Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides , (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system  and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively.Biofilm formation is crucial for bacterial community development and host colonization by  Streptococcus salivarius is one of the early colonizers of oral mucosa surfaces in neonates and is a commensal inhabitant of the oral cavity and digestive tract of healthy adults. S. salivarius is thought to exert a range of biological activities related to host health, in particular through its impact on the stability of microbiota composition, and its interaction with the host , to the salivary pellicle on teeth and to soluble factors, as well as to bacterial cells of the same strain (auto-aggregation) or genetically distinct species (co-aggregation). The ability of rategies . Numeroull lines , variousll lines  and to Mll lines . S. saligregates  and to cllonella  and Prev species , the intucleatum  and Candalbicans , and theorsythia  and Porpngivalis .S. salivarius to biological surfaces remains limited. It has been shown that cell wall-associated fimbriae and fibrils are involved in this adhesion process. Indeed, fimbriae are responsible for adhesion to cervicovaginal epithelial cells and co-aggregation with Prevotella intermedia  or kanamycin (1000 μg/ml) were added to the medium as required. PCRs were performed using Phusion® high-fidelity DNA polymerase . The primers were purchased from Eurofins MWG Operon (Germany) and are listed in Supplementary Table Bacterial strains used in this study are listed in eviously . F. nuclMutants with altered auto-aggregation properties were isolated using Todd Hewitt broth with 0.5% yeast extract with a low (0.03%) concentration of agar . In thesS1 was performed essentially as described previously  were discarded. Clean reads were mapped to the reference JIM8777 strain using Bowtie (g Bowtie  and varig Bowtie . The IllMutant derivatives of strain JIM8777 were constructed by exchanging the coding sequence of a target gene (sequence between the start and stop codon) for an erythromycin or kanamycin resistance cassette devoid of a transcriptional regulatory region , as described previously . After pS. salivarius strains to HT-29 human epithelial cell lines  and MUC2 proteins  was performed as described previously  experiments were conducted as described previously . Images 600) of 0.5 ± 0.05  × 100, where ODt represents the OD600 of the suspension at time t  and ODt0 the OD600 at time zero. To evaluate the impact on auto-aggregation of various pre-treatments of bacteria, cell suspensions prepared in aggregation buffer as described above were treated with EDTA , Ca2+ , proteinase K , trypsin  and by heat . Proteinase K- and trypsin-treated cells were harvested by centrifugation and the pellets washed twice in aggregation buffer before the auto-aggregation assay. To determine percentage co-aggregation, suspensions of S. salivarius strains were combined with an equal volume of a test strain (F. nucleatum) or aggregation buffer (as a control) and incubated at room temperature, without agitation, for at least 6 h. The percentage co-aggregation was calculated using the following equation: 100 × [OD600 /2 - OD600 ]/OD600 /2, where OD600  represents the OD600 of control tubes containing only the S. salivarius or F. nucleatum strain at time t and OD600  represents the OD600 of the mixture of both suspensions at time t. Experiments were repeated at least three times, each in duplicate.The ability of bacterial cells to auto-aggregate and co-aggregate was assessed by a spectrophotometric procedure . Station5 ± 0.05 . To dete® base 96-well plates  and analyzed by confocal laser scanning microscopy (CLSM) as described previously  was used for statistical analyses. A P-value < 0.05 was considered to be statistically significant.Variables are expressed as the means and standard deviations of results obtained from three  or two  independent experiments. Comparisons between groups of variables were analyzed using the student’s S. salivarius were screened for auto-aggregation . Most of the S. salivarius isolates tested under our culture conditions formed aggregates, although their auto-aggregation ability was found to be highly strain specific (ranging from 9 to 56%). We chose one of the most strongly aggregating strains, JIM8777, for further study. The chromosome sequence of JIM8777 has already been determined . We also found that addition of Ca2+ led to an increase in auto-aggregation, whereas heat-treatment, addition of EDTA, proteinase K or trypsin led to a reduction in auto-aggregation , suggesting that proteins are involved in these cell-to-cell interactions.To characterize the environmental factors influencing JIM8777 auto-aggregation and to clarify the nature of the interactions, we evaluated the impact of various treatments on auto-aggregation levels. Auto-aggregation was most efficient at pH 8  agar medium according to chain-forming capacity . In thesFigure 2A, left panel, black arrow). After 140 h of growth, we observed the appearance of “roots” and “caps” from these round colonies, presumably formed by faster- and slower-sedimenting spontaneous mutants, respectively . Samples of the roots and of the caps were then removed and streaked out on agar plates to isolate single colonies of SL medium sedimenting mutants. Analysis of bacterial growth in liquid medium confirmed that selected mutants exhibited altered auto-aggregation phenotypes . Compared to the parental strain JIM8777, the slower-sedimenting mutants (such as JIM9443) showed a hyper-auto-aggregating phenotype in liquid medium, whereas the faster-sedimenting mutants (including JIM9445) showed diminished auto-aggregation capacity .We hypothesized that similarly to chain-forming bacteria, the auto-aggregates of JIM8777 strain will counteract sedimentation, resulting in formation of round colony. In keeping with this reasoning, we found that the auto-aggregates of the JIM8777 strain became immobilized in the SL medium  was inoculated on the top of a flask with SL medium. After 140 h, the faster-sedimenting “roots” appeared and were collected for further analysis . This experiment led to the isolation of 40 additional faster-sedimenting mutants.We then performed a similar experiment in which bacterial cells containing the whole genome-wide random insertion library of S1 from 11 insertion mutants. Altogether, 12 genes were identified from the two independent experiments . Two different spontaneous mutations were identified in the asp1 gene, as well as two different ISS1 insertions in the genes cshA, secA2, gtfG and gtfH. Our independent selection of several mutations in the same gene confirms the importance of these genes in the auto-aggregation phenotype.The gene mutations potentially associated with the altered sedimentation and auto-aggregation phenotypes were mapped by whole genome sequencing of 6 spontaneous mutants and cloning and sequencing of the chromosomal DNA fragments flanking the insertion site of ISTable 1). We found that deletion of the asp1, asp2, bglB, cshA, gtfE, gtfG, gtfH, liaR, secA2, and srtA genes, point mutations in which induced a faster-sedimenting phenotype in SL conditions, led to a decreased auto-aggregation phenotype in liquid medium . As cwpB and cwpK, two genes coding for enzymes predicted to synthetize cell wall polysaccharides (WPS), are located in the same operon , we chose to include only one of these genes (cwpK) for further analyses and confirmed that the ΔcwpK mutant showed an increased auto-aggregation phenotype . Altogether, our results confirmed that deletion of the genes identified as having an impact on bacterial sedimentation also affected the auto-aggregation properties of S. salivarius. This confirms that our method, based on differential sedimentation in SL conditions, provided an efficient tool for the specific isolation of mutants with altered auto-aggregation properties.To confirm the phenotypes of the bacterial mutants and the correlation between sedimentation in SL conditions and auto-aggregation in liquid medium, we constructed a series of mutants in the JIM8777 background carrying deletions of the genes in which we identified the point mutations associated with altered sedimentation , to the mucin 2 (MUC2) glycoprotein  and to the human pathogen F. nucleatum (co-aggregation) . In comparison to the wild-type JIM8777, 5 out of 10 of the deletion mutants with altered auto-aggregation capacity  displayed a significant (P < 0.005) reduction in adhesion to both HT-29 and MUC2 . Moreover, the cwpK (increased auto-aggregation) and srtA (decreased auto-aggregation) deletion strains showed a drastic reduction  in co-aggregation with F. nucleatum compared to the wild-type strain . The co-aggregation ability of the ΔgtfG strain was also altered but to a lesser extent (50% reduction). Similar results were obtained with the spontaneous mutants asp1spont1 (JIM9445), spontasp2 (JIM9447), spontcwpB (JIM9442) and spontcwpK (JIM9443) (data not shown).We investigated the ability of the auto-aggregation mutants to adhere to the human colonic epithelial cell line HT-29 . Biofilms formed by the mutants differed in their architecture from JIM8777 (P < 0.05 compared to wild-type for at least one geometric descriptor of the biofilm structure). With the exception of strain ΔgtfG, all non-auto-aggregative strains  developed compact and uniform biofilms. Hyper-auto-aggregative mutants  formed excessively filamentous biofilms compared to wild-type. This result indicates that the S. salivarius factors implicated in cell-to-cell interactions in planktonic conditions also influence the morphology and spatial organization of bacterial biofilm communities.Cell aggregation and biofilm formation are interconnected bacterial properties . We evalcwpK and asp1 genes in auto-aggregation, we evaluated the cell surface morphology of the corresponding mutants by scanning and transmission electron microscopy . We observed by SEM that the JIM8777 cells exhibited a rough and irregular surface that was partially covered by fibril-like structures . The overall morphology of JIM8777 and Δasp1 was similar, however, the electron-dense layer was completely absent in micrographs of the ΔcwpK and ΔcwpK Δasp1 strains. A similar result was obtained with the spontaneous spontcwpK (JIM9443) mutant, confirming that the point mutation resulted in loss of function , as well as in the asp1spont1 and ΔsrtA strains , suggesting that WPS may hamper glycoprotein formation, exposure, or detection.We have shown previously that the SecA2/Y2 transported glycoproteins form fibril-like structures that can be observed outside of the cell wall of ruthenium red-stained cells . AccordiS. salivarius. Auto-aggregation is