Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.n = 10, supervised moderate-intensity aerobic exercise program) and non-trained . Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls  for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise , and the heart rate recovery (ΔHRR)  and second (ΔHRR2) minutes of recovery after exercise).Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained . In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion , peak speed , peak VO2 , CR , and in ΔHRR1 and ΔHRR2  in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis . SLEDAI-2K scores remained stable throughout the study.The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.NCT01515163. Systemic lupus erythematosus (SLE) is a life-long systemic autoimmune disease with a large variability in its clinical course. The onset of SLE during childhood and adolescence represents 10 to 20% of all SLE cases .Both disease-related  and traditional factors  have been implicated in the increased CVD risk in SLE -7. AdditIn this context, exercise training has emerged as a potential non-pharmacological therapeutic strategy to counteract physical dysfunction in adult SLE ,9,14-16,A few studies have demonstrated the beneficial role of exercise in other pediatric rheumatic diseases, including juvenile dermatomyositis (JDM) ,23 and fThus, the aim of the present study was to evaluate the safety and the efficacy of a 12-week supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.A 12-week randomized trial was conducted between May 2010 and April 2011 at the Laboratory of Physical Conditioning in Rheumatology of the School of Medicine, University of Sao Paulo, Brazil. This trial was registered at clinicaltrials.gov as NCT01515163. The sample consisted of children and adolescents with C-SLE from the outpatient ambulatory of the Pediatric Rheumatology Division of the Children's Institute of the School of Medicine, University of Sao Paulo, Brazil, who were randomly assigned to participate in a three-month supervised exercise training program  or to remain physically inactive . Randomization was accomplished through a block randomization procedure with block sizes of four individuals. Body mass index-, gender- and age-matched healthy children from the surrounding community were recruited as controls (C). The patients fulfilled the revised American College of Rheumatology criteria  for C-SLAt baseline (PRE) and 12 weeks after the intervention (POST), clinical (that is SLEDAI-2K  and SystThe exercise program consisted of 12 weeks of twice-weekly supervised moderate-intensity aerobic exercise training. Training sessions comprised a 5-minute warm-up followed by 20 to 50 minutes of treadmill aerobic training (a 10-minute increment in the aerobic training volume was applied every four weeks), and 5 minutes of cool-down on the treadmill at a low speed followed by stretching exercises. All of the training sessions were monitored by at least one fitness professional and a Rheumatologist monitored adverse events on a weekly basis. The exercise program was performed in an intra-hospital gymnasium. Aerobic training intensity was set at the corresponding heart rate between the ventilatory anaerobic threshold (VAT) and 10% below the respiratory compensation point (RCP). All of the patients were able to achieve the set aerobic training intensity.2) and carbon dioxide output were obtained through breath-by-breath sampling and expressed as a 30-second average using an indirect calorimetry system . Heart Rate (HR) was continuously recorded at rest, during exercise, and at recovery, using a 12-lead electrocardiogram . Peak oxygen consumption (peak VO2), VAT and RCP were determined according to previously described criteria [The cardiorespiratory exercise test was performed on a treadmill , using a maximal graded exercise protocol . The trecriteria .HR response during exercise was evaluated by the chronotropic reserve (CR) as follows :HR recovery was defined as the difference between HR at peak and at both the first (ΔHRR1) and second (ΔHRR2) minutes after exercise. Absolute change (Δ) was used to calculate the difference between the HR at peak exercise and at the first and second minutes after the exercise test. The relative change in HR (Δ%) was calculated for the intervals between rest to VAT, rest to RCP, and rest to peak exercise.2) was determined by dual-energy x-ray absorptiometry (DXA), using a Hologic QDR 4500 Discovery densitometer . Bone area (BA) (cm2) was calculated using the software provided with the densitometer. Bone mass was analyzed in the lumbar spine (lumbar vertebrae L1 to L4), total femur, and whole body. The whole-body analysis was conducted without including the head, as it has been described to increase variability in children [3). BMAD was assessed by dividing the BMD in a given site  by the square root of the corresponding bone area (BA) (BMAD = BMD/√BA) [2 at the lumbar spine, 0.039 g/cm2 at the total femur, and 0.020 g/cm2 at the whole body. Body composition  was assessed through the same densitometer, using pediatric software.BMD (g/cmchildren . To miniBMD/√BA) . All of BMD/√BA) . Least st-test. After the normality and homogeneity of the variance were confirmed, the dependent variables were compared using a mixed model for repeated measures, assuming groups and time as fixed factors and subjects as random factors. Single degree of freedom contrasts were used to determine significant differences between groups. Finally, effect sizes (ES) were estimated for the POST assessments using the pooled SDs of the two independent samples at POST [P < 0.05. All of the analyses were performed using SAS 9.2, SAS Institute Inc., Cary, NC, USA.The data are presented as means and SDs. Fisher's exact test was used to assess baseline differences between the C-SLE groups in the drug regimen. The disease-related parameters  at baseline were compared using the Student's  at POST . The sigP > 0.05). The exercise adherence rate was 94.0 ± 7.03%.Twenty C-SLE patients were randomly assigned to the experimental groups (TR = 10 and NT = 10). One patient from the NT group withdrew from the study due to personal reasons and was excluded from the analysis. Thus, 19 patients were included in the analysis  . In addition, the relative change in HR (Δ%) from rest to VAT, rest to RCP, and rest to peak of exercise, and the HR recovery  were also significantly lower in the C-SLE patients when compared with healthy controls . Importantly, all of the parameters were similar between the TR and NT groups .The exercise and cardiorespiratory parameters at baseline are shown in Table P < 0.05, within-group comparisons). In contrast, the TR group presented significant increases in time-to-exhaustion  and peak speed  and a decrease in resting HR  when compared with the NT group. Additionally, the TR group had increased peak VO2 , CR , relative change in HR (Δ%) from rest to VAT , and rest to peak of exercise , and ΔHRR1  and ΔHRR2  in comparison to the NT group. Moreover, after the aerobic exercise training program, the latter cardiorespiratory responses to exercise were comparable between the trained group and the healthy control group (P > 0.05) .SLEDAI-2K scores remained unchanged throughout the study for both groups . Similarly, the other drugs (type and dose of medications) remained stable throughout the study. Additionally, the levels of ESR, CRP, C3 and C4 and anti-DNAds antibodies were unaltered in both groups throughout the study . Finally, there was no clinical evidence of excessive exhaustion, pain, osteoarticular injury, muscle soreness, or any other adverse event.Prednisone dosage was also unchanged in both groups after the intervention . Additionally, no significant changes were observed in any of these parameters after the intervention   Table .To the best of our knowledge, this is the first randomized controlled trial study aimed at investigating the effects of an exercise training program in a cohort of C-SLE patients. The main finding of this study is that a supervised aerobic exercise training program was safe and effective in improving the cardiorespiratory capacity in C-SLE patients.2) and exercise tolerance  in C-SLE patients when compared with their healthy peers. Moreover, we showed for the first time that C-SLE patients presented an abnormal HR response during and after the cardiorespiratory test , which are considered as important non-invasive markers of autonomic dysfunction [C-SLE is commonly associated with poor physical function and low levels of physical activity ,12, possfunction -39. Our function ,41. Howeet al. [et al. [2max), and quality of life in SLE patients aged 18 to 55 years. Although these studies point out the potential therapeutic role of exercise in SLE, some limitations  preclude definitive conclusions. Moreover, studies aimed at investigating the effects of exercise on physical capacity in pediatric rheumatologic patients are still scarce. Nonetheless, we have recently shown that a three-month combined endurance and resistance exercise training program induced improvements in the cardiorespiratory capacity , muscle strength, and physical function in patients with JDM [2 peak and time-to-exhaustion in a 15-year-old boy with antiphospholipid syndrome and C-SLE [Exercise has been shown to be a potential therapeutic tool in the management of adult SLE. Three pilot studies demonstrated that supervised exercise training can alleviate fatigue in adult SLE patients ,14,15. Fet al.  observed [et al. , reportewith JDM . Furthernd C-SLE . Importa2, peak speed, and time-to-exhaustion in a maximal graded exercise test in C-SLE patients. Noticeably, after the exercise training program, these parameters were comparable with those of their healthy peers, suggesting that the exercise training was effective in offsetting the C-SLE-related impairment in physical function and aerobic capacity.Specifically, we showed that a three-month aerobic training program was effective in improving resting HR and peak VOAccordingly, we demonstrated that the exercise training program was also effective in attenuating the impaired CR and the delayed post-exercise HR recovery in C-SLE patients, supporting the homeostatic role of exercise in C-SLE, particularly regarding the autonomic dysfunction. The present results corroborate the positive effects of exercise training upon autonomic control in adult patients with SLE  and CVD Children display a high bone turnover rate and a more pronounced change in bone mass in response to exercise training than later in life. In fact, we have previously demonstrated that a short-term (12 weeks) exercise training program comprising aerobic and resistance exercises was sufficient to increase BMAD in JDM patients . ConversFinally, it is important to emphasize that the exercise training program did not provoke disease flare, as evidenced by lack of changes in disease activity scores and