariensis Poir, from which it was isolated and structurally characterized for the first time (Rhamnus spp. (Madagascine (3-isopentenyloxyemodin) is a natural compound containing an anthraquinone core linked to a 3, 3-dimethylallyoxy chain  were used to investigate the effects of madagascine-mediated activation of AMPK on vasoconstriction. The porcine coronary arteries were used to investigate the effects of madagascine-mediated activation of AMPK on abnormal constriction induced by SPC because porcine coronary arteries are cholesterol-enriched and cholesterol potentiates the Ca2+ independent of VSM constriction mediated by SPC  for 2 weeks before experimentation. All rats were euthanized by barbiturate overdose  for mesenteric arteries isolation. Porcine coronary arteries  were obtained from a local abattoir.The experimental protocol was approved by Dalian Medical University Animal Care and Ethics Committee at June 8th, 2012, and all animals used were maintained in accordance with National Institutes of Health Guide for Care and Use of Laboratory Animals . Fifty Male Wistar rats (weighing 200–300 g) were obtained from Experimental Animal Center, Dalian Medical University. [Certificate of Conformity: No. SCXK (Liao) 2008-0002]. The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions , AMPK-α1 (phospho T183) +α2 (phospho T172) antibodies (ab72845), eNOS antibodies (ab5589), and eNOS (phospho S1177) antibodies (ab184154) were bought from Abcam (Hong Kong) Ltd. . MYPT1 p-MYPT1 (phospho Ser 695) antibodies (sc-33360) were bought from Santa Cruz Biotechnology, Inc. . MLC antibodies (3672) and phosphor-MLC (Ser 19) antibodies (3671) were bought form Cell Signaling Technology, Inc. (USA). GAPDH (10494-1-AP) antibodies and MYPT1 antibodies (22117-1-AP) were bought form Proteintech Group, Inc. . SPC was bought from Biomol. Unless otherwise indicated, chemicals were obtained from Sigma-Aldrich . The cell HUVECs and HCASMCs were obtained from cell bank of Shanghai Institute . The cells used in this study were evaluated before experiment including the expression of eNOS and AMPK in these cell lines. No significant inter-species variations in AMPK and eNOS signaling which affect the results in this study were observed according to previous publications and pre-experiments.D-glucose 11.1 (pH 7.4)] at a constant flow rate of 5 mL/min with a peristaltic pump . Changes in the perfusion pressure were recorded by BL-420F biological system   . The end, China) . The MRA3, 1.2 KH2PO4, 1.2 MgCl2, 1.25 CaCl2, and 11.5 D-glucose). The arteries were cut into strips (1 mm × 4 mm) without endothelium and adventitia. These strips were mounted vertically at the organ bath filled with PSS, gassed with 5% CO2/95% O2, and maintained at 37°C. The isometric force of VSM strips was measured by a force transducer . Effect of madagascine on contractile force was investigated at the maximum and steady state of SPC or 40 mM K+ induced constriction.Porcine coronary arteries were placed in 4°C physiological salt solution  were simultaneously measured using porcine VSM strips  equipped with a randomized optical fiber system .The HUVECs or HCASMCs cells were transfected with Lipofectamine 2000 (Invitrogen) and AMPK-α1/α2-targeted or a control small interfering RNA (siRNA) oligos  according to the manufacturer’s instructions  CO., LTD). The siRNA sequence was as follows: 50-ACC GAG CUA UGA AGC AGC UGG GUU U-30. The efficiency of gene silencing was confirmed by Western blotting analysis.Data analysis was conducted in a blinded manner according to single-blind study design. The One-Way ANOVA was used where three or more groups of data were compared. Data were expressed as the mean ± SD. The data followed a normal distribution and each group had equal variances. To further evaluate the data, Kruskal–Wallis rank sum test was used. All experiments were repeated for at least six times.+ in a concentration range of 0.3–100 μM. Madagascine-induced vasodilatation was transient and disappeared within about 6 min. Equal volume of vehicle DMSO did not show any inhibitory effect on the constriction of MRAs . Madagascine also relaxed MRAs pre-contracted by methoxamine and endothelin-1. However, madagascine did not significantly change the basal tension . The rat MRAs remained in good condition after madagascine treatment by washing.Based on pre-experiments, 0.3–100 μM madagascine were selected to study its vasodilatory effects. Madagascine reduced constriction of rat isolated MRAs induced by 40 mM K+-induced constriction of rat MRAs without epithelium . In MRAs with an intact endothelium, madagascine exerted vasodilatation effect was significantly blocked by NO synthase inhibitor L-NAME and AMPK inhibitor compound C, respectively. These results suggest that AMPK/eNOS signaling pathway is involved in madagascine-induced vasodilatation . Both AMPK/eNOS and AMPK/AKT/eNOS are known to be related to increase the production of NO . The potential role of AMPK/eNOS pathway was mainly studied in this study.Madagascine did not induced vasodilatation on 40 mM Kon of NO . As showFigure 6). The effects of AMPK knockdown by siRNA in HUVECs were confirmed by Western blotting analysis . After siRNA knockdown of AMPK, the madagascine-induced phosphorylation of eNOS was abrogated, indicating that madagascine-induced phosphorylation of AMPK activated the phosphorylation of eNOS, leading to nitrite-mediated blood vessel relaxation .To validate the involvement of AMPK/eNOS signaling pathway in madagascine exerted vasodilatation, the effects of madagascine on AMPK and eNOS were studied using HUVECs. Madagascine, in a concentration range of 0.3–30 μM, induced the phosphorylation of eNOS and the phosphorylation of AMPK, respectively, in a time dependent manner from 1 to 24 min ; and madagascine induced vasodilatation was abolished by AMPK inhibitor compound C , indicating that madagascine reduced SPC-induced Ca2+ independent vasoconstriction via activating AMPK.As SPC is found to induce Caasospasm , the eff20 in VSM cells with siRNA knockdown of AMPK were studied. The effects of AMPK knockdown by siRNA in HCASMCs were confirmed by Western blotting analysis . The phosphorylation of MYPT1 and the phosphorylation of MLC20 were significantly increased in HCASMCs incubated with SPC . Madagascine significantly decreased both SPC-elevated phosphorylation of MYPT1 and the phosphorylation of MLC20. The inhibitory effects of madagascine on the phosphorylation of MYPT1 and the phosphorylation of MLC20 were abrogated by siRNA knockdown of AMPK , indicating that madagascine-induced phosphorylation of AMPK inhibited the activity of ROCK (the expression of phosphorylate MYPT1 represents the ROCK activity), leading to the vasodilatation of Ca2+-independent vasoconstriction. In order to prevent the false-positive results by ROCK independent phosphorylation of MYPT1 .In this study, madagascine exerted vasodilatory effects in rat MRAs and porcine VSM strips, respectively. The phosphorylation of AMPK was significantly increased by madagascine in a concentration and time dependent manner. The phosphorylation of eNOS in HUVECs was significantly increased and the phosphorylation of MYPT1 in HCASMC cells was significantly decreased by madagascine. These results suggest that AMPK-mediated activation of eNOS in epithelium and inhibition of ROCK/MYPT1 in VSM were involved in madagascine-induced vasodilatation . Madagascine significantly inhibited the VSM constriction induced by SPC and the inhibition was not abolished by compound C, suggesting that AMPK activation is involved in madagascine induced VSM relaxation. In HCASMC cells, SPC-induced increase in the phosphorylation of MYPT1 and the phosphorylation of MLC20 were significantly reversed by madagascine. AMPK knock down block madagascine-induced the inhibition of the phosphorylation of MYPT1 (represents for the ROCK activity) and the phosphorylation of MLC20. The results suggest that madagascine-induced AMPK activation is beneficial for amelioration of vasospasm as SPC-mediated activation of ROCK is found to be involved in pathogenesis of vasospasm (AMPK-mediated relaxation of VSM constriction is also observed where the endothelium is damaged , suggesttriction  and thisasospasm . Althougasospasm , it is bin vivo needs further study. The highest concentration of madagascine used in this study is 100 μM and it was shown that madagascine and its parent compound emodin shows almost no cell toxicity on normal cells but cancer cells (in vivo study. The aim of this study is to study the vasodilatory effect of madagascine on vasoconstriction in vitro, and to uncover weather AMPK activation is involved in madagascine induced vasodilatation. The other roles including potassium channel, alpha adrenergic receptors, and some vasodilatation factors in madagascine induced vasodilatation maybe, respectively, studied in future.This study studied the effects of madagascine on both endothelium and VSM because endothelium and VSM play different pathophysiological roles in vascular diseases , 2014. Fer cells . The ratCardiovascular diseases are often associated with co-occurring atherosclerosis, hypercholesterolemia, and inflammation, and madagascine induced activation of AMPK also is potentially beneficial for the prevention against and amelioration of the co-occurring disorders based on its modulation on VSM and circadian rhythm . MadagasYL, DC, FE, SK, and PS designed the study. DC, BL, PS, SH, and DY performed the tissue research. BL, YX, YJ, and FT performed the cell research. YL, SG, and FE contributed reagents or analytic tools. DC, SK, and YX analyzed data. YL and DC wrote and revised 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.


Interestingly, the long-used anti-alcoholism drug disulfiram (DSF), also known as Antabuse, emerged as the top hit. Enzymatic kinetics analyses revealed that DSF inhibited SNARK kinase activity in a noncompetitive manner to ATP or phosphosubstrates. Comparative in vitro analyses of DSF analogs indicated the significance of the disulfide bond-based molecular integrity for the kinase inhibition. DSF suppressed SNARK-promoted TGF-β signaling and demonstrated anti-HCC effects. The chemical and enzymatic findings herein reveal novel pharmacological effects of and use for DSF and its derivatives, and could be conducive to prevention and inhibition of liver fibrosis and HCC.We recently described that the anti-apoptotic AMPK-related kinase, SNARK, promotes transforming growth factor (TGF)-β signaling in hepatocellular carcinoma (HCC) cells, as a potentially new therapeutic target. Here we explored FDA-approved drugs inhibiting the enzymatic activity of SNARK, using an  Protein kinases are critically involved in nearly all cellular functions encompassing metabolism, gene expression, proliferation, motility, and death. Their dysregulation, contributes to serious diseases such as cancer, hypertension, Parkinson's disease, and autoimmune diseases , which ain vitro luminescence kinase assay system. Interestingly, the top hit in an FDA-approved drug library was disulfiram (DSF), the long-used anti-alcoholism drug also known as Antabuse. Enzymatic kinetics analyses revealed that DSF inhibited SNARK kinase activity in a noncompetitive manner to ATP or phosphosubstrates. While a close DSF analog tetramethylthiuram disulfide (TR) exerted inhibition, its monosulfide analog tetramethylthiuram monosulfide (TMTM) did not, indicating the significance of the disulfide bond-based molecular integrity of DSF for the kinase inhibition. DSF was indeed proved in cell culture to suppress SNARK-enhanced TGF-β signaling monitored through plasminogen activator inhibitor (PAI)-1 activity. DSF also exhibited anti-HCC effects, to which HCC cells were conferred resistance by excessive supply of SNARK. The successful discovery of a SNARK inhibitor here discloses a novel mode to explain the recently recognized anti-cancer effects and anti-fibrogenic potential of DSF , no, no50 42) Figure  while on) Figure , and nei) Figure , a furthM Figure  with milM Figure , wherefoM Figure .in vitro, while no inhibition of luciferase activity itself by DSF was confirmed in HepG2 cells with the overexpression of luciferase protein  in expectation of exploitation as a chemotherapeutic agent. Another attractive feature of DSF is the tolerability and safety demonstrated over years of clinical experience with a large number of patients, and also in the NSCLC patients [50 of the in vitro kinase assay described here, so SNARK inhibitory concentrations are expected to be achieved safely in patients. It should be cautioned that the possible novel clinical use of DSF would be in persons with chronic liver disease at risk for cirrhosis and HCC, and understandably toxicity would need to be carefully monitored especially in persons with advanced cirrhosis, as summarized in disease guidelines [In practice, therapeutic effects of DSF have been observed in metastatic non-small cell lung cancer (NSCLC) patients with an increase in survival  and solipatients . Plasma patients , 38, reaidelines . HoweverHere the enzymatic inhibition of SNARK and resultant anti-TGF-β/HCC effects by DSF provide us with new biochemical and pragmatic insights. The utility and a newly revealed property of DSF were mechanistically suggested and in turn the significance of regulatory sites for SNARK kinase activity as validated drug target can now be comprehensively assessed using DSF as a chemical probe. Also DSF derivatives with better potencies could be examined  in additDSF, and STS, TR, TMTM and Sodium diethyldithiocarbamate trihydrate were purchased from Selleckchem  and Sigma-Aldrich , respectively. DETC-MeSO was purchased from Santa Cruz Biotechnology . TGF-β was purchased from R&D . Antibodies