Glossina pallidipes  to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly's salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.The competence of the tsetse fly G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.SGHV alters the protein expression pattern in  Tsetse fly  transmits two devastating diseases to farmers  and their livestock  in 37 sub-Saharan African countries. During the rainy seasons, vast areas of fertile, arable land remain uncultivated as farmers flee their homes due to the presence of tsetse. Available drugs against trypanosomiasis are ineffective and difficult to administer. Control of the tsetse vector by Sterile Insect Technique (SIT) has been effective. This method involves repeated release of sterilized males into wild tsetse populations, which compete with wild type males for females. Upon mating, there is no offspring, leading to reduction in tsetse populations and thus relief from trypanosomiasis. The SIT method requires large-scale tsetse rearing to produce sterile males. However, tsetse colony productivity is hampered by infections with the salivary gland hypertrophy virus, which is transmitted via saliva as flies take blood meals during membrane feeding and often leads to colony collapse. Here, we investigated the salivary gland secretome proteins of virus-infected tsetse to broaden our understanding of virus infection, transmission and pathology. By this approach, we obtain insight in tsetse-hytrosavirus interactions and identified potential candidate proteins as targets for developing biotechnological strategies to control viral infections in tsetse colonies. Glossina sp.) are found exclusively in sub-Saharan Africa and are efficient vectors of African trypanosomes, causative agents of sleeping sickness in humans and nagana in domesticated animals Glossina pallidipes colonies are vulnerable to infections by a salivary gland hypertrophy virus (SGHV) Tsetse flies  and ten-day old were obtained from the IAEA Insect Pest Control Laboratory Seibersdorf, Austria. To detect the presence of Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) infections in teneral flies, total DNA was extracted from one intermediate excised leg of individual flies using the ZR Genomic DNA kit . The DNA was amplified by PCR using primers and conditions described previously Glossina using SGHV preparations from hypertrophied salivary glands either orally or by injection does not lead to hyperplasia in the same generation (unpublished data). The hyperplasia of the SGs was confirmed microscopically during subsequent dissections. The ten-day old experimental flies were divided into eight groups based on hours post feeding (hereafter referred to as hpf) and whether they were hypertrophied or not. These were groups 1A and B at 0 hpf , and groups of ten-day old flies at 48 (groups 2A and B), 72 (groups 3A and B) and 96 (groups 4A and B) hpf. Flies in groups 1A and B were dissected immediately after PCR results. The flies in the other groups were given a blood meal and maintained in the insectaria in standard rearing conditions Two groups of Harvesting of saliva was performed by adaptation of previously described methods 3 cubes and in-gel protein digestions performed as previously described 2O, followed by 15% acetonitrile (ACN)/1% TFA. The extracts were combined, reduced in volume in a speed vacuum and dissolved in 20 µl of 0.1% formic acid/H2O. The peptides resulting from this digestion were analyzed by LTQ-Orbitrap Nano liquid chromatography coupled to electrospray and tandem mass spectrometry (nanoLC-MS/MS) as previously described To determine the tsetse SGs protein profiles, the saliva samples were separated using SDS-PAGE (12% acrylamide) G. m. morsitans decoy database generated by reversing the protein sequences. The database used for peptide/protein searches was derived from the SGs expressed sequence tag (EST) library available from the International Glossina Genome Initiative (IGGI) (http://old.genedb.org/genedb/glossina/). Protein sequences of common contaminants, e.g. trypsin and keratins were used in MaxQuant's “contaminants.fasta” database. MaxQuant was used with a peptide tolerance of 10 parts per million while all other settings were kept as default with one extra addition of asparagine or glutamine de-amidation as variable modification Raw MS/MS files from the LTQ-Orbitrap were generated by MaxQuant software version 1.1.1.36, supported by Andromeda as the database search engine for peptide identification http://www.blast2go.org/) http://www.cbs.dtu.dk/services/SignalP/).To suggest putative functions for individual tsetse SGs secretome components, the accepted proteins were inputted into Blast2GO v.2.4.8 (http://www.ebi.ac.uk/Tools/pfa/iprscan/). Structural and functional annotations were determined by pasting the single-letter amino acid codes of the proteins into the Sequence Annotated by Structure (SAS) interface (http://www.ebi.ac.uk/thornton-srv/databases/sas/) http://pfam.sanger.ac.uk/).To predict the conserved domains of SGHV-encoded proteins, the viral protein sequences were inputted into InterPro suite  Electrophoretic profiling and identification of tsetse saliva proteins by nanoLC-MS/MS, (2) cataloguing of the MS-supported proteins by gene ontology mapping using Blas2GO suite, (3) confirmation of the presence of N-terminal signal peptide sequences in these proteins by InterProScan and SignalP suites, and (4) prediction of the potential Glossina-SGHV interactions by analyzing the proteins expressed in hypertrophied SGs.For a comprehensive analysis of the The rationale of using 10 and 40 pairs of intact glands from hypertrophied and non-hypertrophied salivary glands respectively, follows from documented reports that hypertrophied salivary glands are enlarged at least four times their normal thickness G. m. morsitans SG EST dataset identified 317 protein hits. Twenty-five of these proteins were SGHV-encoded  SGHV proteins were abundantly expressed in hypertrophied flies relative to non-hypertrophied flies  mapping on the saliva proteins. At least one GO term could be assigned to 285 of the 317 detected saliva proteins. Five (1.8%) Tipula iridescent (TIV) viruses, an enlarged nucleus in target cells was noted to be accompanied by a large increase in nuclear DNA synthesis and a massive enlargement of fat body cells Glossina SG cells, in terms of the formation of vesicles and/or multivesicular bodies associated with various organelles such as the endoplasmic reticulum. Indeed, Sang et al., showed that the cytoplasm of SGHV-infected cells of G. m. centralis are heavily vacuolated, show complete disintegration of cytoplasmic organelles, including the smooth and rough ER and the mitochondria, leaving the nuclei scattered around Glossina origin, as well as cell-to-cell transport.Based on the GO annotations, the MS/MS-supported proteins were grouped into three categories: (1) the broad biological processes (BP) the proteins are involved in, (2) the predicted molecular functions (MF) they perform, and (3) the sub-cellular structures/locations/macromolecular complexes or components (CC) these proteins associate with . The thrGlossina and (6/25) and 24% of SGHV MS/MS-supported proteins contained predicted secretion signals, respectively. Further, SignalP analysis of these proteins confirmed that 23% (65/282) of these Glossina proteins and the 6 SGHV proteins contained N-terminal signal peptide sequences. G. pallidipes saliva proteins with signal peptides predicted by SignalP (hereafter referred to as the SG secretome proteins). Structural and functional annotation of all detected SGHV-encoded proteins revealed that 14 were non-structural (NS) an 11 were structural/capsid (VP) proteins, respectively  of ectively .Glossina proteins may not be synthesized in the SGs. For this, alternative mechanisms for the translocation of proteins into SGs should be taken into account. We expected Glossina to have a large number of saliva effector macromolecules with complex effects due to their unique blood-sucking nature. Perhaps some of the identified proteins and macromolecules are synthesized in other organs and then transported into the SGs through hemolymph, by yet-to-be identified mechanisms. This is likely because SGs are capable of sequestering proteins from the hemolymph and then secreting them It is noteworthy that some of the detected Glossina proteins that are abundantly expressed in the hypertrophied SGs but are either not detectable or are expressed in very low abundance in the non-hypertrophied SGs revealed that approximately 31% (10/32) may be involved in DNA replication  and tsetse antigen 5 protein are of unknown function. It is possible that the need for bulk synthesis of proteins for maturation of the virions in hypertrophied SGs could have led to the down regulation of the larval serum protein-2, which serves as a store of amino acid for synthesis of adult proteins. Additionally, the down regulation of salivary apyrase, which has been postulated to facilitate blood location and blood feeding Glossina SG secretome was based on the assumption that the SG proteins would have N-terminal signal sequences. Two clear trends were inferred from the cataloguing of saliva proteins in this study: (1) a high proportion of Glossina SG secretome proteins indeed have signal peptide sequences, and (2) only a low proportion of the secreted proteins associated with Glossina saliva (∼4.9%) are of unknown function. The percentage of non-annotatable Glossina SG secretome proteins is substantially lower than the average of non-annotatable proteins across other systems such as the pea aphid Acyrthosiphon pisum genome (30%) Glossina saliva in blood feeding.Our approach in establishing the Glossina secretome may have excluded some important proteins from our secretome pool. These proteins do not necessarily need to be excluded from being part of the Glossina secretome. As this is the first study of the secretome of the Glossina SGs, there is insufficient data available to provide insights into the roles of a majority of the identified proteins. However, potential effector roles of many of these candidate proteins, particularly in the interactions between Glossina and SGHV macromolecules, can be predicted based on their homology or similarity to other proteins involved in vector-virus interactions. In this regard, we discuss a selection of the secretome proteins in the context of six categories of functional proteins, and in relation to SGHV infection.It is likely that our stringent analysis and assumption that the