An ancestral genomic locus in Mycobacterium tuberculosis clinical isolates from India hints the genetic link with Mycobacterium canettii.

BACKGROUND: Tuberculosis remains a worldwide public health emergency. To better understand M. tuberculosis and to identify genomic variations characteristic to the Indian clinical isolates by a low-cost method, a genomic subtractive hybridization between M. tuberculosis H37Rv and a clinical isolate from South India was performed. RESULTS: This revealed a novel 0.4-kb subtractive fragment which was used as a handle to pull out a 4.5-kb genomic region characteristic to the clinical isolate and was absent in H37Rv. On further studies, this 4.5-kb region was found to be present in 91% of the M. tuberculosis clinical isolates screened from Kerala, a state in South India. Interestingly, this novel region has 99% identity (with 100% query coverage) with genomic regions of M. canettii. DISCUSSION: The present study hypothesizes that this locus was present in the recent common environmental ancestor of mycobacteria, retained to the maximum extent in M. canettii and ancestral isolates of M. tuberculosis, and later deleted in other modern lineages of M. tuberculosis. Thus, this region may serve as one of the links between the pathogenic mycobacteria and the environmental species. We also propose that the Indian isolates of M. tuberculosis might be closely related to the putative progenitor M. prototuberculosis with respect to this locus. More studies on other genomic loci from different strains of M. tuberculosis are required to establish more links in this direction.

Mycobacterium tuberculosis in Wild Asian Elephants, Southern India.

We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants.

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation.

Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.

Whole-genome sequences of two clinical isolates of Mycobacterium tuberculosis from Kerala, South India.

We report the annotated genome sequence of two clinical isolates of Mycobacterium tuberculosis isolated from Kerala, India.

Comparative analysis of mycobacterial truncated hemoglobin promoters and the groEL2 promoter in free-living and intracellular mycobacteria.

The success of Mycobacterium tuberculosis depends on its ability to withstand and survive the hazardous environment inside the macrophages that are created by reactive oxygen intermediates, reactive nitrogen intermediates, severe hypoxia, low pH, and high CO(2) levels. Therefore, an effective detoxification system is required for the pathogen to persist in vivo. The genome of M. tuberculosis contains a new family of hemoproteins named truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), encoded by the glbO and glbN genes, respectively, important in the survival of M. tuberculosis in macrophages. Mycobacterial heat shock proteins are known to undergo rapid upregulation under stress conditions. The expression profiles of the promoters of these genes were studied by constructing transcriptional fusions with green fluorescent protein and monitoring the promoter activity in both free-living and intracellular milieus at different time points. Whereas glbN showed an early response to the oxidative and nitrosative stresses tested, glbO gave a lasting response to lower concentrations of both stresses. At all time points and under all stress conditions tested, groEL2 showed higher expression than both trHb promoters and expression of both promoters showed an increase while inside the macrophages. Real-time PCR analysis of trHb and groEL2 mRNAs showed an initial upregulation at 24 h postinfection. The presence of the glbO protein imparted an increased survival to M. smegmatis in THP-1 differentiated macrophages compared to that imparted by the glbN and hsp65 proteins. The comparative upregulation shown by both trHb promoters while grown inside macrophages indicates the importance of these promoters for the survival of M. tuberculosis in the hostile environment of the host.

Deciphering sulfoglycolipids of Mycobacterium tuberculosis.

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

Combined use of Amplified Fragment Length Polymorphism and IS6110-RFLP in fingerprinting clinical isolates of Mycobacterium tuberculosis from Kerala, South India.

BACKGROUND: DNA fingerprinting by IS6110-RFLP has shown a high incidence of Mycobacterium tuberculosis isolates having no and low copies of the insertion sequence in Kerala, South India. Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome rather than a few repetitive elements, we thought that this technique would help us in differentiating the large reservoir of isolates from an endemic region. Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) to type clinical isolates. METHODS: Fifty clinical isolates of M. tuberculosis were analysed by conventional radioactive AFLP and IS6110- RFLP. M. bovis, M. bovis BCG and two non tuberculous mycobacteria were also analysed to see species specific differences generated by AFLP. Cluster analysis was performed using the AFLP profile that showed the maximum polymorphism within M. tuberculosis and this was compared to the number of copies of IS6110 insertions. RESULTS: For AFLP, out of ten primer pairs tested, the EO/MC pair generated maximum polymorphism among the clinical isolates of M. tuberculosis. The similarity between the isolates ranged between 88 and 99.5%. Majority (nearly 85%) of the 'low copy' IS6110 isolates clustered together, while the rest clustered irrespective of the copy numbers. AFLP could show rare differences between isolates of M. tuberculosis, M. bovis and M. bovis BCG. The AFLP profiles for non-tuberculous mycobacteria were highly different from those of M. tuberculosis. CONCLUSION: Polymorphism generated by AFLP within the M. tuberculosis species is limited and hence AFLP alone seems to have limited use in fingerprinting the isolates in Kerala. The combined use of AFLP and IS6110-RFLP showed relatively better differentiation of 'high copy' IS6110 isolates, but failed to differentiate the 'low copy' isolates. However, the technique may be efficient in inter-species differentiation, and hence potentially useful in identifying and developing species-specific markers.