to luciferase, HCV NS3, and FLAG and β-actin (ACTB) were purchased from MBL , Abcam , and Sigma-Aldrich, respectively. The FDA-Approved Drug Screen-well Library and Cell Counting Kit (CCK)-8 were obtained from Enzo Life Sciences  and Dojindo , respectively. Cytotoxicities of compounds were determined with the LDH cytotoxicity detection kit . HepG2, PLC/PRF/5, and Huh7 cells were cultured according to the protocols of American Type Culture Collection  and Japanese Collection of Research Bioresources Cell Bank . The cell lines were authenticated by short tandem repeat method  in Jan 2016. PXB cells were purchased from Phoenix Bio . JFH1 virus infection was performed as described previously .pCMV14-SNARK_NCBI-3×FL was constructed by primers EcoRI-SNARK-F 5′-TTGAATTCGGCCACCATGGAGTCGCTGGTTTTC-3′, XbaI-SNARK-ΔTGA-R 5′-AATTCTAGAGGTGAGCTTTGAGCAGAC-3′, SNARK-1323-G-F 5′-GGCAGGCTGCCCCGCTGCTC-3′, and SNARK-1323-G-R 5′-GAGCAGCGGGGCAGCCTGCC-3′, using pCMV14-SNARK-3×FL as a template . SequencNUAK2 Kinase Enzyme System was purchased from Promega . Kinase assay was performed according to the manufacturer's protocol and the luminescence was monitored (n = 4). Briefly, 12.5 ng of recombinant SNARK was incubated with CHKtide and ATP, in the presence or absence of DMSO or compounds, for 60 min at room temperature, followed by ATP depletion and subsequent detection of converted ADP via luminescence.The mode of enzymatic inhibition was calculated bySigmaplot , with the best fit models determined by the Akaike Information Criterion.Renilla luciferase activities from pRL-TK as described previously [Firefly luciferase activity was monitored by a dual-luciferase reporter assay system (Promega) and normalized to eviously . SiNT3 (eviously , with 3 SNARK and JFH1 normalized to that of GAPDH.Relative mRNA and viral RNA levels were quantified as previously described  using thTotal protein was resolved by SDS-PAGE and subjected to western blotting as described previously .


Tetrahymena cells provide such a model system because simple behavioral bioassays, gene knockouts, biochemical analysis, and other approaches can be used with these eukaryotic model cells. This work can contribute to the basic understanding of unicellular sensory responses and provide insights into the evolution of chemoreceptors and possible chemorepellent approaches for preventing infections by some pathogenic protozoa.Although many single-cell eukaryotes have served as classical model systems for chemosensory studies for decades, the major emphasis has been on chemoattraction and no chemorepellent receptor gene has been identified in any unicellular eukaryote. This is the first description of a gene that codes for a chemorepellent receptor in any protozoan. Integration of both depolarizing chemorepellent pathways and hyperpolarizing chemoattractant pathways is as important to chemoresponses of motile unicells as excitatory and inhibitory neurotransmitter pathways are to neurons. Therefore, both chemoattractant and chemorepellent pathways should be represented in a useful unicellular model system.  Tetrahymena thermophila contains a powerful chemorepellent for wild-type cells, and a gene called G37 is required for this response. This is the first genomic identification of a chemorepellent receptor in any eukaryotic unicellular organism. This conditioned supernatant factor (CSF) is small (<1 kDa), and its repellent effect is resistant to boiling, protease treatment, and nuclease digestion. External BAPTA eliminated the CSF response, suggesting that Ca2+ entry is required for the classical avoiding reactions (AR) used for chemorepulsion. A macronuclear G37 gene knockout (G37-KO) mutant is both nonresponsive to the CSF and overresponsive to other repellents such as quinine, lysozyme, GTP, and high potassium concentrations. All of these mutant phenotypes were reversed by overexpression of the wild-type G37 gene in a G37 overexpression mutant. Overexpression of G37 in the wild type caused increased responsiveness to the CSF and underresponsiveness to high K+ concentrations. Behavioral adaptation (by prolonged exposure to the CSF) caused decreases in responsiveness to all of the stimuli used in the wild type and the overexpression mutant but not in the G37-KO mutant. We propose that the constant presence of the CSF causes a decreased basal excitability of the wild type due to chemosensory adaptation through G37 and that all of the G37-KO phenotypes are due to an inability to detect the CSF. Therefore, the G37 protein may be the CSF receptor. The physiological role of these G37-mediated responses may be to both moderate basal excitability and detect the CSF as an indicator of high cell density growth.A conditioned supernatant from IMPORTANCE Although many single-cell eukaryotes have served as classical model systems for chemosensory studies for decades, the major emphasis has been on chemoattraction and no chemorepellent receptor gene has been identified in any unicellular eukaryote. This is the first description of a gene that codes for a chemorepellent receptor in any protozoan. Integration of both depolarizing chemorepellent pathways and hyperpolarizing chemoattractant pathways is as important to chemoresponses of motile unicells as excitatory and inhibitory neurotransmitter pathways are to neurons. Therefore, both chemoattractant and chemorepellent pathways should be represented in a useful unicellular model system. Tetrahymena cells provide such a model system because simple behavioral bioassays, gene knockouts, biochemical analysis, and other approaches can be used with these eukaryotic model cells. This work can contribute to the basic understanding of unicellular sensory responses and provide insights into the evolution of chemoreceptors and possible chemorepellent approaches for preventing infections by some pathogenic protozoa. Tetrahymena integrate sensory information in a manner that is similar to that of neurons in higher organisms. In ciliates such as Tetrahymena and Paramecium, chemorepellents generally cause depolarizations  resulted in not only loss of chemoattraction but also reduced G-protein activity (TTHERM_00633170) that we have named G37. In this report, we describe the altered responses of a G37 gene knockout mutant that we call G37-KO.Many sensory responses in eukaryotic cells are initiated by ligand binding to a G-protein-coupled receptor (GPCR), and several studies suggest that GPCR signaling does exist in ciliates , 7. We hTetrahymena thermophila as a simple model organism for the continuing study of chemosensory responses because of a number of important characteristics. Tetrahymena displays recognizable and reproducible changes in swimming behavior when exposed to chemoattractants   . Voltageswimming , 15. In emotaxis . As in b2+ levels  produced by eukaryotic unicells during growth, but no chemorepellent receptor has ever been matched to a factor in the growth medium. Such factors could be forms of intercellular communication between these unicells. A CMF and several other secreted factors have been described in ostelium . Some ofostelium , 28, butostelium , but no na vorax , and it na vorax . This fana vorax , but theTetrahymena.The objective of the current work was to identify the major phenotypic differences between this G37-KO mutant and the wild type in order to gain insights into the roles of chemorepellent receptors in both the basal and chemosensory responses of TTHERM_00633170 by GOModo is shown in G37 gene (TTHERM_00633170) encodes a protein that shows some homologies with a well-described GPCR from Dictyostelium  (Tetrahymena (GPCR6) (The protein structure predicted from the amino acid sequence of ed CAR1)  and the  (GPCR6) . The perG37 knockout construct to the macronucleus of vegetative wild-type CU427 cells as previously described  was grown under increasing concentrations of paromomycin to allow phenotypic assortment and complete replacement of the endogenous G37 gene with the Neo3 cassette. Genomic PCR confirmed the presence and successful recombination of Neo3 into the target locus by using primers specific to Neo3 and outer flanking sequences of the knockout construct (G37 gene expression was verified by reverse transcription (RT)-PCR of G37 cDNA  mutant . A map of this plasmid can be found at the Tetrahymena Stock Center webpage. This construct recombined into the RPL29 locus and conferred cycloheximide resistance on the transformants. RT-PCR confirmed the presence and expression of the ectopic G37 gene in the G37-KO mutant (To show that the wild-type phenotype could be rescued by the expression of wild-type ) mutant . We alsoO mutant . The var10.1128/mSphere.00182-17.1FIG S1 FIG S1, PDF file, 0.1 MB.Primers used for PCR analysis. Some of these were used to generate Copyright © 2017 Zou and Hennessey.2017Zou and HennesseyCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00182-17.2FIG S2 G37 gene was placed in this Cd2+-inducible ectopic expression plasmid. This construct recombined into the RPL29 locus and conferred cycloheximide resistance on the transformants. RT-PCR confirmed the presence and expression of the ectopic GPCR3-GFP-encoding gene in the G37 mutant. The G37 gene was functionally expressed because it rescued the wild-type phenotype in the G37-KO mutant the same as the G37-OE. It is interesting that there may be localization to the base of the cilia because the orderly rows suggest alignment with the basal bodies. However, we need to do many more colocalization experiments to test this. (A) The black arrows show some of the cilia, but many of the others are outside this focal plane. (B) The white arrow shows the region of the oral groove . Download FIG S2, PDF file, 0.1 MB.Localization of the GFP-tagged G37 protein in the wild type. The pchxMTTGFP plasmid  was used for G37-GFP localization studies. Vegetative CU427 cells were biolistically transformed with a construct where the Copyright © 2017 Zou and Hennessey.2017Zou and HennesseyCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Caenorhabditis cells with ImageJ  that we call Tetratracker .Although the CSF is a powerful chemorepellent of wild-type (CU427) cells, G37-KO mutant cells are insensitive to it. While the CSF-containing fraction reliably changed the swimming behavior of wild-type cells in a concentration-dependent manner, similar to what would be expected from a chemorepellent, it had no effect on the swimming behavior of G37-KO test cells . At a 20+ concentration. In stark contrast to its underresponsiveness to CSF, the G37-KO mutant overresponded to high K+ concentrations over the range tested .We used the same behavioral assay to test the responses of G37-KO to a general depolarizing agent, a high Ke tested . G37-KO + concentrations, they were also overresponsive to all of the other chemorepellents tested  (G37 codes for a dedicated chemorepellent receptor.There have been many reports of possible chemoattractants in ic acid)  and protic acid) . G37-KO ic acid) , furtherG37 expression in G37-KO resulted in both underresponsiveness to CSF and overresponsiveness to other chemorepellents, we wanted to know if both of these phenotypes could be reversed (rescued) by ectopic expression of the wild-type G37 gene in the G37-KO mutant. A Cd2+-inducible expression construct carrying the wild-type G37 gene was used to transform CU427 and G37-KO to create WT-OE and G37-OE cell lines, respectively. Since cells were always washed before testing, this added Cd2+ (0.5 µg/ml) did not interfere with the behavioral assays. These cell lines were tested for their total avoidance response time (TRT). The TRT is the elapsed time from the start of avoidance swimming to the resumption of normal forward swimming to 20% CSF and 5.0 mM KCl. We used this manual TRT measurement because some of the conditions caused continuous backward swimming and this was difficult to quantitate by the Tetratracker program. This TRT measurement provided a more convenient way to make comparisons between groups and also provided a measurement of adaptation and deadaptation times.Since the absence of G37 significantly increased the responsiveness of G37-OE   . The res(G37-OE) . Taken tG37 knockout) cause both a specific loss of response to the CSF and an overresponse to all of the other chemorepellents tested? This might happen if the CSF is always present in the growth medium, constitutively stimulating the wild type but not the G37-KO mutant. This could possibly lead to not only behavioral adaptation to CSF in the wild type but also cross adaptation to any other chemorepellents that use the same downstream response pathways. This general decrease in excitability would be seen in the wild type but not in the G37-KO mutant because the G37-KO mutant cannot sense the CSF. In comparison to the CSF-adapted wild type, the G37-KO mutant could show an overresponse to these compounds. We tested this hypothesis by adapting CU427 to the enriched CSF fraction , but since G37-KO cells do not respond to CSF, we could not use it to assay adaptation in G37-KO. Removal of the cells from the CSF resulted in deadaptation to the CSF . Expression of wild-type G37 in G37-KO (G37-OE) caused a return to wild-type response levels, showing that cross adaptation between CSF and high K+ concentrations can occur in rescued G37-KO mutant (G37-OE) cells  cells .Tetrahymena cultures grown at room temperature by the overnight incubation method. Both wild-type CU427 and G37-KO cultures yielded good CSF-containing supernatants, suggesting that G37-KO had no problem producing CSF.Supernatants containing CSF could be isolated from 3- to 14-day-old Tetrahymena (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA) on the responses to the CSF. External BAPTA (630 µM) eliminated the avoidance behavior of Tetrahymena in the CSF, suggesting that the activity of the CSF requires Ca2+ influx . Each image is a representative result of one experiment. Download FIG S3, PDF file, 0.1 MB.The absorbance spectrum of the CSF fraction and enrichment of the CSF. (A) The peak is around 255 nm, and there is no further absorbance in the visible range. (B) Separation of the enriched CSF. The crude CSF was separated on a Sephadex G25 column, and the enriched CSF eluted before the bulk of the material absorbing at 260 nm. The repellent activity was scored visually as 0 (no response), 1.0 (slow swimming), 2.0 (weak AR), 3.0 (repetitive AR), or 4.0 (CCR), and the average values of several observations were plotted. The repellent activity (orange line) was enriched in the earlier fractions containing ODCopyright © 2017 Zou and Hennessey.2017Zou and HennesseyCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 260) that contained almost all of the active CSF, according to behavioral testing . This suggests two immediate possibilities. Either both the T. vorax transformation factor and our CSF are in this same fraction or they are the same compound. Further purification of the CSF should resolve this.We generated a further enriched CSF preparation by column chromatography. We found that this method reproducibly yielded an early peak in optical density at 260 nm . However, all of the experiments described were done with the original G37-KO line.Verification of the Tetrahymena  these responses in G37-KO mutant cells. Overexpression of wild-type G37 in G37-KO mutant (G37-OE) cells caused them to return to wild-type CSF response levels, and overexpression of G37 in the wild type (WT-OE) caused an increase in the response of the wild type to the CSF. Similarly, the excessive high K+ concentration responses of G37-KO mutant cells were returned to nearly wild-type levels by overexpression of G37 (G37-OE), while overexpression of G37 in the wild type (WT-OE) caused a small decrease in the high K+ concentration responses. This showed not only the genetic linkage between underresponsiveness to the CSF and overresponsiveness to high K+ concentrations but also that the phenotypes caused by the G37 knockout mutation were specific for the G37 gene.Since + concentrations. Cross adaptation to high K+ concentrations does not happen in the G37-KO mutant, consistent with its inability to detect the CSF. Furthermore, rescue of the wild-type phenotype in the G37-KO mutant (G37-OE) also enabled behavioral cross adaptation to the CSF because G37-OE cells could now sense the CSF. This supports not only the idea that G37 is necessary for CSF adaptation but also the idea that this is not a specific CSF receptor event because cross adaptation to other chemorepellents is seen.We found that chemosensory adaptation to the CSF not only caused a decrease in the response of the wild type to the CSF but also cross adaptation to high KG37 knockout mutation and chemosensory adaptation to the CSF cause changes in many different types of depolarizing responses, it is likely that all of these responses have something in common. All of these responses require Ca2+ entry through the voltage-dependent Ca2+ channel. Therefore, it is possible that one of the main actions of the CSF-activated G37 receptor is to modify the activity of this Ca2+ channel.Chemosensory adaptation to the continued presence of the CSF may be the reason why G37-KO mutant cells are overresponsive to the other chemorepellents in comparison to the wild type. Since adaptation to the CSF appears to generally decrease the excitability of wild-type cells, their basal behavior in high-density cultures could be decreased because of the continued presence of the CSF. However, G37-KO mutant cells do not sense CSF and do not have this adaptation response, causing them to show greater basal excitability than the wild type. Since both the 2+ channels are one way to decrease the general excitability in these types of cells , and the G37-KO mutant with G37 overexpression (G37-OE) but not of the G37-KO mutant. Although this suggests that our CSF may be stomatin, it is also possible that the crude stomatin fraction contains both stomatin and our CSF. It is well known that Tetrahymena can release mucocysts and lysosomal enzymes into the growth medium and into a buffered solution  cells were obtained from Cornell University’s Tetrahymena Stock Center. All cells were cultured in 5 ml of SPP medium  supplemented with 250 µg/ml penicillin G and streptomycin sulfate and 1.25 µg/ml amphotericin B. For overexpression mutant cells, the medium was additionally supplemented with 0.5 µg/ml CdCl2 for 24 h to induce gene expression and 50 µg/ml cycloheximide as a selective agent. Long-term stock cultures were kept in the above-described medium at 15°C, whereas working cultures were kept at room temperature (25°C). Cells were washed in wash buffer  prior to behavioral testing.Wild-type G37 (TTHERM_00633170) macronuclear sequence, as well as 1.2 kb of its upstream and downstream flanking sequences, was cloned into the pCR4-TOPO vector. The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce ClaI and BamHI sites near the 5′ and 3′ termini of the G37 gene, respectively. The knockout construct was made by cutting the Neo3 antibiotic resistance cassette  from its vector with the introduced restriction sites and ligating it into the pCR4-TOPO vector to replace most of the G37 gene. This knockout construct was then introduced into vegetative CU427 wild-type cells by biolistic transformation. Macronuclear transformants containing the knockout construct were cultured in increasing concentrations of paromomycin (up to 20 mg/ml) for about 2 months to completely replace the endogenous G37 gene with the Neo3 cassette by phenotypic assortment. Complete gene replacement was confirmed by RT-PCR.Approximately 3.6 kb of DNA containing the G37 overexpression construct was made by cutting out the green fluorescent protein (GFP) sequence with PmeI and XhoI and replacing it with the genomic GPCR3 sequence. All constructs were introduced into vegetative CU427 or G37-KO cells by biolistic transformation. Macronuclear transformants containing the construct were selected with cycloheximide (50 µg/ml).The pChxMTTGFP plasmid  was modified to remove the PmeI restriction site upstream of the MTT1 promoter, leaving only one PmeI site downstream of the MTT1 promoter. To do this, the original MTT1 promoter was cut from the plasmid with PmeI and replaced with a nearly identical MTT1 promoter insert that had an EcoRV site instead of a PmeI site upstream of the promoter. The About 0.5 ml of CU427 from a stationary culture was inoculated into 50 ml of SPP medium in a 500-ml flask. This was cultured at room temperature for 14 days. The 14-day-old cells were washed in wash buffer by centrifugation at 2,500 rpm, and then the pellet was resuspended in 50 ml of wash buffer three times. After washing, the cell pellet was resuspended in 5 ml of wash buffer and gently rocked overnight in a 15-ml conical tube. The overnight suspension was then centrifuged, and the supernatant was decanted into a fresh tube. The tube containing the supernatant was sealed and placed in a boiling water bath for 30 min to allow precipitation of unwanted compounds. After the heated supernatant was again centrifuged at 2,500 rpm to eliminate precipitants, it was loaded onto a 10-kDa concentrator . This was centrifuged at 4,000 rpm for 1 h to completely pass the supernatant through the 10-kDa filter. The filtrate was the CSF-containing fraction. This is the fraction that was used in all of the experiments in this study.O-[γ-thio]triphosphate (GTPγS), and lysozyme were prepared in wash buffer, whereas test solutions of the CSF were prepared in water. To begin behavioral testing, 5 µl of the test solution was pipetted into a single well of a 10-well polytetrafluoroethylene printed slide . A 5-µl volume of test cells was then quickly mixed with the test solution for approximately 4 s, and the resulting mixture was filmed with a Moticam 480 camera  for 10 s at ×40 magnification. The behavior of test cells upon encountering the test solution was analyzed in the second, third, and fourth seconds of the 10-s video with a plugin for ImageJ that we call Tetratracker. Each cell track was analyzed for deviation from linear forward swimming paths, as well as path length. This information allowed calculation of the DCs and swimming speeds of multiple cells .Cells used for behavioral testing were grown in SPP medium for 2 days at room temperature. Prior to testing, cells were washed as follows. A total of 1,000 µl of cells was centrifuged in 1.5-ml microcentrifuge tubes at 7,000 rpm. A 20-µl volume of cell pellet was collected and suspended in 1,000 µl of wash buffer in 1.5-ml microcentrifuge tubes. The washed cells were kept at room temperature for 2 h prior to behavioral testing. Test solutions of KCl, quinine, guanosine 5'-Cells for the chemoattraction assay were washed as described above and then tested in a three-way stopcock assay , 33, 42.Test cells were grown in SPP medium for 2 days at room temperature and washed as described above. After 3 h of starvation at room temperature, 10 µl of the test cells was mixed with 10 µl of the CSF fraction or 10 µl of water for a specific period of adaptation (1 min to 1 h). A 980-µl volume of wash buffer was then added to the cells, and they were centrifuged at 7,000 rpm for 2 min. About 40 µl of the pellet was removed and placed into a fresh tube for testing. To test for cross adaptation to various repellents, 5 µl of 10 mM KCl, 80 µM quinine, 1 mM GTPγS, or 1 µM lysozyme was mixed with 5 µl of cells and the resulting mixture was filmed for 1 min with a Moticam 480 at ×40 magnification. The TRT of each cell was manually measured, and this was defined as the amount of time that elapsed until the cell resumed forward swimming after repellent addition. Deadaptation to CSF was tested by adapting test cells to the CSF for 1 h and then transferring them to wash buffer to be assayed over time in the wash buffer.Tetrahymena.The CSF-containing fraction was chemically treated by being mixed with various concentrations of pronase, proteinase K, RNase I, or DNase I for up to 8 h under manufacturer-recommended incubation conditions. Subsequent mixtures were directly tested on washed cells to determine if the avoidance effect of the CSF was abolished. The various enzymes were also tested on cells to determine whether they have any effect on the basal avoidance of 260 was measured. Each fraction was tested for avoidance effects on washed cells. Fractions with noticeable avoidance effects were combined and concentrated to the original input volume (0.5 ml) by boiling.About 0.5 ml of the crude CSF fraction was loaded onto a Sephadex G25 column  with deionized water as the liquid phase. Fractions of 0.75 ml were collected, and the OD12), uracil, and the active, concentrated CSF fractions from a Sephadex G25 column were sequentially loaded onto a fresh Sephadex G25 column with 0.1 M NaCl as the liquid phase. The elution profile of the MW standards was determined colorimetrically, whereas the elution profiles of uracil and CSF fractions were determined by measuring the OD260.Molecular weight (MW) standards (blue dextran, myoglobin, and vitamin B


The analysis of the PLI and Igeo indicated that site #1 was relatively clean, while sites #2 and #3 were heavily polluted. The results of the RAC showed that PTEs in top soils at sites #2 and #3 were significantly increased (p  <  0.05) for Cd and Zn. The HRA indicated that both As and Pb presented non-carcinogenic risks to children and adults at sites #2 and #3. Our findings can be a reference for risk prevention of industrially abandoned land in rural China.The potential toxic elements (PTEs) pollution problems in many rural industrial wastelands have been observed to be conspicuous. Therefore, 40 top soil samples were collected from the wasteland of a typical rural metallurgy factory in Baoding, China. The total concentrations of six key PTEs were measured. The soil properties and speciation of the PTEs were also identified. Extremely high concentrations of As, Cd, Pb, and Zn were observed in the surface soils. Using the PTEs concentration in the top soils of the rural industrial wasteland, the following indices of pollution were calculated: the pollution load index (PLI), the geo-accumulation Index (I Soils are generally regarded as a sink of potential toxic elements (PTEs) in terrestrial ecosystems. PTEs are one of the thorniest types of pollutants due to their broad distribution, hard degradation, and biological enrichment along the food chain . The excThe high emission of industrial wastes has produced numerous industrial wastelands around the world. Numerous metallurgical wastelands pose significant hazards to the health of the exposed population. For example, there are more than 450,000 sites contaminated with toxic chemicals in the United States (U.S.) .geo), potential ecological risk index (PERI), risk assessment code (RAC), enrichment factors (EF), and mean probable effect level quotient (m-P-Q) [Under environmental supervision by the Chinese government, numerous low-technology township enterprises, such as smelting factories, metal plating workshops, and chemical plants, have been shut down, and many industrial abandoned wastelands have resulted, which are mainly polluted by PTEs. These rural industrial abandoned wastelands not only encroach on large land resources but also bring about high levels of soil pollution, land degradation, and other detrimental effects. The remediation of PTEs contaminated sites is gaining considerable significance. Conventional technologies for site remediation