influenced by a variety of environmental factors and involves bacterial proteins . To identify the proteins determining cell-to-cell interactions of a highly auto-aggregating S. salivarius strain (JIM8777), we developed a screening strategy based on immobilization of cell clusters in SL medium that allowed us to select for mutants with altered auto-aggregation. Twelve proteins involved in different aspects of auto-aggregation in S. salivarius were identified. It has been shown previously that the surface-exposed adhesin, SrpB, mediates auto-aggregation in S. salivarius JIM8777 , which are required for WPS biosynthesis in us mitis . It is peumoniae . InteresS. salivarius auto-aggregation factors: the GtfG and GtfH glycosyltransferases. Glycosyltransferases participate in the maturation and/or synthesis of various extracellular compounds, including glucans, cell wall polysaccharides, lipoteichoic acid, peptidoglycan and glycoproteins. Therefore, mutations in genes encoding the GtfG and GtfH glycosyltransferases may affect some of these compounds, leading to consequences for the auto-aggregation capacity of these mutants.We also identified two additional S. salivarius but further investigations are required for a more comprehensive assessment of how these factors act and interplay.The ability of bacterial cells to form auto-aggregates is considered as an advantageous feature under certain environmental conditions, however, excessively large cell clusters may increase the probability of their clearance . This suS. salivarius to host surfaces, such as MUC2 and HT-29 cells , and co-aggregation with the human pathogen F. nucleatum . As mentioned above, these mutations impact the SRR glycoprotein synthesis pathway. SRR glycoproteins, which are important for colonization and persistence in mice, also mediate adhesion of S. salivarius to host cells and host-derived molecules, such as MUC2   . The actF. nucleatum, a multifaceted bacterium that plays a pivotal role in oral biofilm community development (cwpB and cwpK mutants of S. salivarius. It also suggests that the WPS of S. salivarius act as receptors for the binding of F. nucleatum. This explanation is supported by published data showing that WPS expressed by other oral bacteria are implicated in co-aggregation with F. nucleatum (srtA and gtfG mutants, indicating the existence of at least one more S. salivarius determinant that remains to be identified. This determinant is likely to be a glycosylated SrtA-anchored surface protein, which may function as an adhesin. This unknown protein is not transported by the SecA2Y2 system and is not a LPXTG-containing CshA or SRR protein. Thus, S. salivarius JIM8777 appears to express both a cell surface adhesin and a carbohydrate receptor specific for the interaction with F. nucleatum. This may provide a partial explanation for the strain specificity of the co-aggregation reaction (We also identified mutations affecting co-aggregation with elopment  and is aelopment . It has elopment . The requcleatum . In addireaction .S. salivarius to host biological substrates, including MUC2, the human colonic epithelial cell line HT-29, and F. nucleatum. These auto-aggregation factors may exert key functions in terms of human health and disease by playing roles in bacterial colonization and persistence, microbial community development and pathogen elimination. Identification of these factors paves the way for further molecular studies. In addition, our results demonstrate that the screening method developed in this study can be used to identify the genes involved in bacteria-bacteria and bacteria–host interactions. This method could be especially useful if applied to isolates, for which molecular biology tools have not yet been developed. Our screen may also facilitate molecular studies of microbial community development of pathogenic or probiotic bacteria, for which aggregation is either a virulence trait or beneficial characteristic, respectively.In summary, we have identified several auto-aggregation factors from our screen of a random insertion mutant library and selection of spontaneous mutants in SL medium. All of these factors influence biofilm architecture, demonstrating that cell–cell interactions governing auto-aggregation and biofilm development are interconnected. Some of these factors also contribute to the adhesion of SK and EG designed the experiments and wrote the manuscript. BC, SK, NP, BQ, A-LA, CD, TM, RB, NL, and EG performed the experiments. BC, SK, NP, A-LA, RB, PR, NL, and EG performed the analyses. All of the authors critically reviewed the manuscript and approved the final version.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.


Tuberculosis (TB) has been a leading cause of death for more than a century. While effective therapies exist, treatment is long and cumbersome. TB control is complicated by the overlapping problems created by global inadequacy of public health infrastructures, the interaction of the TB and human immunodeficiency virus (HIV) epidemics, and the emergence of drug-resistant TB. After a long period of neglect, there is now significant progress in the development of novel treatment regimens for TB. Focusing on treatment for active disease, we review pathways to TB regimen development and the new and repurposed anti-TB agents in clinical development. TB is the fifth leading infectious killer of adults worldwide2, the third largest killer of women in their reproductive years3, and a leading infectious cause of death among people with HIV disease5. In recent years, the world has seen a rapidly emerging epidemic of drug-resistant TB, multi-drug-resistant TB, and extensively drug-resistant TB, which is lethal and extremely expensive and complicated to treat6.In 2014, the World Health Organization (WHO) reported 9.6 million incident cases of tuberculosis (TB), 1.1 million deaths from TB among human immunodeficiency virus (HIV)-negative people, and an additional 0.4 million deaths from HIV-associated TB7. Even though more than 250,000 children develop TB each year, inexcusably, most anti-TB agents are not available in suitable pediatric formulations8. While liquid formulations may be easy to administer to young children, they are bulky and more expensive, and some have unacceptable toxicity. Only recently have TB drug dosing recommendations been revised to reflect differences in the way children metabolize drugs and, until recently, most first-line drugs were under-dosed9. Equally important, the last 50 years have only brought about the FDA licensing of two new drugs for TB treatment (rifapentine and bedaquiline)  . There i10, and even shorter courses of treatment are under study .There are some positive developments, though: after a long drought, new drugs are available, and new strategies for the treatment of latent disease and for regimen development in active disease are emerging. A three-month regimen of isoniazid and rifapentine has been approved for the treatment of latent infection in the United States11. Discussed in more detail below, there are novel drugs in new classes approved and/or in clinical trials, including bedaquiline, delamanid, pretomanid, AZD5847, and sutezolid. Used either separately or in novel combinations, these agents are anticipated to shorten and otherwise improve the treatment of drug-resistant and, possibly, drug-susceptible TB.New and old rifamycins are being repurposed at optimized doses. Clofazimine, an iminophenazine dye used to treat leprosy, has attracted renewed interest owing to its ability to target TB. Fluoroquinolones have been studied for shorter treatment durations of drug-susceptible disease, though phase III trial results have been disappointing thus far12. Treatment shortening remains an important goal for the near future, as is the development of more tolerable regimens. Regimens consisting of novel drugs with activity against all TB, irrespective of resistance to drugs currently in use, are under discussion. Future goals in TB drug development also include the identification of relevant biomarkers to act as surrogate endpoints rather than waiting for relapse-free survival13.As important as the emerging new strategies for anti-TB regimen development are regulatory changes to expedite these efforts. Since the 1950s, TB clinical trials have consisted of addition to, or substitution of, an existing drug in a standard regimen. Traditional trial designs use TB cure without relapse as an endpoint, which requires observation for the duration of treatment and then 12–18 months of follow-up. Therefore, large phase III trials often take five or six years to complete and, with multiple compounds now in the pipeline, the development of a novel regimen using this model would require decades. The recent increase in global philanthropy directed at TB and the collaborations amongst major stakeholders have created opportunities to overcome bottlenecks and dramatically shorten the time to regimen development. For example, the Global Alliance for TB Drug Development, which is a not-for-profit product development partnership (PDP), is positioned to leverage a global network of public and private partners to advance TB drug development by developing drug combinations instead of individual drugs14. Surprisingly, the daily dose of 600 mg currently used is likely not on the optimal part of the dose-response curve, and higher doses may be needed to achieve treatment-shortening goals for drug-susceptible TB15. High-dose rifampicin appears to be well tolerated, as shown in the RIFATOX study, for example, which evaluated 20 mg/kg versus 15 mg/kg versus 10 mg/kg of rifampicin16; the HIGHRIF project, which is a series of four trials of high-dose rifampicin (http://www.edctp.org); and an NIH-supported study in Brazil and Peru . The PanACEA MAMS-TB-01 trial found that three months of dosing with 35 mg/kg of rifampin, in addition to standard isoniazid, ethambutol, and pyrazinamide, improved time to stable culture conversion over 12 weeks on liquid (though not on solid) media over the standard drug-susceptible TB treatment 17, without apparent toxicity from the higher rifampin dosage.Rifampicin is a cornerstone of TB treatment, largely because of its sterilizing ability . Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has shown that rifampicin efficiently penetrates sites of TB infection in lung lesions, and even accumulates in necrotic caseum18. The ACTG study 5290 is currently comparing strategies of double-dose lopinavir boosted with ritonavir in combination with rifampicin-based TB treatment versus standard dosing with rifabutin (NCT01601626).For HIV-infected patients requiring protease inhibitor-based antiretroviral therapy, rifabutin is generally recommended where available because of reduced potential for significant drug–drug interactions and is effective and safe in this setting10. The CDC guidelines were revised in 2011 to include this regimen19. An even shorter regimen of rifapentine and isoniazid for one month in HIV co-infected patients is under study by the ACTG .From the same class but with a longer half-life, rifapentine is being evaluated for both latent and active TB. In an 8,000-patient, international study from the TB Trials Consortium (TBTC) funded by the Centers for Disease Control and Prevention (CDC), weekly rifapentine at 900 mg with isoniazid 900 mg weekly for three months was non-inferior to nine months of isoniazid for the treatment of latent infection20. Phase III trials with rifapentine for pulmonary TB are ongoing, with the launch of TBTC study 31 in late 2015. This phase III randomized clinical trial with 2,500 participants will investigate four-month rifapentine-containing (1200 mg daily) treatment regimens compared to the six-month standard-of-care rifampicin-containing regimen (NCT02410772).The TBTC study 29 found similar rates of sputum culture conversion (i.e. from positive to negative for TB) at eight weeks in 381 smear-positive patients with TB using rifampin or rifapentine21. Rifapentine was previously considered a less potent inducer of the metabolizing CYP3A isozyme than rifampin and therefore less likely to cause significant drug–drug interactions. However, data in healthy volunteers at clinically relevant doses using midazolam as a probe found rifapentine to be a more potent inducer of CYP3A than rifampin22. Despite this, no clinically meaningful effect on efavirenz exposure was seen in 87 patients receiving a four-week course of daily rifapentine for TB prevention in ACTG 527923. Rifapentine increased raltegravir exposure in a healthy volunteer study, but this was safe and tolerable; therefore, once-weekly rifapentine can be used in HIV-infected patients on raltegravir24.Unfortunately, all rifamycins induce phase II and cytochrome P450 drug-metabolizing enzymes, resulting in significant drug–drug interactions when used with antiretroviral agents and other drugs. This presents a major obstacle to the treatment of HIV/TB co-infected patients with rifamycins, particularly those who require protease inhibitor therapyin vitro and in a mouse model25. However, three recent phase III trials all failed to show non-inferiority to current standard-of-care regimens when investigating four-month treatment regimens containing moxifloxacin for drug-susceptible TB28. As the four-month regimens worked in a large majority of study participants, fluoroquinolone-containing regimens still warrant additional investigation and several studies are ongoing. TBTC study 31 will include moxifloxacin in one of its investigational treatment arms in combination with high-dose rifapentine, isoniazid, and pyrazinamide. The Global Alliance for TB Drug Development [http://www.tballiance.org] is currently conducting a large, multi-country phase III trial investigating moxifloxacin in combination with PA-824 and pyrazinamide in the STAND Trial (NCT02342886).Although not licensed for TB treatment, fluoroquinolones are often used in the treatment of drug-resistant TB and are also under study as first-line agents. Levofloxacin, moxifloxacin, and gatifloxacin are all activeFluoroquinolones do not have significant drug–drug interactions with antiretroviral drugs, although adverse effects limit their utility in children and pregnant women. Moxifloxacin may also have overlapping toxicity with regard to prolongation of the QT interval.Mycobacterium avium infection. Clofazimine was first synthesized in the 1950s with the intent of treating TB, although its efficacy against TB was difficult to establish. With the discovery of rifampicin, clofazimine failed to find a place in TB treatment but is now being investigated as a possible treatment modality for treatment shortening and against drug-resistant TB. For example, TRUNCATE-TB will use an adaptive design to test several two-month drug-susceptible TB regimens including new and repurposed drugs . The ACTG is also developing a clinical trial for drug-susceptible TB based on preclinical work in the mouse model29.Clofazimine is a drug currently used in combination to treat leprosy as well as in therapy for30. It has potent activity againstMycobacterium tuberculosis isolates regardless of resistance but little activity against other common bacterial pathogens. Bedaquiline was approved by the FDA for the treatment of pulmonary multi-drug-resistant TB in December 2012, largely on the basis of phase IIb data31. Multiple clinical trials are planned or underway to learn how best to use bedaquiline in drug-resistant TB treatment. From the TB Alliance, NC-005 is a two-month phase II study looking at pretomanid, bedaquiline, and pyrazinamide (NCT02193776). The STREAM trial is evaluating the effectiveness of two bedaquiline-containing regimens with the goal of developing an all-oral six-month regimen for multi-drug-resistant TB (NCT02409290). NIX-TB is an ambitious phase III trial utilizing bedaquiline, pretomanid, and linezolid for the treatment of extensively drug-resistant TB in six to nine months and was launched in early 2015 (NCT02333799).Bedaquiline is a diarylquinoline (a drug class not related to fluoroquinolones) with a novel mechanism of action which involves inhibition of the mycobacterial ATP synthase22.Although bedaquiline is a substrate of the metabolizing enzyme CYP3A, problematic drug–drug interactions with antiretroviral agents are not anticipated because bedaquiline is metabolized only by CYP3A and does not induce or inhibit the enzyme. However, the drug has a very long half-life, and unanswered questions remain about long-term safety and tolerability. Concentrations of bedaquiline can be reduced by 50% or more with co-administration with rifamycins, but no significant effect was seen when given with efavirenzin vitro32. In mice, either drug in combination with rifampicin and pyrazinamide shortened TB treatment by at least 2 months34.The next generation of nitroimidazoles shows promise for TB treatment. This includes pretomanid under development by the Global Alliance for TB Drug Development, and the Otsuka Pharmaceutical Company is developing a related compound, delamanid. Both have potent activity against drug-susceptible and drug-resistant TB35. Early bactericidal activity studies measure decline in sputum colony counts per day among patients with sputum smear-positive pulmonary TB. A combination of pretomanid together with moxifloxacin and pyrazinamide in an early bactericidal activity study found that this three-drug combination was more effective than standard TB treatment , a first-ever finding in a two-week early bactericidal activity study36. Though studies conducted in the first two weeks of treatment cannot predict a regimen’s ability to provide cure, these groundbreaking studies were greeted with great interest, and the next wave of trials is currently underway investigating the new paradigm of a universal regimen to treat patients with either drug-susceptible or -resistant TB.In patients with pulmonary TB, the addition of pyrazinamide to pretomanid or bedaquiline significantly increased the early bactericidal activity of both drugs37. Results from this trial led to conditional approval from the European Medical Agency (EMA) in November 2013 for the use of delamanid in the treatment of multi-drug-resistant TB. The approved dose of delamanid is 100 mg twice daily in combination with an optimized background regimen. A larger phase III study of delamanid in adults with multi-drug-resistant TB including subjects on antiretroviral therapy is currently underway with results expected in 2017. Both delamanid and bedaquiline may cause prolongation of the cardiac QT interval, so there is concern about co-administration38. The ACTG is conducting a study to address this issue39. Delamanid appears to be void of clinically relevant interactions with antiretroviral therapy, which may make it an appealing agent to use in HIV/TB co-infected individuals42.Otsuka have completed a phase II, randomized, double-blind trial of delamanid in multi-drug-resistant TB patients receiving either 100 mg or 200 mg twice daily in addition to standardized second-line drugs. The addition of delamanid significantly increased the rates of sputum culture conversion at two months in patients with multi-drug-resistant TB43. This class includes linezolid, sutezolid, and AZD5847.In vitro, linezolid is effective at doses of 1–2 µg/mL against 90% of clinical isolates44 and works well in a mouse model43.Oxazolidinones are protein synthesis inhibitors with a unique mechanism of action against TB46. Ongoing clinical trials are planned to investigate linezolid in patients with multi-drug-resistant and extensively drug-resistant TB.Unfortunately, long-term administration of linezolid is associated with toxicities such as neuropathy and myelosuppression, but the drug has been evaluated retrospectively for the treatment of drug-resistant TB with encouraging results47. Unlike linezolid, sutezolid appears to have sterilizing activity, a key factor in successful TB treatment. Addition of the drug to a standard regimen produced a 2-log drop in bacillary burden in a mouse model and appeared synergistic with rifampin48; however, a two-week study in humans was unable to reproduce these findings49. Unfortunately, the development of this compound has been complicated by licensing issues and remains extremely slow at present50. Unlike linezolid, sutezolid does not appear to cause QT prolongation or bone marrow suppression; however, there are concerns of neurotoxicity and hepatotoxicity associated with its use.Sutezolid, a related oxazolidinone in development formerly from Pfizer but now licensed to Sequella, is superior in preclinical models and phase I bactericidal activity studiesAstraZeneca were developing an oxazolidinone, AZD5847, but the future of the drug is uncertain. A phase II early bactericidal activity study was completed in December 2013, but results have not been presented or published.After a long drought, the pipeline for new TB therapeutics is trickling. Caution still needs to be exercised with regard to identifying resistance to the novel drugs and ensuring that the optimized background regimen offers the maximum protection possible and there remains a bottleneck between preclinical and clinical development. However, there is a global commitment to major improvements and progress over the next decade towards the goal of eliminating TB as a global public health crisis.ACTG, AIDS Clinical Trials Group; CDC, Centers for Disease Control and Prevention; HIV, human immunodeficiency virus; TB, tuberculosis; TBTC, Tuberculosis Trials Consortium.


