Microelectrode array (MEA) devices are frequently used in neural circuit studies, especially in retinal prosthesis. For a high throughput stimulation and recording paradigm, it is desirable to obtain the responses of multiple surface RGCs initiated from the electrical signals delivered to multiple photoreceptor cells. This can be achieved by an high density MEA-tissue-MEA (MTM) sandwich configuration. However, the retina is one of the most metabolically active tissues, consumes oxygen as rapidly as the brain. The major concern of the MTM configuration is the supply of oxygen.We aimed to develop a high density MTM sandwich platform which consists of stacks of a stimulation MEA, retinal tissue and a recording MEA. Retina is a metabolically active tissue and the firing rate is very sensitive to oxygen level. We designed, simulated and microfabricated porous high density MEAs and an adjustable perfusion system that electrical signals can be delivered to and recorded from the clipped retinal tissue.The porous high-density MEAs linked with stimulation or recording devices within a perfusion system were manufactured and the MTM platform was assembled with a retina slice inside. The firing rate remained constant between 25 and 55 min before dramatically declined, indicating that within certain period of time (e.g. 30 min after habituation), the retina condition was kept by sufficient oxygen supply via the perfusion holes in the MEAs provided by the double perfusion system.MTM sandwich structure is an efficient platform to study the retinal neural circuit. The material and arrangement of high density microelectrodes with porous design make this MEA appropriate for sub-retina prosthesis. Finding ways to prolong the recording time and reduce the signal-to-noise ratio are important to improve our MTM prototype. In outer retinal degenerative diseases, such as the age-related macular degeneration (AMD) and hereditary Retinitis Pigmentosa (RP), although photoreceptors are lost, numerous bipolar and retinal ganglion cells (RGCs) are still preserved , 2. RetiWhile RGCs are activated by electrical signals delivered to photoreceptors, point-to-point responses are key to decode the retinal information process. Various in vitro models are developed to solve the puzzle –16. Micr2) level is greater than 45 mmHg. While arterial PO2 values are between 24 and 34 mmHg, the RGC firing rates are increased and then followed by a decrease. If the arterial PO2 value is below 24 mmHg, the RGCs show a train of large firings but a complete cessation afterward within 5 min. Other experiments also indicate that the electrical activity of inner retina is unaffected during systemic hypoxia as long as the arterial PO2 is above 40 mmHg [2 inside of retina is lower than the arterial PO2. The minimum PO2 at the retina is about 17.4 mmHg in dark adapted in the Long-Evans rat retina measured with oxygen-sensitive microelectrodes. Mammals such as cat, Long-Evans rat, mice, and macaque has similar oxygen consumption behavior by the retina [2 required for maintaining the normal response of RGCs. For an MTM platform, if the PO2 within the clipped retinal tissue exceeded the minimal PO2 requirement and was maintained, it could be a useful tool to study the retinal circuits.The major concern of the MTM configuration is the supply of oxygen. The retina is one of the most metabolically active tissues, consumes oxygen as rapidly as the brain –21. The  40 mmHg . Normalle retina –32. The 2 within the retina was exceeded. The distribution of the perfusion pores was also considered to accommodate the size and pitch of the microelectrodes of the MEAs to achieve the high-density resolution which is critical for prosthesis of high visual acuity [Here we report the design, microfabrication and test of an MTM sandwich platform for retinal neural circuit study. The problem of oxygen supply was overcome by the opening of holes in the middle of the high-density MEAs on the top of and below the retina slice. The hole-opening ratio and flow speed of oxygenated fluid were concerned and computer simulation was used to ensure that the minimum POl acuity .−6 ml cm2/(100 g min mmHg) [NR) is ~3.5 To maintain the oxygen supply to the retina tissue within the MEA-tissue-MEA (MTM) sandwich structure, we designed perforated MEAs with uniformly distributed diffusion holes Fig. . We assuin mmHg) . P is th/100gmin –32 was uSR is the oxygen tension at the surface of the retina  and reaction equations were considered with the Newton-laminar flow to build the governing equation.The distribution of oxygen tension in the fluid was simulated using Finite Element Method (FEM) software .a is concentration of oxygen in the medium, The concentration of oxygen in the medium was measured using a dissolved oxygen meter (Lutron DO-5509) and converted to the oxygen tension (P) using Henry LawWe designed and fabricated the MEAs with the distribution of diffusion holes among the high-density electrode array such that the retina tissue could be under adequate oxygen supply during the in vitro study. Perforated MEA samples were fabricated on the silicon, as illustrated in Fig. 2PO4; 1.3 MgCl2; 26 NaHCO3; 2.5 CaCl2; 10 glucose, pH 7.5 ± 0.2) with carbogen- bubbled (95 % O2–5 % CO2) at room temperature. Visual streak was cut into three pieces and mounted flat on filter paper (Millipore) with a 1 mm-diameter hole. The paper was flipped and the RGC layer was facing down and contacted with the recording MEA (rMEA) as illustrated in the Step 1 of Fig. Male C57BL/6 mice of 4–6 weeks old were used in this study. All animal experiments were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. The dark-adapted mice were sacrificed in a darkroom. The eyes were hemisected, and lens and vitreous were removed, preserving the visual streak in an artificial cerebrospinal fluid  at a constant volume rate, 100 ml/h. The retina slice was allowed to settle and adapt to the new environment  for 25 min before recording.The stimulating electrode was designed as small as 11.5 m in diameter. Pt black was electroplated on the surface to provide a high charge injection capacity. The charge injection capacity of each electrode is limited by the electrode material used and the size of electrode. In this implementation, a single Pt electrode used for stimulation does not have adequate charge injection within the water window to achieve RGC spiking; however, the RGC spiking was achieved within the water window when 7 (1 center and six surrounding) electrodes were simultaneously used with the same electrical stimulation waveform shown as Fig. Then the stimulation signals of two-fold of the threshold voltage (1.8 V) were created by programmable waveform generators  in constant voltage mode (1.5 Hz with 5 ms duration) were delivered to the sMEA by 42 multi-channels. The data acquisition system consisted of a 16-channel pre-amplifier , backend amplifier, data acquisition card  and user interface .2 were used to activate the retina from the photoreceptor layer simultaneously. Sixteen electrodes aligned in two rows were used to collect RGC response (n = 5 retina slices). To analyze the raw data, an off-line spike sorting software  was used to detect and quantify the spikes. Numerical values were given as mean ± standard error of the mean.Seven stimulation electrodes within a hexagon area about 60 × 60 μmFor an ideal MTM platform, the retina tissue clipped by two MEAs should be supplied with sufficient oxygen that the proper physiological condition could be maintained. Furthermore, the density of microelectrodes on the MEAs should be great that the precise point-to-point stimulation-recording relationship could profit the decoding of retinal neural circuits. However, these two concerns are somewhat contradictory, although the problem of oxygen supply might be overcome by the opening of holes in MEAs that could also reduce the density of microelectrodes on the MEAs. Since the central region of the MEA is crowded with microelectrodes and metal wires, the hole size and open area ratio (OAR) in the central area should be small. Accordingly, different sizes of diffusion holes were designed in different regions. In the center, middle and outer regions of the MEA, the diameters of diffusion holes were 10, 50 and 100 μm with 10, 20 and 20 % OAR, respectively.inlet) was 583 mmHg. Based on these parameters, oxygen tension distribution in the fluid was simulated using FEM  in check point 1 (the outer surface the MEA), 144 mmHg (PSR) in check point 2 (the surface between retina and MEA) and 37 mmHg (PIR) in check point 3 (the middle of the retina) , the oxygen tension in the inlet  0.16, better than the level of legally blind VA 0.1 . Besides, Pt black has excellent electrochemical property such as low impedance. Together, the material, diameter and density of the MEA make it suitable for retinal neural circuit study and retinal prosthesis. The opening of perfusion holes was also included in the fabrication process. In the central region of the MEA, the oxygen supply through the 10 μm holes with 10 % OAR from the dual perfusion paths were simulated to be sufficient. This, to the best of our knowledge, is the first attempt to simulate the oxygen tension within the clipped retina tissue. The perfusion system not only provide the flow of oxygenated fluid for gas supply but also a negative pressure to pull the retina close to the MEAs, that the noise signals were reduced during recording.Generally using whole-cell recording technique, the condition of a RGC can be held for up to 1 h. The recording time can be even prolonged using loose patch or extracellular recording paradigm. Using our MTM platform, the activity of RGCs can be kept for about half an hour before its decline. It is not satisfactory. We are still fine-turning the preparation of retina slices, constituents of perfusion fluid and flow rate. Sufficient recording time is important for large-scaled multiple stimulation and recording paradigm to decode the retinal information processes.Using the small-diameter high density rMEA, different types of RGC were characterized based on the electrically evoked responses , 10. It For our present MTM platform and stimulation/recording system, there is still room for improvement, especially in reducing the signal-to-noise ratio (SNR) and prolonging the recording time. Out of 16 recording electrodes, RGC responses can only be analyzed from those that had higher SNR. The numbers were only 6 electrodes dependent upon the condition of the retina. There are several possible ways to increase the SNR. For examples, by increasing the perfusion flow rate that negative pressure is raised to draw near the MEAs and retina. Alternatively, developing lower-impedance electrodes may be another route to increase SNR. It is likely that the retina tissue deteriorated during the in vitro operation and limited the recording of neural activity to ~30 min. Reducing the preparation-and-positioning time of the in vitro operation, and using a milder negative pressure to hold the tissue in the future may prolong the tissue survival time.MTM sandwich structure consists of two perforated microelectrode arrays, for stimulation and recording, respectively, and a clipped retinal tissue in between. It is the most efficient platform to study the retinal neural circuit. The material and arrangement of high density microelectrodes with porous design make this MEA appropriate for sub-retina prosthesis. Finding ways to prolong the recording time and reduce the signal-to-noise ratio are important to improve our MTM prototype.