laboratory parameters of inflammation. Additionally, the drug regime was virtually unchanged throughout the study and no other adverse events were reported. Collectively, these findings suggest that a supervised moderate-intensity aerobic training program may be safe for C-SLE patients, supporting previous studies with adult SLE patients ,9,14,15.This study presents some limitations. First, our sample may be considered heterogeneous since the patients presented a large range of active disease scores. The selection of patients with different active disease scores was intentional and aimed at increasing the external validity of our study. However, we acknowledge that the number of patients enrolled in this trial did not allow us to compare the potential differential responses of active and non-active subjects to the exercise training. Therefore, further studies with larger samples comparing the exercise responses as a function of active disease are indeed necessary. Second, the healthy children did not participate in the exercise training program, thus precluding any comparison between patients and controls with respect to adaptive responses to exercise . Finally, this was a short-term intra-hospital supervised intervention. Further studies should investigate the efficacy, safety and feasibility of other training program . Moreover, it is imperative to investigate the long-term effects of exercise  in more robust outcomes .In conclusion, this study demonstrated for the first time that a three-month supervised moderate-intensity aerobic exercise training program can be safe and effective in ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients. These findings stress the potential role of exercise training in the management of C-SLE, strengthening previous evidence of the beneficial effects of exercise in other pediatric rheumatic diseases.2: oxygen consumption; VO2 peak: peak oxygen consumption.BA: bone area; BMAD: bone mineral apparent density; BMC: bone mineral content; BMD: bone mineral density; BMI: body mass index; C: healthy control group; C3: complement 3; C4: complement 4; CR: chronotropic reserve; CRP: C-reactive protein; C-SLE: childhood-onset systemic lupus erythematosus; CVD: cardiovascular disease; ΔHRR: heart rate recovery; ΔHRR1: heart rate recovery at the first minute; ΔHRR2: heart rate recovery at the second minute; DXA: dual-energy x-ray absorptiometry; ES: effect size; ESR: erythrocyte sedimentation rate; HR: heart rate; JDM: juvenile dermatomyositis; NT: non-trained C-SLE group; POST: 12 weeks post-intervention; PRE: pre-intervention (baseline); RCP: respiratory compensation point; RER: respiratory exchange ratio; SLE: systemic lupus erythematosus; SLEDAI: systemic lupus erythematosus disease activity index; SLICC/ACR-DI: Systemic Lupus International Collaborating Clinics/ACR Damage Index; TR: trained C-SLE group; VAT: ventilatory anaerobic threshold; VOThe authors declare that they have no conflicts of interest.Study conception and design: DMLP, CAS, BG, ALSP, EB, and HR. Acquisition of data: DMLP, CAS, AMES, RMRP, APH, and FBB. Analysis and interpretation of data: DMLP, CAS, AEMS, FBB, RMRP, ALSP, APH, BG, EB, and HR. All of the authors were involved in drafting the article or revising it critically for important intellectual content, and all of the authors approved the final version to be submitted for publication. The authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.


Trypanosoma brucei. We show here that TbSAXO is a microtubule stabilizing protein with properties similar, upon cold and nocodazole treatment, to those of the microtubule-stabilizing Mn domains of the MAP6 proteins, thus identifying the first MAP6-related protein in a protozoan. Further, we demonstrate, in the parasite, that TbSAXO is an axoneme-associated protein, which plays a role in flagellum motility. We also show that TbSAXO is the first member of a group of MAP6-related proteins (that we named SAXO proteins) present only in organisms with centrioles / cilia / flagella and ranging from protozoa to mammals, suggesting potential roles of the SAXO proteins in cilia and flagella function.The microtubules (MTs) of most vertebrate tissue cells will disassemble at low temperature, but some remain cold-stable or resistant to drugs such as nocodazole. It has been shown that MT cold- and nocodazole-resistance is largely due to the association with the class of Microtubule Associated Proteins (MAP) known as MAP6 (previously named STOP for Stable Tubule Only Polypeptide) . MAP6 prhttp://mcmp.aquitaine.cnrs.fr/mfp/team_bct_en.php


Within the last 30 years, a substantial number of interventions for alcohol use disorders (AUDs) have received empirical support. Nevertheless, fewer than 25% of individuals with alcohol-related problems access these interventions. If several intensive psychosocial treatments are relatively effective, but most individuals in need do not access them, it seems logical to place a priority on developing more engaging interventions. Accordingly, after briefly describing findings about barriers to help-seeking, we focus on identifying an array of innovative and effective low-intensity intervention strategies, including telephone, computer-based, and Internet-based interventions, that surmount these barriers and are suitable for use within a stepped-care model. We conclude that these interventions attract individuals who would otherwise not seek help, that they can benefit individuals who misuse alcohol and those with more severe AUDs, and that they can facilitate subsequent help-seeking when needed. We note that these types of low-intensity interventions are flexible and can be tailored to address many of the perceived barriers that hinder individuals with alcohol misuse or AUDs from obtaining timely help. We also describe key areas of further research, such as identifying the mechanisms that underlie stepped-care interventions and finding out how to structure these interventions to best initiate a program of stepped care. Within the last 30 years, a number of psychosocial interventions for alcohol use disorders (AUDs) have received empirical support. Evaluations of these interventions have employed well-controlled randomized trials involving large numbers of individuals and substantial follow-up periods. These trials provide strong support for such interventions as motivational enhancement, cognitive-behavioral treatment, and 12-step facilitation treatment ), computer, or the Internet. Both original articles and meta-analyses were deemed suitable. Our review was performed using the electronic databases of the US National Library of Medicine (PubMed) and the American Psychological Association (PsycInfo) to identify relevant articles published from 1990 through September 2011. The term “alcohol” was combined with the following search terms: web-based intervention, Internet intervention, online intervention, bibliotherapy, text message, book intervention, telephone intervention, remote intervention, computer intervention, and self-help intervention (excluding Alcoholics Anonymous [AA] or 12-step). The term “intervention” was added to most search terms to identify the most relevant articles. In addition to searching electronic databases, we examined potentially relevant articles cited in the identified articles’ reference section .Using these procedures, we identified a total of 686 articles via PubMed and an additional 382 articles via PsycInfo (1068 unique articles). The titles of all identified articles were examined for correspondence with the study inclusion criteria. This led to the identification of 77 articles of potential interest for further review. The content of these 77 articles was carefully examined by two reviewers who further excluded studies that had the following characteristics: provision of face-to-face care, an insufficient follow-up rate to evaluate the intervention including failure to account for missing data, participant age <18, college samples , or usep2) were obtained directly from the original study calculations in all but four cases [Toolkit for Practical Meta-Analysis[Effect sizes presented  yielded more heterogeneous outcomes, only some of which tended to support the use of bibliotherapy.Sobell and colleagues  used medDrink Wise: How to Quit Drinking or Cut Down[Cunningham and colleagues  assessed Cut Down was chos Cut Down. At six-Feedback, Responsibility, Advice, Menu of options, Empathy, and Self-efficacy) with a goal of motivating individuals to begin the drinking change process prior to initiating treatment. Rates of treatment entry were 10% higher for the leaflet than the no-leaflet group, although the effect was not statistically significant . This study is the only one included in this review to specifically investigate the impact of a low-intensity intervention on treatment entry.A study by Bamford and colleagues  examinedWild and colleagues  studied 2(1) = 7.46, p = 0.006). Participants cut their drinking almost in half but continued to drink at fairly high levels, with men reporting 27 drinks per week and women 14 drinks per week. Even so, the reduction in drinking is notable given that the average AUDIT score at intake was 22.3, indicating moderate alcohol dependence.Kavanagh and Connolly  evaluateTelephone interventions increase accessibility of care by eliminating the need for travel to a treatment center . We idenA second telephone study  randomizThe benefits of computer-based interventions include the potential for remote access, the ability for individuals to choose content they prefer, and increased appeal of multimedia applications. Hester and coworkers  evaluateAnother study  evaluate2 = 6.67, p = 0.01). At six-month follow-up, the intervention effects were less strong, with drinking within recommended limits reported by 46% and 37% of the intervention and control conditions, respectively .A third study  evaluateThe Internet offers another method for reaching individuals with AUDs. One form of Internet-based intervention involves assisting individuals in assessing and evaluating their own drinking. Cunningham and colleagues  tested tA study by Pemberton and colleagues  comparedComplex web-based alcohol treatment involves more elaborate forms of engagement beyond feedback interventions, such as communication between users, communication with clinical personnel, and/or content meant to be perused over weeks or months. The web-based “Drinking Less” intervention  includesDiagnostic and Statistical Manual of Mental Disorders, 4threvision (DSM-IV) criteria for mild alcohol dependence, and 76% reported never having received treatment for their alcohol problems.An e-therapy alcohol intervention  includedAnother intervention study of problem drinkers, conducted by Blankers and colleagues , evaluatA study of the “Down Your Drink” web-based intervention  compared2(1) = 28.3; p = 0.001; OR, 9.4; 95% CI, 3.7-23.9). The