secretome proteins must have N-terminal sequences to substantiate their inclusion into the Glossina hypertrophied SG secretome. HSP induction by viruses is not the result of a meager “canonical” HSP-mediated heat shock response (HSR), but rather the effect of virus-controlled transcriptional/translational switches, sometimes involving individual viral products A high abundance of heat shock proteins (HSPs) was identified in the E1A of Adenovirus T ag) of Simian vacuolating virus 40 IE2 of human cytomegalovirus, and nuclear antigen 3 (EBNA3) of EBV hsp70 by direct interaction with different components of the basal transcription apparatus. Additionally HSP70-cognate 4 (HSP70-4) plays an important role in the homeostasis and suppression of O'nyong-nyong virus (ONNV) replication and in the establishment of latent infections in the mosquito Anopheles gambiaeGlossina, this protein may play roles in the establishment of asymptomatic SGHV infections in GlossinaMany viruses interact with HSPs at different infection stages. In Epstein-Barr Virus (EBV), virus attachment at cell membrane receptors activates signal transduction pathways interfering with the heat shock response (HR) Drosophila melanogaster and An. gambiaeWe also identified some members of inducible secreted polypeptides, including C-type lectins (CTLs) . CTLs plADP-ribosylation factor (ARF) is an abundant protein that reversibly associates with Golgi membranes, and is implicated in the regulation of membrane traffic through the secretory pathway Six types of serine proteases were detected in the secretome of hypertrophied SG but not in the non-hypertrophied SG. Additionally, a precursor of phosphenol oxidase activating factor of the prophenol-oxidase-activating system (proPO-AS), an important component of the innate immune response in insects RNA polymerase II general transcription factor (BTF3), the translation elongation factor EF-1 gamma (EF1γ), and the ATP-dependent RNA helicase were among the factors detected in the secretome of hypertrophied SG. BTF3 is a general transcription factor necessary for activation of a number of viral promoters by RNAP II Most viruses use components of the host cytoskeleton to move within cells. Upon virus infection, virions or sub-viral nucleoprotein complexes are transported from the cell surface to the site of viral transcription and replication. During viral escape, particles containing proteins and nucleic acids move again from the site of their synthesis to that of virus assembly and further to the plasma membrane Glossina cellular metabolism. In the following sections, we briefly discuss potential roles played by these SGHV proteins and their possible interactions with Glossina SG secretome proteins during the different facets of the viral infection cycle. Inferences below are drawn from other nuclear-replicating DNA viruses, including adenoviruses, hepadnaviruses, herpesviruses, papillomaviruses, and polyomaviruses.The movement and/or replication of viruses in insect vectors require specific interactions between viruses and host components. DNA viruses have evolved mechanisms to evade the host restrictions at entry, cytoplasmic transport, replication, protein synthesis, innate  recognition, and egress from the infected cells. The SGHV genome is a circular double-stranded (ds) DNA molecule per os infectivity factor proteins (PIFs) have been shown to be involved in viral attachment to the host cells. The stepwise entry of DNA viruses into host cells requires viral attachment to cell surface receptors and lateral movements of the virus-receptor complex to specialized sites on the plasma membrane Glossina hypertrophied SGs, and suggest their participation in viral trafficking towards the nucleus. Finally, although the role of spectrins in cytoplasmic transport is not clear, this study identified SGHV010 to have spectrin repeat domains, indicating its potential involvement in SGHV anterograde trafficking.DNA viruses that replicate their genomes in the nucleus use microtubule motors for trafficking towards the nucleus and the periphery during egress after replication Cytoplasmic transport is followed by viral genome docking and uncoating at the nuclear pore complex (NPC), a stepwise programme involving partial proteome degradation of incoming capsid or tegument proteins Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) establish centers for transcription, DNA replication and progeny nucleocapsid assembly Glossina protein encoded by GMsg-6444 (a SUMMO-binding protein). The ubiquitin-like protein SUMO is a partner protein to viral replication center and are dramatically enhanced by interferons Glossina and SGHV proteins in the hypertrophied SGs are an indication that SGHV genome associates with the periphery of PML-NBs, and that viral replication compartments would develop from these sites, as has been observed in other viral systems for instance in HSV-1 Upon infection, some viruses such as O-glycosyl hydrolase) protein, which like the chitinases family-18 proteins may be involved in virus maturation (see 2+) and ion (S2O42−)-binding protein with multiple TMs, thus likely to be part of SGHV ion-channel proteins. Newly assembled enveloped viruses recruit periphery directed motors, and are transported to the plasma membrane on the microtubules upon binding of the outer membrane SGHV049 detected in this study was predicted to be a pre-mRNA splicing factor-9-like protein. The WD40/G-β-repeats represent in this protein is a signature domain for proteins that associate with the spliceosome Glossina pallidipes species is found in several African countries and rearing facilities have been established in Kenya, Ethiopia and Tanzania with the aim of strengthening the fly eradication campaigns in African. Salivary gland hypertrophy virus infection leads to drastic negative effects on the productivity and stability of the G. pallidipes colonies and in certain cases to colony collapse. In addition, a recent study G. pallidipes which further complicates the tsetse eradication campaigns because the mass-reared colonies are normally established from flies collected from the target wild populations. Due to this negative effect, it is necessary to develop virus management strategies to enable sustenance of a healthy and productive colony size for the fly eradication campaigns. Designing a strategy that would interrupt the replication/transmission cycle of the virus in the colonies requires a comprehensive understanding of the mechanism involving the vector-virus interactions. The aim of our study was to establish an extensive protein map of G. pallidipes salivary gland secretome proteins and SGHV proteins in hypertrophied salivary glands, using stringent mass spectrometry criteria to validate the potential proteins, and to establish possible Glossina-SGHV interactions. The substantial differences between the protein profiles of the secretomes of hypertrophied and non-hypertrophied salivary glands, and the analysis of the nanoLC-MS/MS-supported secretome proteins data obtained demonstrate that a large proportion of the proteins identified are indeed secreted.The G. pallidipes salivary gland secretome encompasses a wide spectrum of proteins that may be required for the different facets of the SGHV infection cycle from viral attachment to egress of the virions from infected Glossina cells. On the basis of our previous proteomic analysis of GpSGHV virions Glossina and SGHV proteins can be predicted (per os infectivity factors (PIFs) were only detected in very low abundance, and although they are not included in our final pool of the salivary proteins, they probably play a vital role in the oral infection of Glossina sp. One other SGHV protein that was noted to be present in very low abundance was the baculovirus ODV-E66 (PIF4) homolog. This protein is known to be involved in the initial attachment of the baculovirus in the mid-gut epithelia of infected insects, and it is likely to play a similar role in the case of Glossina.The data presented in this study demonstrates that the redicted . It is tGlossina mid-gut and subsequent movement of the virus into the fly hemocoel. Such chemically-synthesized oligopeptides or the phage display library expressing the active peptides could be supplemented in the flies' blood meals and, thereby, upon ingestion of the meal, the peptides would out-compete the viral homologs (ODV-e66) in the attachment to the mid-gut receptors Taken together, the identification of putative host-viral protein interactions opens novel avenues for the development of mitigation strategies against GpSGHV infections. Such strategies could include immune-interventions whereby virus-specific antibodies against PIF proteins could be supplemented in the blood meals used in the membrane-feeding in tsetse production facilities. This would lead to immuno-complexion of SGHV virions in the blood meals, which would block the horizontal transmission of SGHV from fly to fly. In addition, phage display-selected gut epithelia-binding peptides such as derived from the ODV-e66 protein (PIF-4) homolog could be designed to impede the attachment of SGHV to the Glossina and SGHV proteins identified in this study need to be experimentally established. Still to be investigated are questions particularly with regard to Glossina specificity and how SGHV overcomes various transmission barriers in the tsetse fly. Also to be resolved are the roles played in Glossina specificity by SGHV proteins, Glossina proteins, virus receptors, Glossina symbionts, as well as the role of hemolymph and other tissues in the viral transmission process. In addition, the barriers to transovarial  transmission of SGHV in tsetse remain grossly under-investigated. We hope that with the cataloguing of Glossina salivary glands secretome proteins, the detection of SGHV proteins in hypertrophied salivary glands and the advent of new molecular technologies, that such roles can be elucidated further and eventually exploited to initiate novel strategies for controlling SGHV infections in tsetse mass rearing facilities.The roles of Table S1Sixty-five salivary gland secretome proteins of G. pallidipes. Sixty-five G. pallidipes salivary gland secretome proteins supported by nanoLC-MS/MS, confirmed by gene ontology (GO) annotation, presence of signal peptide sequences and by Blasts on the NCBI and G. m. morsitans databases.(DOC)Click here for additional data file.Table S2Twenty-five SGHV-encoded proteins detected in the G. pallidipes hypertrophied salivary glands.(DOC)Click here for additional data file.


Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins.Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.Here we use tandem mass spectrometry-based proteomics to show that treatment of Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. In both cases the enrichment led to significant increase in ratio of spectra assigned to oxidised Met peptides to all assigned spectra (Table2-based enrichment step enhanced further increase in the number of oxidized peptides identified as compared to oxidized Met peptides enriched in the absence of DHB. This is consistent with a report that shows that Met oxidised peptides co-enrich with phosphopeptides because the affinity for the TiO2 (in the presence of DHB) is stronger in oxidised as compared to non-oxidised isoforms[The debate of whether methionine (Met) oxidation of proteins is a purely chemical consequence of cellular oxidative damage or a protective mechanism against oxidative damage, or indeed a post-translational modification that can act as a specific cellular signal and/or response, is ongoing-3. To shn Figure. We therg medium following medium and procra Table. Moreoveisoforms.In our proteomic analysis we considered a peptide as containing oxidised Met residue when it was identified with high confidence (≥ 95%) in at least two biological replicates. A total of 385 cGMP-dependent methionine oxidised proteins were identified  ratio of 15.9994 that corresponds to the average mass of an oxygen atom. For example, the peptide fragment (DHDKPIQQVIAEMTDGGVDR) of the alcohol dehydrogenase 1 (AT1G77120) in non-oxidised form has the m/z ratio of 850.3723 [-02) in categories including 'response to stress', 'response to abiotic stimuli', 'response to oxidative stress', and 'response to oxygen and ROS metabolic process'  analysis (Fatigoipf.es/) of the uProteins enriched in the GO categories: 'response to oxidative stress', 'response to ROS', 'response to oxygen', and 'ROS metabolic process' showed four main patterns. (1) Loss of peptides or proteins containing oxidised Met residue(s) and reduction in the number of peptide copies after treatment, e.g. glyceraldehyde-3-phosphate dehydrogenase  and 60S acidic ribo-somal protein (AT2G27720). Loss of Met oxidised peptides can occur due to degradation of copies of proteins with oxidised Met residues or the reduction of oxidised Met residues. (2) Increase in the number of oxidised peptide fragments after treatment, e.g. peroxidase . This may imply that cGMP either indirectly induces over-expression of specific proteins with peptide fragments susceptible to oxidation or preferentially induces oxidation of Met residues in specific proteins without necessarily inducing transcription and/or translation. (3) New peptides detected with Met residues oxidised after treatment, e.g. ATP synthase (AT5G08670), and (4) multiple Met residues becoming oxidised after treatment, e.g. in the heat shock protein 70 (AT3G12580)  comprised only one oxidised Met residue, while 86 proteins  had ≥ 2 oxidised Met residues  with 235 oxidised fragments representing ≈ 30% of the identified peptides  residues, five proteins had both residues oxidised and eight proteins - only one. Thirdly, of the 575 oxidised Met residues detected, 75 had a glutamic acid (Glu) and 68 had an aspartic acid (Asp) as an immediate C-terminal neighbour and this bias is likely, at least in part, due to preferential enrichment of these AAs by TiO2[Given that Met oxidation can profoundly alter cellular responses, the question is if there is evidence for site selectivity and if so, what determines it. In order to address this question, we have subjected all 385 proteins with one or several oxidised Met to further analysis and noted the following. Firstly, the average Met frequency in the set of proteins containing oxidised Met is 0.027 as compared to the other amino acids (AA) in the complete proteome where it is 0.025[ by TiO2, howeverThe rapidly evolving field of redox proteomics provides new evidence supporting the notion that oxidation of Met residues may have a great impact on protein activity, regulation of biochemical pathways and cellular function in response to changing environmental conditions. This is consistent with the observed Met oxidation accumulation in plants under low temperature conditions and the fact that plant methionine sulfoxide reductase (MSR) confers increased tolerance to freezing. It is aThe authors declare that they have no competing interests.CG conceived the project. CM, IT, BP, LT and KSL designed and performed the experiments, BJ performed the bio-informatics analyses, and CG wrote the manuscript with critical input from all authors. All authors read and approved the final manuscript.The file contains the list of methionine oxidised peptides and their assigned spectral counts following in vitro cGMP treatment experiment.Click here for fileThe file contains the lists of all methionine oxidised proteins and fragments and the result of the gene ontology analysis.Click here for file


Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells.Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In  Drosophila melanogasterTissue homeostasis depends on adult stem cells that constantly self-renew and produce differentiated cells Drosophila testis, the germline cells and their somatic support cells are arranged in a spatio-temporal order along the apical to basal axis. The germline stem cells (GSCs) are attached to a single group of post-mitotic, apical hub cells and enclosed by cytoplasmic extensions from two somatic stem cells, the cyst stem cells  and the Hedgehog signaling pathways to induce and maintain stem cell fate in the CySCs Drosophila, germline differentiation is dependent on EGF signaling, a highly conserved pathway that plays multiple roles in development and has been associated with many forms of human cancers spi or stet mutant animals, germline cells and cyst cells were present but the cyst cells did not enclose the germline cells In both genders of egfr and in spi led to defects in germline enclosure, as multiple cyst cells were associated with one cluster of germline cells. In both situations, the germline cells accumulated at early stages and failed to differentiate raf, which encodes a Mitogen Activated Protein Kinase (MAPK) that can act downstream of the EGFR raf contained somatic cells in the CySC position that expressed late stage cyst cell markers. Though it was not addressed whether the cyst cells in raf mutant testes enclosed the germline cells, the testes contained over-proliferating germline cells similar to testes from egfr and spi mutant animals spi, egfr, or raf mutant animals was due to defects in germline-soma association or due to a direct requirement for EGF signaling in the cyst cells for the production of these signals.It appears essential that exactly two cyst cells enclose the germline cells. The two cyst cells express different molecular markers and eventually become morphologically distinct, as one of them will develop into a relatively small head cyst cell and the other will develop into a much larger tail cyst cell To separate the role of EGF signaling during germline enclosure from the potential role of EGF past the enclosure event, we specifically addressed the behavior of the germline cells when properly enclosed by cytoplasmic extensions from exactly two early-stage cyst cells, as normally seen in wildtype testes. We discovered that EGF signaling has a dose-dependent effect on cyst development. When we reduced EGF signaling in testes, the germline cells appeared to be properly enclosed by cyst cells but were trapped in TA-divisions. When we increased EGF signaling in testes, the germline cells also appeared properly enclosed by cyst cells but the cysts entered terminal differentiation before the germline cells completed all four rounds of TA-divisions. These observations strongly suggest that EGF signaling from the germline to the cyst cells normally increases as the cysts develop and that this increase in EGF signaling leads to the production of different return signals with different effects on the germline. We further observed that simultaneous over-expression of EGF and EGFR promoted germline differentiation, while high levels of either EGF or EGFR alone had no effect on germline development. We conclude that the doses of EGF signaling are controlled at the level of both the active ligand produced by the germline cells and the available receptor molecules on the cyst cells as these two cell lineages develop. We propose that the increase in EGF signaling provides a temporal signature that guides the cysts through the early steps of development. This is a novel concept that may shed light on the principles how stem cell daughters differentiate into highly specialized cells.Drosophila to different temperatures. When Drosophila are exposed to a temperature of 18°C, the Gal4 transcription factor has low activity. In contrast, when Drosophila are exposed to a temperature of 29°C, Gal4 is highly active To investigate a dose-dependent effect of EGF signaling we used the