Tenebrio molitor and Tribolium castaneum have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion.Larvae of the tenebrionids T. castaneum, 25 genes and one questionable pseudogene encoding cysteine peptidases were identified, including 11 cathepsin L or L-like, 11 cathepsin B or B-like, and one each F, K, and O. The majority of transcript expression was from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502). For cathepsin B, the major expression was from genes on chromosome 3 (LOC663145 and LOC663117). Some transcripts were expressed at lower levels or not at all in the larval gut, including cathepsins F, K, and O. For T. molitor, there were 29 predicted cysteine peptidase genes, including 14 cathepsin L or L-like, 13 cathepsin B or B-like, and one each cathepsin O and F. One cathepsin L and one cathepsin B were also highly expressed, orthologous to those in T. castaneum. Peptidases lacking conservation in active site residues were identified in both insects, and sequence analysis of orthologs indicated that changes in these residues occurred prior to evolutionary divergence. Sequences from both insects have a high degree of variability in the substrate binding regions, consistent with the ability of these enzymes to degrade a variety of cereal seed storage proteins and inhibitors. Predicted cathepsin B peptidases from both insects included some with a shortened occluding loop without active site residues in the middle, apparently lacking exopeptidase activity and unique to tenebrionid insects. Docking of specific substrates with models of T. molitor cysteine peptidases indicated that some insect cathepsins B and L bind substrates with affinities similar to human cathepsin L, while others do not and have presumably different substrate specificity.High throughput sequencing was used to quantify and characterize transcripts encoding cysteine peptidases from the C1 papain family in the gut of tenebrionid larvae. For These studies have refined our model of protein digestion in the larval gut of tenebrionid insects, and suggest genes that may be targeted by inhibitors or RNA interference for the control of cereal pests in storage areas.The online version of this article (doi:10.1186/s12864-015-1306-x) contains supplementary material, which is available to authorized users. Tribolium castaneum (red flour beetle) and Tenebrio molitor  are pests of cereal grains and stored products. Over 20 years of combined research by our group and others have revealed many similarities in the digestive processes of these insects, and yet they are distinct in physical parameters and developmental period  family is part of clan CA of cysteine peptidases containing catalytic Cys25 (hereinafter papain numbering) and His159 residues in the active site . CysteinA large subset of C1 family cysteine peptidases is lysosomal, described mostly in mammals and human . Human lT. molitor larval anterior midgut [T. castaneum larval cysteine peptidases contribute up to 97% of the total anterior midgut proteolytic activity [The importance of digestive cysteine peptidases in insect pests has been described in beetles from the infraorder Cucujiformia, some hemipterans, and thrips -11. For r midgut , T. castactivity .T. molitor larvae have identified at least six fractions of cysteine peptidase activities [T. molitor, cysteine peptidases also are important in processing the major glutamine-rich dietary proteins in cereals, prolamins, functioning as post-glutamine hydrolyzing enzymes [T. castaneum larval gut [Biochemical studies of digestive peptidases in tivities ,16,18 witivities . In T. m enzymes . At leasrval gut ,21.T. castaneum genome, we identified 25 potential cysteine peptidase genes, including cathepsins B, K, L, and O, and some were predicted to be inactive homologs due to a lack of sequence conservation in critical conserved residues [T. castaneum cysteine cathepsin genes and related sequences in other species. Many of the cathepsin B and L peptidases are expressed in the T. castaneum larval gut to varying levels, according to gene expression microarrays [During the annotation of the residues . Four liroarrays .T. molitor and T. castaneum. These data allow us to refine our model of C1 peptidase genes and predict relative transcript expression in each insect. Furthermore, we have used modeling and substrate docking to speculate on the peptidase structure relative to function in the larval gut of each insect species. These data provide the most comprehensive dataset for coleopteran digestive peptidases to date. In the analysis of peptidase structures, we describe for the first time a new subset of C1 peptidases.To further study the expression and activity profile of cysteine peptidases in tenebrionid larvae, we accumulated new sequencing data from the midgut of T. molitor maintained on a diet of 50% oat flakes, 2.5% brewer’s yeast, and 47.5% wheat flour at 28°C, 75% R.H., in darkness. Approximately five week old larvae with an average weight of 5.1 mg from three independent biological replicates were fasted overnight and were placed on a diet consisting of 85% stabilized wheat germ, 10% wheat flour, and 5% brewer's yeast for 12 h. For each replicate, the midgut was extracted from 4-7 larvae and placed in room temperature RNAlater . For RNA isolation, excess RNAlater was blotted, and pooled midguts were ground with a plastic pestle in 1.5 ml microfuge tube containing liquid nitrogen. Total RNA was isolated using the Absolutely RNA Kit with DNase on-column treatment . The resulting total RNA was sent to a sequencing facility , where mRNA was isolated by polyA, standard libraries were made, and paired-end sequencing was performed on a Illumina HiSeq 2000  using standard protocols from the manufacturer. We obtained approximately 240 million sequence reads, with an approximate 250 bp insert.The Center for Grain and Animal Health Research  has laboratory colonies of T. castaneum were reared at CGAHR on a diet of 95% wheat flour and 5% brewer's yeast at 28°C, 75% R.H., in darkness. Neonate larvae from three independent biological replicates were placed on 85% stabilized wheat germ, 10% wheat flour, and 5% brewer's yeast and reared under normal rearing conditions for 14 d. Total RNA was extracted as with T. molitor (above). Sequencing of cDNA was by the High Throughput Genomics Center, Seattle, USA, and paired-end sequencing on the Illumina HiSeq 2000. We obtained 344,476,216 sequence reads, >95.6% = Q30 post filtering, with a mean quality score of 37.39, and insert size approximately 250 bp.T. molitor sequences combined from all replicates was made by NCGR, resulting in 197,800 contigs . For T. molitor sequences, we also combined the replicate data and included previous databases of Sanger sequencing [de novo assemblies with SeqManNGen  and custom assembly programs. For T. castaneum sequences, we used SeqManNGen to map sequences to the T. castaneum genome , as well as Galaxy [T. molitor contigs using custom software.A custom assembly of quencing  and pyros Galaxy . PotentiT. molitor were used as query sequences (Cont-08879 and Cont-00890) [T. molitor and T. castaneum mature enzyme sequence was identified by sequence homology through alignment with mature human cathepsin L and cathepsin B.BLAST  and custt-00890) . We const-00890) . ORFs tht-00890)  and custt-00890)  to detect-00890)  to compamature human cathepsin L complexed with a peptide inhibitor (3OF8)  to modelmature rat cathepsin B with a peptide inhibitor (1THE)  - for thHomologous modeling of the predicted mature enzyme with a ligand (inhibitor) was with Modeller . The folHomologous modeling of tertiary structures of the proenzyme and the mature enzyme was with RaptorX ,36 usingThe simulation of 3D structures of cysteine cathepsin proteins was obtained by two different approaches:Both methods resulted in similar structures; therefore, method 2 was used for analysis. Model quality was evaluated by Ramachandran plots using RAMPAGE . StructuT. molitor and T. castaneum were identified by comparison to model sequences using ClustalW [Amino acids corresponding to S1 and S2 subsites in ClustalW  and struClustalW . PyMol sClustalW  was usedClustalW  using PDClustalW .secondarily optimized by MOPAC [The distance between the C atom of arginine and the cysteine S atom of the active center should not exceed 4 Å;The orientation of the substrate shall be as described in the literature: N-terminal phenylalanine must be located in the subsite S2, and C-terminal should be in the S1’ subsite;2 and-COOH groups of the substrate must be sterically accessible and not be immersed in the enzyme.-NHDocking was used to model the enzyme-substrate complexes of cysteine peptidases and classical substrates of cathepsins, the tripeptides phenylalanine-arginine-phenylalanine (FRF) and lysine-arginine-phenylalanine (LRF). 3D structures of substrates were obtained by ChemSketch  using spby MOPAC . Dockingby MOPAC , using sT. castaneum, we used reads mapped to the genome of T. castaneum (Tcas3) to obtain expression values for contigs by normalized reads per kilobase per million mapped reads (RPKM) [To analyze the expression of peptidase transcripts in s (RPKM) , using ST. molitor, about 40 million reads were assembled from each of three datasets of RNA-Seq data. BLAST, MAFFT, and custom scripts were used to obtain all nucleotide sequences in the contigs potentially encoding peptidases. The contigs were used to assemble and refine sequences of complete peptidase mRNAs. Refined peptidase mRNA sequences were used to identify contigs in each assembly with at least 97% sequence identity, which were used for expression calculations. If a contig aligned only partially to the mRNA, its contribution to expression was proportional to the aligned part if it was more than 50% of the contig length. RPKM was used to calculate the number of reads mapped to a contig, calculating each multiread as one unit. As an additional approach, we used a “rescue” method [T. molitor, the method of rescue probably gives a better estimate of mRNA expression, because repetitive and overlapping contigs may have been overrepresented, and calculation of each multi-mapped read as one unit can give an inflated expression. To compare expression levels within each insect, we took the RPKM for each predicted peptidase and divided by the sum of RPKM for all peptidase transcripts × 100.For ” method . The resT. castaneum was used as the outgroup for the tree. Based on the cladogram, pairs of sequences from T. molitor and T. castaneum were considered orthologous if they formed a single clade. If more than two sequences formed a clade that could not be separated into orthological pairs, this set of sequences was considered an orthological group.A cladogram was constructed with MegAlign, using ClustalW alignment ,47 accorT. molitor and T. castaneum that were similar to peptidases from the C1 papain family, and calculate relative expression values. Different approaches were taken because of the availability of a reference genome for T. castaneum and lack of a sequenced genome for T. molitor. These comparisons identified similarities and differences in the complement of C1 cysteine peptidases in the two tenebrionids.Using different algorithms to analyze RNASeq data, we were able to predict cysteine peptidase genes in sequences from the larval gut of T. castaneum genome as part of the annotation project, 24 genes were identified that encode enzymes similar to the C1 papain family peptidases [In our previous bioinformatic study of cysteine peptidases in the T. castaneum genome (Tcas3) were supported by transcriptome data  also clustered, suggesting that they diverged prior to the separation of the tenebrionid lineage.The location of T. castaneum had the highest expression levels of all peptidase genes, NP_001164001 and NP_001164314 in T. castaneum and TmL13 in Т. molitor  than the T. molitor major cathepsin L (about 62%) (Tables T. molitor) and XP_971698 (T. castaneum), about 6% and 2% of the total cysteine peptidase expression, respectively. TmL5 was previously characterized as digestive cathepsin L  by [T. molitor  and five from T. castaneum, three included in clusters , and two single genes (XP_969833 and XP_001814509). The expression levels correlated to our previous biochemical data, demonstrating the greater significance of cysteine peptidases in the digestive process for T. castaneum than for T. molitor [In ) Tables  and 3. O5023) by . AnotherT. castaneum and T. molitor demonstrated sequence conservation of the active site residues QCHN in the majority of the sequences . There were five SNPs in the N-terminal region of XP_966750; most other SNPs were random and not found in conserved residues.QCHN active site residues were conserved in all except TmB32, XP_968689 and XP_967834, in which C → S  and H → T (XP_968689) were found; we consider these sequences inactive homologs  and consists of about 25 amino acid residues similar to human cathepsin B. The models of “typical” cathepsin B peptidases show a conserved structure in subsites S1 and S2 , it was previously suggested that the site of AH in the occluding loop participates as an exopeptidase [For two eptidase . HoweverSchistosoma mansoni mature cathepsin B (3S3R), to identify amino acid residues in the S1 and S2 subsites that presumably play the most important role in the substrate specificity of C1 peptidases (data not shown). We found five amino acid residues whose radicals were located within 5 Å of the substrate-like inhibitor and thus could affect the substrate specificity of S2 subsite - 67, 68, 133, 157, 205, similar to another report [We compared the 3D structures of complexes of a peptide-like inhibitor and model cysteine peptidases from the C1 family: papain and human mature cathepsins L1 (3OF8), mature cathepsin S (1NQC), and r report . We alsor report . Our lisT. molitor and T. castaneum peptidases, alignments were made of the predicted sequences of cathepsin L and B peptidases with human cathepsin L1 (P07711) [T. molitor with 3D structures of human mature cathepsin L2  and mature cathepsin B , respectively. Cathepsin L-like sequences of both insects contained a significant amount of variability in the substrate binding sites, especially at the S2 subsite  lacked homology in the S1 subsite (Table To determine residues in the binding subsites of (P07711)  and L2 ((P07711) , and B ((P07711) , respectT. castaneum  and orthologs TmL13 and TmL1 in T. molitor, respectively, were closest to human cathepsin L in the S1 and S2 subsites  and a related cathepsin L from chromosome 3, and orthologous T. molitor cathepsins TmL4 and TmL6, have basic residues in the 205 position . Position 157 in this cluster was mostly occupied with a large aromatic Phe residue. Mammalian cathepsin L peptidases contain nonpolar Ala205 and Met157 (HsCath1) or Leu157 (HsCath2) residues in the S2 subsite, so tenebrionid cathepsin L from this cluster may have different substrate specificity. This cluster contains the transcripts with moderate expression levels . One group of specific T. molitor transcripts  lacked homology in both S1 and S2 subsites and may have specific regulatory functions. Orthologous cathepsin O and F in both insects had similar substrate binding sites, except for acidic Asp 67 and Glu205 in both cathepsin O peptidases.T. castaneum  and orthologs in T. molitor  contained negatively charged amino acids  at position 205 in S2 subsites, consistent with the Glu205 in mammalian cathepsin B . In most atypical cathepsin B-like sequences with a shortened occluding loop, located in two clusters on chromosome 8, position 205 was occupied by polar uncharged residues . Therefore, affinity to substrates with Arg at position P2, characteristic for human cathepsin B, is different in predicted atypical cathepsin B-like peptidases of tenebrionids. TmB28 differed substantially from other B-like cathepsins, not only in the S1 subsite (Ser65), but also in the S2 subsite with a positively charged His in the 205 position. T. castaneum has two cathepsin B peptidases (previously characterized as pseudogenes XP_008196465 and XP_008196467 in the Tcas3 genome build) with atypical occluding loops containing Glu205, and these peptidases may have similar substrate specificity as mammalian cathepsins B.Most cathepsin B sequences, including inactive homologs (XP_008195352 and TmB32), contained typical Gly residues in the S1 subsite, except for TmB28 and inactive XP_968689, which contained Ser65  and cathepsins with changes in the S1 subsite . Mature enzyme models were constructed for all of the chosen peptidases. All models were evaluated by Ramachandran plots  with crystal structures of corresponding mutant procathepsins 3QJ3 and 3QT4 , respectT. molitor sequences TmL2, TmL5, Tm7, TmL8, TmL13, TmB18, and TmB33. Sequences TmL2, TmL5 and TmL13 demonstrated FRF substrate affinity, and free binding energies of the substrate were similar to each other and with the binding energy of the substrate with human cathepsin L  were predicted to freely form an enzyme-substrate complex, with a preference for the substrate FRF with an aromatic amino acid at position P2 , encoded by neighboring genes on chromosome 10 (LOC659441 and LOC659502), which are highly expressed in the gut and are most similar to the mammalian cathepsin L in the structure of substrate binding site. The T. molitor larval gut contains only one highly expressed orthologous cathepsin L, TmL13, and the impact of cathepsin B transcripts as a percentage of total peptidase transcripts (21%) is higher than in T. castaneum. However, we did not find a correlation between the expression profile and the structure of the peptidase clusters. Therefore, the regulation of expression in tandem genes may be independent.The data on C1 peptidase expression, together with biochemical  and protT. castaneum and T. molitor major digestive peptidases and for human cathepsin L2, with the only substitution Leu67. The other eight cathepsin L peptidases in T. castaneum and 11 cathepsin L peptidases in T. molitor have substitutions in S2 subsite, and levels of their transcripts expression are much lower under normal dietary conditions. However, we know that the expression of tenebrionid peptidases can change in response to dietary inhibitors or toxins [We propose that the structure of substrate binding S2 subsite, containing residues Trp67, Met68, Ala133, Leu157, Ala205, provides the most effective hydrolysis of proteins, because it is characteristic for r toxins ,27.T. castaneum compared to T. molitor may be, at least in part, due to the overwhelming role of cathepsin L in food digestion, and this can be one of the factors facilitating the distribution of these larvae around the world.As C1 cysteine peptidases in tenebrionids perform the most important initial steps in protein digestion, we speculate that the 5-6-fold increased speed of larval development in T. castaneum and their orthologs or homologs in T. molitor  may be lysosomal enzymes. These enzymes have increased diversity in the structure of substrate binding sites, suggesting the possibility to hydrolyze a wide variety of substrates. The remaining transcripts of cathepsin L, B, and also F and O with low level of expression in the larval guts of both insects most probably are involved in regulatory processes. Transcripts of cathepsin L and B with negligible level of expression, like TmL15, TmL8, TmB22 and their cathepsin L orthologs (homologs) mainly from chromosome 7, XP_971868, XP_971975, XP_971752, XP_969833, as well as cathepsin B from chromosome 8, XP_966663, may have specific regulatory roles or are not expressed in the gut and may be an artifact of sequence assembly or tissue contamination. Cathepsin K, which is found in the T. castaneum genome, is expressed primarily in the embryo (data not shown).We predict that cathepsin L and B peptidases with moderate expression in the gut of tenebrionids, like XP_971698, NP_001164088 (both cathepsin L), XP_974244, XP_966750 (both cathepsin B) in in silico primary and tertiary structures of the predicted peptidases, we were able to demonstrate that peptidases similar to cathepsin B can be divided in two groups: those with typical cathepsin B structures and containing occluding loops, and cathepsin B-like proteins with a short loop lacking two His residues, that are apparently unable to function as exopeptidases. Cathepsin B transcripts with the highest expression levels in both insects were orthologs and belong to the cluster of typical cathepsin B . The structure of the substrate-binding site in cathepsins from this cluster (chromosome 3 in T. castaneum) was similar to mammalian cathepsin B, and the enzymes contained acidic residues in position 205 of the S2 subsite, which enables the hydrolysis of peptide bonds after basic amino acid residues in the substrate. Cathepsin B-like peptidases from another cluster (chromosome 8) with short occluding loops presumably will not accept basic residues in the P1 position due to the absence of acidic residues in position 205.By examining the T. molitor cathepsin L and B supported the analysis of the primary structures and 3D models of enzymes. Cathepsins TmB18, TmB33 and TmL13, with a typical binding site structure, formed complexes with these substrates with binding energy comparable to mammalian homologs. Enzymes with slightly altered S2 subsites, TmL5, TmB18 and TmL2, showed lower or no affinity to LRF, while enzymes with an altered S1 and S2 subsites, TmL7 and TmL8, did not bind these substrates in the proper position, and thus have entirely different specificity, or may be inactive. These data suggest that a Gly-Gly pair in the S1 subsite is crucial for substrate binding, and proteins with changes in this pair will lack typical substrate affinity.Docking characteristic peptide substrates FRF and LRF to the active center of T. castaneum larvae. Using transcriptome data acquired in this and previous studies, we found 14 complete predicted peptidase sequences similar to cathepsin L and related peptidases, and 13 cathepsin B and B-like peptidases transcripts in the T. molitor larval gut. In addition, there were sequences encoding cathepsin F and O in both insects, but expression levels were low, and cathepsin K was found only in the T. castaneum genome. The most highly expressed peptidases in both insects were orthologous cathepsin L peptidases, with an additional highly expressed cathepsin L in T. castaneum. The expression of cathepsin B peptidases was much lower than cathepsin L in both insects. Most cysteine cathepsin B and L peptidases had considerable variability in the substrate binding sites, consistent with the hypothesis that evolution in cysteine cathepsins has enabled the insects to survive diets high in proline and glutamine, as well as seed inhibitors of peptidases. A new group of atypical cathepsin B-like peptidases was described with shortened occluding loops. These data provide unique perspectives of protein digestion in these tenebrionids and the most comprehensive data for coleopteran peptidases to date.Three main groups of cysteine peptidases were identified in the gut of tenebrionid insects: cathepsin L, cathepsin B, and a new group of cathepsin B-like peptidases that lack an additional active site in the occluding loop. Using genomic, transcriptome, and microarray data from this and previous studies, we have identified 11 cathepsin L and 11 cathepsin B-like peptidases transcripts in the gut of