are based on physical, chemical, and biological methods . However (m-P-Q) . These m (m-P-Q) , anthrop (m-P-Q) ,13, and  (m-P-Q) ,15. Howe (m-P-Q) ,17.geo; (3) the RAC and a health risk assessment (HRA) were used to estimate the environmental risk and human exposure risk to PTEs in a typical rural wasteland in North China. The findings will be particularly useful for risk prevention and the ecological reclamation of industrial waste sites in rural North China.To update the comprehensive understanding on the PTEs pollution of rural abandoned wastelands in North China, we chose a typical rural wasteland in southeast Baoding where rural industrial enterprises are densely distributed, and we evaluated the pollution levels and eco-risk by multiple assessment methods. The primary objectives of this study were: (1) to determine the spatial distributions and total concentrations of arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), and zinc (Zn) in the rural industrial abandoned site in North China; (2) to identify anthropogenic contamination of heavy metal in topsoil using the PLI and IThe study sites are in a suburb with an abandoned factory in southern Baoding, Hebei province, China, where small non-ferrous metallurgy enterprises are densely distributed. Three sites were investigated: site #1, site #2, and site #3 shown in . Based o2 exchange followed by compulsive MgSO4 exchange. Total nitrogen (TN) was measured by the Kjeldahl procedure. Organic matter (OM) concentration was determined by the Walkley-Black method. Particle-size distribution was measured using the dry sieving method to determine the sand, silt, and clay percentages. Then, the undersize soil samples were passed through a plastic sieve with a mesh aperture of 0.154 mm. Each time, 0.1 g of soil was poured into a polytetrafluoroethylene jar and digested with a guarantee reagent mixture HF-HClO4-HNO3 in an automatic digestion instrument . The digested solution was cooled, filtered, and diluted to 25 mL. The PTE concentrations  were determined by an axial view inductively coupled plasma-atomic emission spectrometer . A procedural blank and a standard reference material GBW07401  were included for quality assurance and quality control (QA/QC) (one blank and one standard for ten samples). The modified Community Bureau of Reference (BCR) sequential extraction procedure was used for PTE form analysis of all the soil samples. The four extracted fractions of PTEs were defined as the water/acid soluble fraction (F1), reducible fraction (F2), oxidizable fraction (F3), and residual fraction (F4) [Eight top soil samples 0–20 cm) from site #1 and 16 top soil samples from both sites #2 and #3 were collected using the random point method. These samples were placed in self-locking polyethylene bags and transferred to the laboratory. The soil samples were air-dried, ground, and passed through a 2 mm nylon sieve to remove roots, debris, glass, stones, and other impurities. Next, all samples were subjected to physico-chemical analyses. Soil pH was measured in a 1:2.5 soil:water suspension. Cation exchange capacity (CEC) was determined in triplicate by BaClion (F4) ,19,20. cm from 1 × PI2 × PI3 × … × PIn) 1/n. The pollution index (PI) is defined as PI = Ci/Ci0, where Ci is the concentration of the ith element in soil samples (mg/kg) and Ci0 is its corresponding reference concentration (mg/kg). The values of Ci0 used in this article are the national first-level standard values provided in GB15618-1995 [The integrated PLI reveals the overall pollution status of a sample. The PLI can be calculated from , where Cn is the measured concentration of the element (mg/kg), and Bn is the background value of the element (mg/kg). The constant factor of 1.5 in the equation indicates very small anthropogenic influence on the contents of investigated PTEs in the natural environment [geo are given as follows: no pollution (Igeo ≤ 0), no-to-moderate pollution (0 < Igeo ≤ 1), moderate pollution (1 < Igeo ≤ 2), moderate-to-heavy pollution (2 < Igeo ≤ 3), heavy pollution (3 < Igeo ≤ 4), heavy-to-extreme pollution (4 < Igeo ≤ 5), or extreme pollution (Igeo > 5) [Iironment ,25. In tgeo > 5) .RAC assesses the availability of metals in a solution by applying a scale to the percentage of PTEs present in the water/acid-soluble fraction (F1) ,28. Fiveing), dermal contact , and inhalation (ADDinh) for both adults and children were estimated as follows:The HRA of topsoil quantitatively reveals both carcinogenic and non-carcinogenic risks of the three exposure pathways  to humans . The aveThe detailed explanation of the exposure parameters and values used to estimate the risks ,32 are gThe non-carcinogenic effects of PTE were assessed using the hazard quotient (HQ). The HQ is the ratio of the ADD of a PTE to its reference dose (RfD)  for the −6), acceptable or tolerable (1 × 10−6 < RI < 1 × 10−4), and high (RI > 1 × 10−4).HQ or HI ≤ 1 indicates that non-carcinogenic risks are unlikely even for sensitive populations, whereas the potential for adverse effects may be a concern when HQ or HI > 1 . As, Cd,p < 0.05 as significant in the top soils of three abandoned sites.The experimental data were analyzed using a one-way ANOVA (SPSS 16.0). The Turkey’s test was used to test the difference between various soil physico-chemical factors, taking On the basis of previous research, the pH, OM content, nitrogen content, and particle size distribution of the three sites are shown in The statistics of six PTEs in top soils are presented in The toxicity and mobility of PTEs were not only related to their total concentrations, but also their chemical speciation, their binding state, the metal properties, environmental factors, and soil properties like pH, OM content, and soil type . The F1 The dominant phase of Pb in the top soils of sites #1 and #3 was F3, indicating its integration with OM and lower leaching rate under prevailing environment conditions. However, the dominant phase of Pb in the top soil of site #2 was F2, indicating the anthropogenic Pb was mainly accumulated in the oxide fraction. This result is consistent with the findings indicating that Fe and Mn hydrous oxides have a strong adsorption capacity for Pb ions in soils and have an important effect in reducing its mobility in edatope .The PLI is proposed as a standardized system for assessing the overall pollution status that permits a comparison of pollution levels between different sites. It was first used for quantifying the estuarine quality in the simplest way  and is gThe average PLI for site #1 was slightly higher than 1, suggesting that site #1 was basically unpolluted to moderately polluted by the tested trace metal(loid) elements. The average PLI for site #2 was less than 5, indicating the top soils of site #2 were highly polluted by lead smelting slag. However, the average PLI for site #3 substantially exceeded 