UAS/Gal4- system that allows for temporal control of tissue-specific expression of target genes by exposing 1stet mutant animals (stet/stet) are short and contain only cells with small, bright DAPI-stained nuclei  are indistinguishable from wildtype testes when raised at 29°C , raised at semi-permissive temperature (data not shown). These findings strongly suggest that EGF signaling within the cysts is required for the production of a return signal that allows the germline cells to undergo all four rounds of TA-divisions. They further suggest that the dose of EGF signaling required for the production of this return signal is higher than the dose of EGF signaling required for germline enclosure.All cells within each enclosed cluster of over-proliferating germline cells in  compare  [55]. Thne cells ′′, blue ne cells , severalne cells ′, or smaλtop and UAS-EGFRA887T, cause hyperactivity of the EGF pathway when expressed in Drosophila ovaries or eyes C784-Gal4 and tj-Gal4 (cyst cell-caEGFR-testes and cyst cell-sSpi/EGFR-testes). To circumvent expression of the constructs during development, animals were raised at 18°C and also carried a ubiquitously expressed, temperature sensitive Gal80 , a known negative regulator of Gal4, that has high activity at 18°C and low activity at 29°C To generate cysts with increased EGF signaling we also took advantage of the UAS-Gal4-system. Flies carrying constructs for the expression of constitutively active versions of the EGFR (caEGFR), UAS-EGFR-caEGFR-animals, non-shifted cyst cell-sSpi/EGFR-animals, animals carrying only the Gal4-transactivators or only the UAS-constructs shifted to 29°C for seven days) did not display observable defects in spermatogenesis. They contained Vasa-positive GSCs  but were not found in testes from 1118w-animals (n = 80), in testes from cyst cell-sSpi/EGFR-animals prior to the temperature shift (n = 37), or in control testes from animals carrying only the Gal4-transactivators (n = 42) or only the UAS-constructs (n = 58) shifted to 29°C. Thus, their appearance in the testes of cyst cell-sSpi/EGFR-animals shifted to 29°C was specifically due to the increase in EGF signaling. Conversely, over-expression of sSpi and EGFR in the germline via the nanos-Gal4 transactivator did not result in any observable defects in the testes , and α-spectrin. Wildtype testes contain a wide area of cells that co-express Vasa and the spermatogonial marker Bam in the apical region  compared to control-animals (n = 50) at one, two, or seven days after the shift to 29°C on the basis of the cell death assay, TUNEL (not shown).Testes from cyst cell-sSpi/EGFR-animals already contained fewer germline cells in the apical region after only two days at 29°C, based on expression of the molecular germline markers Vasa, l region ′, arrow l region ′, arrow.l region . Cyst cel region . The dif+, Eya+-cyst cells, note that the testis in +, Eya+-cyst cells in one focal plane). Testes from cyst cell-sSpi/EGFR-animals shifted to 29°C also contained Tj+, Eya+-cyst cells yet their numbers changed dependent on how long the animals were at 29°C. At 36 hours after the temperature shift, the cyst cell-sSpi/EGFR-testes (n = 20) contained more Tj+, Eya+-cyst cells than the control testes . This shows that over-expression of sSpi and EGFR increased the number of cysts that have entered the growth phase. By 48 hours after the temperature shift cyst cell-sSpi/EGFR-testes (n = 20) contained fewer Tj+, Eya+-cyst cells . This suggests that after 48 hours at 29°C most of the cyst cells that were associated with germline cells have differentiated into Tj-negative, Eya-positive late-stage cyst cells. These observations suggest that a high dose of EGF either directly or indirectly promotes cyst cell differentiation.Communication between germline cells and cyst cells did not appear to be disrupted in cyst cell-sSpi/EGFR-testes as the germline cells and the cyst cells remained properly associated. The early-stage germline cells at the apical tip ′ and thest cells , E′ with-sSpi-testes) or the EGFR (cyst cell-EGFR-testes) alone using either of the two cyst cell Gal4-transactivators did not have an effect on the cysts  over-proliferated beyond the 16-cell stage, producing large clusters of interconnected spermatogonia decapentaplegic (dpp) dpp over-expression divided in synchrony, as large cysts were detected in which all of the spermatogonia expressed the mitosis marker pHH3 stet/stet; germline-sSpi-animals shifted from 29°C to 18°C and in testes from 77-20spi- animals raised at semi-permissive temperature, large cysts were detected as well. However, the germline cells within a single cyst divided out of synchrony as we observed that only a few of them were positive for pHH3. It has not been revealed yet how the decision of the gonialblast to divide synchronously is regulated. Synchronous versus asynchronous cell division could depend on morphology changes of the fusome. When a GSC or a gonialblast divides, the fusome morphology changes from spherical to dumbbell-shaped. During the division of a GSC, the fusome breaks and one spherical fusome is formed in each of the resulting daughter cells. When a gonialblast divides, the dumbbell structure of the fusome is maintained in the resulting 2-cell stage spermatogonia Here we propose that a temporal increase in EGF signaling between germline cells and surrounding cyst cells regulates cyst development. We present genetic evidence that a low dose of EGF signaling is required for the early stages of germline development, the progression of the germline cells through TA-divisions, and that a high dose of EGF signaling promotes the entry of germline and cyst cells into terminal differentiation. Reduction in EGF signaling led to the accumulation of cyst cell-enclosed, tumor-like aggregates of early-stage germline cells. The germline proliferation defects in EGF mutant testes were different from the germline proliferation defects reported for other mutants displaying germline tumors. For example, germline cells mutant for bam and the loss of nucleoporin98-96 (nup98-96). While loss of bam caused the proliferation of spermatogonia beyond the 16-cell stage, over-expression of Bam in the germline cells caused the differentiation of spermatogonia into spermatocytes at the 8-cell stage. This suggested that the germline-intrinsic levels of Bam regulate the number of TA-divisions nup98-96, the germline cells differentiated into spermatocytes at the two-, four-, and eight-cell stage instead of the 16-cell stage. This indicated that nup98-96 plays a role in preventing the transcription or transport of differentiation factors into the nuclei of the spermatogonia bam and nup98-96 mutant animals and cyst cell-sSpi/EGFR-animals suggests that a mechanism exists that prevents the entry of spermatogonia into the spermatocyte-stage prior to the completion of exactly four rounds of TA-divisions and that the EGF pathway plays a role in this mechanism. However, how EGF signaling from the germline to the cyst cells ties into germline-intrinsic activities of Bam and Nup98-96 is yet to be explored.Conversely, a high dose of EGF signaling may decrease stem cell characteristics of the spermatogonia. We observed that germline cells in cyst cell-caEGFR-testes and in cyst cell-sSpi/EGFR-testes bypassed TA-divisions and developed into spermatocytes prior to the 16-cell stage. A similar effect on the germline cells was previously described for two other genetic backgrounds, the over-expression of Together, our phenotypic analyses suggest that EGF signaling plays a rather complex role in the developing cysts. Based on the mutant phenotypes caused by the loss, reduction, and increase of EGF signaling, we propose that EGF signaling between germline and cyst cells increases as the cysts develop and that different doses of EGF signaling, in turn, induce distinct responses in the cyst cells: germline enclosure, the production of return signals that allow for the germline cells to progress through the early steps of development, the spermatogonia TA-divisions, and the production of return signals that promote terminal differentiation of the cysts . It is pDrosophila tissues, a spatial EGF gradient is generated by the diffusion of the ligand away from the source Different doses of EGF signaling also guide the development of other tissues, for example photoreceptor development in the developing eye Drosophila tissues, such as the eye and the nervous system. In the eye, for example, the cleaved Spi ligand is retained in the endoplasmatic reticulum to regulate the range of EGF signaling How could such an increase in ligand and receptor be regulated? A simple explanation for an increase in EGF would be that its amount depends on the number of germline cells. Within one cyst, the number of spermatogonia that can produce the active ligand increases during TA-divisions. At the 16-cell stage, the amount of ligand may be sufficient to induce the production of a signal in the cyst cells that promotes spermatocyte differentiation. However, such a simple mechanism is not likely as