To evaluate the therapeutic effects in terms of disease clearance and hearing improvement of canal wall up mastoidectomy in combination with Type I tympanoplasty in otitis media.A total of 78 patients (81 ears) with otitis media were treated by canal wall up mastoidectomy in combination with Type I tympanoplasty. The postoperative tympanic membrane morphology, average of pure-tone hearing thresholds and average air-bone gap were used as the indices for evaluating therapeutic effects.The patients were followed up for two years in average. All the tympanic membranes recovered, with the ear canals being dry. There were five cases (5 ears) of tympanic membrane retraction and one case of otitis media recurrence. Hearing was effectively recovered by 76.54% (62/81) after surgery.Combining canal wall up mastoidectomy with Type I tympanoplasty can treat otitis media safely and effectively due to high postoperative dry ear canal rate, satisfactory reconstruction of hearing and maintenance of ear morphology. Mastoidectomy preserving the bony external auditory canal and tympanoplasty are referred to as closed technique or combined approach tympanoplasty that was first proposed by Sheehy and Janson,4With the development of ear microsurgical techniques, infectious foci can be removed while preserving acoustic features-related structures and reconstructing hearing. After rational selection of indications, 78 patients (81 ears) with otitis media were treated by canal wall up mastoidectomy in combination with Type I tympanoplasty. They were then followed up to analyze the overall treatment outcomesOur objective was to evaluate the therapeutic effects in terms of disease clearance and hearing improvement of canal wall up mastoidectomy in combination with Type I tympanoplasty in otitis media.The surgical procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975. Written consent was obtained from all patients. A total of 78 patients (81 ears) with otitis media who were enrolled in our hospital from March 2009 to January 2012 were treated by canal wall up mastoidectomy in combination with Type I tympanoplasty.When otitis media, otoscopic examination through the external auditory canal showed the eardrum was inflammatory, manifested as redness, swelling and extrusion; reflection of the otoscopic light by the eardrum surface disappeared or attenuated; the eardrum had a white interface upon middle-ear hydrops; age older than 18 years.Pregnant women; lactating women; patients complicated with severe cardiac, lung, liver or kidney diseases.Before surgery, all patients were subjected to regular HRCT scan of the temporal bone, pure tone audiometry (or multi-frequency steady-state and bone-conduction auditory brainstem response) and Eustachian tube function  examinations.Myoperiosteal flap on the muscle pedicle was prepared by using temporal fascia after making a postauricular incision. With the posterior ear canal wall preserved, the mastoid was contoured. Then the posterior ear canal wall was worn thin to gradually open the attic forward and to expose the incudomalleolar joint. Afterwards, tissues with pathological changes in the mastoid cavity and the attic were carefully removed. In the meantime, the posterior external ear canal wall was worn thin downward to expose the incudostapedial joint and stapes, and to eliminate the infectious foci in the posterior tympanum. Then the internal tympanum was exposed by lifting and pushing the tympanic flap of ear canal skin through the external auditory canal, after which lesions were cleared from two sides of the tympanum and mastoid . The ossThe tympanic membrane was assessed postoperatively according to the following criteria. 1) Cured: The tympanic membrane did not congest after surgery, and purulent secretions in the external ear canal and otitis media symptoms disappeared; 2) remitted: the tympanic membrane still mildly congested after surgery, but purulent secretions in the external ear canal and otitis media symptoms were significantly mitigated; 3) ineffective: the tympanic membrane was still congested after surgery, and purulent secretions or clinical symptoms were unalleviated or even aggravated.All patients were followed up, i.e. otoscopic examination in the postoperative 1st and 3rd months as well as pure tone audiometry in the postoperative 6th month. One year later, pure tone audiometry was performed once every six months. The outcomes of lesion removal, preservation of posterior ear canal wall structures, tympanic membrane and hearing were recorded. Averages of air-conduction thresholds and average air-bone gaps at 0.5, 1, 2 and 4 kHz detected in the last follow-up were used to evaluate the postoperative hearing. In addition, the hearing changes were observed by comparing the values with the pure-tone hearing thresholds before surgery.There were 42 males and 36 females between 13-56 years of age [average: (35.6±7.3)]. Disease course: 5 months ~ 30 years . LesionsOssicular chains in 48 ears were preserved, partial ossicular replacement prostheses were placed in 31 ears, and total ossicular replacement prostheses were placed in two ears. There were various inflammatory lesions  in 51 ears and restricted cholesteatoma in 30 ears. Hearing was rendered as effective increase when air-bone gap was decreased by 15 dB after surgery. The preoperative and postoperative pure-tone hearing thresholds are summarized in Otologists have endeavored to better treat otitis media, a common otological disease. Canal wall up mastoidectomy, which modifies the classical method, is devoted to preservation or improvement of the physiological and auditory functions besides elimination of infectious foci. This protocol, although capable of both radical treatment and functional reconstruction, requires accurate selection of indications, adeptness in anatomy and surgical operations, as well as sophisticated apparatus. In this study, the long-term effects of this protocol were evaluated by following up eligible patients who were free from lesions with reconstructed hearing for two years in average. In addition to complete removal of infectious foci in all the selected patients (ears), the normal anatomical structures of the posterior external ear canal wall were preserved and the rate of effective hearing increase reached 76.54% (62/81), similar to the results of a previous study.5Similarly, it has been reported that the recurrence rates of canal wall up mastoidectomy ranged between 5%-40%,8Moreover, indications were rationally selected based on temporal bone CT and hearing examinations together with clinical characteristics and intraoperative changes. Canal wall up mastoidectomy in combination with tympanoplasty is applicable to the treatments of all kinds of chronic otitis media in the case of intact external auditory canal bone wall, Eustachian tube patency and relatively restricted lesions. However, open surgery and ossicular chain reconstruction should be conducted instead for middle ear-involved external auditory canal cholesteatoma, widely invasive cholesteatoma and infectious foci, intracranial and extracranial complications, and necrotic osteitis, aiming to allow dry ear canal by eliminating foci.10Meanwhile, elimination of lesions should be given first priority over surgeries that can retain the original ear anatomical structures. More importantly, even if open surgery in combination with ossicular chain reconstruction can basically be comparable to canal wall up mastoidectomy due to the application of artificial ossicles and the advance in surgical techniques,The novel protocol is generally performed within narrow cavities under a small visual field, which thus easily causes complications in the absence of sufficient knowledge and skilled surgeons. The complications commonly include facial nerve injury, damage of the labyrinthine segment, cerebrospinal fluid leakage, cholesteatoma recurrence and middle ear adhesions, etc.When indications are rationally selected, retaining the bony external auditory canal by skilled surgeons is more conducive to preservation of ear morphology and reconstruction of normal tympanic and acoustic structures compared with traditional radical treatment protocols.


Objective. To evaluate Auditory Steady-State Responses (ASSR) at high intensities in pediatric cochlear implant candidates and to compare the results to behavioral tests responses. Methods. This prospective study evaluated 42 children with suspected severe-to-profound hearing loss, aged from 3 to 72 months. All had absent ABR and OAE responses. ASSR were evoked using binaural single frequency stimuli at 110 dB HL with a 10 dB down-seeking procedure. ASSR and behavioral test results were compared. Results. Forty-two subjects completed both ASSR and behavioral evaluation. Eleven children (26.2%) had bilateral responses. Four (9.5%) showed unilateral responses in at least two frequencies, all confirmed by behavioral results. Overall 61 ASSR responses were obtained, most (37.7%) in 500 Hz. Mean thresholds were between 101.3 and 104.2 dB HL. Among 27 subjects with absent ASSR, fifteen had no behavioral responses. Seven subjects showed behavioral responses with absent ASSR responses. No spurious ASSR responses were observed at 100 or 110 dB HL. Conclusion. ASSR is a valuable tool to detect residual hearing. No false-positive ASSR results were observed among 42 children, but in seven cases with absent ASSR, the test underestimated residual hearing as compared to the behavioral responses. As universal newborn hearing screening programs are established in numerous countries, more children will be diagnosed in early childhood with some degree of hearing loss.Early detection and intervention during the critical period for language and cognitive development can improve individual performance .Children with severe-to-profound bilateral hearing loss are candidates for cochlear implantation (CI) and require specific audiologic evaluation prior to intervention. As early age of indication and presence of residual hearing are important factors for postimplant speech perception and language development, this has resulted in further decrease of minimum age of surgery –7.In these very young children, behavioral audiologic evaluation can be challenging, may not be obtained in children younger than 6 months, and usually does not assess each ear separately. Thus the audiologic evaluation of pediatric cochlear implant candidates relies more and more on electrophysiological measures.The most widely used electrophysiological procedure for estimating hearing thresholds in young children is click and tone burst auditory brainstem responses (ABR). Due to the transient nature of the stimuli used to evoke ABR, maximum output levels are 95 dB hearing level (HL). In view of that, the possibility of residual hearing at severe-to-profound levels cannot be investigated with ABR .Hearing assessment of children, using the Auditory Steady-State Responses (ASSR), is made by frequency specific continuous modulated tones and allows increased levels of stimulation intensity. Therefore, ASSR can provide ear and frequency specific threshold information at elevated intensity levels up to 120 dB HL and higher, providing better and more reliable investigation of ears with minimal residual hearing . FurtherFor such reasons, ASSR is a unique tool for auditory assessment of young cochlear implant candidates.Some authors , 11 haveSolutions have been implemented by the manufacturer to reduce artifacts at high-intensity stimulation .Few papers have been published since 2004. One report evaluated 15 children with severe-to-profound hearing loss by ASSR, but behavioral thresholds were obtained for only one subject . As cochPreviously, we performed two studies at the University of São Paulo. One of them evaluated adults with severe-to-profound hearing loss. The responses of pure tone audiometry (PTA) and ASSR were compared. Patients' subjective perception of ASSR stimuli was also evaluated and compared to PTA test results, and no systematic extra-auditory ASSR responses at high intensities were observed .The other study evaluated children with severe-to-profound hearing loss from 6 to 65 months. Most ASSR responses (48%) were found at 500 Hz .The aim of this study was to evaluate Auditory Steady-State Responses (ASSR) at high intensities in pediatric cochlear implant candidates and to compare the results to behavioral test responses.This prospective study evaluated 58 children with suspected severe-to-profound hearing loss, aged from 3 to 72 months. All children