Reserve refers to the phenomenon of relatively preserved cognition in disproportion to the extent of neuropathology, e.g., in Alzheimer’s disease. A putative functional neural substrate underlying reserve is global functional connectivity of the left lateral frontal cortex . Resting-state fMRI-assessed global LFC-connectivity is associated with protective factors (education) and better maintenance of memory in mild cognitive impairment (MCI). Since the LFC is a hub of the fronto-parietal control network that regulates the activity of other networks, the question arises whether LFC-connectivity to specific networks rather than the whole-brain may underlie reserve. We assessed resting-state fMRI in 24 MCI and 16 healthy controls (HC) and in an independent validation sample (23 MCI/32 HC). Seed-based LFC-connectivity to seven major resting-state networks  was computed, reserve was quantified as residualized memory performance after accounting for age and hippocampal atrophy. In both samples of MCI, LFC-activity was anti-correlated with the default-mode network (DMN), but positively correlated with the dorsal-attention network (DAN). Greater education predicted stronger LFC-DMN-connectivity (anti-correlation) and LFC-DAN-connectivity. Stronger LFC-DMN and LFC-DAN-connectivity each predicted higher reserve, consistently in both MCI samples. No associations were detected for LFC-connectivity to other networks. These novel results extend our previous findings on global functional connectivity of the LFC, showing that LFC-connectivity specifically to the DAN and DMN, two core memory networks, enhances reserve in the memory domain in MCI. The reserve theory proposes that individuals with favorable cognitive and lifestyle factors such as education, IQ and occupational complexity can transiently maintain relatively high cognitive performance when developing Alzheimer’s disease (AD), the most common cause of dementia in elderly people . While rThe major aim of the current resting-state fMRI study was to test our hypotheses that (1) higher education is associated with higher LFC-connectivity to other major resting-state functional networks, and (2) higher connectivity between the LFC and other networks is associated with higher memory-related reserve. To address the questions set out by the current study, we computed the global LFC-connectivity to seven major brain networks  . EstimatFor the current study we included two independent samples, each comprising both amnestic MCI subjects and healthy controls (HC). The first sample was recruited at the Technische Universität München (TUM) in Munich Germany (henceforth referred to as TUM sample) and encompassed 24 MCI patients and 16 HC. Here, MCI was diagnosed following the recommendations of the National Institute on Aging and the Alzheimer’s Association . All MCIThe second sample comprised 23 MCI and 32 HC recruited at the Institute for Stroke and Dementia Research (ISD sample), Ludwig-Maximilian University of Munich. Here, MCI was diagnosed according to the Petersen criteria , i.e., sBoth studies were approved by the ethics committees of the respective institutions and conducted in accordance with the 1964 Helsinki declaration and its later amendments. Written informed consent was obtained from all subjects.Scanning was performed on a Philips 3T MRI scanner system using an 8-channel phased-array head coil. A structural high-resolution T1-weighted MPRAGE image was recorded with an isotropic voxel resolution of 1mm. fMRI data were recorded using a gradient EPI sequence with a TR/TE = 2000/35 ms, a flip angle of 82°, with an in-plane resolution of 2.75 mm and a slice thickness of 4 mm without an interslice gap. The resting-state fMRI scan comprised a total of 300 volumes, during which subjects were instructed to keep their eyes closed and not to fall asleep.All scans were acquired on a Siemens Verio 3T MRI scanner using a 32-channel head coil. Initially, a structural image was obtained using a high-resolution T1-weighted MPRAGE sequence with 1mm isotropic voxels. Resting-state fMRI data were recorded using a EPI pulse sequence with a TR/TE = 2580/30 ms, a flip angle of 80° and 3.5 mm isotropic voxel resolution without an interslice gap. The overall scan comprised 180 volumes prior to which subjects were instructed to keep their eyes closed.1). For each subject, high resolution T1-weighted structural images were segmented into probabilistic grey matter (GM), white matter (WM) and cerebrospinal fluid (CSF) maps via the new-segment approach implemented in SPM12  = -0.790, p = 0.433; TUM: t(39) = -1.664, p = 0.104]. For spatial normalization to MNI space, the non-linear DARTEL and affine registration parameters that were estimated during preprocessing of the T1-weighted images were combined and applied to the coregistered EPI volumes. All EPI images were subsequently smoothed using an 8 mm FWHM Gaussian kernel, detrended and band-pass filtered, using a frequency band of 0.01–0.08 Hz. We further regressed out the 6 motion parameters  and the BOLD signal averaged across the WM and CSF masks that were created during preprocessing of the T1-weighted images. We did not apply global signal regression since it can artificially introduce anti-correlations in the BOLD signal . We further tested whether diagnostic groups (HC vs. MCI) showed differences in head motion. To this end we computed the average frame-wise displacement for each individual following a previously described protocol . When coD signal .x = -42, y = 6, z = 28; see Figure 1 for the ROI location), and used as a seed region for all subsequent functional connectivity analyses.The location of the LFC ROI was based on our previous study in patients with MCI due to AD, where we showed that greater resting-state global LFC-connectivity was associated with more years of education and allowed to maintain memory performance relatively well in the face of AD-related posterior parietal FDG-PET hypometabolism . In brieFigure 1, left panel) were defined independent of the current study based on a widely used brain network parcellation scheme assessed on 1000 subjects   .Figure 2 illustrates the principle of this residualized reserve measure.As a measure of memory performance, we used the delayed free recall subscale of the word-list learning test included in the CERAD-Plus battery. The test includes a list of 10 unrelated words for examining memory and is thus suitable for older individuals and cognitively impaired patients for whom longer lists would be too taxing. The words are shown at a rate of 2 s each and presented in a different order in three learning trials. The tested individual is instructed to read out every word to ensure word registration. After each learning trial, the tested individual is asked to recall the list. After a 3 to 5 min delay, retention is tested by free recall . Using tt-tests for continuous measures and Chi-square tests for categorical measures. To map the spatial pattern of LFC-connectivity and to assess whether the LFC exhibits connectivity predominantly to the fronto-parietal brain regions and the DMN similar to previous reports  we computed the spatial correlation . We further tested whether associations between LFC-connectivity and memory reserve were specific to the stage of MCI or whether these associations also applied to HC. To this end, all models were computed in an equivalent fashion in the HC groups.For each sample, group demographics, cognitive scores and hippocampal volume were compared between MCI and HC using two-sample  reports , we mapprelation  and R2 bty index  on binarSupplementary Figure S1) that divides the 7 networks into smaller sub-networks. The rationale for including this additional analysis was to test whether associations between memory reserve and LFC-connectivity to the DMN or DAN were driven by connectivity to specific sub-parcels of these networks.In an exploratory analysis, we repeated all above delineated analysis steps this time using the 17-network parcellation  violations of linear model assumptions were found.All linear regression models were computed using the freely available statistical software package R . Linear Table 1. In the ISD sample, the MCI group was significantly older than the HC group. Consistent across both samples, the MCI groups showed significantly lower MMSE and word-list delayed recall scores as well as lower hippocampal volume. In the TUM sample, LFC to DAN connectivity was significantly reduced in MCI when compared with HC .Group demographics, cognitive scores and average LFC-connectivity measures are displayed in Figure 1 shows the pattern of significant LFC functional connectivity, as assessed via voxel-wise one-sample t-tests pooled across diagnostic groups in each sample. Visual inspection of the figure reveals that the LFC exhibits significant positive connectivity to regions belonging to the DAN, fronto-parietal control network, VAN and somatomotor network as well as the visual network. Negative connectivity is found predominantly to midline regions belonging to the DMN, as well as inferior temporal brain regions . Overall, this pattern of LFC connectivity is in line with previous research  and dice similarity index of 0.72. When recomputing the spatial correlation using a less restrictive threshold  thereby including a higher number of voxels showing lower connectivity, the correlation  and dice similarity index of 0.65 among both samples remained high.research . Comparit(22) = -2.569, B/SE = -31.23/12.16, p = 0.0088; Figure 4A, left panel] and ISD sample . When computing the average LFC-connectivity only to those voxels that surpassed the group level t-test against 0 , we obtained congruent associations between LFC-connectivity to the DMN and memory reserve . In the HC groups, LFC to DMN connectivity was not associated with memory reserve, neither in the TUM sample , nor when tested in the ISD sample .Next we tested our hypothesis that higher LFC-connectivity to the DMN and DAN predicts greater memory reserve. For the DMN, results of the regression analysis showed that stronger (negative) connectivity between the LFC and the DMN was associated with higher memory reserve in MCI. This association was consistently detected for MCI patients of the TUM  and ISD sample . When assessing LFC-connectivity to the DAN based on only those correlations that surpassed the group-level t-test against 0 , we obtained a similar result including significant effects of LFC to DAN connectivity on memory reserve . Again, no associations between LFC to DAN connectivity and memory reserve were found for HC subjects .For the DAN, higher (positive) connectivity between the LFC and DAN was associated with greater memory reserve in MCI consistently across the TUM  and higher (positive) LFC to DAN connectivity  in the ISD sample. In the HC group, no significant associations between education and LFC-connectivity were detected .In a last step we aimed to extend previous findings, that LFC-connectivity to those networks for which we found significant associations between LFC-connectivity and memory reserve is associated with years of education as a common reserve proxy. As shown by our regression analyses, greater education predicted higher (negative) LFC to DMN [Our major findings were that in MCI greater LFC-connectivity specifically to the DMN as well as the DAN was associated with higher memory-related reserve, i.e., relatively high memory performance when accounting for the level of brain pathology. These findings suggest that resting-state connectivity levels of the LFC to particular other networks contributes to memory-related reserve in MCI.We showed that the LFC exhibits widespread functional connectivity to fronto-parietal and DMN networks, where higher LFC activity was associated with higher activity within the DAN regions, and lower activity within the DMN. These results are consistent with previous findings of the LFC as a hub of the task-positive network including the DAN, which is anti-correlated with the DMN .Both the DMN as well as the DAN have been consistently shown to be fundamentally involved in memory , where hFor HC, we did not detect any associations between resting-state LFC-connectivity and memory-related reserve. Possible explanations include ceiling effects in memory performance levels in the HC subjects or absence of AD pathology and ensuing aggravated hippocampal atrophy. Note that the delayed recall memory measure that was used to compute the residualized memory reserve score is tailored for the clinical detection of cognitive impairment and may thus be less amenable to detect slight cognitive decline in cognitively asymptomatic controls. Thus the absence of the detection between an association of LFC-connectivity and residualized memory performance in the HC groups of the current study may have been due to ceiling effects. As an alternative explanation for the current results, in the HC groups neurodegeneration may not be advanced to a level at which memory maintenance becomes reliant on high levels of resting-state connectivity of the LFC hub. We point out, however, that when the same HC subjects in the ISD sample were challenged with a difficult face-name association task, task-related LFC-connectivity was associated with higher education and memory reserve as assessed on task performance levels . Thus, wA strength of the current study is that we found an identical pattern of associations between LFC-connectivity to the DMN and DAN and reserve in the memory domain in two independent samples and when using different network parcellations, which guards against overfitting of statistical models in a given sample and Type I error in the face of multiple testing. However, when interpreting the results of the current study, several caveats should be considered. Since education was only available in one of our samples (ISD sample), the associations between education and LFC-connectivity in MCI could not be independently validated. However, our current results are in line with the findings of our previous study based on data from the Alzheimer’s disease neuroimaging initiative , showingIn order to establish LFC-connectivity as a functional measure of reserve, test–retest reliability of LFC-connectivity estimation is fundamental. Previous studies have reported that test–retest reliability of centrality measures such as global connectivity or regional homogeneity is highest in heteromodal regions like the LFC and in higher order networks such as the DAN and DMN . HoweverOverall, our results provide novel insight in how the LFC supports reserve in MCI via connectivity to memory-related functional networks. These findings could act as a starting point to assess modifiability of LFC-connectivity via cognitive and pharmacological interventions or brain stimulation . PreviouNF, study concept and design, data collection, statistical analysis, interpretation of the data, and drafting the manuscript. JG, TG, AD, MÁ-C, AT, LS-V, KB, CC, DJ, CM, MD, CS: data collection and critically revising the manuscript. ME, study concept and design, interpretation of the data, and drafting the manuscript.Outside the submitted work TG reported having received consulting fees from Actelion, Eli Lilly, MSD; Novartis, Quintiles, Roche Pharma, lecture fees from Biogen, Lilly, Parexel, Roche Pharma, and grants to his institution from Actelion and PreDemTech. The other 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.


To characterize the patterns of brain atrophy and perfusion as measured by arterial spin labeling (ASL)-MRI, in amyotrophic lateral sclerosis (ALS) patients with varying levels of cognitive deficit, including ALS with frontotemporal dementia (FTD).n = 27), ALS with cognitive impairment , and ALS-FTD (n = 11). Voxel-based comparisons of gray matter (GM) changes and cerebral blood flow (CBF) were conducted among the subgroups.A total of 55 ALS patients and 20 healthy controls (HCs) were included, and all participants underwent neuropsychological assessments and MRI scans. According to their cognitive performance, ALS patients were further subclassified into ALS with normal cognition (ALS-Cn, The whole-brain comparisons of GM changes and CBF among ALS-Ci, ALS-Cn, and HCs were not significantly different. However, the ALS-FTD patients demonstrated a similar pattern of GM loss and hypoperfusion with more significant alterations in the left frontal and temporal lobe compared with the HCs, ALS-Cn, and ALS-Ci patients. Decreased CBF was found in many of the same brain areas wherein structural alterations occurred, although isolated GM loss and hypoperfusion were also observed. In addition, for both GM and CBF abnormalities, a similar pattern of changes was found in the comparisons of ALS-FTD vs. ALS-Ci, ALS-FTD vs. ALS-Cn, and ALS-FTD vs. HCs, with the differences being most significant between ALS-FTD and HCs.The cognitive status of ALS patients is associated with different patterns of GM changes and cerebral perfusion. ASL-MRI might be a useful tool with which to investigate the pathological burden of ALS and to disclose the early signature of possible cognitive impairment. C9orf72 gene can be detected in both diseases. Thus, the clinical, pathological, and genetic overlap between ALS and FTD is widely recognized. Advances in the understanding of the spectrum of frontotemporal dysfunction in ALS have mandated the revision of the Strong criteria, and the term “ALS frontotemporal spectrum disorders” has now been adopted to characterize the expanded syndrome  is a progressive neurodegenerative disease involving both upper and lower motor neurons with no effective cure. In addition to motor impairment, studies have found extramotor abnormalities in ALS including cognitive deficit, autonomic disorders, and extrapyramidal signs . Specifisyndrome . Overallsyndrome –8. Thesesyndrome .in vivo pathological insights into the involvement of frontotemporal and limbic regions in ALS, thus lending support to a neurostructural overlap with FTD. Several imaging techniques, including voxel-based morphometry (VBM) or surface-based morphometry (SBM), diffusion tensor imaging (DTI), single-photon emission computed tomography (SPECT), and 18F-2-fluoro-2-deoxy-d-glucose-PET (FDG-PET), have helped to identify brain changes related to cognitive impairment in patients with ALS  perfusion is a non-invasive, rapidly repeatable method for quantitatively measuring cerebral blood flow (CBF). While ASL-MRI provides information comparable to FDG-PET , it doesThis study was approved by the Research Ethics Committee of Peking Union Medical College Hospital. Patients with ALS who agreed to undergo both neuropsychological assessment and MRI scanning were recruited after informed written consent had been obtained from themselves or their guardians. All patients fulfilled the revised El Escorial diagnostic criteria for clinically definite, probable, or lab-supported probable ALS , 20. ALSDemographic and clinical characteristics of included patients, including the age at the time of MRI, gender, site of onset, education level, and disease duration  were collected. Disease severity was assessed by the revised ALS functional rating scale . All parImages were acquired on a 3.0 T magnetic resonance system  with an 8-channel phase array head coil. A sagittal T1-weighted three-dimensional (3D) fast spoiled gradient echo sequence  was applied to acquire structural images of the whole brain. Images of 3D pseudocontinuous ASL (PCASL) were collected using the following protocol: 8 arms with 512 points in each spiral arm, bandwidth = ±62.5 kHz, slice thickness = 4 mm, postlabel delay = 2,025 ms, image reconstruction matrix = 128 × 128, FOV = 24 cm, and number of excitations = 2. Fast axial T1 FLAIR with the same section as ASL and other conventional sequences  were also performed to exclude major neurological disorders such as tumor, stroke, or advanced WM disease.t-tests between paired groups, considering sex, age at MRI scan, and education years as text covariates. When we compared the CBF differences among the groups, GM maps were also considered as image covariates to control for the GM loss. All comparisons were tested for significance at p < 0.05 voxel-wise-corrected for multiple comparisons using false discovery rate (FDR). If significant clusters were not found, a more liberal voxel-wise threshold at p < 0.001 uncorrected with an extent threshold of 100 voxels was adopted.Imaging data were postprocessed using SPM12 implemented in Matlab 2015a. VBM was