reductions in drinking remained at three-month follow-up.The “Drinking Less” intervention described earlier was also tested via a television-supported platform . ProblemA variety of low-intensity interventions can engage individuals and effectively reduce drinking. Moreover, these strategies offer easier access and flexibility to individuals who misuse alcohol and circumvent some of the barriers to entry into traditional treatment. They also offer the potential for greater privacy, although strong encryption and other safeguards are needed to ensure that individuals’ data remain private and confidential for online interventions. In the following section, we summarize three key findings from existing studies of low-intensity interventions and identify three important areas for future research on stepped-care intervention models.Low-intensity interventions attract individuals who have not previously sought help. Several of the studies discussed herein included participants who had never sought treatment previously or who expressed disinterest in formal treatment options but accepted the low-intensity intervention. Mail [on. Mail , Interneon. Mail ,48, and on. Mail  intervenon. Mail ,35. Noneon. Mail  than wheon. Mail . AnotherLow-intensity interventions can benefit individuals with more severe AUDs. Meta-analytic studies suggest that brief interventions conducted in settings such as primary care tend to be more effective for individuals with less severe AUDs [ere AUDs . Similarere AUDs  found there AUDs , anotherere AUDs , and twoere AUDs -49—despiLow-intensity interventions may lead to subsequent help-seeking. Low-intensity interventions may inspire a self-change attempt which, if unsuccessful, leads to subsequent help-seeking [-seeking . Almost -seeking . Almost -seeking  sought s-seeking  indicate-seeking . Thus, c-seeking -21, low-Assessing subsequent help-seeking in the context and aftermath of low-intensity interventions could provide valuable data to inform stepped-care interventions. We need to know whether individuals seek help after a low-intensity intervention because they resumed problematic drinking  and/or whether low-intensity interventions help dispel concerns about treatment and thereby increase individuals’ motivation to enter into it.How can low-intensity interventions be structured to initiate a stepped-care intervention? In general, low-intensity interventions appear well-suited as the first step of a stepped-care intervention [rvention ,47,48. HAnother potential implementation pathway for low-intensity interventions is as population-based interventions that focus more on the overall impact on groups of individuals than on efficacy for each individual. Within this framework, the “impact” of an intervention is not only its efficacy, such as a 5% decrease in hazardous alcohol use, but the efficacy multiplied by the number of participants . The effIn countries such as the US, where higher intensity treatment options are widely available, a variety of low-intensity interventions can be included as part of population-based stepped intervention, as currently exists with “AlcoholScreening.org” . SimilarThe success of smoking quitlines offers another model for population-based alcohol interventions . SmokingWhat are the mechanisms that underlie the benefits of low-intensity interventions? Gaining a better understanding of how low-intensity interventions work, particularly within the process of seeking help, can inform the creation and implementation of stepped-care interventions. We know that alcohol-related problems, particularly multiple problems, predict help-seeking and reductions in drinking. All of the low-intensity interventions in this review provided online or telephone feedback about negative consequences associated with drinking [drinking ,48, autodrinking , or selfdrinking ,47. Withdrinking , all of drinking . SupportWe also need to examine whether certain aspects of low-intensity interventions, such as feedback about negative consequences of drinking or providing information about normative drinking, are especially likely to lead to subsequent treatment seeking. Is an initial assessment and evaluation sufficient to motivate help-seeking, or is participation in the intervention and an unsuccessful quit attempt the key to help-seeking? Gaining a better understanding of how low-intensity interventions help individuals recognize the severity of their alcohol-related problems offers the hope of telescoping the normal help-seeking process and thereby averting a considerable degree of alcohol-related harm.Are some low-intensity interventions more beneficial for some groups  than others, and are there additional low-intensity interventions that should be considered? Design of stepped-care interventions should take into account the potential for low-intensity interventions to be more or less effective for some groups. More information is needed about the extent to which low-intensity interventions are effective across diverse population groups. As noted, Brown and colleagues [lleagues  found thlleagues  than whelleagues . It may lleagues  to modiflleagues , future A recent report by the Pew Research Center’s Internet and American Life Project  indicateRather than developing new forms of intensive treatments for AUDs when current treatments work reasonably well, more effort should focus on expanding the reach of treatment by developing more accessible interventions and exploring how to integrate them into existing alcohol treatment systems. Low-intensity interventions are flexible and can be tailored to address many of the perceived barriers that hinder individuals with AUDs from obtaining timely help. Substantial evidence indicates that low-intensity interventions can engage individuals who shun current treatment options, reduce problematic alcohol use, and may even motivate individuals who need it to engage in more intensive treatment. Given the existence of effective low- and higher intensity interventions to address AUDs and the low levels of treatment uptake, greater attention needs to be focused on implementation-oriented aspects of stepped-care interventions. Several issues regarding the implementation of stepped-care interventions still need to be addressed by the literature, such as identifying the best structure for stepped-care models, understanding the impact of stepped-care interventions on motivation for changing alcohol use, and comparing the effectiveness of stepped-care models across diverse populations.The authors declare that they have no competing interests.JM and RM collaborated on the conception of the manuscript. JM wrote most of the manuscript with the exception of the Methods section and table that was written by JA. JA carried out all literature reviews for the paper. RM provided editorial feedback. All authors read and approved the final manuscript.


Recent work has extended these in vitro applications to the cell or to whole organisms. We review recent advances in intein-mediated protein expression and modification, post-translational processing and labeling, protein regulation by conditional protein splicing, biosensors, and expression of trans-genes.Intein-mediated protein splicing has become an essential tool in modern biotechnology. Fundamental progress in the structure and catalytic strategies of  Protein splicing is a post-translational process by which an intervening polypeptide, called an intein, catalyzes its own excision from the flanking polypeptides, or exteins, as well as ligation of the exteins Figure A.Many inteins are interrupted by homing endonuclease domains similar to those found in mobile introns. However, the homing endonuclease domain can be deleted from the intein without complete loss of splicing activity and is absent in a class of inteins called mini-inteins.The mechanism of splicing for canonical inteins is a four-step process Figure 1,2]. F,2. F1,2]Two side reactions can occur if the splicing process is disrupted Figure  B and C.trans [Intein-based methods have been developed to facilitate purification and post-translational modifications of recombinant proteins. Expressed protein ligation (EPL) and protein ng (PTS)-9 can prng (PTS),7,10-13.EPL is a method to modify the C terminus of a recombinant protein,15  ensures that PTS results in ligation of the internal polypeptide fragment. The cyclization of the target proteins results in enhanced stability and bioactivity[in vivo generation of large libraries of genetically-encoded cyclic peptides for high throughput screens[C-protein-IN precursor may result in polymerization of the target protein[Protein and peptide cyclization can be facilitated by inteins by two methods [Mtu RecA intein that is active only under specific pH conditions[Inteins can facilitate the overexpression of toxic proteins. For instance, the e (HpaI). Anothernditions,37.Expression of antibodies using a single open reading frame was achieved by fusing the genes for antibody heavy and light chains with an intein Figure F. This fin vivo, site-specific protein cleavage[Ssp DnaB S1 split intein was inserted between two target sequences and used as a cleavage site that is recognized by the C-terminal intein fragment. This C-terminal fragment is called an intein-derived protease (IP), because on co-expression of IP and complementation with the N-extein fragment, the target protein is cleaved via N-terminal cleavage of the reconstituted split intein. Site-specific protein cleavage by the IP was demonstrated in bacterial and eukaryotic cells. In contrast to the relatively low substrate specificity of other commonly used proteases, this intein-derived protease has very limited unintended proteolysis of endogenous proteins resulting in minimal cellular toxicity. The authors suggested utilization of the IP as a molecular tool to provide control of protein cleavage inside living cells.Split inteins can be used to facilitate cleavage  construct was used for a single step internal labeling of calmodulin with GFP in yeast.Inteins can facilitate  markers  and eGFP in vivo. The target proteins are expressed as a C-terminal fusion to the Ssp DnaB intein, and are designed to have an N-terminal Cys after intein C-terminal cleavage. The target proteins are then labeled by a cell-permeable, thioester-containing small molecule tag such as biotin or a fluorophore[Ssp or Npu DnaE intein fragment, while the fluorophore was part of the C-extein. Therefore, the quencher and fluorophore were part of the same molecule before splicing. On PTS, the fluorophore is ligated to the protein of interest and dissociated from the IC-linked quencher.One such intein-based labeling method uses native chemical ligation, in both bacterial and mammalian systems, to label glutathione-orophore. Howeverorophore  domain was performed using the  embryos. The auti-intein. This woi-intein.in vivo, it must splice conditionally, either in cis or in trans. CPS is activated by a trigger, such as a small molecule, light, temperature, pH or change in redox state (reviewed in[For protein splicing to regulate the activity of a protein iewed in). For CPiewed in. Rather,Sce VMA intein was split and fused to rapamycin binding domains FKBP12 and FRB, such that the addition of rapamycin induces intein reassociation and PTS[in vivo applications such as the controllable generation of firefly luciferase in cultured cells and in Drosophila melanogaster[PTS facilitated by ligand-induced dimerization domains allows for activation of splicing by small molecules Figure A. The Sc and PTS,57. Thisanogaster, and PTSanogaster. A mutatanogaster. Recentlanogaster.cis-protein splicing  residue in the N-extein. This intein could facilitate N-terminal cleavage only under reducing conditions in E. coli and allows for purification of uncleaved precursor and subsequent in vivo cleavage after addition of reducing agents[Pyrococcus abyssi has a native disulfide bond also between Cys-3 and Cys1 that can control intein activity[Pab PolII intein has a disulfide bond between Cys1 and Cys + 1 that prevents splicing, and the influence of flanking extein residues on both splicing activity and disulfide bond formation appears to be linked[Mma PolII intein is dependent on an internal intein disulfide bond, and shows differential splicing activity based on the redox state of the E. coli strain or localization to the periplasm or cytoplasm[Lately, there has been evidence that this disulfide-bond control of splicing activity may have physiological relevance. Belfort, Callahan and coworkers designed a redox trap into the fused, g agents. This ing agents. They alactivity. The Pabe linked. The actytoplasm.CPS permits splicing in response to a specific trigger and lays a foundation for the development of intein-based sensors. Most of these sensors have three functionally and structurally distinct modules: a sensing module, an output module and intein-derived signal transducer Figure A. Signalin vivo systems ranging from E. coli to transgenic animals. In their original work, an E. coli-based biosensor was developed to monitor binding between calmodulin and its target peptide M13, using GFP reconstitution as the reporter, mediated by the artificially split Sce VMAI intein[Ssp DnaE intein[Intein biosensors for protein-protein interactions utilize PTS facilitated by split intein fragments that have low binding affinity for each other. Design of these biosensors involves creation of two fusion proteins, each containing one protein binding partner, a split intein fragment and a fragment of a reporter protein Figure B. InteraI intein. Next, aE intein. Then, tE intein. To incrE intein,103. In Similar biosensor design was utilized in living cells for reporting sequence specific changes in DNA methylation via luminescence. The bioSsp DnaE intein fragment and a fragment of a reporter protein (GFP or luciferase). In addition, one of the fusion proteins has a target protein and the other is designed to localize in a specific compartment, such that PTS can only occur in the appropriate cellular compartment. This sensor design was used to study translocation of protein to the nucleus[Intein biosensors can be used to perform high-throughput screens to identify protein localization in a specific compartment. Design of these biosensors involves creation of two fusion proteins, each containing a split  nucleus or mitoc nucleus, as well nucleus.Npu DnaE intein-mediated reconstitution of luciferase[In addition to the detection of protein subcellular localization, protein splicing has been used for quantitative analysis of G-protein-coupled receptor (GPCR) internalization. In this case, interaction between activated GPCR and intracellular beta-arrestin2 results in ciferase.Ssp DnaE intein and split luciferase. The second is a nucleus-localized fusion of the N-terminal fragments of the intein and luciferase. Upon corticosterone binding, the glucocorticoid receptor is translocated into the nucleus, facilitating intein fragment complementation and splicing and therefore activation of luciferase.A protein localization-dependent sensor also was developed for corticosterone detection in animals. Again, Mtu RecA intein. The intein is fused to the E. coli maltose binding protein and a T4 bacteriophage thymidylate synthase reporter  and thyroid hormone (TRβ-1) receptors were used to develop highly sensitive methods for detection of nuclear hormone receptors ligands[Allosteric intein biosensors can be used to detect small molecules. Wood and coworkers have designed sensors for ligand detection by human nuclear hormone receptors utilizing allosteric effects induced by ligand-receptor binding. The sensor does not rely on protein splicing; the intein is used as an allosteric transmitter that allows communication between the hormone receptor and the reporter. The sensors exploit a four-domain fusion protein in which a nuclear receptor of interest is inserted in a loop region of the non-splicing  Figure C. The bi ligands. Later,  ligands. Subtype ligands and thyr ligands receptor ligands. This stE. coli-based estrogen detector[Sce VMA(ER) intein that was generated by replacement of the endonuclease region with the human estrogen receptor α. The VMA(ER) gene was inserted in the constitutively expressed chromosomal lacZ gene. The principal difference of this sensor from that discussed above is that the detection here relies on estrogen-dependent intein splicing and activation of the reporter protein.A splicing-dependent allosteric intein biosensor was employed by Liu and coworkers for construction of an detector. The senin vivo intein-promoted protein cyclization[Ssp DnaE split intein. In the absence of caspase activity, the activity of the cyclized luciferase was diminished due to steric hinderance. However, the activity of luciferase is fully restored upon caspase-dependent cleavage, enabling real-time quantitative sensing of caspase-3 activity in mice.A biosensor for protease activity was developed based on lization  was determined by rational design, and trans-splicing to produce herbicide-resistant ALS was demonstrated in E. coli[Salmonella typhimurium 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was selected by a library-based approach in E. coli, and the split intein segments facilitated EPSPS activity via fragment reassociation, to create an active split EPSPS protein, with PTS not required[in vivo. Full-length EPSPS was later generated by PTS, with expression directed to the chloroplast, in Nicotiana tabacum[Pseudomonas fluorescens both in E. coli and in N. tabacum[Inteins have been used to control  in 2005. Brieflyn E. coli. The splrequired,118. Thi. tabacum.Arabidopsis thaliana, both by PTS and by intein-mediated reassociation of GUS fragments[A split intein was also used to generate functional, transgenic β-glucuronidase (GUS) in ragments. The splragments.Bacillus amyloliquifaciens can be facilitated by the split version of the Ssp DnaB intein[. The split genes were placed under the control of a promoter for the tapetum, such that reconstitution of the barnase results in male sterile plants, given that the ribonuclease activity of the barnase is toxic to the tapetum cells in the anther[N. benthamiana or to result in male sterility in transgenic A. thaliana. Rather than select for the split barnase genes with separate genetic markers, each fragment was genetically linked to a split gene for an acetolactate synthase (ALS)-intein fragment fusion[A. thaliana[Triticum aestivum[Functional reconstitution of the barnase from B intein. The sple anther. The splt fusion. Therefot fusion. Althoug thaliana. This wo aestivum,125.Dictyoglomus thermophilum with the DnaE-1 intein from Thermus thermophilus[Intein-mediated and thermoregulated control of transgenic maize has been recently described with a split xylanase, which when active breaks down plant cell walls. Buildinrmophilus. Random A split DnaB intein was used to facilitate polymerization of spider silk flagelliform protein in tobacco plant leaves by both stable and transient transfection Figure C.trans-genes by adenovirus delivery vectors. The split fragments of the Ssp DnaE intein were fused to heavy and light chain genes for the B-domain deleted factor VIII, and delivered to mammalian cells or mice by separate viral vectors[in vivo generation of proteins too large to be delivered by traditional viral vectors. The split Ssp DnaE intein also was used to facilitate split Cre reconstitution in mice. In short, fragments of split Cre recombinase can be fused to separate promoters that drive expression under different conditions. Under conditions where both Cre fragments are expressed, Cre is reconstituted and facilitates expression of genes under control of the Cre-LoxP system. The Ssp DnaE intein was shown to help improve functional Cre fragment complementation[Recently, PTS in mammalian cells and in mice has been used to test delivery of  vectors,128. Splentation.Npu DnaE intein[in vivo protein manipulation using intein catalysis also promises advancement in intein-based tools for systems and functional biology.It is exciting to observe that biotechnology applications of protein splicing have begun to move from proof-of-concept experiments to productive applications in which the intein is the tool rather than the object of study itself. However, the variability in how inteins behave in heterologous contexts can be a limiting factor in the general applicability of intein-based biotechnology. Going forward, recent advances in the understanding of the role of flanking extein residues in the splicing process-136 may E intein also mayS-transferase; GUS: β-glucuronidase; HC: heavy chain; Hay: Haemophilus parainfluenzae; IC: C-terminal intein fragment; IN: N-terminal intein fragment; IP: intein-derived protease; LC: light chain; mRFP: the monomeric red fluorescent protein; MHC: major histocompatibility complex; Mtu: Mycobacterium tuberculosis; Pch: Penicillium chrysogenum; PH: plextrin homology; POI: protein of interest; PTS: protein trans-splicing; Sce: Saccharomyces cerevisiae; Ssp: Synechocystis sp. PCC6803; TR-1: human thyroid receptorACP: acyl carrier protein; ALS: acetolactate synthase; CHO: Chinese hamster ovary; CPS: conditional protein splicing; eGFP: enhanced GFP; EPL: expressed protein ligation; EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase; ER: human estrogen receptor; GB1: domain B1 of the immunoglobulin binding protein G; GFP: green fluorescent protein; GPCR: G-protein-coupled receptor; GST: glutathione-The authors declare that they have no competing interests.NT and KM co-authored the article and read and approved the final manuscript.NT is a post-doctoral scholar in the lab of Professor Marlene Belfort at the University at Albany; KM is associate professor and chair of the chemistry department at the College of the Holy Cross.


Recurrent tuberculosis suggests potentially modifiable gaps in tuberculosis treatment and control activities. The frequency of late recurrences following treatment completion has not been well-studied. We determined the frequency of, and risk factors associated with, tuberculosis that recurs at least one year after completion of anti-tuberculosis therapy in California.The study population included culture-positive, pulmonary tuberculosis patients reported to the California tuberculosis case registry from 1993 to 2007 who completed anti-tuberculosis therapy. A person with late recurrent tuberculosis was defined as an individual that appeared in the registry more than once, determined by match on name and date-of-birth, with at least one year between treatment completion of the first episode and treatment initiation of the second episode.Among 23,517 tuberculosis patients, 148 (0.63%) had a late recurrence. Independent risk factors for recurrence included: infection with a pyrazinamide mono-resistant isolate ; initiation of an isoniazid- and rifampin-only treatment regimen ; sputum smear-positive disease ; human immunodeficiency virus infection ; and birth in the United States . Infection with an isoniazid mono-resistant isolate was protective .The low frequency of late recurrent tuberculosis in California suggests that local TB control programs are largely successful at preventing this adverse outcome. Nonetheless, we identified subpopulations at increased risk of late tuberculosis recurrence that may benefit from additional medical or public health interventions. Recurrent tuberculosis (TB) within a population suggests potentially modifiable gaps in TB treatment and control activities. We examined TB recurrence in California to assess its frequency and to identify populations at increased risk that may benefit from specific interventions. A recurrence of TB can be due to relapse or re-infection Although clinical trials have demonstrated the rarity of TB relapse and re-infection in the U.S. and Canada The study population included culture-positive, pulmonary TB cases reported in California from January 1, 1993 to December 31, 2007. Cases were excluded if they did not complete treatment or if initial drug susceptibility test results were not recorded. Persons with late recurrent TB were those who had more than one episode of culture-positive, pulmonary TB with at least one year between the date treatment was completed for the first TB episode and the date that treatment was initiated for the second TB episode. We identified late recurrent TB by a deterministic match using first name, last name, and date of birth. Minor mismatches were tolerated in names or date of birth if sex, race, ethnicity, and country of origin matched.M. tuberculosis isolates from patients with late recurrent TB, the national TB genotyping surveillance database, initiated in 2004 The local and state TB case registries were matched to the local and state Acquired Immunodeficiency Syndrome (AIDS) registries to determine HIV status of TB cases. To identify genotypes of pairs of We assessed changes in the frequency of late recurrences over time with the Joinpoint Regression Program A multivariate Cox model was constructed by forwards stepwise selection, and included, a priori, the variable for age at TB report and covariates known to be associated with TB relapse: HIV co-infection, and sputum smear-positive disease. Additional covariates were added if, in univariate analysis, p≤0.2 for association with late recurrence. Age at TB report was retained in the multivariate model. All other covariates were retained in the multivariate model if their p-values were less than 0.05. We tested interaction terms to determine whether they significantly improved the models. We used a log-rank test of the likelihood ratio to determine best model fit. Person-time-at-risk for late recurrence was the time between the date therapy was completed for the first (or single) episode, and the date that therapy started for the second episode or the censor date, December 31, 2006  .We tested the proportional hazards assumption by comparing cumulative hazards of the multivariate model for strata of each dichotomous variable and by Schoenfeld residual analysis for age. To illustrate the increased risk of late recurrent TB for various subpopulations, we modeled the proportion and timing of recurrences for a 30-year-old population with a progressive increase in numbers of risk factors.Among 23,517 culture-confirmed, pulmonary TB patients who completed anti-TB therapy and were reported in California during 1993–2007, 148 (0.63%) had a second episode of TB reported at least one year after completion of therapy . The majM. tuberculosis complex; and HIV co-infection  had identical isoniazid and rifampin susceptibility test results for first and second TB episodes: four were MDR; two were isoniazid mono-resistant; and 127 were sensitive to isoniazid and rifampin. Fourteen pairs of isolates (10%) demonstrated changes in initial drug susceptibility patterns from first to second TB episodes; 13 demonstrated increasing resistance, indicating possible re-infection or acquired resistance . Substituting cavities on diagnostic chest radiograph for sputum smear-positive disease did not substantially change the model .The final model identified the following independent risk factors, present in the first TB episode, for late recurrent TB: pyrazinamide mono-resistance (adjusted hazards ratio (aHR) = 2.93, 95% confidence interval (95% CI) = 1.19–7.19, p-value (p) = 0.0190); an initial treatment regimen of isoniazid and rifampin only ; sputum smear-positive disease ; birth in the U.S. ; and HIV co-infection . An independent protective factor in the multivariate model was infection with an isoniazid mono-resistant The median time-on-treatment was significantly longer for the initial episode of persons with late recurrences than persons with one episode of TB . Also, pWe modeled the risk of late TB recurrence, and its timing after treatment completion, among a subpopulation of 30-year-old TB patients with a progressive increase in numbers of risk factors . The proThis study is the first large population-based assessment of recurrent TB in the U.S. Of 23,517 pulmonary, culture-positive TB cases in California from 1993–2007, at least 148 (0.63%) recurred after one or more years following treatment completion. Populations at high risk for late recurrence represent a TB control opportunity. Extending patient treatment may prevent recurrent disease and detecting a second TB episode promptly may avert transmission. Risk factors for late TB recurrence, present in the first TB episode, included: infection with a pyrazinamide mono-resistant isolate, initial treatment with isoniazid and rifampin only, sputum smear-positive disease, HIV co-infection, infection with an isoniazid sensitive, rather than an isoniazid mono-resistant isolate, and birth in the U.S.The proportion of late recurrent TB in California was similar to findings from other surveillance studies and clinical trials in which 0.24% to 0.71% of TB patients had recurrences after twelve months of completing treatment for the first TB episode The importance of using recommended TB treatment regimens is confirmed in this study; TB patients who were initiated on only isoniazid and rifampin instead of the recommended four-drug treatment regimen had a 2.5-fold increased risk of recurrence. The use of the recommended four-drug treatment regimen increased during the study timeframe; the proportion of TB cases initiated on the recommended four-drug regimen was 62% in 1993 and 95% in 2006. Since 2000, less than 1% of the study population was initiated on a two drug-only treatment regimen. The increased use of the recommended initial regimen may have also contributed to the decline in late recurrent TB during the study timeframe.M. bovisM. tuberculosis, M. bovis is associated with increased mortality M. bovis, including relapse and death, may be warranted.The independent risk factor with the highest hazard ratio was pyrazinamide mono-resistance, a surrogate marker of tuberculosis due to Our study demonstrated that factors associated with relapse soon after treatment completion were also associated with late recurrence: sputum smear-positive disease, cavities on chest radiograph, and HIV infection Our study identified U.S. birth as a risk factor for TB recurrence. Birth in the U.S. may have been a proxy for increased risk of non-adherence to anti-TB therapy M. tuberculosis were more likely to have a longer time-on-treatment than persons without these characteristics. Thus, the absence of evidence for time-on-treatment as a predictor of late recurrent TB in this population may reflect the limitation of the available data rather than the actual lack of influence of treatment duration in the first TB episode. Finally, the HIV status determination may have been incomplete, leading to an underestimate of HIV co-infection frequency in this population. However, we expect no difference in the extent of TB/HIV co-infection underestimation for first episodes of individuals with recurrent TB compared to individuals with one episode of TB.Our findings should be interpreted in light of a number of study limitations. We had no information about the true length of follow-up (person-time at risk) for individuals who had a single TB episode. The use of the censor date provided an approximation that may have overestimated length of follow-up for the TB patient population, resulting in an increasing underestimate of the hazard of late recurrent TB over time. Also, the absolute number of persons who developed a late recurrence is an underestimate because some unmeasured number of persons may have developed a second episode of TB after moving out of California. The absence of sufficient genotyping data to assess the relative frequency of relapse and re-infection in this population limits inferences about acquired drug resistance and the evidence base for recommending specific prevention interventions. Because this study used the time between treatment initiation and treatment completion dates as a surrogate for the time-on-treatment covariate, the extent and duration of treatment interruptions were unknown and likely differed by risk groups. Indeed, persons with sputum smear-positive disease, HIV infection, or infection with pyrazinamide mono-resistant isolates of How should healthcare providers address the elevated risk of TB recurrence in specific subpopulations? Post-treatment monitoring beyond one year may facilitate early TB case-finding among those at high risk of recurrence, but clinical trial evidence to support this intervention is limited. Implementation of post-treatment monitoring varies in California, by local TB programs, and elsewhere in the U.S. In one public health jurisdiction, TB clinic appointments at six- but not twelve- months after treatment completion facilitated early case-finding On the other hand, there is precedent for routine post-treatment follow-up of TB patients at increased risk of recurrence. Individuals who complete treatment for MDR-TB are monitored for at least 24 months to identify potential relapse In summary, this study provides a minimal estimate of the frequency of late TB recurrence in a low incidence setting, and suggests populations that may benefit from additional medical and public health interventions or further investigation. Additionally, our findings raise questions about the adequacy of treatment for HIV-positive TB patients and TB patients with pyrazinamide mono-resistant isolates. Investigation of treatment extension and the utility of extended follow-up in specific patient groups at high risk of recurrence may be warranted.


Bi-directional gene pairs have received considerable attention for their prevalence in vertebrate genomes. However, their biological relevance and exact regulatory mechanism remain less understood. To study the inner properties of this gene organization and the difference between bi- and uni-directional genes, we conducted a genome-wide investigation in terms of their sequence composition, functional association and regulatory motif discovery.We identified 1210 bi-directional gene pairs based on the GRCh37 assembly data, accounting for 11.6% of all the human genes owning RNAs. CpG islands were detected in 98.42% of bi-directional promoters and 61.07% of unidirectional promoters. Functional enrichment analysis in GO and GeneGO both revealed that bi-directional genes tend to be associated with housekeeping functions in metabolism pathways and nuclear processes, and 46.84% of the pair members are involved in the same biological function. By fold-enrichment analysis, we characterized 73 and 43 putative transcription factor binding sites(TFBS) that preferentially occur in bi-directional promoters from TRANSFAC and JASPAR database respectively. By text mining, some of them were verified by individual experiments and several novel binding motifs were also identified.Bi-directional promoters feature a significant enrichment of CpG-islands as well as a high GC content. We provided insight into the function constraints of bi-directional genes and found that paired genes are biased toward functional similarities. We hypothesized that the functional association underlies the co-expression of bi-directional genes. Furthermore, we proposed a set of putative regulatory motifs in the bi-directional promoters for further experimental studies to investigate transcriptional regulation of bi-directional genes. According to the orientation and status of the 5’ end, the adjacently located genes can be arranged in convergent, divergent or tandem configuration. Among tConsiderable attention has been focused on bi-directional genes in recent years. Examples including LRRC49/THAP10, COL4A1/de novo purine synthesis[The expression data obtained from biotechnologies such as SAGE and microarray indicated a correlated expression profile between bi-directional genes-13. Basesynthesis and carcsynthesis. DespiteMore recent studies have suggested an intrinsic difference in nucleotide composition of bi-directional promoters compared to uni-directional ones,2,13,16.In the present study, we have undertaken a genome-wide survey of gene organization in the human genome. To reveal functions collectively performed by such bi-directional genes, we mapped them to the Gene Ontology (GO) and GeneGO pathways. We also explored the functional similarity between the genes on the plus strand and those on the minus strand. We devoted our effort into exploring the binding preference of transcription factors on the bi-directional promoters and statistically identified a set of over-represented transcription factor binding sites(TFBS) in bi-directional promoters, the research scheme is shown in figure We calculated the distances between the transcription start sites of nearest gene neighbors for four kinds of combination of the clusters and the result was showed in table et al.[et al. [There have been contradictory observations on the CpG island frequency in bi-directional promoters. Adachi et al.,16 consi.[et al.  reported.[et al. , CpG isl.[et al.   whose plus and minus genes share at least one GO term, with SARS2/MRPS12 having most GO terms in common. Number and percentage of gene pairs that overlapped for various GO terms were showed in Figure We also provided separate estimates for each of the Gene Ontologies. We obtained 337 annotated pairs in subcategory "cellular component", 185 pairs in "molecular function" and 146 pairs in "biological process" respectively. It’s observed that, in general, bi-directional gene products are more likely to perform coordinated roles in the same cellular component, compared to the other two subsystems. Figure etc. In molecular function, the GO terms involved in DNA-directed RNA polymerase activity, RNA methyltransferase activity, purine NTP-dependent helicase activity, NAD or NADH binding, NADH dehydrogenase (quinone) activity, etc. are significantly over-represented as compared to others. In Cellular Component, we found that bi-directional genes tend to be tightly associated into the same class of organelle, organelle envelope, nucleus, nucleoplasm, nucleolus, membrane-bounded or non-membrane-bounded organelle, etc. Interestingly, almost all the items shared by the two divergent genes are related to metabolism and energy transfer. We proposed that genes involved in functions including metabolism, are more likely to be organized in the head-to-head configuration.Then we set out to find out the GO terms that represent coordinated functions of bi-directional pairs. In Biological Process, the GO terms related to metabolic process and its branch such as primary metabolic process, cellular process and biopolymer biosynthetic process topped the list of both gene pair members. Their child nodes were focused on RNA  metabolic process, cellular (macromolecule or biopolymer) catabolic process, organelle organization, mitotic cell cycle Based on the P values from MetaCore™, totally we found 45 pathways that are significantly enriched with bi-directional genes out of the total 451 distinct pathways. According to the different classification criterion, the 45 pathways were assigned to 18 regulatory processes, 8 protein function, 4 disease maps and 15 metabolic maps. Extreme enrichment occurred for, in order of descending significance level, NHEJ mechanisms of DSBs repair, Oxidative phosphorylation, Nucleotide excision repair and GTP-XTP metabolism, Chromosome condensation in prometaphase, Role of Brca1 and Brca2 in DNA repair. Enriched pathways are further clustered into larger functional categories according to the GeneGO annotation. Regulatory processes/Cell cycle and Regulatory processes/DNA-damage ranked among the top enriched functional categories. Table So far we have been analyzing the level of gene function enrichment using two function annotation schemes respectively. The GO results show a clear agreement with those derived from the GeneGO pathways. For example, the GO terms that are significantly enriched include genes that are engaged in processes such as DNA metabolic process, which correspond to the Metabolic maps/Metabolic maps (common pathways)/Nucleotide metabolism pathway in GeneGO; Cell cycle, which corresponds to the same pathways in GeneGO; response to DNA damage stimulus, which corresponds to Regulatory processes/DNA-damage in GeneGO. This agreement is also apparent in that ‘‘DNA repair’’ is the most enriched GO term and ‘‘* DNA damage_Nucleotide excision repair,’’ which corresponds to the Regulatory processes/DNA-damage pathway, is one of the top enriched pathways in GeneGO as well.We characterized the enrichment of known motifs from TRANSFAC and JASPAR in bi-directional promoters relative to background uni-directional promoters. Based on the Jaspar PSSM information, we categorized 43 transcription factors as over-represented and 6 as under-represented. In the TRANSFAC database, 73 TFBSs found increased presence in bi-directional promoters. Complete lists of over-represented motifs and their enrichment folds are provided as Additional file We further investigated the experimental evidence supporting the roles of these transcriptional factors in regulating certain bi-directional genes. Table Interestingly, the over-represented recognition sequence for MYC, ELK1, NF-Y, SP1, ATF, GABPA, SREBP-1, NF-E2, STAT5A, NF-1 as well as SOX-9 rank among the most conserved motifs found in human promoters.Given the enrichment of these motifs in bi-directional promoters and their strong evolutionary conservation across mammalian promoters, we assume that the predicted TFBSs located within bi-directional promoters are more likely to be functional in co-regulation than other TFBSs. Interestingly, it would appear that TFs within the same family tend to have similar binding preference. A TFBS is either over-represented or under-represented in parallel with other family members. These observations suggest a common mode of expression across the family members of transcription factors.et al.[In this study, 11.6% of the human genes were shown to be arranged in a head-to-head fashion, and this proportion is slightly larger than most of the previous report, except et al. reportedet al., brain det al. and oncoet al..Our study provided insight into the function constraints of bi-directional genes. Functional enrichment analysis in GO and GeneGO both revealed that bi-directional genes are often associated with housekeeping functions. GO terms, including metabolic process, such as DNA, RNA, biopolymer or macromolecule metabolism, as well as nuclear processes, such as DNA repair and replication or cell cycle regulation are significantly enriched. The GeneGO pathways that are involved in growth or proliferation, such as those engaged in Energy metabolism, Nucleotide metabolism, Vitamin and cofactor metabolism, tend to be more enriched with bi-directional genes. Pathways in genetic information processing  and cell cycle tend to be enriched as well. To summarize, bi-directional genes are significantly enriched in housekeeping functions such as metabolism pathways and nuclear processes.Further analyses revealed that the significant functional categories are more likely to be shared by bi-directional genes. This indicated that the bi-directional genes are strongly biased toward functional similarities and coordinated regulation. We postulate that for bi-directional genes involved in basic biological processes, coordinated regulation ensures their synchronized action and thus minimizes transcriptional error. In contrast, genes with less coordinated regulation may be involved in pathways that are more flexible in responding to environmental changes.We compared the TFBSs between bi- and uni-directional promoters according to their rate of occurrence. We discovered several transcription factors that preferentially regulate bi-directional promoters. Some of the TFBSs matched well with experimentally determined ones and several novel binding motifs were also identified. These bi-directional gene associated motifs may be envisaged as the best candidates for functional regulatory elements. In addition, the motif search result could help identify novel genes, which is linked to a known gene via a bi-directional promoter. And these genes probably perform important conserved functions.We are also aware of some limitations in our analysis. The motifs for the identification of TFBSs are still incomplete, and the evolutionary importance of the over-representation of TFBS remains to be elucidated. Although some of their function are indicated by functional categories (GO terms) of experimental verified motifs, conclusive evidence of the role played by regulatory factors in the co-regulation of the two genes will be tested in experiments. Eventually, the combination of computational and experimental approaches will permit us to construct mechanistic models of regulatory transcription networks of bi-directional genes. It would be interesting, as a future endeavor, to examine these regulatory elements in other species in a similar fashion and compare the results to those obtained herein. Comparative analyses of these regulators across multiple species will validate our predictions by their appearance in another species. A related work is still in progress.In this work, we conducted a systematic investigation of bi-directional gene organization focusing on sequence features, functional association and regulatory motif discovery. We confirmed known properties of bi-directional gene organization and also provided new observations. We found that bi-directional gene pairs show a higher probability to be functionally associated, formulating hypotheses that the requirement for co-regulation of functionally related genes is a possible cause for the observed co-expression of bi-directional genes. We also proposed that a special set of motifs in the bi-directional promoters play a role in transcriptional regulation of bi-directional genes. Our data also provide the putative regulatory motifs for experimental studies to investigate how the expression of divergent gene pairs is regulated.ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh37/Primary_Assembly/assembled_chromosomes/FASTA/). Gene annotation (NCBI Build36) was retrieved from the NCBI Entrez Gene ftp site (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/). The transcript annotation including transcription orientation, strand, starting site (hg19) was downloaded from hg19 RefGene table from UCSC Genome Browser (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/). A total of 45,408 genes  and 31,357 transcripts were collected and filtered for redundancy. This resulted in 44,293 non-redundant items of RefSeqs transcripts. Genes without clear mRNA information  were filtered to ensure the exact transcription of all the genes. The 28520 mRNAs were collapsed into 21757 unique and non-overlapping clusters, which were further ranked according to their chromosome position and TSS coordinates to determine the adjacent gene pairs. Discrimination of bi-directional gene pairs and uni-directional genes was performed by a perl script according to the definition by Trinklein. et al[Human genome assembly GRCh37, released as NCBI Build36 and Ensemble release 55, was downloaded from Genome Reference Consortium .Based on the mapping information of gene and its transcripts, possible multiple TSSs were assessed. The intergenic regions between bi-directional genes’ TSS were taken as bi-directional promoters. For uni-directional genes the region of 1000 bp upstream of the TSS were extracted as promoter. Promoter regions were extracted from the chromosome fasta files of the latest GRCh37 version genome assembly datasets.  and a commercial software MetaCore-GeneGO Pathway Maps (http://www.genego.com/metacore.php) to group functionally related genes and to contrast the functional distribution of bi-directional genes to the average distribution in the whole genome. The analysis of over-represented GO terms for bi-directional genes was performed by the GOEAST[We utilized Gene Ontology (GO) categories  is also applied to correct for multiple testing.Genes were then mapped to GeneGO database by MetaCore™ tools to infer pathways preferentially targeted by bi-directional genes. In MetaCore™, the statistical significance of the enriched pathways is indicated by a http://biomed.org.ua/COTRASIF/). TFBSs within bi-directional promoters were categorized as over-represented, shared or under-represented at 2-fold threshold. Over-represented TFBS was defined as whose normalized number of binding sites in bidirectional promoters is 2-fold larger than those in unidirectional ones while under-represented means the normalized number of binding sites in bidirectional promoters is 2-fold smaller than the number of sites in a single unidirectional promoter. Shared motif is the intermediate state. A total of 18840 uni-directional promoters was used to give a contrast of bi-directional genes.Putative TFBS in promoter regions were searched for matches to the position-weight matrix(PWM) in the JASPAR,25 and TThe authors declare that they have no competing interests.BL carried out the promoter compositional analysis, participated in the TFBS categorization and drafted the manuscript. JC participated in the functional enrichment analysis, performed the statistical analysis and draft the manuscript. BS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Identification and statistics of GO terms enriched with bi-directional genes.Click here for fileFunctional similarities for plus and minus genes within annotated bi-directional gene pairs.Click here for fileThe enrichment of TFBS in bi-directional promoters relative to uni-directional promoters.Click here for file


A key early step in embryogenesis is the establishment of the major body axes; the dorsal-ventral (DV) and anterior-posterior (AP) axes. Determination of these axes in some insects requires the function of different sets of signalling pathways for each axis. Patterning across the DV axis requires interaction between the Toll and Dpp/TGF-β pathways, whereas patterning across the AP axis requires gradients of bicoid/orthodenticle proteins and the actions of a hierarchy of gene transcription factors. We examined the expression and function of Toll and Dpp signalling during honeybee embryogenesis to assess to the role of these genes in DV patterning.Drosophila are expressed in an AP-restricted pattern in the honeybee embryo, including Dpp and its receptor Tkv. Components of the Toll pathway are expressed in a more conserved pattern along the ventral axis of the embryo. Late-stage embryos from RNA interference (RNAi) knockdown of Toll and Dpp pathways had both DV and AP patterning defects, confirmed by staining with Am-sna, Am-zen, Am-eve, and Am-twi at earlier stages. We also identified two orthologues of dorsal in the honeybee genome, with one being expressed during embryogenesis and having a minor role in axis patterning, as determined by RNAi and the other expressed during oogenesis.Pathway components that are required for dorsal specification in We found that early acting pathways (Toll and Dpp) are involved not only in DV patterning but also AP patterning in honeybee embryogenesis. Changes to the expression patterns and function of these genes may reflect evolutionary changes in the placement of the extra-embryonic membranes during embryogenesis with respect to the AP and DV axes. Drosophila melanogaster, which has a derived mode of development compared to other insects [Drosophila is the fusion of the extraembryonic membranes, which arise as a single tissue, the amnioserosa [zenknüllt (zen), a Hox3 gene that has been co-opted, during insect evolution, into defining the serosal membrane [A critical early step in establishing the future body plan of any animal is the formation of the major body axes, the anterior-posterior axis (AP), and the dorsal-ventral axis (DV). Determination of these axes is established by an initial, often maternally driven, symmetry-breaking event, and as the process continues it requires precise zygotic control of gene expression across the body axes via cell-signalling pathways. DV patterning has been well studied in  insects ,2. One uioserosa . In the ioserosa . A consemembrane ,7,8. In membrane .zen expression in Drosophila embryos, requires the expression of decapentaplegic (Dpp) at the dorsal midline. Dpp is a member of the TGF-β family of signalling molecules, which activate the MAD pathway on the dorsal midline of the blastoderm embryo in a gradient-like fashion. Short gastrulation (Sog) acts by binding to and inhibiting Dpp activity, thus promoting formation of the presumptive neuroectoderm. In dorsal regions, Tolloid  cleaves Sog to release Dpp, enhancing its activity [Megaselia abdita, a basal Cyclorraphan fly, also requires Dpp and zen expression for development of distinct amnion and serosa tissues [Tribolium castaneum, Dpp expression mirrors where zen-1 mRNA is found, and where the serosa-embryo border is positioned [Apis mellifera) model, we previously found that Dpp mRNA was maternally expressed and localized to the dorsal side of the oocyte, rather than the embryo, and the MAD pathway was activated in overlying follicle cells, implying that the Dpp-MAD pathway may pattern the DV axis of the oocyte, rather than the embryo [Patterning of the dorsal epidermis and refinement of activity . This liactivity -12, and  tissues ,5. In thsitioned , indicate embryo .D. melanogaster. The first symmetry-breaking event is movement of the oocyte nucleus to an anterior-dorsal location [gurken mRNA, encoding a TGF-β-like ligand, which in turn activates the EGF receptor in the overlying follicle cells [pipe (pip) is restricted to ventral follicle cells, triggering a ventral protease cascade that cleaves and activates the signalling molecule Spätzle (Spz) [gurken in most other insect genomes [Tribolium, Nasonia, and honeybee, but only localized to the anterior-dorsal side of the oocyte in a similar manner to gurken mRNA in Nasonia and the honeybee [DV axis formation is initiated in the oocyte prior to fertilization and oviposition long before localized expression of Dpp in location . This rele cells . Expressle (Spz) . Shortlyle (Spz) . While t genomes -21, therhoneybee ,21.Tribolium and Drosophila as, in Tribolium, Tc-Dorsal activates transcription of Tc-cact to negatively regulate its own function [A key target of the Toll pathway is Cactus. Phosphorylation of Cactus results in its degradation on the ventral side of the embryo ,23. Cactfunction .. The homeodomain-containing gene orthodenticle (otd) is required for anterior patterning in long and short germ-band arthropods [Tribolium and this requires Tc-otd[. Otd, in honeybee embryos, controls formation of the serosa via regulation of Am-zen expression [hunchback (hb) specifies correct zen expression in dorsal regions of the honeybee embryo [There is also emerging evidence for ‘cross-talk’ between AP and DV pathways in some insect groups, coordinating positional information across the body axesthropods ,26-28. Des Tc-otd. Otd, inpression . Additioe embryo .Drosophila[in situ hybridization, we demonstrate that, while patterning of the ventral neuroectoderm via snail and twist expression and their necessity in refining zen expression is conserved, early-acting components of DV patterning are expressed differently to other insects, and RNAi knockdown of those components results in defects across both AP and DV axes.These differences in the regulatory networks that define DV fates, combined with the relatively conserved nature of the molecules involved, make DV patterning an interesting model system in which to study the evolution of developmental pathways. To further understand how this pathway has changed over the course of insect evolution we examined the expression of some of the key orthologues of DV patterning genes during development of the honeybee embryo. Honeybee embryos follow a long germ-band mode of development that may have evolved independently from long germ-band development in rosophila. Using RDorsal homologues were identified by tBlastN searches of insect genomes . Multiplin situ hybridization probes. Digoxygenin (DIG)-RNA probes were synthesized and used in in situ hybridization experiments as described previously [Am-Dpp RNA probe cloning has been described elsewhere [PCR-amplified cDNA fragments for all genes of interest  using the MEGAscript RNA kit (Ambion). dsRNA was injected at 2.5 mg/mL in reverse osmosis-purified H20 into freshly laid honeybee embryos. For each target, between 300–500 embryos were injected, with 15–20% surviving the injection process. Injected embryos were incubated for 30 h (stage 4) or 48 h (stage 9) at 35°C (80% humidity) before collection for in situ hybridization or DAPI staining.Targeted mRNA knockdown was performed as described by Wilson and Dearden ,37. BrieDrosophila dorsal-ventral pathway. The BMP/Dpp pathway is a cascade, conserved in evolution, involved in DV patterning in both invertebrates and vertebrates [Am-Dpp is expressed maternally and that its mRNA is localized to dorsal regions of the oocyte [Am-Dpp mRNA is restricted to cells in the posterior two-thirds of the embryo prior to gastrulation  and hoverfly , have similar dorsal expression domains of Dpp[Clogmia albipunctata (moth midge) and Anopholes gambiae (mosquito) have a different pattern of Dpp expression; Ca-Dpp is expressed at the anterior and posterior ends of the blastoderm embryo [Ag-Dpp mRNA is initially expressed as a broad medio-lateral band extending from the anterior to the posterior of the embryo corresponding to the presumptive amnion, but absent from the dorsal midline region where the presumptive serosa is patterned (as determined by Ag-zen expression) [Tribolium Dpp (Tc-Dpp) is initially expressed throughout the blastoderm embryo, with higher expression at the anterior pole. Tc-Dpp expression at the anterior pole is later lost and Tc-Dpp mRNA is detected at the border between serosa and germ rudiment in a stripe along the AP axis [Schistocerca gregaria, Sg-Dpp RNA is detected at the posterior of the germ band and head lobes at 15% development as well as in necklace cells that include presumptive serosa cells [This expression pattern is unusual when compared with ioserosa ,39. Two ns of Dpp,40. The m embryo , while Aression) . Triboli AP axis . In the sa cells . These dAm-Dpp, we also examined the expression of a Dpp receptor, thickveins (Am-tkv), in honeybee embryos. Knockdown of both maternal and zygotic tkv in Drosophila produces a phenotype identical to loss of Dpp[Am-tkv mRNA is detected initially throughout the posterior two-thirds of the blastoderm embryo .Sog acts as an antagonist to the BMP/Dpp pathway by binding to, and trafficking, the Dpp protein . Apis som Figure E. In conmovement . In Droselopment . No orthequences ; howeverPannier mRNA, encoding a zinc finger transcription factor, is upregulated in dorsal regions of the embryo by Dpp signalling in Drosophila. Am-pnr is not expressed during early to mid-stages of embryogenesis  mRNA is detected throughout honeybee embryonic ectoderm  [A key component of the ventral pathway is Pipe. Restriction of s Figure A, and nos Figure B. This ee cells) .Drosophila and Tribolium[Am-cact RNA is expressed maternally ) genomes contain between two to three Dorsal-like genes  all contain a rel homology domain required for DNA binding and dimerization . They cas Figure F.Apis Dorsal-like genes, GB19066 and GB18032. Am-GB19066 mRNA is maternally expressed and detected throughout the oocyte from mid-oogenesis  results in severe anterior defects and disrupted patterning along both AP and DV axes. Thoracic and anterior abdominal segments are fused and, while tracheal pits are visible, the segments are indistinguishable  gene, an immediate downstream target of the Toll pathway, is required for mesoderm specification, being expressed as a ventral domain in most insects . Am-eve mRNA in honeybee is maternally expressed, and in early embryos is detected throughout the syncytial blastoderm and early cellularized embryo [Am-eve is expressed as seven broad stripes that later split into 14 stripes to establish the parasegment boundaries during segmentation  than Am-zen, which has little effect on Am-eve stripe expression [Am-eve stripe expression during segmentation. This influence is in addition to their roles in DV axis establishment, and occurs independently of Am-zen.pression . This imA. mellifera, during early embryo development. Comparisons of the expression and function of individual components of the DV patterning pathways in insects reveal the roles of some factors are more highly conserved, particularly those acting later in these pathways. This is consistent with evolutionary changes in segmentation and axis formation in insects, where earlier acting components are evolving more rapidly than downstream factors [sim, twist, and snail are expressed along the DV axis of the long and short germ-band embryos and a zen gene is expressed in regions where extraembryonic membranes will form. The most striking differences in spatial expression are found during early development. In honeybee embryos, the expression of many factors, including Am-Toll, Am-Dpp, and Am-cact, appears to be oriented with respect to the AP axis of the early embryo. This may be partially due to the way in which the extraembryonic membranes are determined in different insects (Figure Tribolium (Figure Drosophila, aminoserosa tissue originates at the dorsal midline following zen expression (Figure Dpp and zen as described in Figure This study set out to describe the expression and function of the Toll, Dorsal, and Dpp pathways in the long germ-band insect,  factors ,27,36,61Drosophila have led us to understand.Our data implies that signalling pathways have considerable influence over cell fate determination across both axes. Loss of honeybee Toll and Dpp function not only resulted in loss of DV patterning but also disruption to patterning across the entire ectoderm. This suggests that patterning of both major body axes may be more co-dependent than previous studies in Am-even-skipped; Am-TollRNAi: Am-Toll by dsRNAi; AP: Anterior-posterior; DV: Dorsal-ventral; Dpp: decapentaplegic; hb: hunchback; otd: orthodenticle; pip: pipe; RNAi: RNA interference; Sog: Short gastrulation; spz: spätzle; Tc-Dpp: Tribolium Dpp; tkv: thickveins; twi: twist; WT: Wildtype; zen: zenknüllt.Am-eve: PKD is an editor of EvoDevo.MJW conceived of the study, performed most of the experiments, and co-wrote the manuscript. PKD co-wrote the manuscript. NJK performed some of the cloning experiments and co-wrote the manuscript. All authors read and approved the final manuscript.


Although the association between cancer and sarcoidosis or sarcoid reaction is known, sarcoid reaction associated with Merkel cell carcinoma is rare.We report the case of a 57-year-old Japanese woman with Merkel cell carcinoma in the inguinal area associated with sarcoid reaction. Fluorodeoxyglucose positron emission tomography demonstrated elevated fluorodeoxyglucose uptake by mediastinal lymph nodes and at the carcinoma site. Histopathologically, the mediastinal lymph nodes contained no Merkel cell carcinoma components. Sarcoid lesions were identified. Systemic examinations returned no sarcoidosis-specific findings.Fluorodeoxyglucose positron emission tomographic scans can be used to assess neoplastic lesions and depict sarcoidosis. Sarcoid reactions must be considered in the interpretation of fluorodeoxyglucose positron emission tomographic scans.Sarcoidosis, a common systemic disorder of unknown etiology, is characterized by the formation of non-caseating epithelioid cell granulomas. The lungs, lymph nodes, liver, spleen, skin, eyes, small bones of the hands and feet and the salivary glands are most often affected . A diagnA 57-year-old Japanese woman presented to our hospital with a painless, firm, palpable mass 3 cm in diameter of five months' duration in the left inguinal area. She was a non-smoker and had no particular respiratory symptoms. The resected inguinal mass was a dermal tumor consisting of small to intermediate-sized cells with scant cytoplasm and regular nuclei with dusty chromatin and no nucleoli Figure . ImmunohThe association of sarcoid reaction with Merkel cell carcinoma is rare. Our literature review uncovered a case of an 84-year-old woman with Merkel cell carcinoma and chronic sarcoidosis . SarcoidMerkel cell carcinoma, composed of small, round blue cells, should be distinguished from small cell lung carcinoma, which has a similar pathological appearance. Their differentiation is possible because antibodies to cytokeratin 20 and thyroid transcription factor 1 are specific to Merkel cell carcinoma and small cell lung carcinoma, respectively .In our patient, the immunohistochemical findings regarding the inguinal mass and the absence of tumor cells in the mediastinal lymph node led to a diagnosis of Merkel cell carcinoma arising in the inguinal region. Her sarcoid reaction was mediastinal. Although sarcoid reactions are most commonly observed in cancer-draining lymph nodes, they can occur in non-regional tissues ,6. They et al. [et al. [18F]-α-methyltyrosine PET (18F-FMT PET) in combination with FDG-PET can distinguish sarcoidosis from malignancy. Sarcoid reaction must be considered in the evaluation of cancer metastasis, and the acquisition of 18F-FMT PET scans is desirable.FDG-PET, widely used to assess neoplastic lesions, depicts the glucose avidity of tissues. Although FDG-PET images reflect the different utilization of glucose by normal and malignant tissues, they fail to differentiate malignancy from inflammation reliably. Increased glucose uptake has been reported in patients with benign disorders ,11, and et al.  proposedet al. ,14 or saet al. , FDG-PET [et al.  reportedWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.YH drafted the manuscript and reviewed the literature. KM, MS, MH and AB obtained informed consent from the patient, assisted in data collection and coordinated and helped to draft the manuscript. SM and MY carried out the patient's surgery and revised the manuscript. TK was responsible for the diagnosis, patient management and review. All authors read and approved the final manuscript.