Downregulation of vimentin in macrophages infected with live Mycobacterium tuberculosis is mediated by Reactive Oxygen Species.

Mycobacterium tuberculosis persists primarily in macrophages after infection and manipulates the host defence pathways in its favour. 2D gel electrophoresis results showed that vimentin, an intermediate filament protein, is downregulated in macrophages infected with live Mycobacterium tuberculosis H37Rv when compared to macrophages infected with heat- killed H37Rv. The downregulation was confirmed by Western blot and quantitative RT-PCR. Besides, the expression of vimentin in avirulent strain, Mycobacterium tuberculosis H37Ra- infected macrophages was similar to the expression in heat-killed H37Rv- infected macrophages. Increased expression of vimentin in H2O2- treated live H37Rv-infected macrophages and decreased expression of vimentin both in NAC and DPI- treated heat-killed H37Rv-infected macrophages showed that vimentin expression is positively regulated by ROS. Ectopic expression of ESAT-6 in macrophages decreased both the level of ROS and the expression of vimentin which implies that Mycobacterium tuberculosis-mediated downregulation of vimentin is at least in part due to the downregulation of ROS by the pathogen. Interestingly, the incubation of macrophages with anti-vimentin antibody increased the ROS production and decreased the survival of H37Rv. In addition, we also showed that the pattern of phosphorylation of vimentin in macrophages by PKA/PKC is different from monocytes, emphasizing a role for vimentin phosphorylation in macrophage differentiation.

Subunit Protein Vaccine Delivery System for Tuberculosis Based on Hepatitis B Virus Core VLP (HBc-VLP) Particles.

Despite the development of modern medicine, tuberculosis (TB), caused by the pathogenic bacterium, Mycobacterium tuberculosis (Mtb), remains one of the deadliest diseases. This bacterium can lay dormant in individuals and get activated when immunity goes down and has also shown considerable prowess in mutating into drug resistant forms. The global emergence of such drug resistant Mtb and the lack of efficacy of Bacille Calmette Guérin (BCG), the only vaccine available so far, have resulted in a situation which cries out for a safe and effective tuberculosis vaccine.Number of different strategies has been used for developing new anti-TB vaccines and several protective antigens have been identified so far. One strategy, the use of protein subunits, has the potential to develop into a powerful tuberculosis vaccine, not only because of its efficacy and safety, but also because they are economical. The proper delivery of protein subunit vaccines with adjuvants or novel delivery systems is necessary for inducing protective immune responses. The available adjuvants or delivery systems are inadequate for generating such a response. In the present method, we have constructed a vaccine delivery system for tuberculosis based on Virus-Like Particles (VLPs). Hepatitis B Virus core antigen gene was recombinantly modified using Overlap Extension PCR (OEPCR). The final construct was designed to express HBc-VLP carrying external antigen (fusion VLP). Mycobacterium tuberculosis antigen CFP-10 was used for the construction of fusion VLP. The recombinant gene for the construct was cloned into a pET expression system and transformed into E. coli BL21(DE3) and induced with IPTG to express the protein. The fusion protein was purified using the Histidine tag and allowed to form VLPs. The preformed VLPs were purified by sucrose density gradient centrifugation. The VLPs were characterized using Transmission Electron Microscopy (TEM).

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR.

Rv3334 protein of Mycobacterium tuberculosis belongs to the MerR family of transcriptional regulators and is upregulated during hypoxia and other stress conditions. Employing GFP reporter constructs, mobility shift assays and ChIP assays, we demonstrate that Rv3334 binds to its own promoter and acts as an autorepressor. We were able to locate a 22 bp palindrome in its promoter that we show to be the cognate binding sequence of Rv3334. Using chase experiments, we could conclusively prove the requirement of this palindrome for Rv3334 binding. Recombinant Rv3334 readily formed homodimers in vitro, which could be necessary for its transcriptional regulatory role in vivo. Although the DNA-binding activity of the protein was abrogated by the presence of certain divalent metal cations, the homodimer formation remained unaffected. In silico predictions and subsequent assays using GFP reporter constructs and mobility shift assays revealed that the expression of ketosteroid regulator gene (kstR), involved in lipid catabolism, is positively regulated by Rv3334. ChIP assays with aerobically grown M. tuberculosis as well as dormant bacteria unambiguously prove that Rv3334 specifically upregulates expression of kstR during dormancy. Our study throws light on the possible role of Rv3334 as a master regulator of lipid catabolism during hypoxia-induced dormancy.

Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase.

We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment.

Identification of moaA3 gene in patient isolates of Mycobacterium tuberculosis in Kerala, which is absent in M. tuberculosis H37Rv and H37Ra.

BACKGROUND: Tuberculosis is endemic to developing countries like India. Though the whole genome sequences of the type strain M. tuberculosis H37Rv and the clinical strain M. tuberculosis CDC1551 are available, the clinical isolates from India have not been studied extensively at the genome level. This study was carried out in order to have a better understanding of isolates from Kerala, a state in southern India. RESULTS: A PCR based strategy was followed making use of the deletion region primers to understand the genome level differences between the type strain H37Rv and the clinical isolates of M. tuberculosis from Kerala. PCR analysis of patient isolates using RD1 region primers revealed the amplification of a 386 bp region, in addition to the expected 652 bp amplicon. Southern hybridization of genomic DNA with the 386 bp amplicon confirmed the presence of this new region in a majority of the patient isolates from Kerala. Sequence comparison of this amplicon showed close homology with the moaA3 gene of M. bovis. In M. bovis this gene is present in the RvD5 region, an IS6110 mediated deletion that is absent in M. tuberculosis H37Rv. CONCLUSION: This study demonstrates the presence of moaA3 gene, that is absent in M. tuberculosis H37Rv and H37Ra, in a large number of local isolates. Whether the moaA3 gene provides any specific advantage to the field isolates of the pathogen is unclear. Field strains from Kerala have fewer IS6110 sequences and therefore are likely to have fewer IS6110 dependent rearrangements. But as deletions and insertions account for much of the genomic diversity of M. tuberculosis, the mechanisms of formation of sequence polymorphisms in the local isolates should be further examined. These results suggest that studies should focus on strains from endemic areas to understand the complexities of this pathogen.

Mycobacterium tuberculosis Infection Induces HDAC1-Mediated Suppression of IL-12B Gene Expression in Macrophages.

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live, but not killed, virulent MTB. Additionally, we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live, virulent MTB, and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases, and by knockdown of crucial transcriptional regulators, we demonstrate that protein kinase-A (PKA), CREB, and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis, we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live, virulent MTB to subvert the immune system to survive and replicate in the host.

Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles.

Nano-sized hepatitis B virus core virus-like particles (HBc-VLP) are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10) is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.

Dry powder cationic lipopolymeric nanomicelle inhalation for targeted delivery of antitubercular drug to alveolar macrophage.

Excipients having self-assembling properties are less explored in the field of dry powder inhalation (DPI) technology. An amphiphilic lipopolymer system was developed using stearic acid (SA) and branched polyethyleneimine (BPEI) (1800 Dalton), at different proportions by covalent conjugation. A molecular dynamic (MD) simulation tool was employed for predicting the carrier behavior in a polar in vivo condition. The structural characterization was carried out using nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared (FTIR) spectroscopy. The physical nature of the lipopolymer was analyzed by differential scanning calorimetry. Determination of zeta potential and diameter of the micelles showed existence of cationic particles in the nano size range when a lower number of primary amino groups of BPEI was grafted with SA. The rifampicin (RIF)-loaded lipopolymer was also formulated further into spray-dried microparticles. Powder X-ray diffraction (PXRD) studies revealed that the RIF API (active pharmaceutical ingredient) exists as molecular dispersion in spray-dried microparticles. Topological analysis of the spray-dried nanomicelle was carried out using scanning electron microscopy (SEM). A large population of the drug-carrying particles were found to be under the inhalable size range (fine particle fraction 67.88% ± 3%). In vitro drug release kinetics from spray-dried nanomicelles were carried out at lung fluid pH.

Efficient discrimination by MIRU-VNTRs of Mycobacterium tuberculosis clinical isolates belonging to the predominant SIT11/EAI3-IND ancestral genotypic lineage in Kerala, India.