performed to observe GM changes using the DARTEL method, including the standard procedure of segmentation template creation based on all participants, normalization to Montreal Neurological Institute space, and smoothing with an 8 mm full width at half maximum (FWHM) filter, following the VBM tutorial.2 test or Fisher’s exact test for discrete variables and a two-sample t-test for continuous variables following a normal distribution. For variables that did not follow a normal distribution, Mann–Whitney U tests were carried out.Using the SPSS 22.0 statistical package, demographic and clinical data of patients in different groups were compared using the χA total of 55 patients with ALS were included: 27 patients were diagnosed with ALS-Cn, 17 with ALS-Ci (12 with executive dysfunction and 5 with non-executive dysfunction), and 11 with ALS-FTD. The demographic and clinical features of included patients are summarized in Table p < 0.05 FDR-corrected, Figure p < 0.05 FDR-corrected, Figures p < 0.001 uncorrected). Please refer to Data Sheet S2 in Supplementary Material for details regarding anatomical regions and brain coordinates for each contrast.The ANCOVA revealed a significant main effect of cognitive diagnosis, and regions of GM differences include the frontal lobe, temporal lobe, precentral gyrus, cingulate gyrus, and thalamus, all bilaterally (p < 0.05 FDR-corrected, Figure p < 0.05 FDR-corrected, Figure p < 0.05 FDR-corrected, Figures There was a significant main effect of cognitive diagnosis in the ANCOVA, and regions of CBF differences include the bilateral frontal lobe, insular lobe, corpus callosum, and caudate, predominantly in the left hemisphere (Figure To date, this is the first study using ASL to assess the voxel-based CBF of ALS patients with varying levels of cognitive deficit. Combined with the VBM study, we identified a similar pattern of frontal and temporal lobe hypoperfusion and GM loss among ALS-FTD patients compared with the patterns observed in HCs, ALS-Ci, and ALS-Cn patients, and the alterations were more significant in the left hemisphere. Perfusion abnormalities were detected in many of the same brain areas wherein structural alterations occurred, although isolated hypoperfusion and atrophy were also detected in some GM areas.A voxel-wise meta-analysis of VBM studies demonstrated that the entire cohort of ALS patients exhibited a significant GM volume reduction in the bilateral precentral gyrus and the frontal and temporal lobes . HoweverWhen the comparisons were conducted within the ALS cognitive subgroups, we found no difference between ALS-Cn and ALS-Ci, while previous investigations have reported that ALS-Ci patients exhibited intermediate cortical atrophy and thinning between pure ALS and ALS-FTD , 12. HowIn keeping with MRI studies of patients with ALS-FTD, a distributed GM volume reduction across the whole brain was observed in the ALS-FTD patients in our study, regardless of the comparative group. In addition, in all brain regions, there was a trend toward a more severe GM loss compared with, in increasing order, ALS-Ci patients, ALS-Cn patients, and HCs, particularly in the left frontal and prefrontal lobes and the bilateral temporal lobe. Degeneration of the frontal and prefrontal cortices is associated with executive dysfunction and behavioral disorders. The temporal lobes subserve various cognitive domains, including processing of color information and word and face recognition. Atrophy of the limbic system might lead to emotional dysregulation and memory deficits. All of the symptoms mentioned earlier were noted in the majority of the ALS-FTD patients in this study. We also detected significant subcortical GM loss within the basal ganglia, which are directly connected to the key cortical regions involved in ALS pathology. This finding is in agreement with a previous MRI study that highlighted widespread basal ganglia involvement along the ALS/FTD spectrum and suggested that the degree of subcortical GM loss in ALS is closely associated with cognition . The cerSince CBF is closely related to brain metabolism, ASL-MRI might depict functional deficiencies in a manner similar to FDG-PET. Similar patterns of hypoperfusion and hypometabolism in regions typically associated with AD and FTD were identified using this technique, indicating that ASL-MRI is in good accordance with FDG-PET . HoweverWe also examined whether the CBF results could be influenced by regional GM changes. FTD is known to be associated with widespread GM loss in many brain regions, primarily the frontal and temporal regions. GM loss may lead to an artificial reduction in measured blood perfusion. When comparing CBF differences among the cognitive subgroups and controls, this issue could potentially be crucial due to group differences in the degree of regional atrophy. In this study, after GM correction, we found that the CBF results were slightly altered, primarily in areas of involvement in the left frontal and prefrontal lobe that were smaller, but the patterns were approximately consistent with those before correction. This implies that our CBF results could reflect the intrinsic brain perfusion alterations in these patients.In our study, regions with reduced CBF detected in patients with ALS-FTD in extramotor regions, particularly the left prefrontal and temporal cortices, correlated with the structural changes observed in VBM studies. Interestingly, these regions are considered to be particularly vulnerable in patients with FTD. Our findings are supported by the results of a study comparing structural brain MRI and metabolic FDG-PET changes in the cortical regions of patients with ALS-FTD, in which significant reductions in cerebral glucose metabolism rates and structural changes in brain regions were concomitantly observed . It shouHowever, isolated GM loss and CBF decrease were also detected in ALS-FTD patients in our study. Such local discordance between brain perfusion/metabolism and structural changes was observed in patients with ALS and the behavioral variant FTD as well , 46–48 . The finDespite the promising results, the limitations of this study should be considered. First, the main limitation is the relatively small numbers of patients with ALS-Ci and ALS-FTD. It would be beneficial to replicate these findings in larger independent samples and to establish the clinical value of ASL in managing ALS or FTD. Second, although we did not select the tests requiring hand function for a great number of patients who were affected by motor disability, and verbal tests were selected instead, we did not adjust for patients with bulbar dysfunction, whose scores might be lowered consequently. In addition, to avoid patient fatigue, we did not include the assessment of premorbid IQ, which is an important predictor of cognitive reserve, despite its minor impact on executive function. Third, as this is a cross-sectional study, patients were studied at a single time point only, and brain alterations of ALS cognitive subgroups over time remain largely unexplored. Nevertheless, ALS patients with cognitive deficits, including ALS-FTD patients, have shorter lifespans than patients without cognitive or behavioral impairment , 34, whiIn conclusion, the cognitive status of ALS patients is associated with different patterns of GM atrophy and cerebral perfusion, lending support to the hierarchical brain changes observed in patients on the ALS/FTD spectrum. Our findings also highlight the complex relationships and regional heterogeneity between GM atrophy and hypoperfusion in ALS-FTD patients. While the majority of the perfusion alterations largely overlapped with the structural changes, greater GM atrophy than hypoperfusion might suggest that the brain tissue loss did not involve metabolically active cells or that the perfusion of the remaining cells was higher than expected. On the other hand, greater hypoperfusion than GM atrophy could reflect either a diaschisis mechanism or an early stage of metabolic neuronal failure before cell death. Finally, our study emphasizes that any single imaging technique alone does not allow a complete understanding of the brain alterations in ALS. ASL-MRI can identify early regional spreading of pathological changes and is beneficial for exploring the pathophysiological basis of cognitive impairment in ALS along the disease course.This study was carried out in accordance with the recommendations of Good Clinical Practice and applicable local regulations with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Research Ethics Committee of Peking Union Medical College Hospital.Study concept and design: LC and FF. Acquisition of data: DS, BH, YX, BC, PP, XL, HT, HF, KZ, SL, JG, and ML. Analysis and interpretation of the data; drafting of the manuscript: DS and BH. Study supervision: JG, ML, FF, and LC. Critical revision of the manuscript: LC.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.