5, indicating the top soils of site #3 were seriously polluted by the Zn residue dumping. The different pollution levels of the three sites demonstrated by the PLI relate to the metal concentration levels and different pollution sources.geo considers the human factors, geochemical background values, and the impact of diagenesis on background values, which directly reflect the degree of PTEs and the metal enrichment in soil. As shown in geo indicate no pollution by Cr or Cu, low As pollution, partial median pollution by Pb and Zn, and partial serious Cd pollution. These results indicate that Cd, Pb, and Zn concentrations are heavily affected by anthropogenic inputs, surface runoff, and dust from nearby contamination sources, while Cr and Cu were mainly of geochemical origin. At site #2, the mean Igeo of six PTEs indicate no Cr pollution, partial median Cu pollution, as well as partial serious-to-severe pollution by four PTEs (Cd > Pb > Zn > As > Cu). At site #3, the mean Igeo of four PTEs is greater than 4 (Pb > Cd > Zn > As > Cu), indicating significantly high pollution levels; the mean Igeo of Cu is 2.02, and the mean Igeo of Cr is slightly higher than zero  between sites #2 and #3. The results indicated that Cd at sites #3 showed geo-accumulation in an acid condition. Fine grain sediments and more OM facilitate the accumulation of more PTE contents. Therefore, sites #2 and #3 with higher OM may increase the Pb accumulation [Ihan zero . The resl origin . The resmulation .Generally, PTEs in the F1 are bound to carbonates by relatively weak electrostatic interactions and could be more rapidly absorbed by biota . Therefogeo). Although the total concentrations of As and Pb showed heavy contamination according to the Igeo, the lower RACs of As and Pb came from their low level of association with the F1. These results indicated that the release of As and Pb in the top soil of two industrial wastelands were unlikely under prevailing environmental conditions, and so these elements showed a low toxicity to the surrounding area. However, the highest RAC value of Cd at site #3 was more than 50%, suggesting a very high environmental risk to the ecosystem. The RAC of Zn at site #3 was the largest among three sites, with a value of 24.69%, indicating a medium risk to the environment. The RAC method has been reported in previous studies, and similar evaluation results for Igeo and RAC have been reported by several authors [geo based on the total contents of pollutants. The low background levels of As and Pb . Compared with site #1 (control plot), the RAC values of PTEs at sites #2 and #3 significantly increased (p  <  0.05) for Cd and Zn, suggesting that there was serious contamination of Cd and Zn in the top soils due to historical industrial activities. PTEs introduced by anthropogenic activities in the Chinese rural abandoned waste sites carry a potential risk to the ecological environment in terms of speciation  and thus deserve more attention.The RACs for As, Cr, Cu, and Pb among the rural industrial abandoned sites indicate a low risk (LR) to the environment, which was not in accordance with other evaluation methods  for children decrease by the exposure pathway in the following order: ingestion > inhalation > dermal contact, and the HQs of Cd change in the following order: ingestion > dermal > inhalation contact, which is the same as in other studies [−6, except for As, where the RI value for children was slightly higher than 10-6, indicating its slight carcinogenic risk at site #2. In addition, the toxicity of Cr is mainly dependent on its valence state, and the Cr (VI) is more toxic to biota than Cr (III) [At site #2, the HQ studies . These r studies ,55. The Cr (III) . The totCr (III) .ing, with 99.98% for children and 99.67% for adults, suggesting that ingestion is the main exposure pathway that threatens human health. This conclusion is consistent with other studies [−6, suggesting the carcinogenic risks of As and Cr at site #3 can not be neglected, and children are faced with more health risks in daily life than adults via the unconscious ingestion pathway. However, the RI value of Cr may also be overestimated in the top soils at site #3 because the total mean concentration of Cr was used, which was lower than the second-class National Soil Environment Standard (pH < 6.5).At site #3, the HQs of As and Pb are larger than 1, which indicated there were adverse health effects on both children and adults via ingestion. For the six PTEs, the contributions to HI  are the highest for HQ studies ,58. The geo, and RAC results, the evaluations using different approaches can provide some valuable recommendations for government regulators and environmental workers to discern priority control PTEs, develop an economically restoration strategy, and manage the rural abandoned industrial wasteland effectively in rural China [Although sites #2 and #3 were two different kinds of metallurgy slag polluted land, both were similar in their HRA results for their total concentration of PTEs. Previous studies have reported that the most dangerous exposure route of non-cancer risk is direct oral ingestion ,60. Chroal China .geo suggested that As, Cd, Pb, and Zn are key toxic elements and priority control PTEs at both sites #2 and #3. For site #1, there were almost no health risks for adults because the HQs, HIs, and RIs were all lower than the threshold levels. However, the HQing and HI values of As and Pb for children and adults were above the threshold levels, indicating that the health of children and adults may be affected by As and Pb after long-term exposure to sites #2 and #3. The main exposure pathway for both children and adults was ingestion, followed by dermal contact and inhalation. The RI values of As and Cr for children were slightly higher than 10−6, suggesting that carcinogenic risks at site #3 could not be neglected. Thus, risk recognition and further enhanced natural revegatation measures are needed to reduce exposure risks for susceptive groups in hazardous environments. Based on the site risk assessments in this research, safe landfill of high concentration slags combined with in-situ phytostabilization by indigenous species may be an economical and ecological solution for rural industrial abandoned sites remediation in North China in the future.A typical metallurgical wasteland in a rural area of North China was chosen to represent the high pollution and widely distributed industrial abandoned wastelands in vast rural areas of China. The concentrations, pollution levels, and health risk assessment of PTEs in the topsoil at three sites of an abandoned rural factory in Baoding were thoroughly investigated. In general, the results of the PLI showed that the abandoned industry wasteland (sites #2 and #3) was heavily polluted by toxic trace elements compared with site #1. Furthermore, the results of the I