cysts in testes with reduced EGF signaling contained a large number of germline cells that eventually should have produced a high enough dose of EGF for differentiation, yet these germline cells still over-proliferated. More likely, the amount of EGF may depend either on transcriptional up-regulation of signaling molecules within the developing germline cells or on an increase of ligand secretion from the developing germline cells. Ligand secretion is a critical step during development of other In summary, our data strongly support the idea that EGF signaling between germline cells and cyst cells increases as they transition from early to later stages of development, and that this increase guides early steps of gametogenesis. It will be interesting to learn if threshold doses of signaling molecules guide differentiation of stem cell lineages in mammals as well.1118white, tub-Gal80ts, UAS-Rolled-RNAiTRIP.HMS007, and balancer chromosomes are as described in C784-Gal4 nanos-Gal4 TRIP.JF02283 and UAS-EGFR-RNAiTRIP.JF02284 were obtained from the Harvard Drosophila RNAi Screening Center, UAS-Spi-RNAi103817 from the Vienna Drosophila Research Center, tj-Gal4 from the Kyoto Stock Center, UAS-A887TEGFRλtopstet-alleles are described in 77-20spi-allele is described in . The UAS-sSpi-construct Flies were raised on standard cornmeal molasses agar medium. Crosses were set up either at 18°C or at 29°C and the animals were shifted to different temperatures as outlined in the results.Immunofluorescence experiments were performed as previously described Testes were dissected in Tissue Isolation Buffer


Cancer borealis stomatogastric ganglion, and examine whether this synapse exhibits homeostatic plasticity.Homeostatic synaptic plasticity is a method employed by some neuronal networks to regulate their activity levels to stay within normal bounds. In this work, we investigate one particular synapse in the central-pattern-generating pyloric network of the crab The pyloric network of the crustacean stomatogastric ganglion consists of an intrinsically bursting pacemaker kernel – one anterior burster (AB) neuron and two pyloric dilator (PD) neurons that all share electrical connections – which rhythmically inhibits the non-intrinsically-bursting follower neurons – one lateral pyloric (LP) neuron and several pyloric (PY) neurons – to participate in a characteristic triphasic oscillatory pattern. The lone feedback to the pacemaker kernel is inhibitory and comes from the LP neuron. To test whether this synapse exhibits homeostatic plasticity, we measured the synaptic conductance of the pacemaker-feedback synapse (LP-to-PD) before, during, and after a strong perturbation of pyloric network activity. The perturbation consisted of either a depolarizing or hyperpolarizing voltage-clamp step of 25 mV from baseline (-60 mV) into one of the PD neurons and was considered a strong network-wide activity manipulation because many network activity features  were affected along with the post-synaptic PD membrane potential. We observe in several trials that the conductance of the LP-to-PD synapse changes significantly in response to the perturbation. Surprisingly, this conductance change seems not to oppose the PD membrane potential perturbation as we hypothesized. For instance, we hypothesized that the inhibitory feedback synapse to the pacemaker would strengthen if the PD neuron were depolarized in order to oppose the depolarization in a homeostatic manner through increased inhibition, but instead we observe a marked weakening of the feedback synapse in response to post-synaptic depolarization.To further investigate what possible network activity effects could arise from this marked change in synaptic conductance, we turned to a previously described  computat


Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes. Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a groundbreaking tool to study protein interactions in living cells Many processes influence the fluorescence decay kinetics of fluorophores in living cells e.g. Förster Resonance Energy Transfer (FRET), excited-state quenching processes, pH concentration as well as the local refractive index and changes of the cells' auto-fluorescence during the exposure time Recently, Controlled Light-Exposure Microscopy (CLEM) has been introduced to minimize the excitation energy dose on the sample in laser-scanning confocal microscopy In the current work, we present a novel approach to perform multi-parameter FLIM in long-term observations of living cells: Wide-Field Multi-Parameter FLIM (WFMP-FLIM). The WFMP-FLIM setup is based on a conventional fluorescence microscope and a space sensitive photon-counter, the Quadrant Anode photomultiplier (QA) WFMP-FLIM offers a broad range of possible applications in long-term live cell imaging. Furthermore, any fluorescence microscope equipped with pulsed-laser illumination sources can be upgraded into a multi-parameter FLIM system by adding a position sensitive photon-counter in combination with a laser synchronization unit.The used data analysis technique is a blend of well established methods, i.e. MLE and LS see  section.The algorithm was extensively tested on simulated data-sets. The fit, mainly performed on bi- and tri-exponential fluorescence decay models at variable number of photons, lifetimes and amplitudes, were always convergent to the true set of parameters. Accuracy tests of the MLE and LS methods on synthetic data sets were already published e.g. FLIM images of a Rhodamine 6G solution in n-butanol were acquired. The dye decayed with a mono-exponential kinetic when the fluorescence emission was monitored through a polarizer oriented at 54.7The choice of n-butanol as a solvent was motivated by the obtained fast component of 0.5 ns. In fact, it approximated the quenched decay time of the eGFP in presence of FRET as presented in the following sections, providing a suitable calibration method for the FLIM setup.The data sets were analyzed by selecting variable number of photons per pixel (from 20 to 1000) in order to estimate mean and standard deviations of the pre-exponential factors and average lifetimes as a function of the number of photons. The precision in the determination of the two fluorescence lifetimes in the FLIM image was negligible due to the total number of photons used  and simultaneously retrieve complex fluorescence decay dynamics, HeLa cells transfected with a tagRFP-5AA-eGFP chimeric protein were measured. The construct consisted of a binary composition of one eGFP and one tagRFP molecule linked by a flexible chain of 5 amino acids. A strong FRET signal was obtained due to the favorable distance between the donor (eGFP) and acceptor (tagRFP) molecules.Firstly, the photobleaching of the tagRFP-5AA-eGFP construct at several illumination intensities was investigated. (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester or The photo-production of ROS at typical excitation intensities required by the WFMP-FLIM setup was also monitored. HeLa cells expressing the tagRFP-5AA-eGFP construct were additionally loaded with a ROS sensing dye he cells .Multi-exponential lifetime analysis of the fluorescence decay of tagRFP-5AA-eGFP molecules after expression in HeLa cells clearly revealed that FRET occurred within the construct. The fluorescence decay kinetics in the 473GFP and 473RFP FLIM channels see , 532RFP The experimental fluorescence decay data were subsequently analyzed according to the delta-function iterative re-convolution technique. For the current experiments, an Erythrosine B water solution with a mono-exponential decay of 86 ps was used as the reference fluorophore The parameters of the multi-exponential models were linked as already suggested by Laptenok et al. The observed eGFP decay kinetic was heterogeneous. However, several groups reported multi-exponential fluorescence decay kinetics of purified eGFP aqueous solutions . K. Suhling and coworkers The apparent FRET efficiency The pre-exponential factors of ch pixel . These pch pixel . The perWFMP-FLIM is capable to achieve the optical diffraction limit. To demonstrate the high spatial resolution of our setup for live cell imaging, we performed several tracking experiments on mitochondria-tagRFP transfected neurons. Neuronal processes of cells expressing moderately tagRFP-mito were continuously illuminated by PIE at 473 nm and 532 nm and the mitochondrial traffic was monitored for up to 10 h uninterruptedly . We co-tWide-field FLIM by the Quadrant Anode detector is a valuable technique to measure the fluorescence decay of fluorophores at very high temporal resolution. The unique feature of the detector, i.e. the combination of a position sensitive photomultiplier and TCSPC, makes it a very sensitive tool for FLIM experiments in living cells The detection system has been extended to feature the simultaneous acquisition of multiple parameters of the fluorescence radiation: four simultaneous FLIM channels were obtained by combining Pulsed Interleaved Excitation (PIE) and multi-spectral detection.Instead of PIE, an ALternating-Laser