The meta-analysis was performed to investigate the clinical efficacy of spleen-preserving distal pancreatectomy with splenic vessel preservation (SPDP-SVP) and spleen-preserving distal pancreatectomy with splenic vessel resection (SPDP-SVR).P< .05 or I2>50% indicated significant heterogeneity. The random-effects model is used to pool data if significant heterogeneity exists; otherwise, the fixed-effects model is used. Publication bias was evaluated by Begg's funnel plot.Potential articles were searched on the databases of Pubmed, Embase, and Chinese National Knowledge Infrastructure (CNKI) from January 1988 until March 2017. Weight mean difference (WMD) with 95% confidence interval (CI) was applied to compare the efficacy of SPDP-SVP and SPDP-SVR. Odds ratio (OR) with 95% CI was calculated to figure out the risks for complications. P = .636).Thirteen eligible articles were obtained in the meta-analysis. SPDP-SVP seemed to relate with reduced operative time and blood loss, prolonged hospital stay, and less complications; however, the effects were not statistically significant. Meanwhile, we found that SPDP-SVP was closely related with the reduced rate of splenic infarction and gastric varices . No publication bias was observed in the analysis (SPDP-SVP seems to show superiority than SPDP-SVR in reducing the rate of splenic infarction and gastric varices. Hence, spleen-preserving distal pancreatectomy (SPDP) is frequently applied to decrease the occurrence of sepsis and improve patients’ survival.Distal pancreatectomy with splenectomy has been demonstrated to be an effective therapy for managing benign or low-grade malignant lesions in the body or tail of the pancreas.,5 For this technique, a small breakage in the splenic vessels could result in massive intraoperative bleeding, which makes SPDP difficult. To control bleeding from the main splenic vessels, combined spenectomy is always suggested. Another one is spleen-preserving distal pancreatectomy with splenic vessel resection (SPDP-SVR). It involves resection of the splenic vein and artery before distal pancreatectomy, and conservation of the short splenocolic and gastric vessels to keep normal blood flow of spleen. During the procedure, not only the 2 main splenic vessels need to be controlled, but also fine resection near the splenic hilum is always required. Moreover, the risks of perigastric varices and spleen-related morbidities should be bewared.,7 Up to now, the superiority of SPDP-SVP or SPDP-SVR in managing the pancreatic lesions is still debatable.Two surgical techniques for SPDP have been described. Spleen-preserving distal pancreatectomy with splenic vessel preservation (SPDP-SVP) preserves the main splenic artery and vein and excises the tail of the pancreas and those small, short vascular connections to the body.The current meta-analysis was initiated to compare the clinical efficacy between SPDP-SVP and SPDP-SVR in the management of the pancreatic lesions. In the analysis, laparoscopic, robotic and open surgery SPDP were all considered. The outcome contributes to improving the treatment of patients with benign or low-grade malignant lesions in the body or tail of the pancreas.22.1We searched Pubmed, Embase, and Chinese National Knowledge Infrastructure (CNKI) databases for potential articles from January 1988 until March 2017. The search terms included distal pancreatectomy, spleen, vessel, preservation/conservation, and Kimura technique. The references of obtained articles were manually searched to identify possible studies.2.2These obtained articles were selected according to inclusion criteria and exclusion criteria. The studies comparing the clinical efficacy of SPDP-SVP and SPDP-SVR will be considered in the current meta-analysis, whatever approach  was used. For the studies with overlapped data, the recent published one was selected.The exclusion criteria were as follows: review articles; case reports; the studies only focused on the clinical efficacy of SPDP-SVP or SPDP-SVR and not compared SPDP-SVP and SPDP-SVR; and the articles not reported available data.2.3Two authors were responsible for extracting data from included articles. The data were: name of first author, publication year, country, sample size, study design , surgery type , operative time, blood loss, hospital time, overall complications (minor complications and major complications), pancreatic fistula, splenic infarction, and gastric varices of each group. Minor complication is graded as Dindo grades I-II and major complication is listed as Dindo grades III-IV. The debatable issues were discussed with a third author.2.4P< .05 or I2> 50% indicated significant heterogeneity. The random-effects model is used to pool data if significant heterogeneity exists; otherwise, the fixed-effects model is used. Sensitivity analysis was performed to evaluate the robustness of overall results. Possible publication bias was evaluated by Begg's funnel plot and Egger's regression analysis.The current meta-analysis was performed with Stata 12.0 software. Weight mean difference (WMD) for continuous outcomes and odds ratios (ORs) for dichotomous outcome are provided. WMD with 95% confidence interval (CI) was used to figure out the influences of SPDP-SVP in operative time, blood loss, and hospital stay, compared with SPDP-SVR. OR with 95% CI was calculated to represent the risks for overall complications. 33.1–18,28,29 About 122 relevant articles were obtained after rough search. And 81 articles were excluded after screening titles and abstract. For the remaining 41 articles, 31 articles were removed for not comparing the efficacy of SPDP-SVP and SPDP-SVR (n = 20) and unavailable data (n = 8). The selection process was exhibited in Figure –18,28,29We carefully selected the obtained articles according to the inclusion and exclusion criteria. At last, 13 eligible articles were obtained.3.2Random-effects model was used to comparing the efficacy of SPDP-SVP and SPDP-SVR in operative time, blood loss, and hospital stay Table –4. We foIn addition, the outcome indicated that SPDP-SVP was related with less overall complications  and the relationship was not statistically significant Table .3.3Sensitivity analysis was performed to assess the robustness of pooled outcome. The analysis suggested the pooled outcome was robust.3.4P = .636, complications analysis)  Fig. .4 Besides, it may increase the postoperative platelet count. In 1943, Mallet-Guy and Vachon firstly described SPDP technique. It is commonly applied in patients with benign or low-grade malignant tumors in the body and tail of the pancreas. In 1994, Soper et al developed an animal model for laparoscopic distal pancreatectomy, which is demonstrated to be safe and effective. In 1996, Gagner et al suggested a spleen-preserving laparoscopic distal pancreatectomy procedure with preserving splenic artery and vein, which was performed in patients with cystic tumors, neuroendocrine tumors, and chronic pancreatitis. In 1999, Vezakis et al demonstrated that spleen-preserving laparoscopic distal pancreatectomy could be performed with splenic vessel resection as well.Distal pancreatectomy for malignant tumors of the tail or body of the pancreas needs a splenectomy. Whereas, splenectomy usually results in postsplenectomy infection in 1% to 5% patients and cause 50% of mortality rate.–27As for SPDP-SVP, it could conserve the splenic artery and vein, maintain the blood supply to spleen, and ultimately reduce the risks of abscess formation and splenic necrosis. However, SPDP-SVP is time-consuming. Moreover, the technique is difficult because of delicately dissecting the small branches of splenic vessels. During SPDP-SVR procedure, dissecting the splenic vessels may be difficult when large tumors compress and distort the course of the vessels. SPDP-SVR has been demonstrated to be faster and less technically demanding compared with SPDP-SVP. thought that the short-term benefits of SPDP-SVP compared with SPDP-SVR may lead to an increased preference for this technique. In the study of Zhou et al, there were no significant differences between SPDP-SVP and SPDP-SVR groups in blood loss and operative time. However, the rates of splenic infarction were 16.0% in the SPDP-SVP group and 52.5% in the SPDP-SVR group at 3 days after surgery. At 6 months, the rates of gastric varices were 1.9% in the SPDP-SVP group and 35% in the SPDP-SVR group. These data also indicated the superiority of SPDP-SVP than SPDP-SVR. Beane et al reported that SPDP-SVP procedure was related with less blood loss than SPDP-SVR. In addition, SPDP-SVP resulted in fewer grade B or C pancreatic fistulas and splenic infarctions, and shorter post-operative length of stay. They concluded that SPDP-SVP was preferred when SPDP was performed. On the contrary, the study by Matsushima et al suggested that SPDP-SVR (Warshaw) could be used as the more appropriate procedure in cases whose tumors are relatively large or close to the splenic vessels.In recent years, more attention has been paid to compare the clinical efficacy of SPDP-SVP and SPDP-SVR in managing the pancreatic lesions. Adam et al reported that operative time, blood loss, postoperative complications, pancreatic fistula rates, and hospital stays were comparable between SPDP-SVP and SPDP-SVR. However, SPDP-SVR was related with higher incidence rates of splenic ischemia and gastric/perigastric varices. Another meta-analysis by Partelli et al concluded that the 2 procedures were comparable in terms of intraoperative blood loss and rate of pancreatic fistula. SPSP-SVR did not affect the risk of perigastric collateral vessels and submucosal varices. Compared with the 2 meta-analyses, the selected articles were searched on CNKI database and much more Chinese population was analyzed, which may contribute to obtaining much more comprehensive conclusion. In the analysis, both of overall complications and every complication were investigated, which may help for obtaining much more clear conclusions.In the current meta-analysis, we found that SPDP-SVP seemed to reduce operative time and blood loss compared to SPDP-SVR; however, the effects were not statistically significant. Besides, SPDP-SVP was related with prolonged hospital stay (WMD: 0.21), but the relationship was not statistically obvious. Meanwhile, we found that SVP was related with less complications  and the relationship was not statistically significant. Moreover, we found that SPDP-SVP was closely related with the reduced rate of splenic infarction and gastric varices . The results were calculated based on 13 articles and they were credible. One meta-analysis by Tang et al and Fernandez-Cruz et al). Meanwhile, no selected studies were designed as randomized controlled trials (RCTs). The doctors must respect the intention of patients and informed consent must be signed by patients before surgery, which may be one reason for no RCTs.However, there existed limitations in the analysis. First, there existed significant heterogeneity in the analysis of operative time, blood loss and hospital time, which might result from the differences in characteristics of patients and operation manipulation. Second, specific type of complications was not analyzed in the current analysis because of the lack of sufficient data. Third, most studies were conducted with retrospective design in addition of 2 studies (Worhunsky et alIn conclusion, SPDP-SVP seems to show superiority than SPDP-SVR for patients with benign or low-grade malignant tumors in the body and tail of the pancreas. It shows close relationship with reduced rate of splenic infarction and gastric varices.


The available public and controlled access data sets from different sources are curated and re-analysed using standardized, open source pipelines and made available for queries, download and visualization. As of August 2017, Expression Atlas holds data from 3,126 studies across 33 different species, including 731 from plants. Data from large-scale RNA sequencing studies including Blueprint, PCAWG, ENCODE, GTEx and HipSci can be visualized next to each other. In Expression Atlas, users can query genes or gene-sets of interest and explore their expression across or within species, tissues, developmental stages in a constitutive or differential context, representing the effects of diseases, conditions or experimental interventions. All processed data matrices are available for direct download in tab-delimited format or as R-data. In addition to the web interface, data sets can now be searched and downloaded through the Expression Atlas R package. Novel features and visualizations include the on-the-fly analysis of gene set overlaps and the option to view gene co-expression in experiments investigating constitutive gene expression across tissues or other conditions.Expression Atlas ( Its primary remit continues to be serving as a value-added database, providing information on where and under what conditions different genes are expressed on RNA and protein level. To achieve this, Expression Atlas enables gene- and condition-based queries across tissues, cell types, developmental stages, disease states and other conditions across multiple data sets. Expression Atlas aims to serve a wide research community by providing data sets from different organisms, including plants and metazoans.Since the last update in 2016, Expression Atlas . Expression Atlas has also analysed and will disseminate expression data from the Pancancer Analysis of Whole Genomes (PCAWG) project (https://www.ebi.ac.uk/gxa/experiments?experimentSet=Pan-cancer).Since our last update, the number of studies in Expression Atlas has grown two-fold. A quarter of studies relate to plants, now spanning across twenty different species. The first multi-omics study including RNA-Seq and a proteomics data set in human induced pluripotent stem (iPS) cells, generated within the HipSci project , has beehttps://www.bioconductor.org/packages/release/bioc/html/ExpressionAtlas.html). Finally, users are now able to perform gene set overlap queries that result in a list of comparisons from differential experiments enriched for the group of genes of interest.Since the last release, programmatic access has been made available through a new R package in Bioconductor (Sus scrofa), green monkey (Chlorocebus sabaeus), wild cabbage (Brassica oleracea), potato (Solarum tuberosum), zebrafish (Danio rerio), tomato (Solanum lycopersicum). The majority of studies continue to be of differential design, consisting of 2998 data sets, studying samples in 8391 differential comparisons, in 33 different organisms.At the time of writing, Expression Atlas contains 3127 studies comprising 113 148 assays, including 1101 studies on human, 2193 on mammals (including human) and 731 studies in plants. Table http://www.ebi.ac.uk/gxa/experiments?experimentSet=BLUEPRINT), HipSci (http://www.ebi.ac.uk/gxa/experiments?experimentSet=HipSci), Genotype-Tissue Expression (GTEx) version 6 (http://www.ebi.ac.uk/gxa/experiments/E-MTAB-3871), Human Developmental Biology Resource (HBDR) for prenatal brain development (http://www.ebi.ac.uk/gxa/experiments/E-MTAB-4840), PCAWG—PanCancer Analysis of Whole Genomes Project (https://www.ebi.ac.uk/gxa/experiments?experimentSet=Pan-Cancer), which will be openly available with the PCAWG publication. Expression Atlas continues to re-annotate and re-analyse data sets with each new Ensembl . Experiments can be searched via direct accessions, if known, or by using meta-data such as species, experimental conditions or tissues. The search uses ontology (based on EFO) term expansion, for instance, a search for ‘cancer’ will also retrieve experiments annotated as ‘leukemia’. Raw and normalized counts per experiment, as well as data annotations are available for download, for both RNA-Seq and microarray experiments.Since the previous update we have released an R package in Bioconductor, which enables users to search and download pre-packaged data from all studies in the Expression Atlas database into their R session  found in each comparison/study in the same species. The P-values from these tests are corrected for multiple tests using the Benjamini–Hochberg procedure. This functionality is available via the web-interface (front page) but also via a REST API. The implementation is available through https://github.com/gxa/atlas_gsa.Expression Atlas now enables users to check if a set of genes of their interest overlaps significantly with the sets of differentially expressed genes in Atlas experiments of the same species. This enables users to test the results of their own experiments and investigate whether there is evidence that their set of differentially expressed (DE) genes is also DE in other experimental settings, involving similar or different experimental conditions, diseases or stresses. In a nutshell, the gene set overlap is implemented using the Fisher-exact test to check if the user's set of genes overlaps significantly with the DE genes (adjusted https://bioconductor.org/packages/release/bioc/html/clusterSeq.html). ClusterSeq (manuscript in preparation) investigates patterns of expression by comparing k-means clusters of gene expression across conditions, between each pair of genes, for different values of k. The results can be viewed through single gene queries within a baseline experiment, showing those similarly expressed genes to the original query, ranked by similarity of expression patterns  and can be integrated in any third-party site, provided that the bioentity identifiers match those of Expression Atlas.Baseline expression data from Expression Atlas continue to be automatically included in Ensembl, Ensembl Genomes, Gramene , EnsemblWe are actively working on curation efforts, coupled with the development of analysis pipelines and appropriate visualization tools for single cell RNA-Seq data. In the future, we will release a new web-service to accommodate and visualize these data sets.We will be adjusting our analysis pipelines and web interfaces to process alternative transcript quantifications and visualize dominant transcripts for each gene .In the future, we will integrate pathway diagrams to enable users to view gene expression across the components of a pathway and how this is affected across different experimental conditions.Currently, through our keyword search, we enable users to implicitly view gene expression across orthologous genes, amongst other related genes. We aim to develop a query and visualization tool that will present orthologous gene expression explicitly in the user interface.We will be testing methods for batch correction of expression data sets from different studies, with the view of releasing aggregated, cross-study results on baseline and differential views of the data.Future plans involve expanding the number of proteomics data sets included in Expression Atlas. We are developing analysis and QC pipelines and the additional needed infrastructure for enabling the routine integration of high-quality data sets, now that the availability of proteomics data in the public domain has become the norm ,31. FurtFinally, we are always seeking feedback from our users, through our website, training courses, conference attendance or usability testing. Plans for new features on our web-site and R package are continuously evaluated and adjusted through our interactions with our users.


Although membrane proteins represent most therapeutically relevant drug targets, the availability of atomic resolution structures for this class of proteins has been limited. Structural characterization has been hampered by the biophysical nature of these polytopic transporters, receptors, and channels, and recent innovations to in vitro techniques aim to mitigate these challenges. One such class of membrane proteins, the ATP-binding cassette (ABC) superfamily, are broadly expressed throughout the human body, required for normal physiology and disease-causing when mutated, yet lacks sufficient structural representation in the Protein Data Bank. However, recent improvements to biophysical techniques  have allowed for previously “hard-to-study” ABC proteins to be characterized at high resolution, providing insight into molecular mechanisms-of-action as well as revealing novel druggable sites for therapy design. These new advances provide ample opportunity for computational methods  to catalyze the discovery of novel small molecule therapeutics that can be easily translated from computer to bench and subsequently to the patient's bedside. In this review, we explore the utility of recent advances in biophysical methods coupled with well-established in silico techniques towards drug development for diseases caused by dysfunctional ABC proteins.  The adenosine triphosphate- (ATP-) binding cassette (ABC) protein superfamily is comprised of transmembrane proteins that utilize the energy generated from ATP binding and hydrolysis to translocate physiological solutes or ions across the lipid bilayers of certain cell types throughout the body . Based oThe human ABC protein superfamily is comprised of 49 genes . Most ofABC proteins responsible for multidrug resistance in human cancers include the multidrug resistance protein 1 (MRP1/ABCC1), P-glycoprotein (P-gp/ABCB1), and the breast cancer resistance protein (BCRP/ABCG2) , 27–29. Design of small molecule modulators for other disease-relevant ABC protein targets , and nuclear magnetic resonance (NMR) spectroscopy. Each technique has its own set of advantages and disadvantages, and these will be explored in more detail in Using solved structures of disease-associated ABC proteins, many of the traditional in silico structure-based tools can be used to facilitate drug development . In geneHowever, as previously mentioned, most ABC proteins that have been structurally characterized and reported in the Protein Data Bank to date are from non-human species . Thus, hBy using these high-quality, human disease-relevant biophysical structures, best-in-class therapies can be developed. Appropriate medicinal chemistry approaches facilitated by well-established in silico techniques, including homology modeling , MD simuAnother computational tool, which has provided much insight into the conformational dynamics, ATP-dependent transport/gating cycles, and putative mechanism-of-action of small molecule modulators of certain ABC proteins, is that of MD simulations . This meLastly, any small molecules identified using these aforementioned in silico approaches must abide by previously established (and predictive) drug development algorithms, including Lipinski's “Rule of 5”  and the As previously described, ABC proteins are found in all three domains of life , playing many key physiological roles required for normal biology. ABCB1 is no exception; however, when this ABC transporter is overexpressed it is disease-causing in humans. ABCB1 was the first ABC protein to be identified and cloned, as it was highly expressed in the tumours of cancer patients . ABCB1 i CFTR gene in conjunction with the discovery of CFTR as a phospho-regulated chloride channel was seminal in the development of high-throughput, target-based initiatives for Cystic Fibrosis drug discovery [Another ABC protein, CFTR (ABCC7), is the most unique member of the ABC superfamily. Cloning of theiscovery , 73–75. iscovery . Althougiscovery , 77. Furiscovery .In addition, the transporter associated with antigen processing (TAP1/ABCB2) is an important ABC family member required for adaptive immunity . TAP1 trFinally, as more ABC proteins become structurally characterized in multiple conformations using state-of-the-art biophysical techniques, our understanding of similarities (and differences) between each unique family member will be further elucidated. This may facilitate identification of druggable binding sites which are only present in the mutant form of each target of interest, thereby allowing for efficient in silico virtual screening and SBDD initiatives aimed at developing ABC- and mutation-specific therapeutics. This approach may also assist with the repurposing of drugs within the ABC superfamily. One recent example is the repurposing of the CFTR/ABCC7-specific potentiator, ivacaftor , for patients with mutations in a homologous ABC protein: ABCB4 . Both inABC proteins are required for normal physiology and can cause disease when mutated. Biophysical techniques aim to structurally characterize this family of membrane proteins, and recent improvements to certain techniques, mainly cryo-EM, have elucidated the tertiary topology of several disease-relevant ABC proteins. This structural information provides a foundation for in silico-based drug discovery and drug development initiatives  intended to repair misfolded and/or dysfunctional mutant ABC variants. Thus, potentially druggable sites can be identified and investigated with respect to the compatibility of small molecule therapeutics aimed at improving structural defects caused by various mutations, and further, specific modulators can be designed using various well-established in silico approaches. Taken together, the advent of high resolution structures of these human disease-relevant proteins provided ample opportunity to translate findings from the lab into the clinic.