referred to this institution for pediatric cochlear implant evaluation between January and December 2011 were enrolled. included only children with normal external and middle ear conditions.We excluded patients with severe neurologic disorders who did not permit behavioral evaluation. We also excluded patients who showed responses on either ABR or OAE or who did not achieve noise ratio under 30 microvolts during ASSR.WeOverall 16 patients were excluded: three had severe neurologic disorders and could not complete behavioral evaluation, one showed responses on click ABR, one had bilateral absent click ABR and normal DPOAE (possible auditory neuropathy spectrum disorder), one showed high noise levels during ASSR, and ten did not show up for behavioral evaluation.The procedure was a routine assessment for pediatric cochlear implantation at the Department of Otolaryngology, University of São Paulo School of Medicine. The study was reviewed and approved by the Hospital's Ethic Committee (number 38954) and written informed consent was obtained from all parents.In a unique session using light general anesthesia with Sevoflurane, all children were examined by otomicroscopy and tympanometry followed by click auditory brainstem responses (click ABR), bone conduction ABR, distortion-product otoacoustic emissions (DPOAE), and ASSR.All ABR, OAE, and ASSR recordings were obtained in a sound-treated room.The tests were run using the Navigator Pro SCOUT and AEP  software.EEG activity was recorded using gold disk electrodes placed on the earlobe and Fpz. The contralateral earlobe was used as ground. Interelectrode impedance was less than 2 KOhm.Click stimuli (duration: 100 milliseconds) were presented with ER3A insert earphones at the maximum level of 90 dB HL at a rate of 21.1/s with rarefaction and condensation polarity. The responses were considered to be absent when both rarefaction and condensation waves showed no responses at 90 dBHL. Bone conduction was tested with a standard bone vibrator at the maximum intensity of 55 dB HL using alternated click stimuli and contralateral masking of the same intensity.For DPOAE we applied the diagnostic 750 to 8000 Hz test protocol . Responses greater than 6 dB over background noise  at five out of eight frequencies were considered as Pass criteria.The ASSR test was completed in the same session as ABR and OAE. ASSR were evoked using binaural single frequency stimuli at 110 dBHL with a 10 dB down-seeking procedure. Test stimuli were 0.5, 1, 2, and 4 kHz tones modulated in amplitude and frequency. Stimuli were 20% frequency and 100% amplitude modulated at 65 Hz for all tones in the left ear and at 69 Hz for all tones in the right ear according to the default specifications of the ASSR system . Modulation rates of 65 Hz or higher were used to ensure acceptable signal-to-noise ratio for response detection. Test stimuli were presented through insert earphones (ER3A) previously calibrated for each frequency as suggested by the manufacturer. Measurements were made with a Brüel & Kjaer sound level meter DB-0138 which conforms to ANSI S3.7-1995.P < 0.05). If a sweep contained more than 80 nV of electrophysiological noise, it was rejected. Thresholds were repeated to guarantee reliable results, completing 10 sweeps in each run. The same physicians performed all tests.The ASSR assessments were performed by a dichotic single frequency technique. This implies that a single frequency was offered to both ears simultaneously. Electrode disks were fixed with electrolytic paste to the scalp at Cz (active), midline posterior neck (reference), and Fpz (ground). All electrode impedances were below 5 KOhm, and the interelectrode impedance values were kept below 3 KOhm. A maximum of 10 sweeps containing 16 epochs each were recorded per trial. Each epoch was 1.024 seconds. The electrophysiological recording was converted by means of a fast Fourier transform after each sweep. The response was accepted with an F ratio, comparing the fast Fourier components at the stimulus modulation frequencies to determine whether the difference was significantly different from the background noise  for mid-low frequencies (1000–3000 Hz), and metallic rattle and bell for high frequencies (over 3000 Hz) [The stimuli were uncalibrated sounds, presented at low 55–80 dB SPL, medium 70–88 SPL, and high intensities 80–115 dB SPL, depending on the instrument. Three frequency ranges were evaluated. We used drums for the low frequency spectrum (125–500 Hz), wooden rattle and3000 Hz) , 17. A r3000 Hz) , in one 3000 Hz) .Children had behavioral evaluation within three months of the electrophysiological test battery.We analyzed four ASSR frequencies in each ear . The percentage of present or absent responses at each frequency was determined. We considered present ASSR responses, if responses could be obtained in at least two frequencies in one or both sides.For the behavioral test, we evaluated if the participant had present or absent responses to the frequency range of low, mid-low, and high frequencies. No ear specific responses were obtained in this test condition. (1)Present responses in both tests. (2)Absent responses in both tests. (3)Present responses in ASSR and no responses in behavioral evaluation. (4)No responses in ASSR and present responses in behavioral evaluation.ASSR and behavioral test results were compared, regarding present or absent responses. Therefore, we divided the subjects into four groups.Association between ASSR and behavioral test results was evaluated by Kendall's rank correlation tau and Cohen's kappa coefficient. The software used was “R statistical computing.”From 58 children enrolled in the study, forty-two completed both ASSR and behavioral evaluation.After all we studied 42 subjects (20 girls and 22 boys) between 6 and 60 months .Fifteen subjects (35.7%) showed ASSR responses in two or more frequencies. Eleven had bilateral and 4 had unilateral responses .Most responses (37.7%) were obtained at 500 Hz, 29.5% at 1000 Hz, 21.3% at 2000 Hz, and 11.4% at 4000 Hz .Right and left ear responses are presented in At behavioral evaluation we found 34 responses (48.6%) in the low frequency, 24 (34.3%) in mid-low frequency, and 12 (17.1%) in the high frequency range. Twenty-four children had responses to more than one instrument. Seven subjects of 42 (16.6%) had absent responses to all frequency ranges.Fifteen subjects had responses in both tests (group 1), fifteen had absent responses in both tests (group 2), no subject had present ASSR and absent behavioral responses (group 3), and twelve had absent responses in ASSR and present responses at the behavioral test (group 4) .In group 4, five subjects had single frequency responses on ASSR  but were considered nonresponders for data analysis.Overall, in 30 subjects (71.4%), both tests showed consistent results.Accurate diagnosis of a severe-to-profound bilateral sensorineural hearing loss remains the primary and most basic requirement for implantation . The ASSThe possibility of an objective evaluation at this age is very important for the correct decision of cochlear implantation and of the side to be implanted, in case of a unilateral CI. Still, behavioral responses should be obtained and correlated with ASSR responses.Our results demonstrate a strong correlation between ASSR and behavioral test results in 71.4% of the subjects. Moreover, no false-positive ASSR responses were seen in this casuistic, differing from other authors , 11.In this study we excluded patients with external and middle ear disorders, because the focus was on pure severe-to-profound sensorineural hearing loss. Children with suspected auditory neuropathy spectrum disorder (ANSD) or neurologic disabilities were also excluded, as behavioral testing may be very difficult or even unreliable in some cases. Previous reports showed that ASSR thresholds in ANSD may be substantially higher than pure tone thresholds , so currIn all frequency range both tests showed consistent results, as subjects responded best to low frequencies and less in the high frequency range. Since instruments used for the behavioral test are not frequency specific but encompass a wider frequency range, it is not surprising to observe higher number of responses in this test. This is especially true for the low frequency range, because 250 Hz was not tested during ASSR, but may have contributed to the higher number of behavioral responses in this frequency range (125 to 500 Hz).Stimulation of the low frequency range in high intensities may evoke responses due to vibration and may not reflect the auditory status . So cautN = 10) was composed of older children (mean age: 13 years and 4 months), old enough to perform pure tone audiometry. These results are not comparable to the present study with younger patients (mean age: 2 years and 5 months) who were evaluated by an instrument-based behavioral test, since visual reinforcement audiometry is not a test tool for infants aged 3 months. It seems that ASSR has been unpopular for evaluating pediatric cochlear implant candidates. A reason may be that not all equipment permits ASSR stimulation above 100 dB HL, as it is the case for newer equipment, using the Chirp stimulus for ASSR assessment. To our knowledge, there are no published data about ASSR threshold obtained with the Chirp stimulus in high-intensity levels.All subjects with absent behavioral responses had no responses in ASSR. In other words, no subject had spurious or extra-auditory ASSR responses in our cohort, differing from other studies , 11. FewNevertheless, high-intensity ASSR is a promising tool to evaluate residual hearing in children with severe-to-profound hearing loss and absent responses to click ABR. The continuous amplitude and frequency-modulated tones make it possible to determine frequency specific thresholds at high intensities by means of an objective evaluation, with minimal interference of the examiner. Thus, it is a unique electrophysiological technique to get frequency specific thresholds at intensities exceeding 95 dB HL, where tone ABR rarely will detect responses, due to the transient nature of the tone burst stimulus. Moreover, tone ABR depends on visual wave identification by the clinician who should be well trained and cautious.As ear specific information is available, ASSR thresholds can be used for hearing aid fitting before eventual cochlear implantation. Furthermore, in case of unilateral responses, four of 42 subjects in this study, the results may assist the surgical team to choose the side to be implanted, if unilateral CI is the option.As in other reports , 23, mosThe Navigator Pro MASTER software permits simultaneous stimulation of both ears at one test frequency even at high intensities (>80 dB HL), so overall test time is reduced. Although test time was not the scope of our study, the whole procedure  did not exceed 60 minutes per patient. In our institution these tests are performed by the same physicians for more than four years and are part of routine evaluation before pediatric cochlear implantation, so this may have contributed to the acceptable overall test time.We considered present ASSR responses, if they could be obtained in at least two frequencies in one or both sides to prevent spurious or extra-auditory responses at single frequencies. However, this decision turned ASSR less sensitive than the behavioral evaluation, where one instrument encompasses a wide acoustic spectrum and stimulates a range of frequencies.Unlike Swanepoel and Hugo  we did nTherefore, a complete test battery including electrophysiological and behavioral measures is still the best means to correctly evaluate hearing thresholds, even in cases of severe-to-profound hearing loss.ASSR is a valuable tool to detect residual hearing in young children with absent ABR and DPOAE. No false-positive ASSR results were observed among 42 subjects, but in seven cases with absent ASSR, the test underestimated residual hearing as compared to the behavioral responses.


Resting metabolic rate (RMR) is the component of energy expenditure that explains the largest proportion of total daily energy requirements. Since RMR is determined largely by fat-free mass and a low RMR predicts weight gain in healthy adults, identifying the role of muscle atrophy following stroke on RMR may help identify ways to mitigate the development of obesity post-stroke.Thirty-nine stroke survivors with chronic hemiparesis  underwent DXA scans for measurement of body composition, including total, paretic, and non-paretic leg lean mass and fasted, 30-min indirect calorimetry for measurement of RMR.Predicted RMR was calculated by the Mifflin-St Jeor equation, which considers weight, height, and age for both men and women. RMR was 14% lower than predicted . Total , paretic  and non-paretic  leg lean mass predicted RMR.These data indicate that muscle atrophy post stroke may lead to a reduced RMR. This substantiates the need to attenuate the loss of lean mass after a stroke to prevent declines in RMR and possible weight gain common post-stroke. Stroke is the leading cause of long-term disability . We haveTotal daily energy expenditure (TDEE) consists of resting metabolic rate (RMR), the thermic effect of food, and physical activity. Resting metabolic rate (RMR) is the component of energy expenditure that explains the largest proportion of total daily energy requirements. Individuals with a low RMR are at higher risk of significant weight gain, relative to those with a high RMR ,10. AlthThis cross-sectional study included 39 stroke survivors, between the ages of 45–80 years, which were recruited from the Baltimore area for participation in exercise rehabilitation studies. Participants were in the chronic phase of stroke recovery (at least six months prior) and had residual hemiparetic gait deficits. All volunteers signed University of Maryland Institutional Review Board approved informed consent forms.Participants underwent a health history and physical examination, which included height, weight, blood pressure, and a resting electrocardiogram. Dual-energy X-ray absorptiometry (DXA) scans  were conducted to determine total body fat (%) and total, paretic, and non-paretic leg lean tissue mass.Subjects received two weeks of heart healthy diet  counseled by a Registered Dietitian prior to RMR testing. Subjects reported to our lab first thing in the morning following a 12 hour fast. RMR was measured by indirect calorimetry , while participants rested quietly in the supine position under a ventilated canopy for 30 minutes in a thermo-neutral and comfortable environment. Energy expenditure was calculated by the Weir equation  and exprDescriptive statistics were analyzed using SPSS . Results are expressed as mean ± SEM. Student’s t-tests were used to compare predicted to measured RMR. Linear regression was used to assess whether leg lean mass predicts RMR. Statistical significance was set at a two-tailed P < 0.05.Subject characteristics may be viewed in RMR was not associated with younger age  or latency from stroke . However, we observed that total body (r=0.70) and total leg r=0.73,  lean tisChronic hemiparesis is present in nearly 50% of all long-term ischemic stroke survivors . In addiOur observation that RMR is lower than predicted is relevant to stroke recovery since studies suggest that lower RMR is a predictor of greater sequential weight change . FurtherDespite the small sample size, this study is strengthened by the inclusion of a diverse study population, including a wide range of ages (middle aged to elderly), ethnicities, and body composition . Since the incidence of stroke is higher in men than women (aged less than 75 years) , we beliResearch efforts for effective treatment strategies to maintain a healthy body weight focus on diet and exercise programs. While we have previously reported improvements in body composition (muscle hypertrophy and decreased fat mass) following the initiation of exercise during the chronic phase of stroke –34, how In summary, we observed that stroke survivors are at risk for a low RMR in the chronic phase of stroke. It appears that preserving overall lower extremity muscle mass may contribute in the maintenance of RMR after a stroke. This substantiates the need to attenuate the loss of lean mass, possibly with lifestyle interventions, after a stroke to prevent declines in RMR and possible weight gain common post-stroke.


Production of membrane-associated cell surface receptors and their ligands is often a cumbersome, expensive, and time-consuming process that limits detailed structural and functional characterization of this important class of proteins. Here we report a rapid method for refolding inclusion-body-based, recombinant cell surface receptors and ligands in one day, a speed equivalent to that of soluble protein production. This method efficiently couples modular on-column immobilized metal ion affinity purification and solid-phase protein refolding. We demonstrated the general utility of this method for producing multiple functionally active immunoreceptors, ligands, and viral decoys, including challenging cell surface proteins that cannot be produced using typical dialysis- or dilution-based refolding approaches. Cell surface immunoreceptors, ligands and viral decoys (receptors and ligands) belong to a major class of membrane receptors that play vital roles in a variety of biological processes. These include molecular recognition, viral infection, and immune defense. The successful expression and purification of these host and microbial surface proteins in active forms are critical for biochemical, structural, and functional studies of receptor-ligand molecular recognition in the context of host-pathogen interactions. Among the variety of challenges associated with the study of this class of molecules, obtaining purified functional receptor and ligand proteins is often a long and labor-intensive process.E. coli. This makes inclusion body-based receptor/ligand refolding often a required step to obtain functionally active cell surface proteins , bacteria hosts represent the most economical system in terms of associated costs, speed, and ease of use. However, cell surface proteins partition to the inclusion body (IB) fractions in majority cases when overexpressed in One approach to cope with technical challenges in protein refolding is to apply high throughput screening. Developments of such refolding screenings have been reported, based on analytical detection of reverse phase-fast protein liquid chromatography (rp-FPLC) or differential scanning fluorimetry (DSF)12We sought to develop a rapid and general method for refolding cell surface receptors and ligands that will fill a much needed technology gap for an important class of biomacromolecules in structural biology, functional genomics, and chemical biology applications. Built upon works from literature and our systematic efforts to refold a variety of cell surface immuno- and viral receptors and ligands, we report here a rapid and less expensive method that successfully refolded membrane-associated receptors and ligands with different classes of structures and functions.To solve the problems associated with regular refolding methods, we sought to dramatically enhance the efficiency of the steps used to purify the inclusion bodies. To bypass the slow and labor-intensive washing steps of inclusion bodies in their solid-state form, we chose to directly solubilize the inclusion bodies in a chaotrope solution after cell disruption. We then coupled affinity purification of chaotrope-dissolved proteins and refolding steps with IMAC via a Ni-NTA column to streamline the entire operation. Seamless connection between on-column purification and on-column refolding is possible in part due to that Ni-NTA or similar resins used for IMAC purification can tolerate the chaotropic buffers and mild redox conditions. An additional advantage of these procedures is that the elution volumes of refolded targets can be easily controlled  to avoid an extra concentration step for the final preparative protein liquid chromatography .6-tag in their amino-termini or carboxyl-termini to facilitate affinity chromatographic purification  and fully folded state (0 M urea). These steps are intended to stabilize intermediate folding or unfolded states of the proteins, which may in turn facilitate the refolding reactions to be more productiveE. coli host is a reducing environment, proteins that require disulfide bonds cannot be made efficiently in typical competent E. coli strains. In certain instances, secondary structure formation may be so favorable that the correct cysteine residues spontaneously bind and only weakly oxidizing conditions are required. However, if this is not the case, stronger oxidizing conditions may be required. We have tested a number of reducing or reducing/oxidizing reagents in our system. Three redox pairs, cysteamine/cystamine, cysteine/cystine, and reduced glutathione (GSH)/oxidized glutathione (GSSG) successfully refolded target proteins  to evaluate the secondary structures of all seven cell surface proteins . As expeWe also tested if purified receptor molecules are functionally active. We performed two sets of experiments. First, we used two cell surface ligands, MICA and ULBP3, in conjunction with their target receptor NKG2D as examples, because these receptor-ligand interactions have been well characterized44The method described in this report has several distinct advantages. First, our method efficiently couples modular on-column immobilized metal ion affinity purification and on-column protein refolding. We purposely avoid purification of inclusion bodies in their solid-state, which requires considerable amounts of time and reagents in inclusion body washing and centrifugation. In addition, the elution volume of refolded targets can be easily controlled to facilitate the concentration steps necessary for the follow-up purification step by size exclusion chromatography or a second affinity chromatography step. The entirety of the refolding and purification steps can be completed in one day, duration equivalent to that of the purification of soluble proteins. This is a significant improvement in efficiency compared to regular solid-state inclusion body purification followed by dilution and/or dialysis or approaches. In comparison with several cases of on-column refolding of proteins reported in the literature, our method provides an improvement in experimental efficiency and a general approach that works for a variety of membrane-associated receptors and ligands. Secondly, the format of on-column immobilization allows convenient follow-up processing of the refolded proteins. For example, if the refolded cell surface proteins are in the form of tagged or fusion proteins, it is facile to implement an enzymatic cleavage step. Thirdly, the current approach can be easily adapted to a high throughput format, for example, a 96-well plate or multi mini-column format. Such formats may have versatile applications in structural biology, chemical biology, and functional proteomics studies. Lastly, as demonstrated in the case of the UL37 exon 3 protein, we are able to refold this cell surface protein with our new method, but not with traditional “dialysis” or “dilution” approaches. Presumably, the on-column refolding format facilitates the refolding process, as reported in the case of EGFR domain refoldingin situ. The fluorescence-based assay can rapidly inform us about the optimal conditions for refolding cell surface proteins of interest. This refolding method can also couple with protein microarray and a means of analytical detection  to identify new binding ligands when applied to a collection of rapidly refolded cell surface receptors. Refolded receptors may be immobilized on carrier surfaces by affinity capture, noncovalent adsorption, or covalent binding6-tagged refolded receptors can be immobilized on Ni-NTA-coated glass slides. Receptor hits may be identified by label-dependent detection, such as Cyanine-3/5-labeled ligand of interest, or by label-free detection, such as surface plasmon resonance.We envision that our rapid receptor/ligand refolding method can incorporate other high throughput methods to develop powerful screening assays. A recent study reported DSF guided refolding as a novel method of protein productionIn summary, this study presents a rapid method for refolding and purifying host and microbial surface receptors and ligands. The method takes advantage of immobilized metal ion affinity purification and on-column solid-phase protein refolding. It skips lengthy inclusion body washing steps and dialysis steps associated with traditional refolding protocols. The robustness of our method is supported by the evidence that multiple classes of receptors with diverse structures may be refolded with comparable yields to those of classical dialysis or dilution methods. This method may be readily adapted into high throughput format, manufactured into a commercial kit, or adapted into automatic procedures. In the future, it will be particularly interesting to test our method with integral membrane proteins. We envision that this method will have significant applications in structural biology, functional proteomics, and chemical biology investigations.6-tagged construct  RIL CodonPlus strain . UL144 and UL146 were expressed in E. coli strain BL21 (DE3). Bacteria were grown in 1L LB media containing 100 μg/ml ampicillin and/or 25 μg/ml chloramphenicol in 2.8 L Fernbach flasks. Cultures were grown at 37 °C until OD600 reaches 0.5–0.8 and were induced with 1 mM IPTG. UL37 × 3 culture was induced at 37 °C overnight. All the other proteins were induced at 37 °C for 4 hours. A 250-μl sample was taken immediately before and after induction for SDS-PAGE. Volume of the induced sample was determined based on its OD600. The sample was centrifuged at 15,000 rpm for 1 minute and the supernatant was discarded. Pellet was then re-suspended in 25 μL H2O, mixed with 25 uL 2xSDS sample buffer, denatured at 95 °C for 5 minutes. Freeze the sample at −20 °C until needed for running on SDS-PAGE. Cultures were collected by centrifugation at 9000 rpm for 30 minutes at 4 °C. The pellet was then re-suspended in 20–60 mLs of sonication lysis buffer  with freshly added 5 mM EDTA, and 5 mM DTT). The suspension was lysed using a Fisher sonication dismembrator , and then incubated with 10 mM MgSO4 at room temperature for 20 minutes. The lysate was collected by centrifugation at 9000 rpm for 20 minutes at 4 °C and the pellet was saved for rapid refolding and traditional dialysis-based refolding.NKG2D was refolded and purified according to the literature2PO4, pH 8.0, and 8 M urea) for 2 hours at 4 °C while stirring. Dissolved inclusion bodies were cleared by centrifugation at 9000 rpm for 30 minutes at 4 °C. The supernatants were filtered through 110 μm Waterman filter membrane and were mixed with adequate volumes of Ni-NTA agarose resin  for 1 hour at 4 °C. The mixture was loaded onto a column. The resin was washed with 40 times volume of dissolving buffer and 40 times volume of washing buffer . Refolding was performed on column with 40 times volume of refolding buffers containing 3 M urea and 1.5 M urea respectively. The resin was washed with sufficient volume of phosphate buffer saline (1xPBS) and eluted with elute buffer . The eluted proteins were concentrated and injected into HiLoad 16/60 Superdex 75 gel filtration column attached with ÄktaFPLC system . The fractions were collected and used for protein quantification and for quality check by running SDS-PAGE.For our rapid refolding method, the pellet was dissolved in dissolving buffer  overnight at 4 °C while stirring. Dissolved inclusion bodies were cleared by centrifugation at 9000 rpm for 30 minutes at 4 °C. The supernatants were filtered through 110 μm Waterman filter membrane and were mixed with adequate volumes of N-NTA agarose resin for 1 hour at 4 °C. The mixtures were loaded onto a column. The resins were washed with 15 times volume of dissolving buffer and 15 times volume of washing buffer . The inclusion bodies were eluted with elusion buffer . The eluted, purified inclusion bodies were diluted to 0.3 mg/ml and loaded into a dialysis tubing with suitable molecular weight cut-off for refolding. Refolding and purification processes were followed by a regular dialysis-based refolding protocol as described previously4For traditional dialysis-based refolding of ULBP3 and UL146, both were expressed and lysed as described above. The lysates were collected by centrifugation at 9000 rpm for 20 minutes at 4 °C. The pellets were washed three times with washing buffer A  and once with buffer B . The washing/resuspending-centrifugating steps were repeated multiple times  until the inclusion bodies were 90–95% pure estimated by SDS-PAGE. The pellets were dissolved in dissolving buffer  following literatureThe spectra were acquired using a JASCO J-815 circular dichroism spectrometer over the wavelength range from 190 to 250 nm. All measurements were performed at 20 °C. The spectra were acquired using 5-20 μM protein concentrations in 10 mM potassium phosphate buffer (pH 7.0) in a 1 mm path-length quarts cell . Buffer spectra were subtracted from the raw data. The ellipticity was calculated following reference formulas: Ellipticity, [θ], in deg cmOptimal concentrations of 20 μM of protein and 3 μM Sypro Orange dye  were used in the protein samples. The samples were added to a 96-well plate and the spectra were acquired using a Bio-Rad real-time PCR detection system . The melting temperature range was set from 4–95 °C with increment of 6 °C per minute. The fluorescence was monitored using standard excitation/emission wavelengths, and the protein Tm was determined using the manufacturer’s software. Thermo-unfolding profile of a sample of buffer only was also acquired and was used as background control to be subtracted in data analyses. The RFU values from different data sets were normalized to a common scale of 0–1 as described in the literatureFor MICA and NKG2D binding assay, 0.8 nmol of MICA and 1.6 nmol of NKG2D (molar ratio 1:2) were mixed together and injected into Superdex 75 10/300 gel filtration column attached with ÄktaFPLC chromatographic system. Elution profile of the complex was compared with those of MICA alone or NKG2D only. For ULBP3 and NKG2D binding assay, different amounts of ULBP3  and 5 nmol of NKG2D were mixed to give ULBP3:NKG2D molar ratios of 1:2, 1:3, 1:4, 1:6, and 1:8. The mixtures were analyzed using similar procedures described for the MICA-NKG2D complex. The mixture peaks were deconvoluted using programs from OriginPro  and the deconvulated peak intensities and peak elution volumes were compared.How to cite this article: Zhai, L. et al. A Rapid Method for Refolding Cell Surface Receptors and Ligands. Sci. Rep.6, 26482; doi: 10.1038/srep26482 (2016).


Data on the secretory profile of TCs and the bidirectional shuttle vesicular signalling mechanism between TCs and CSCs are scarce. We aimed to characterize and understand the in vitro effect of the TC secretome on CSC fate. Therefore, we studied the protein secretory profile using supernatants from mouse cultured cardiac TCs. We also performed a comparative secretome analysis using supernatants from rat cultured cardiac TCs, a pure CSC line and TCs-CSCs in co-culture using (i) high-sensitivity on-chip electrophoresis, (ii) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and (iii) multiplex analysis by Luminex-xMAP. We identified several highly expressed molecules in the mouse cardiac TC secretory profile: interleukin (IL)-6, VEGF, macrophage inflammatory protein 1α (MIP-1α), MIP-2 and MCP-1, which are also present in the proteome of rat cardiac TCs. In addition, rat cardiac TCs secrete a slightly greater number of cytokines, IL-2, IL-10, IL-13 and some chemokines like, GRO-KC. We found that VEGF, IL-6 and some chemokines  are secreted by cardiac TCs and overexpressed in co-cultures with CSCs. The expression levels of MIP-2 and MIP-1α increased twofold and fourfold, respectively, when TCs were co-cultured with CSCs, while the expression of IL-2 did not significantly differ between TCs and CSCs in mono culture and significantly decreased (twofold) in the co-culture system. These data suggest that the TC secretome plays a modulatory role in stem cell proliferation and differentiation.Telocytes (TCs) are interstitial cells that are present in numerous organs, including the heart interstitial space and cardiac stem cell niche. TCs are completely different from fibroblasts. TCs release extracellular vesicles that may interact with cardiac stem cells (CSCs)  However, the most plausible assumption is that TCs are involved in intercellular signalling, during which they cooperate with stem cells The presence of regulatory molecules, cytokines, chemokines and growth factors in the CSC environment played crucial roles in stem cell growth and differentiation, especially in the heart We report the protein/peptide secretory profile of TCs from rodent hearts, which confirmed the presence of VEGF, IL-6 and MIP-2 in the supernatants of TCs in culture. In addition, MIP-1α, MCP-1 and GRO/KC, along with several less abundant cytokines , were identified. Our results indicate that TCs could sense and re-direct the cellular microenvironment to increase the renewal capacity of CSCs.ad libitum access to food and water. The rodents were killed by cervical dislocation and immobilized in the supine position with their necks extended. The thoracic cavity was then cut open to expose the hearts, which were consequently removed from the body.Adult C57B6 male mice  weighing 25–30 g (10–12 weeks old) and male adult Wistar rats weighing 150–200 g (8–10 weeks) were used in this study. The rodents were housed in air-conditioned rooms (22–24°C) under a 12-hrs light–dark cycle at a standard facility with This study was approved by the Bioethics Committee of ‘Victor Babeş’ National Institute of Pathology, Bucharest according to the institutional guidelines and the European Union standards for the care and use of experimental animals.3, followed by enzymatic dissociation. The tissue fragments were incubated for 15 min. on a rocking table in 250 U/ml collagenase II (Sigma-Aldrich) at 37°C; the supernatant, which contained the cells, was collected, and the collagenase activity was inhibited with ice-cold Hank’s Balanced Salt Solution (HBSS). This procedure was repeated twice. The suspension, which contained the dissociated cells was washed and centrifuged, and the cells were re-suspended in DMEM/F12 medium (Sigma-Aldrich) that was supplemented with 10% foetal bovine serum  and 100 U/ml penicillin - 100 μg/ml streptomycin (Sigma-Aldrich). The resulting cell suspension was plated in 25 cm2 Petri dishes in DMEM/F12 medium that was supplemented as above and then cultivated in an incubator at 37°C in a humidified atmosphere that contained 5% CO2. The medium was changed every 24 hrs for the next 7 days or until the cells covered more than 80% of the culture plate. Similar conditions were applied to isolate and culture rat myocardial TCs. The cells were dissociated with trypsin-ethylenediaminetetraacetic acid, washed and centrifuged, re-seeded in DMEM-F12 that contained 10% FBS in 25 cm2 flasks and cultivated as described above for passages 2 and 3 at a cell density of 5 × 103 cells/cm2. Similar cultures were maintained in FBS-supplemented or serum-free medium for 72 hrs, and the supernatants were collected for analysis. Serum-free medium was used to avoid interference from the FBS in the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and ‘on-chip’ electrophoresis analyses. The fibroblast cell line BALBc/3T3 (ECACC No. 85022108) was used as a control for cell secretion.The procedures used to isolate TCs from myocardial tissue and subsequently culture them were previously described for mice and rats The rat CSCs were a generous gift from Prof. Piero Anversa .3 cells/cm2 in 24 mm Transwell cell culture inserts with a 0.4 μm pore clear polyester membrane placed in 6-well plates that contained CSCs 5 × 103 cells/cm2  in F12K culture medium. This system permits the free passage of molecules but restricts physical contact between the two cell populations Cell suspensions that were obtained from rat cell cultures were plated at 5 × 10Control wells contained only stem cells in the lower compartment or TCs in the upper compartment. The medium was collected after 48 hrs and stored at −20°C.The serum-free supernatants from mouse TCs and 3T3 cell cultures were separated with on-chip electrophoresis using the High-Sensitivity – Protein 250 kit on an Agilent 2100 Bioanalyzer  according to the manufacturer’s instructions The cell culture supernatants from mouse TCs and BALBc/3T3 cells were analysed in triplicate on a weak cation-exchange (CM10) ProteinChip array . The chips were prepared and the samples were loaded according to the manufacturer’s instructions for low-stringency buffer conditions. The concentrations of all samples were equalized by adding binding buffer , and 1 μl of saturated energy absorbing molecule solution  was added twice and allowed to air dry.The samples were analysed using time-of-flight mass spectrometry on a PBS II Protein Chip Reader . The spectra were collected using the low-setting protocol: focus mass 5000 D, matrix attenuation 1000 D, sampling rate 800 MHz, energy 5000 nJ. The spectra were analysed using the ProteinChip Data Manager Software version 3.0.7 . To improve the detection, the protein concentration was enhanced by freeze-drying the samples and reconstituting them in MilliQ ultrapure water .The cytokine/growth factor levels in the supernatants from serum-free and serum-supplemented mouse TCs and 3T3 fibroblast cell cultures were analysed in triplicate using xMAP technology. The cytokines secreted by mouse TCs were analysed on a Luminex200  using MILLIPLEX MAP Mouse Cytokine/Chemokine Panel-9 plex – IL-1β, IL-2, IL-4, IL-6, IL-12, tumour necrosis factor (TNF)-α, VEGF, MIP-2 (CXC motif chemokine 2) and granulocyte-macrophage colony stimulating factor (GM-CSF). An enhanced panel was used to analyse the secreted molecules in the rat TC and CSC experiment: MILLIPLEX MAP Rat Cytokine/Chemokine Magnetic Bead Panel, which included G-CSF, Eotaxin, GM-CSF, IL-1β, Leptin, MIP-1α, IL-4, IL-1α, IL-6, epidermal growth factor (EGF), IL-13, IL-10, IL-12p70, interferon (IFN)-γ, IL-6, IL-17, MCP-1, IP-10, GRO/KC, VEGF, Fractalkine, lipopolysaccharide induced CXC chemokine (LIX), MIP-2, TNF-α, IL-2, IL-18, and RANTES. These kits, all from Millipore, Billerica, MA, USA. were used according to the manufacturer’s specifications P-values of less than 0.05 were considered to indicate significant differences using Student’s t-test.Briefly, the beads, which were provided within each kit, were incubated with buffer, cytokine standards (included in the kit) or samples in a 96-well plate at 4°C overnight in triplicate. All further incubations with detection antibodies and streptavidin - R-Phycoerythrin Conjugate (SAPE) were performed at room temperature in the dark with shaking at 500 r.p.m. Millipore multiscreen plates were used together with the Millipore filtration system. Multiplex data acquisition and analysis were performed using STarStation software version 2.3 ; the calibration curves were generated with a 5-parameter logistic fit. The data are presented as the mean ± SD of three experiments, and two-tailed On-chip electrophoretic analysis consistently revealed the same protein bands throughout all passages of mouse TCs (in cultures that were maintained in serum-free medium), indicating stability between passages. The overall electrophoretic profile is shown in FigureSELDI-TOF MS protein profiling was used to analyse and compare several differentially expressed peptide and protein peaks that were identified in the mouse TC culture supernatants, the culture medium and the 3T3 fibroblast culture supernatants. Additionally, several differentially expressed peptide and protein peaks in TCs supernatants (maintained in serum-free medium) were demonstrated by MS. Mass peaks between 1.5 and 25 kD were analysed and the most significant peaks were estimated at m/z values of 11.7 estimated to be most significant. Significant peaks were identified between 13.8 and 14.8 and at 22.5 kD , and this level did not significantly differ by passage. Cells grown in serum-free medium secreted significantly more IL-6 (8- to 19-fold increase) than the corresponding 3T3 culture. Mouse TCs secreted significantly more proteins in serum-free conditions than 3T3 fibroblasts (fourfold increase in mouse TCs at first passage (P1) compared with fibroblasts at the same passage, P < 0.05), and this secretion level increased the most  in the third passage (P3) . Mouse TCs secreted significantly more IL-6 than fibroblast cultures in both serum-free and -supplemented media. In serum-supplemented medium, mouse TCs secreted more than threefold the level of IL-6 than 3T3 fibroblasts (P < 0.05), and this level increased further  in the TCs of P3 , threefold in the second passage (P2) and 5.5-fold in the third passage (P3) compared with the fibroblast culture. In serum-supplemented medium, TCs secreted 1.5-fold more VEGF in P1 than fibroblasts. Moreover, TCs secreted significantly more VEGF in P2 than in P1 . This method permitted the highly accurate detection and quantitation of proteins in supernatants of cells that were cultivated in normal (FBS-supplemented) medium.i.e., TCs expressed a fivefold higher level than CSCs  was threefold higher than in TC and 20-fold higher than in CSC mono-culture Fig..Although IL-2 was well-expressed in the individual rat TC and rat CSC cultures, it appeared to be negatively regulated in the TC-CSC co-culture (approximately twofold reduction compared with mono-culture).Consistent with the secretory profile of MIP-2, we found MCP-1 to be highly expressed in TC culture (threefold higher than in CSC culture), and this protein was also expressed at high levels in the rat TC-CSC co-culture  were high in the TC monoculture, moderate in the TC-CSC co-culture and low in the CSC mono-culture supernatants.Interleukin-10, an important anti-inflammatory molecule, was significantly expressed in all cultures , while IL-5 was moderately expressed in mono-culture (TC and CSC) and unaffected by the co-culture conditions.The expression of IL-13 was up-regulated in the TCs-CSCs co-culture compared with the TC mono-culture.The rat cardiac TCs data were consistent with the mouse TC data. Luminex-xMAP identified a larger array of proteins than the entries in the UniProtKB database This study primarily aimed to assess the protein/peptide secretory profile of TCs from rodent hearts. The results reported herein show that the levels of several cyto- and chemokines , as well as VEGF, were significant in mouse and rat TC supernatants. These findings suggest the potential regulatory effect of TCs on other cell types  and their role in the control of cell growth/myocyte differentiation and angiogenesis. However, further advanced proteomics studies or antibody arrays are needed to confirm these findings.via Tps and over short distances via EVs via EVs, which transport crucial cardioprotective agents Recent studies have shown that TCs actively participate in intercellular communication over long distances via the engagement of the JAK-STAT3 axis. In vitro, cytokines activated JAK-STAT3 signalling and increased the expression of the STAT3 target genes hepatocyte growth factor and VEGF In vivo studies showed that JAK-STAT3 signalling was activated and growth factor/cytokine production was increased in MSC-injected animals. The paracrine actions of these host tissue-derived factors activated the endogenous cardiac repair mechanisms in the diseased heart mediated by Akt, ERK and JAK-STAT3 The IL-6 cytokine family includes LIF, CT-1 and IL-11, which activate downstream signalling pathways in cardiac myocytes and contribute to cytoprotection and vessel formation in the heart e.g. IL-6, MIP-1α, MIP-2, MCP-1 and IL-5, which suggests that TCs play a role in paracrine regulation. In addition, the co-culture experiments suggest that TCs and CSCs act synergistically to control the secretion of proteins, as illustrated by the levels of MIP-1α, MIP 2 and IL-13 (up-regulated) as well as the level of IL-2 (significantly down-regulated). As expected, the levels of these chemokines are up-regulated, because they are regulated by IL-6 in vivo findings of the role of TCs in myocardial regeneration.The levels of several secreted molecules are higher in rat TCs than in CSCs, et al. showed that VEGF secreted by MSCs improves myocardial survival and the engraftment of implanted MSCs in infarcted hearts, which promoted the recruitment of stem cells via the paracrine release of myocardial stromal cell-derived factor-1α We also detected high levels of VEGF in the TC mono-culture and TC-CSC co-culture experiments. Tang et al., who demonstrated the role of VEGF in the differentiation of stem cells into cardiomyocytes via delivery systems in regenerative medicine is well-known via bi-directional paracrine cross-talk with CSCs, an additional argument for the potential application of TCs in myocardial regeneration. In addition, the co-culture experiments demonstrated the bi-directional modulation of TCs and CSCs, as demonstrated by the modification of the secretory profile, which indicated that MCP-1 and MIP-1α production were enhanced in co-culture compared with TC or CSC mono-culture. These findings suggest that these chemokines may mediate the role of TCs in directing the formation of cardiomyocytes. MIP-1α and MCP-1 were shown to play similar roles in the formation of smooth muscle in the airway We correlated the secretion of VEGF by TCs reported herein with a report by Song in vitrovia paracrine intercellular signalling.We suggest that TCs, which secrete more growth factors and intercellular communication cytokines than other cell types, can deliver signals to stem cells that drive them to differentiate into myocardial cells and participate in the complex process of myocardial regeneration. Moreover, the heart regeneration process, similar to those of other organs, depends on the integrity of the extracellular matrix, specifically that of fibronectin versus CSC mono-culture.We report here, for the first time, the secretome of cultivated TCs that were isolated from rodent hearts. These data further support the shuttle mechanism of vesicle-mediated signalling between stem cells and TCs. The secreted forms of some chemokines, cytokines and growth factors identified in TC primary cultures suggest that these secretory molecules may be useful in cardiac repair. The panel of cytokines/chemokines identified in this study suggests that IL-6 drives cells towards a proliferative phenotype. In-depth studies of the future therapeutic applications of TCs should focus on mechanisms that control the intercellular communication. Thus, TCs could be key players in the renewal and repair of some organs, including the heart, and TC-CSC co-culture may be a superior option for therapy