Excitation (ALEX) scheme could have been implemented to obtain a two-laser excitation of the sample The presented Wide-Field Multi-Parameter FLIM setup (WFMP-FLIM) provides several advantages compared to the available FLIM techniques concerning the long-term observation of living cells.Laser Scanning Microscopy(LSM)-FLIM based systems expose the sample to higher peak intensities which might accelerate photobleaching or the photo-production of ROS species Aequorea victoria, as shown elsewhere Entacmaea quadricolor. tagRFP was chosen due its superior properties concerning brightness, high quantum yield and pH-stability compared to other available monomeric fluorescent proteins emitting in the red-part of the visible spectrum. Furthermore, it shows a favorable Förster radius  the donor fluorescence shows a fast quenching component The simultaneous detection of the lifetime component with a negative amplitude (or rising component) in the 473RFP channel is very important. It increases the estimation accuracy of the FRET efficiency. In fact, the auto-fluorescence contribution to the signal, which spectrally overlaps with the eGFP fluorescence emission, might affect the values of the calculated parameters during the data analysis process. A bias in the obtained average donor-acceptor distance is then expected Sensitized emission as described by Gordon and coworkers Considering that living cells are not static systems, only the simultaneous observation by multi-parameter FLIM can provide an accurate direct measurement of the FRET efficiency. The proposed WFMP-FLIM is conceptually equivalent to the intensity based FRET measurement via The capability of WFMP-FLIM to follow subcellular structures was demonstrated by the tracking experiments on tagRFP labeled mitochondria. Long-term observations of these tiny organelles ranging in size from treaming . Furthertreaming  of co-trIn summary our novel wide-field multi-parameter FLIM method, based on interleaved synchronized laser excitation of multiple fluorophores in combination with TCSPC by a space sensitive photomultiplier provides several advantages compared to current time-domain FLIM systems.It allows long-term observation of living cells H or flavin cofactors as FMN and FAD). Moreover, incorporating a laser source for activation or switching of photoconvertible dyes into the PIE regime has the potential to combine WFMP-FLIM with super resolution imaging techniques like PALM, STORM and the dual Color Localization Microscopy (2CLM) The WFMP-FLIM setup  is based(2) The collimated interleaved trains of 473 nm and 532 nm laser pulses were merged and focused on the back focal plane of the objective to produce an uniform illuminated field of view. A dual band dichroic mirror  was used to discriminate between the excitation and the fluorescence signal. Several emission filters  removed the stray laser signals which were transmitted through the dichroic mirror. (3) The fluorescence signal collected via the objective was split into two spectral bands by a wide-field dual-imaging system . A dichroic cube  and two band pass filters  were used to select the spectral channels. (4) The split images were projected on a Quadrant Anode (QA) photomultiplier . Four spectral channels were simultaneously detected as summarized in instrumental response function (irf) of 150 ps at a spatial resolution of about (5) The original electronics of the QA detector has been extended to store a set of parameters for each collected photon in addition to the spatial coordinates. The synchronization signals from the OCFs were used to mark each detected photon with the wavelength (473 nm or 532 nm) of the last laser pulse which had illuminated the sample. A sequence of FLIM images with The photobleaching experiments were performed illuminating the sample by a 150W Mercury lamp. Two Nikon filter sets, FITC  and TRITC , provided the required illumination and detection spectral channels.The 100X Plan Apo objective was only used in the mitochondria tracking experiments.instrumental response function (irf) of the TCSPC system and a decay model. The most commonly used model in the data analysis of FLIM decays is a linear combination of exponential functions or multi-exponential model function. The number of exponential functions , whose decay kinetic has been previously characterized, instead of the irf by scattered laser light. The fluorescence signal of the ref dye is measured in all the detection channels as the probe. The irf of the reference dye and of the probe do not differ anymore because they are measured in the same spectral regions. In addition, the method requires that the reference compound decays following a mono-exponential kinetic. The non linear functions ref and a modified model function (eq. 6).In each pixel subset, the fluorescence decay (Each pixel of the FLIM image is labeled by an unique index average lifetime (or intensity-averaged lifetime) of the fluorescence decay.The sum of the amplitudes provide the number of the detected photons per pixel mean lifetime (or amplitude-averaged lifetime) to be distinguish from the average lifetime of eq. 9. The parameters The average lifetime is also obtained by fitting a mono-exponential function to each fluorescence decay trace. However, in many applications especially the determination of the FRET efficiency from a donor with a multi-exponential decay kinetic, a different normalization of the amplitudes is required The estimation of the model parameters .The pixels of the FLIM image are sorted in subsets by applying a segmentation algorithm as proposed by Pelet et al. The fluorescence decay signals are summed in each pixel subset into a single decay trace whose total number of photons is very large. A multi-exponential decay model is fit to the sum of the data. The Once the lifetimes are obtained, the pre-exponential factors are independently estimated in each pixel by minimization of the multinomial deviance bwlabel). The least-square problem was solved by using the lsqnonlin function based on the Levenberg-Marquardt algorithm. The minimization of the multinomial deviances fmincon function based on a reflective Newton method. During the minimization process, the pre-exponential factors have been constrained to obtain The analysis of the FLIM data was executed on a laptop computer (Acer Aspire 5930G with a Intel Centrino 2 P8600 (2.4 GHz) and 3 GB of RAM) in MATLAB , which provides different optimization functions. An intensity based discrimination was performed to identify different regions of the image and to remove the background pixels which contain no valuable information. A threshold value (between 30 to 50 photons per pixel) was applied during the segmentation procedure and the complementary regions were identified by a connected component labeling algorithm , these experiments are not regarded as animal experiments and, therefore, do not need an approval of an ethics committee.The DNA of the plasmids was obtained by PCR encoding of tagRFP from pTagRFP-C vector , adding a Kozak consensus sequence (accatggtg), the restriction site BamHI to the N-terminus and a short linker (SELRS) and the restriction site SacI at its C-terminus. DNA encoding eGFP with a short linker and the restriction site SacI at the N-terminus, the stop codon and the restriction site EcoRI at the C-terminus was obtained by PCR from pcDNA3-eGFP. Both fragments were digested with the indicated enzymes . In a second step, the mammalian expression vector pcDNA3  and the bacterial expression vector pRSET-B  were digested with BamHI and EcoRI. Finally, the digested PCR fragments encoding tagRFP and eGFP were ligated into the pcDNA3 and pRSET-B vectors. All constructs were verified by nucleotide sequencing. Cells from a model cancer cell line HeLa  were culFor long-term experiments the cells were measured in the described DMEM medium. For low background experiments, the culture medium was replaced 24–48 hours after transfection with Hanks Balanced Salt Solution supplemented with 1 g/L glucose and 10 mM Hepes (pH 7.4).Photobleaching experiments were performed on paraformaldehyde-fixed cells (2% PFA in 0.1 M PBS (pH 7.4) for 10–15 minutes) to avoid any movements of the cells or metabolically related changes of the fluorophores (e.g. protein synthesis or degradation).Preparation and culturing of the hippocampal neurons were performed according to the method of Banker et al. Video S1Long-term WFMP-FLIM experiment on living HeLa cells. The acquired FLIM channels are simultaneously shown. On the right side, fluorescence decay kinetics integrated over a region of interest.(MOV)Click here for additional data file.Video S2Measurement of mitochondria trafficking by WFMP-FLIM. Neuronal cell expressing tagRFP-mito marked mitochondria. The mitochondria trafficking was continuously monitored for 10 hours. Two kymograms were produced by selecting distinct processes. Anterograde and retrograde transport as well as oscillating and static phases are present. Mitochondria fissions and fusions are frequently visible.(MOV)Click here for additional data file.


Clinical outcomes after robotic training are often not superior to conventional therapy. One key factor responsible for this is the use of control strategies that provide substantial guidance. This strategy not only leads to a reduction in volitional physical effort, but also interferes with motor relearning.We tested the feasibility of a novel training approach (active robotic training) using a powered gait orthosis (Lokomat) in mitigating post-stroke gait impairments of a 52-year-old male stroke survivor. This gait training paradigm combined patient-cooperative robot-aided walking with a target-tracking task. The training lasted for 4-weeks . The subject’s neuromotor performance and recovery were evaluated using biomechanical, neuromuscular and clinical measures recorded at various time-points .Active robotic training resulted in considerable increase in target-tracking accuracy and reduction in the kinematic variability of ankle trajectory during robot-aided treadmill walking. These improvements also transferred to overground walking as characterized by larger propulsive forces and more symmetric ground reaction forces (GRFs). Training also resulted in improvements in muscle coordination, which resembled patterns observed in healthy controls. These changes were accompanied by a reduction in motor cortical excitability (MCE) of the vastus medialis, medial hamstrings, and gluteus medius muscles during treadmill walking. Importantly, active robotic training resulted in substantial improvements in several standard clinical and functional parameters. These improvements persisted during the follow-up evaluation at 6 weeks.The results indicate that active robotic training appears to be a promising way of facilitating gait and physical function in moderately impaired stroke survivors. There has been a growing interest in using robotic therapy to improve walking ability in individuals following hemispheric stroke ,2. The pSeveral studies have evaluated the efficacy of robot-aided gait therapy in improving gait and physical function after stroke -7. WhileIn this case study, we tested the feasibility of a novel robotic gait training paradigm on a moderately impaired stroke survivor. The paradigm consisted of two components: (1) a patient-cooperative robot that allows participants to actively control the motion of the limb while providing minimal assistance, (2) a target-tracking task that involved matching the ankle position to a particular movement template. The findings from this case study suggest that active robotic training may be a promising means of mitigating gait impairments in stroke survivors.The subject was a 52-year old male stroke survivor (7 months post-stroke) who suffered right hemiparesis due to a left corona radiata infarct. Immediately after his stroke, he was admitted to an acute care hospital for five days, which was followed by 3-weeks of inpatient physical and occupational rehabilitation. He then received outpatient rehabilitation three times a week for the next four months. Before the initiation of the intervention program, the patient was able to ambulate in the community with the assistance of a cane and an ankle-foot orthosis. The subject revealed no major sensory or cognitive deficits . He hadThe subject underwent active robotic training for 12 sessions (3 sessions per week for 4 weeks). During this period the subject did not receive any other therapy or participate in any other research opportunities. An orientation session was provided before the start of the intervention to familiarize the subject with the training conditions and with robot-aided walking. Each training session lasted for about 90 minutes (excluding set-up time of ~ 10–15 minutes) with several periods of rest provided, as needed. During each training session, the subject walked with the aid of a patient-cooperative robot and practiced a gait pattern that necessitated greater hip and knee excursion during swing phase of the gait cycle. No body-weight support was provided during training.Active robotic training consisted of two components: (1) walking in the patient-cooperative control mode in the Lokomat that reduced the amount of guidance from the robot and (2) performing a target-tracking task during walking that involved matching the ankle trajectory  to a target-template in order to facilitate active participation from the subject. These components are described in detail below.The Lokomat is a robotic device that has been widely used for gait rehabilitation in several movement disorders -18. The Recently, a patient-cooperative controller has been developed for the Lokomat, in which the stiffness of the robot can be programmatically controlled (from complete assistance to no assistance) so that the robot allows active participation of the patient ,21. Addinot to the movements of the subject’s ankle joint. The ankle position is uniquely determined by the hip and knee joint angles (see below for details) and therefore changes in this pattern will require changes in the hip and/or knee joint angle. Visual feedback of the subject’s ankle trajectory was provided during the training on a computer monitor placed in front of the subject [Active participation was also facilitated by including a target-tracking task with the aid of visual feedback -29. Once subject . The desx a, ya) of the subject was computed from the hip and knee joint angles recorded with the potentiometers inbuilt in the Lokomat using the following equation:The instantaneous ankle position .where The target-template was changed depending on the subject’s familiarity with walking in the Lokomat. During the initial 5 visits of training, when the gait inside the Lokomat was highly variable, the desired ankle trajectory was based on patterns built into the Lokomat .th visit onwards, we provided a target-template that was based on the subject’s own gait pattern. The target-template was constructed as follows: First, we computed the baseline trajectory by ensemble averaging the paretic leg ankle trajectory at the beginning of each training session when the subject walked in the Lokomat for two minutes. Second, the target-template was set so that it corresponded to a gait pattern which required increasing the hip and knee joint angle of the baseline gait by a specified scaling factor. Because the ankle trajectory is constrained by the ground during the stance phase of the gait, the increase in hip and knee angle was performed only during the swing phase of the gait. A Hanning window was used to prevent abrupt changes in ankle trajectory due to this increase in hip and knee angle during the initial and final part of the swing phase. The target-template was obtained from the baseline trajectory by using the following formula:From the 6xhw, yhw represent the Hanning-windowed version of the baseline trajectories  and s was the scaling factor  for about 20 minutes. This second template was used to practice gait patterns that were closer to normal gait as the information regarding the optimal gait pattern to train a stroke subject to achieve maximal recovery is currently not known.where r Figure B. The scWe chose the greater hip and knee excursion pattern for most part of the training due to three reasons. First, we wanted to target the characteristic stiff-knee gait (which arises due to difficulty in producing adequate hip and knee flexion during swing phase of the gait) that is seen in many stroke survivors including the subject that participated in our study. Second, we believed that this pattern will not only increase active participation, but would also increase the physical effort required by the subject to walk in the Lokomat thereby preventing the “slacking phenomenon” and promoting strength and endurance. Finally, previous research that has incorporated a similar approach of training has shown positive outcomes in patients’ walking capacity in the treadmill .The progression in training was achieved by increasing the subject’s walking speed in the Lokomat and by reducing the visual feedback during target-tracking Table . The speNeuromotor performance and recovery were assessed at multiple levels – biomechanical, neuromuscular and clinical. All testing was performed without the use of cane and ankle-foot orthosis.At the biomechanical level, we evaluated target-tracking performance and kinematic variability of ankle trajectory during robot-aided treadmill walking. We also assessed if the training transferred to overground walking by measuring the ground reaction forces (GRFs) generated during overground walking.The ability of the subject to accurately track a given target-template trajectory was evaluated by determining the area that was not common to both the reference trajectory  and the actual trajectory during each gait cycle Figure C. The arIn addition to the error, we also computed the kinematic variability of the ankle trajectory from the aforementioned target-tracking data (target-tracking variability) and from a 2-minute baseline walking data (baseline variability) collected during the pre, post, and follow-up testing sessions. The variability was quantified similar to the error computation described above, except that instead of the target-template, the ensemble average of the ankle trajectory during baseline walking (for baseline variability) or target-tracking (for target-tracking variability) was used as the reference. The resulting area from each gait cycle was normalized to the area of the reference trajectory to determine% deviation. The values from all the gait cycle were averaged to find the overall% deviation.th order Butterworth digital recursive filter. Four to six trials were collected for each leg and were ensemble averaged for further analysis.GRFs were measured during the stance phase of the gait cycle for both the paretic and the non-paretic legs while the subject walked at his self-selected speed along a 10 m walkway equipped with instrumented force plates . The GRF data were sampled at 1000 Hz and low-pass filtered at 10 Hz using an 8At the neuromuscular level, we evaluated muscle coordination using surface electromyography (EMG) and motor cortical excitability (MCE) during treadmill walking.In order to assess muscle coordination, bipolar EMG electrodes were placed over the muscle bellies of vastus medialis (VM), rectus femoris (RF), medial hamstrings (MH), lateral hamstrings (LH), tibialis anterior (TA), medial gastrocnemius (MG), soleus (SO), and gluteus medius (GM). A common ground electrode was placed over the patella.nd order Butterworth filter (4 Hz).The EMG signals from each muscle were amplified (×1000), filtered (20-500 Hz) and stored for off-line analysis using an AMT-8 Bortec EMG system . These were then digitally high-pass filtered (40 Hz), rectified, and smoothed using a 2The muscle coordination during walking before and after training was analyzed using dimensionality reduction techniques. The EMG from each stride was interpolated into 101 points and the resulting N x 8 matrix  was subject to a principal component analysis (PCA) followed by a varimax factor rotation. A minimum of 25 strides were collected during each session. The PCA analysis effectively expresses the muscle activity as the linear combination of a smaller set of muscle modes, each having time varying activations ,32. In eIn order to examine if training induced motor cortical plasticity, we assessed MCE by comparing the motor evoked potentials (MEPs) elicited with transcranial magnetic stimulation (TMS) before and after the intervention. MEPs were not assessed during the follow-up evaluation due to technical difficulties. MEPs were recorded from the subject’s paretic leg with the EMG electrodes on the 8 muscles mentioned above.TMS was delivered using a Magstim 200 stimulator (110 mm double-cone coil) over the primary motor cortex (M1) of the left lower-extremity as the patient walked on a treadmill at 2 km/hr. The coil was centered 2 cm posterior and 1 cm to the left of the vertex and was securely attached to the subject’s head using Velcro ,35. A puIn addition to our biomechanical and neuromuscular measures, subjective and objective clinical outcome measures were assessed at 3 time points: (1) prior to training, (2) after completing 12 training sessions, and (3) 6-weeks after training (follow-up visit) using standardized procedures. These measures included the self-reported recovery in Stroke Impact Scale (SIS), Timed Up-and-Go (TUG) test, 6-minute walk test for distance, self-selected and fast walking speed, single leg balance test, and lower-extremity Fugl-Meyer score. Self-reported recovery in SIS was evaluated by having the patient report on a scale of 0 to 100 (with 100 representing full recovery and 0 representing no recovery) how much he has recovered from his stroke. Walking speed was measured while the subject walked on a Gait Mat II computerized measuring system (3.84 m x 0.6 m) at self selected and maximum possible walking speeds. Three trials were performed and the average of the three trials was used in the analysis.We first describe the changes observed at the biomechanical and neuromuscular levels followed by the changes observed at the clinical level.The target-tracking error decreased with practice during early- and post-training testing sessions Figure A. With pFigure 2 = .91) compared to before training (R2 = .85), showing that the motor output was highly coordinated after training.There was also a change in the composition of the muscle modes after training, with the muscle modes after training resembling healthy controls (r = 0.93) more than before training (r = 0.71). The main changes after training were due to the first and fourth muscle modes Figure . The varThe mean TMS elicited MEPs of the lower-extremity muscles during treadmill walking are provided in Figure There were large improvements in all of the measured subjective and objective clinical outcome measures Table . The subIn this case study, we tested whether facilitating active participation of the patient while receiving robot-aided gait therapy would help to mitigate gait impairments after hemispheric stroke.potentially effective approach to minimizing post-stroke gait impairments.Our preliminary evaluation indicated that four weeks of active robotic training led to substantial improvements in several clinical and functional parameters and these improvements persisted during the follow-up evaluation. Importantly, the improvements were much larger than the measurement errors  and wereIn addition to improvements that were specific to training (like reduced tracking error and variability), our results also showed improvements in abilities that were not specifically trained. First, our subject showed an improvement in single-leg balance on the paretic leg, going from 1 s before training to 15 s after training. This improvement was also sustained during the follow-up exam. The increase in single-leg balance time may be important from the viewpoint of reducing the risk of falls ,41. An iWe also used several novel biomechanical and neuromuscular metrics to complement the typical clinical measures, which allowed us to objectively quantify the mechanisms underlying the neuromotor deficits and monitor changes with training. At the biomechanical level, we found that training not only improved the propulsive forces generated by both the paretic and non-paretic leg muscles, but also improved the symmetry of the antero-posterior GRFs between the paretic and non-paretic legs Figure . This fiAt the neuromuscular level, we found changes both in muscle coordination and motor cortical excitability during treadmill walking. In terms of muscle coordination, we found that the muscle modes obtained after training were closer to healthy controls, suggesting that part of the gait improvements were due to improvements in the timing of muscle activity. This was particularly evident in the timing of activation of the GM and TA muscles. In addition, the motor cortical excitability of the VM, MH, and GM muscles reduced substantially after training. The reduction in motor cortical excitability is probably due to improvements in lower-extremity muscle strength after training as previous studies indicate that strength training decreases motor cortical excitability -48. WhilWhat are the critical components of the current training paradigm that may have facilitated the positive outcomes observed? As described earlier, the prevalent hypothesis for the suboptimal outcomes in several robotic interventions is that the guidance provided by the robot results in a lack of active participation during training . Indeed,We would like to point out some of the limitations of this study. First, the results reported are from a single subject and it is possible that our subject may not be representative of the typical stroke population at large. He had only moderate levels of lower-extremity impairment, good upper-extremity function, and was highly motivated. As a result, he was able to cope well with the training regimen and perform the target-tracking task without much difficulty. However, stroke subjects with higher levels of impairment may not have adequate knee/hip flexor strength to perform this task. Although the guidance force exerted by the robot can be tuned specifically to match the recovery state and functional capacity of each subject, it remains to be seen whether these improvements will be observed in patients with higher impairment levels. Second, some of the benefits observed in our subject could have been due to natural biological recovery, although this seems unlikely given the evidence that most of neurological and functional recovery plateaus after 3–4 months following stroke . FurtherIn this case study, we report results from a novel robotic gait training approach that aimed to facilitate active participation and mitigate post-stroke gait impairments. Four weeks of patient-cooperative robot-aided walking with a target-tracking task resulted in clinically meaningful improvements in several of the measured locomotor outcomes. These improvements persisted during a follow-up evaluation that was performed at 6-weeks after the completion of training. The promising positive outcomes of this case study suggest that combining patient-cooperative robot-aided walking with a target-tracking task is a feasible approach to improve post-stroke walking function. Further research is necessary to identify whether this approach would be feasible in patients with varying levels of impairment and also to verify whether similar results can be obtained from a larger cohort of stroke population.GRF, Ground reaction force; EMG, Electromyography; MCE, Motor cortical excitability; VM, Vastus medialis; RF, Rectus femoris; MH, Medial hamstrings; LH, Lateral hamstrings; TA, Tibialis anterior; MG, Medial gastrocnemius; SO, Soleus; GM, Gluteus medius; PCA, Principal component analysis; MEP, Motor evoked potential; TMS, Transcranial magnetic stimulation; SIS, Stroke impact scale; TUG, Timed-up-and-go.The authors declare that they have no competing interests.CK and RR contributed equally. All authors contributed to the conception of the study. CK, RR, and SSK collected, analyzed, and interpreted the data. CK performed the training. All authors contributed to the writing of the manuscript. All authors read and approved the final version of the manuscript.