The present study evaluated the ability of MIRU-VNTRs to discriminate Mycobacterium tuberculosis (MTB) clinical isolates belonging to the SIT11/EAI3-IND ancestral genotypic lineage, which is highly prevalent in Kerala, India. Starting from 168 MTB clinical isolates, spoligotyping (discriminatory index of 0.9113) differentiated the strains into 68 distinct patterns, the biggest cluster being SIT11/48 SIT11 (n=48). The present study shows that 12-loci MIRUs and 3 ETRs allowed an efficient discrimination of these isolates (discriminatory indexes of 0.7819 and 0.5523, respectively).

Molecular epidemiology of Mycobacterium tuberculosis isolates from Kerala, India using IS6110-RFLP, spoligotyping and MIRU-VNTRs.

Tuberculosis (TB) continues to be a major health problem in India, and there is very little information about the prevalent genotypes of tubercle bacilli that cause TB in India, especially in Kerala. Our aim was to study the different circulating strains of Mycobacterium tuberculosis (MTB) that are prevalent in Kerala, India. We analyzed 168 MTB isolates from as many pulmonary TB patients using IS6110-RFLP, spoligotyping and MIRU-VNTRs. The results of IS6110-RFLP revealed that majority of isolates had null copy (10.89%) or single copy (44.87%) of IS6110 insertion. Low copy (<6) isolates accounted for 71.5% in the isolates studied. Genotypic clade designations were done by comparing with the SITVIT2 database which showed 68 patterns; of which 51 corresponded to different shared types whereas 17 patterns were orphans. Among the 51 SITs recorded, 42 SITs matched a preexisting SIT in the SITVIT2 database, whereas 9 SITs were newly-created. Majority of the isolates (64.28%) belonged to the ancestral East-African Indian (EAI) lineage. MIRU-40 and 31 (HGDI=0.6555 and 0.6524) showed highest discrimination, while MIRU-2 and 20 (HGDI=0.0354 and 0.0696) had the least discriminatory power. ETR-A and B (HGDI 0.7382 and 0.6743) discriminated better as compared to other MIRU loci. The overall HGDI for MIRU-VNTRs at 0.9735 (calculated for 166 isolates) showed a better discriminatory power than spoligotyping used alone. This study of MTB genotypic diversity was useful by providing a first snapshot of circulating MTB genotypic clones in Kerala.

Bioconversion of sodium dodecyl sulphate to rhamnolipid by Pseudomonas aeruginosa: a novel and cost-effective production strategy.

The utility of rhamnolipids in industry is currently limited due to the high constraints in its economic production. In this scenario, the novel utility of sodium dodecyl sulphate (SDS) as carbon source could serve as promising cost-effective strategy. Screening of effective SDS biodegraders led to the isolation of Pseudomonas aeruginosa S15 capable of concomitant SDS degradation and biosurfactant synthesis. SDS-based rhamnolipid production was proved on SDS minimal agar plates using cetyl trimethylammonium bromide-methylene blue method and optimised in SDS-based minimal salt (SBS) medium. SDS proved to be an ideal carbon source for rhamnolipid synthesis with a high substrate to product conversion rate yielding 6.9 g/l of rhamnolipids from 1 g/l SDS in 5 days. Fast atom bombardment mass spectroscopy analysis of the purified biosurfactant proved the presence of mono- and di-rhamnolipids, viz., Rha-C(10)-C(10), Rha-C(10)-C(12) and Rha-Rha-C(10)-C(10) with surface active properties. The secreted rhamnolipids were not utilised by S15 as a carbon source, but it caused a dispersion of bacterial biofilms in SBS medium. To the best of our knowledge, this is the first report on bioconversion of synthetic detergent to biodetergent. This SDS-based novel methodology presents a more economised mode of rhamnolipid synthesis utilising SDS as sole carbon source.

Standardization and evaluation of a tetraplex polymerase chain reaction to detect and differentiate Mycobacterium tuberculosis complex and nontuberculous Mycobacteria--a retrospective study on pulmonary TB patients.

The clinical presentation of pulmonary tuberculosis by members of Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) cannot be differentiated using the available standard diagnostic procedures. A single-tube tetraplex polymerase chain reaction (T-PCR) was designed to simultaneously amplify 4 well-known DNA targets of MTC. Taguchi's protocol was followed for the optimization of the conditions and was then tested on 288 pulmonary TB patient samples. The analytical sensitivity of the T-PCR was 100 fg of purified mycobacterial DNA, and specificity was found to be 100% in being able to distinguish MTC and NTM in all the cases tested. The results correlated well when validated with hsp65 PCR restriction analysis and sequencing of the 16S-23S internal transcribed spacer region, hsp65, and rpoB. The T-PCR described here is a quick, valuable, and cost-effective tool for determining whether the causative organism is MTC or NTM, and thus is useful for disease surveillance.

A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways.

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs.