The role of the renin–angiotensin system (RAS) in the biology of infantile hemangioma (IH) represents an emerging paradigm, particularly the involvement of renin, angiotensin converting enzyme, and angiotensin II. This study investigated the expression of cathepsins B, D, and G, enzymes that may modulate the RAS, in IH.The expression of cathepsins B, D, and G was examined using immunohistochemistry, enzyme activity assays, mass spectrometry, and NanoString gene expression assay in IH samples at different phases of development.Immunohistochemical staining showed the expression of cathepsins B, D, and G in proliferating and involuted IH samples. This was confirmed at the transcriptional level using NanoString gene expression assays. Mass spectrometry confirmed the identification of cathepsins D and G in all three phases of IH development, whereas cathepsin B was detected in 2/2 proliferating and 1/2 involuting lesions. Enzyme activity assays demonstrated the activity of cathepsins B and D, but not G, in all phases of IH development.Our data demonstrated the presence of cathepsins B, D, and G in IH. Their role in modulating the RAS and the biology of IH offers potential novel targets for the management of this tumor. Infantile hemangioma (IH) affects 4–10% of Caucasian infants . It typiRecent data have implicated IH as an embryonic developmental anomaly due to aberrant proliferation and differentiation of a hemogenic endothelium  derived The classical RAS pathway involves the initial conversion of angiotensinogen to angiotensin I (ATI) by renin, with its subsequent conversion to ATII by angiotensin converting enzyme (ACE)  Figure . Recent We have previously characterized the mast cells within IH and shown their abundant expression of chymase , thus prIn this study, we investigated, within IH, the presence and localization of other non-renin and non-ACE pathways involving cathepsin B, an enzyme that converts pro-renin to active renin , 20; catn = 6), involuting (n = 3), and involuted (n = 6) IH samples were obtained from patients aged 4–7 months, 2–4 years, and 6–12 years, respectively, according to a protocol approved by the Central Health and Disability Ethics Committee .Proliferating (n = 6) and involuted (n = 6) from 12 patients were used for immunohistochemical (IHC) staining. Antigen retrieval was performed using sodium citrate (Leica) at 95°C for 15 min. All sections underwent single 3,3-diaminobenzidine (DAB) staining for the primary antibodies, cathepsin B, 1:200 (Santa Cruz); cathepsin D, 1:200 (Leica); cathepsin G, 1:200 (Santa Cruz); tryptase, ready to use (Leica), CD34, ready to use (Leica); and GLUT-1, 1:200 (Cell Marque) with detection using the bond polymer refine detection kit (Leica). To confirm dual expression of two proteins, selected representative slides of each phase of IH underwent immuno-fluorescent (IF) IHC staining with the same primary antibodies at the same concentrations, but using an appropriate secondary antibody for detection . All antibodies were diluted in Bond primary antibody diluent (Leica), and all DAB and IF IHC staining were performed on the Leica Bond Rx auto-stainer . IF IHC stained slides were mounted using vectashield hardset medium with DAPI .Four μm-thick formalin-fixed paraffin-embedded IH sections of proliferating (n = 2), involuting (n = 2), and involuted (n = 2) IH tissues from six patients of the original cohort was used for mass spectrometry. The tissues were homogenized in 150 μL of ice cold RIPA buffer (Sigma-Aldrich) containing 1× Complete Protease inhibitor cocktail EDTA-free (Roche Life Science) using a Teflon-coated dounce homogenizer. After protein quantitation , 100 mg of total protein from each sample was precipitated overnight at −20°C . The washed protein pellets were re-suspended in 50 μL of 5% sodium deoxycholate (SDC), 10 mM dithiothreitol, 100 mM TEAB buffer (pH 8.5) (Sigma Aldrich), and incubated for 30 min at 80°C. Samples were then alkylated for 60 min in the dark using 40 mM iodoacetamide, and diluted to 500 μL final volume in 100 mM TEAB buffer (pH 8.5). Proteins were digested overnight at 37°C using 4 mg trypsin per sample . SDC removal was achieved by formic acid precipitation  and centrifugation for 30 min at 13,000 × g, and the peptide supernatants transferred to fresh microcentrifuge tubes. The SDC precipitates were washed with 200 μL of 1% formic acid and each wash solution was combined with the previously recovered peptide supernatant. Samples were then concentrated to ~10 μL, reconstituted to 500 μL in 0.1% formic acid, purified using OMIX C18 pipette tips (Agilent Technologies), and prepared for liquid chromatography (LC)–MS/MS (mass spectrometry) in 4% acetonitrile: 0.1% formic acid.Total protein extracted from proliferating (−1 flow rate), and peptides were eluted and analyzed using data-dependent MS/MS acquisition . Raw MS/MS data files were searched against a complete human protein database  using Proteome DiscovererTM V1.4 (Thermo Scientific) and Scaffold 4.0 (Proteome Software) to establish protein identification and relative protein abundance by spectral counting. Peptide assignments were accepted above 90% confidence, and protein identification parameters were: protein threshold, 1.0% FDR; minimum total spectrum count, 2; peptide threshold, 1.0% FDR.Liquid chromatography–MS/MS  was performed using an UltiMate 3000 HPLC system (Dionex) connected to a LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Thirty-five μL per sample per injection were loaded onto an Acclaim PepMap100, C18 column  (0.3 μL minn = 3), involuting (n = 3), and involuted (n = 3) IH samples from nine patients of the original cohort, using cathepsin B activity assay kit , cathepsin D activity assay kit (Abcam), and cathepsin G activity assay kit (Abcam), respectively. All steps of the procedure were performed according to the manufacturers’ protocol. Fluorescence was measured in 96-well plate format using the Varioscan plate reader (ThermoFisher).Enzymatic activities of cathepsins B, D, and G were determined fluorometrically in snap-frozen proliferating (n = 6) and involuted (n = 6) IH from 12 patients were used to isolate total RNA for NanoString nCounter™ Gene Expression Assay . The RNA was extracted from frozen tissues using RNeasy Mini Kit (Qiagen) and quantitated by the NanoDrop 2000 Spectrophotometer (Thermo Scientific). RNA samples with A260/A280 ≥1.9 and A260/A230 ≥1.8 were subjected to the NanoString nCounter™ gene expression assay as performed by New Zealand Genomics Ltd. , according to the manufacturer’s protocol. Probes for the genes encoding cathepsin B , cathepsin D , cathepsin G , and the housekeeping genes CLTC (NM_004859.2), GUSB (NM_00181.3), HPRT1 (NM_00194.1), and PGK1 (NM_000291.3) were manufactured by NanoString Technologies . Raw data were analyzed by nSolver™ software  using standard settings and were normalized against the housekeeping genes.Snap-frozen samples of proliferating . All bright field images were captured using the Olympus BX53 microscope fitted with an Olympus DP21 digital camera .p < 0.05 considered statistically significant.Statistical analyses were performed using the Kruskal–Wallis one-way analysis of variance for independent samples, using IBM SPSS (Version 22). This assigns ranks to the data, with thick arrows), expressing CD34 , in both proliferating  in the differences between the levels of activity in involuting compared to both proliferating and involuted IH samples used. Analysis of the enzyme activity of cathepsin D revealed statistically significant (p = 0.021) increase in the levels of activity from proliferating to involuting and from involuting to involuted phases of IH. Interestingly, the enzymatic activity of cathepsin G was barely detectable in all IH tissues examined (data not shown).Enzymatic assays performed to determine the presence of activities for cathepsins B, D, and G in IH samples in all three phases of IH development confirmed the activity of both cathepsins B and D Figure . Comparip = 0.007) amounts of corresponding mRNA compared to proliferating samples.To support the translational abundance of cathepsins B, D, and G examined in this study, we used transcriptional profiling of the tissue samples for corresponding levels of mRNA at all phases of IH development. The levels of mRNA for both cathepsins B and D were relatively high. This was in comparison with significantly lower levels for cathepsin G mRNA Figure . No statWe have recently demonstrated the crucial role for ATII in the biology of IH . ATII, aMore recent reports have shown variable effects of systemic β-blockers for a given dosage  on IH. HThe final production of ATII results from the classical RAS pathway that depends on the presence of both renin and ACE but also the non-renin/non-ACE pathways involving a number of proteases , 27. ChyOur finding of the presence of cathepsins B, D, and G at both transcriptional and translational levels within IH, and the previous demonstration of the abundance of chymase  and ACE The presence of cathepsins B, D, and G and chymase offers possible explanations that certain lesions are relatively more refractory to β-blockade and ACE inhibition. ACE inhibition results in an accumulation of ATI, which can potentially be converted to ATII by chymase produced by mast cells. This study demonstrates the presence of potential shunting/shuffling of upstream precursor peptides, through these non-classical RAS pathways, which we infer, may subsequently play a more prominent role once the classical RAS undergoes blockade.This report reveals the expression of cathepsins B, D, and G in IH and offers novel insights into their roles in the RAS pathway, in this tumor. It provides understanding of the potential multiple pathways contributing to the ultimate levels of the vasoactive peptide, ATII, and highlights the complexity of β-blockade or ACE inhibition in the treatment of this tumor. Further investigation of the functional roles of cathepsins B, D, and G, and chymase, in the complex interplay of enzymes involved in the RAS pathway is needed to unravel their precise role of RAS in the biology of IH.This report highlights the presence of cathepsins B, D, and G in infantile hemangioma (IH).Cathepsins B, D, and G within IH may play a crucial role in the production of angiotensin peptides in proliferating lH.The presence of potential bypass mechanisms for the production of angiotensin peptides highlights the role for the RAS in proliferating IH.TI, PFD, and STT performed the basis of the research. TI, PFD, and STT designed the research study. TI performed the IHC experiments and analysis. DAC performed enzyme activity experiments and analysis of the enzyme activity and NanoString data. JCD performed the mass spectrometry experiments and analysis. TI and STT drafted the manuscript. All authors contributed to the preparation of this manuscript. All authors read and approved the manuscript.There was no source of funding for the article. The authors declare that there is no source of financial or other support, or any financial or professional relationships, which may pose a competing interest. The content of this article has not been submitted or published elsewhere.http://journal.frontiersin.org/article/10.3389/fsurg.2015.00026The Supplementary Material for this article can be found online at Click here for additional data file.


To review the roles of the different vascular beds nourishing the inner retina [retinal ganglion cells (RGCs)] during normal development of the human eye, using our own tissue specimens to support our conclusions.An extensive search of the appropriate literature included PubMed, Google scholar, and numerous available textbooks. In addition, choroidal and retinal NADPH-diaphorase stained whole mount preparations were investigated.The first critical interaction between vascular bed and RGC formation occurs in the sixth to eighth month of gestation leading to a massive reduction of RGCs mainly in the peripheral retina. The first 3 years of age are characterized by an intense growth of the eyeball to near adult size. In the adult eye, the influence of the choroid on inner retinal nutrition was determined by examining the peripheral retinal watershed zones in more detail.This delicately balanced situation of RGC nutrition is described in the different regions of the eye, and a new graphic presentation is introduced to combine morphological measurements and clinical visual field data. Some of the latest techniques for detecting micro vessels include adapted fluorescence angiography  and broaden our understanding of the nutrition and vascular supply of the peripheral retina. This is interesting for both the development of the retinal ganglion cells (RGCs), its vascular beds, and the adult neuronal tissue, which requires oxygen and glucose to survive. The complex aspects of aging are not included here and will be presented in a subsequent publication.To the best of our knowledge, there is no review to date covering the blood supply of the peripheral retina. Recent reviews on retinal vasculature specialized either on morphological subentities . Textbooks and monographs related to this subject were also used in this literature review.Many publications related to various keyword combinations were discarded if they (a) did not contain human results, (b) described only pathological situations, or (c) did not contain original research. The time period chosen for the literature search was not restricted and non-English publications were also included.Using data from the literature, the Goldmann visual field overlay map was modified and superimposed with morphological aspects of the retinal and choroidal vasculature. The graphic modeling was performed manually and by computer. To verify metric numbers given in the literature, our own measurements were performed using tissue samples from previous studies (see below). Because of the small number of these specimens, we did not perform statistics but restricted their use to graphic illustration.Retinal and choroidal whole mounts, stained with the NADPH diaphorase technique, were reevaluated from a previous publication . No new The staining procedure of the retinae was parallel to that of the choroidal whole mounts, as described previously : the tisMeasurements and analysis of the whole mounts were performed on different magnified micrographs recorded with a Digital Sight DS-Fi1 camera connected to an Optiphot-2 light microscope (Nikon™).Nutrition of the RGCs is a critical factor throughout life. Simple diffusion is limited to the early stages of the embryo and together with the formation of the third germ layer; a specialized vascular system develops specifically in each region of the developing fetus.The first vessels appearing in the human eye are the hyaloid vessels supplying the lens. The earliest cells to differentiate in the fetal retina are the Müller cells during the sixth week of gestation (wog) . Müller By the seventh (six to eight) wog the RGCs begin to differentiate and migrate from the inner neuroblastic layer along the vertical scaffolding of the Müller’s processes toward the ILM where they will form the RGC layer and sprout axons that grow toward the optic nerve. Groups of adjacent RGCs and their axons (fascicles) are separated from neighboring clumps of RGCs by the Müller cell scaffolding which Radius and Anderson refer to as tunnels . We prefWith further differentiation of the RGC layer, there is a loss of the early uniform density of RGCs with a greater concentration occurring at the posterior pole. The total RGC population swells to 2.2–2.5 million by the 18th–30th wog and then decreases rapidly to 1.5–1.7 million cells at birth . The losThe RGCs receive their initial nourishment from the primary hyaloid vasculature  but with the involution of the primary vitreous from the developing CC. Between the third and the fourth month of gestation, the main perfusion of the retina is from the CC. Bruch’s membrane appears with its two collagen layers by the 10th–12th wog , 18, wheThe superficial retinal capillary bed (SCB) begins to form at fourth month of gestation at the base of the hyaloid artery on the optic disk , 21. CruAt birth, the SCB has extended nasally to the ora but temporally only beyond the equator . The DCBThe illustration of the vascular and RGC development Figure  demonstrAfter birth, the temporal SCB grows from beyond the equator to 1–2 mm short of the ora serrata by the fifth month of life , 29. ThiPericytes in the capillary walls begin to appear by the second month after birth and are present in all the capillaries by the third to fifth month except in the extreme retinal periphery . Both reThe fetal eyeball grows rapidly and reaches a sagittal diameter of 12–14 mm at birth. There is continued rapid growth until 3 years of age, when the sagittal diameter reaches 23 mm. Over the next 10 years, it increases only 1–24 mm . In esseBy the age of 13 years, the temporal retina attains its adult dimensions with the temporal macula enlarging 1.39 mm, the equator 6.45 mm, and the very peripheral retina posterior to the ora 1.61 mm . It is tUsing different techniques to estimate the number and density of RGCs in the retina –34 and tVrabec measured the diameter of the RGC axons bundles (fascicles) in the temporal macular region and found them to be ~60 μm in diameter . These aEach of the four quadrants of the retina centered on the optic nerve is perfused by a single retinal artery (end artery) whose SCB ends 1–2 mm behind the ora serrata. Each retinal artery is drained by a single retinal vein with infrequent arteriovenous anastomoses or direct communications with the underlying choroid or the peripheral ciliary body blood vessels. The superior hemiretina is separated from the inferior hemiretina by a horizontal raphe present in the nasal and temporal retina. The temporal horizontal raphe excludes the macula and the papillomacular bundle (PMB). The horizontal raphe crosses the equator ~18.5 mm nasally from the CRA on the optic disk and 22.5 mm from the optic disk temporally . The posFunctional considerations allow the subdivision of retinal vessels into three capillary beds.The SCB traverses the RGC layer at varying levels within this layer and provides nourishment for both the RGCs and the RGC axons in the retinal nerve fiber layer . The diaThe DCB lies in the INL encompassing the posterior pole where it anastomoses freely with the SCB. From the center of the optic disk, it extends 14.5 mm in the lateral horizontal axis , 43. Thiperipapillary capillary bed supplies the inner aspect of the NFL extending from both the superior and inferior poles of the optic disk in an arc along the great vessel arcade to a greater extent temporally than nasally. It arises from the retinal circulation adjacent to the optic disk, which also supplies the capillaries on the surface of the optic nerve head. These surface optic disk capillaries communicate with the capillaries below in the prelaminar region, which arise from the short posterior ciliary arteries. [For a detailed description of the human optic nerve vascular anatomy, see a most recent review by Mackenzie and Cioffi . Concerning the peripheral retina, which is supported only by the SCB of the retinal vessels, two different parts can be distinguished: an inner part, reaching from the end of the central retina toward the equator is characterized by a normal architecture of the CC  but showed a smaller extension in the superior and inferior quadrants .central region of the retina, the proper retinal capillary density is secured by the DCB, which projects between the SCB loops. When zooming through different layers of the central retina  change their vascular supply from a vitreal to a choroidal and then a mixture of both retinal and choroidal blood vessels. This change is not equal for all regions of the retina, because the RGCs of the central retina received their nutrition from the SCB and the DCB, but the peripheral RGCs remain dependent for their nutrition on the CC and the SCB. Most clinical visual field investigations  focus on the central retina; consequently, they fail to record information from the peripheral retina and its interacting with the underlying choroid. This information is crucial for a better understanding of inner retina aging. Virtually, little data exist at present about changes of the peripheral inner retina concerning age or diseases affecting the inner retina (such as glaucoma). It is tempting to speculate that with more comprehensive peripheral retinal examinations , one might better understand the early changes occurring in the peripheral ganglion cell layer, which appears to be more vulnerable, because of its critical dependence for nutrition on both the retinal and choroidal capillaries. New techniques are on its way to include the more peripheral regions, e.g., the 60-4 Humphrey visual field test , the 60°PR initiated the study, had the ideas of the new graphic presentation, and wrote the manuscript. C-AM made profound research, added the original micrographs, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genome evolution in intracellular microbial symbionts is characterized by gene loss, generating some of the smallest and most gene-poor genomes known. As a result of gene loss these genomes commonly contain metabolic pathways that are fragmented relative to their free-living relatives. The evolutionary retention of fragmented metabolic pathways in the gene-poor genomes of endosymbionts suggests that they are functional. However, it is not always clear how they maintain functionality. To date, the fragmented metabolic pathways of endosymbionts have been shown to maintain functionality through complementation by host genes, complementation by genes of another endosymbiont and complementation by genes in host genomes that have been horizontally acquired from a microbial source that is not the endosymbiont. Here, we demonstrate a fourth mechanism.Acyrthosiphon pisum endosymbiosis with Buchnera aphidicola. Using quantitative analysis of gene expression we present evidence for complementation of the Buchnera pantothenate biosynthesis pathway by host genes. Further, using complementation assays in an Escherichia coli mutant we demonstrate functional replacement of a pantothenate biosynthesis enzyme, 2-dehydropantoate 2-reductase (E.C. 1.1.1.169), by an endosymbiont gene, ilvC, encoding a substrate ambiguous enzyme.We investigate the evolutionary retention of a fragmented pathway for the essential nutrient pantothenate (vitamin B5) in the pea aphid, Buchnera symbiosis from amino acid metabolism to include vitamin biosynthesis.Earlier studies have speculated that missing enzyme steps in fragmented endosymbiont metabolic pathways are completed by adaptable endosymbiont enzymes from other pathways. Here, we experimentally demonstrate completion of a fragmented endosymbiont vitamin biosynthesis pathway by recruitment of a substrate ambiguous enzyme from another pathway. In addition, this work extends host/symbiont metabolic collaboration in the aphid/The online version of this article (doi:10.1186/s12915-014-0110-4) contains supplementary material, which is available to authorized users. Buchnera from the aphid, Cinara cedri, is only 416 kbp, approximately 11-fold smaller that its closest free-living relative Escherichia coli [Extensive genome reduction in obligate endosymbiotic bacteria is a hallmark of intracellularity -3. Recenhia coli . Currenthia coli ,9,10. Elhia coli .Table 1Recent genome sequencing of holosymbionts (hosts + their symbionts) reveals that fragmented biosynthesis pathways can be completed by three processes: (1) complementation by host genes with overlapping function -15; (2) et al. [Buchnera amino acid biosynthesis pathways are mediated by other (unidentified) Buchnera enzymes with overlapping activity [Chlamydia is functional because of the recruitment of promiscuous and adaptable enzymes from other biosynthetic pathways [A fourth process for completion of fragmented biosynthesis pathways was proposed by Shigenobu et al.  when theactivity ,14. In aactivity -18. Suchactivity -18. Extractivity -24. Impoactivity -18,21,22activity -27. For pathways .Acyrthosiphonpisum with the gammaproteobacterium Buchnera, we reveal that the fragmented Buchnera pantothenate (vitamin B5) biosynthesis pathway can be completed using a Buchnera substrate ambiguous enzyme recruited from another pathway. We demonstrate using complementation assays in E. coli mutants that retention of a substrate ambiguous enzyme in the massively reduced genome of Buchnera facilitates the collaborative biosynthesis of pantothenate (vitamin B5) in the A. pisum/Buchnera holosymbiont.Here, working with the best-characterized holosymbiosis, that of the pea aphid E. coli, the obligate endosymbiont Buchnera from the pea aphid, A. pisum, has a fragmented pantothenate biosynthesis pathway that is missing panD, panE and ilvE  those from species such as Baizongia pistaciae, C. cedri and Uroleucon ambrosiae that have lost the whole pantothenate biosynthesis pathway [A. pisum, Myzus persicae and Schizaphis graminum that retain identically fragmented pathways characterized by loss of panD, panE and ilvE [Buchnera lineages of identically fragmented pantothenate biosynthesis pathways that include two genes (panB and panC) that only function in pantothenate biosynthesis, led us to hypothesize that the pantothenate biosynthesis pathway in these species is functional.Compared to E Figure . Gene id pathway ,28,29; aand ilvE ,30,31 .To begin the investigation of our hypothesis that evolutionary retention of identically fragmented pantothenate biosynthesis pathways indicates retention of pantothenate biosynthesis capacity we characterized expression of s Figure A. FunctiA. pisum and other insects synthesize β-alanine via two main pathways: (1) the uracil degradation pathway [Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway map 00410), we propose that β-alanine biosynthesis in A. pisum is now additionally required to supply Buchnera. pathway  and (2)  pathway  . Previous work in Salmonella typhimurium [E. coli [ilvC, a gene encoding ketol-acid reductoisomerase (E.C. 1.1.1.86) that mediates reactions in the biosynthesis of the essential amino acids isoleucine and valine and production of 2-oxoisovalerate from pyruvate in pantothenate biosynthesis, also has limited 2-dehydropantoate 2-reductase (E.C. 1.1.1.169) activity. While ilvC can reduce 2-dehydropantoate to pantoate, the activity of ilvC in S. typhimurium to function in pantothenate biosynthesis is 20-fold lower than its activity as ketol-acid reductoisomerase [E. coli that the ilvC knockout mutant was indistinguishable from wild-type K12 in its reduction of 2-dehydropantoate to pantoate [E. coli pantoate auxotrophs requires generation of panE, ilvC double mutants, we hypothesized that Buchnera ilvC has sufficient 2-dehydropantoate 2-reductase activity and, thereby, critically maintains functional pantothenate biosynthesis in Buchnera that retain fragmented pantothenate biosysnthesis pathways.Given host supply of β-alanine, functional pantothenate biosynthesis in himurium  and E. c[E. coli  has showsomerase  and so lpantoate . HoweverBuchnera ilvC has 2-dehydropantoate 2-reductase activity sufficient to complete pantoate synthesis we utilized a 2-dehydropantoate 2-reductase negative strain of E. coli , rescue of E. coli FE8 was only achieved by complementation with E. coli panE and Buchnera ilvC  established robust colonies  and funC Figure A. When c et al., . As a neBuchnera panC. Buchnera panC shares only 48% amino acid sequence identity with E. coli panC. Despite this high level of sequence divergence, Buchnera panC retains pantoate-β-alanine ligase activity  functionally rescue E. coli AT1371 (panC- strain) when grown on a minimal media without pantothenate supplementation. Negative control E. coli AT1371 transformed with pUC19 empty expression vector was unable to grow on minimal media without pantothenate supplementation. With pantothenate supplementation, growth of AT1371 transformed with Buchnera panC, E. coli panC and negative control cells is observed  activity of .2.1 actiBuchnera ilvE  is not critical to the integrity of the pantothenate biosynthesis pathway in Buchnera because Buchnera with fragmented pathways retain ilvH/I, ilvC and ilvD and are predicted to synthesize 2-oxoisovalerate from pyruvate . Symbiont loss of ilvE was first reported in Buchnera APS from A. pisum [A. pisum was published [et al., [Buchnera APS likely utilized the activity of another transaminase to complete branched chain amino acid biosynthesis, members of the Pea Aphid Genome Consortium offered an alternate explanation [Buchnera loss of ilvE encoded branched-chain-amino-acid transaminase is complemented by the host genome encoded branched-chain-amino-acid transaminase  encoded by ilvC as 2-dehydropantoate 2-reductase (E.C. 1.1.1.169)  Figure .Figure 4A. pisum synthesize β-alanine in bacteriocytes via the uracil degradation pathway  and 2-dehydropantoate 2-reductase (E.C. 1.1.1.169) activity. The gene product of ilvC functions as a substrate ambiguous reductase in branched chain amino acid biosynthesis and pantothenate biosynthesis. Functional replacement of panE encoded ketopantoate reductase with a substrate ambiguous ilvC encoded ketopantoate reductase is not without precedent since loss of panE and functional replacement by ilvC has previously been demonstrated in the gram-positive soil bacterium Corynebacterium glutamicum [Notably, we demonstrate that utamicum . In the utamicum .There are currently four mechanisms that facilitate retention of metabolic functionality concomitant with gene loss in the primary endosymbionts of insects. All four mechanisms can operate within a single genome and all four contribute to explaining why fragmented and disconnected biosynthesis pathways, that on first observation appear incomplete and non-functional, are retained across diverse obligate endosymbiont taxa. As more holosymbiont genomes become available, metabolic reconstruction of obligate intracellular symbionts must consider a complex evolutionary patchwork that comprises host metabolic complementarity, symbiont replacement, horizontal transfer of complementary biosynthesis genes to the host genome and now also protein multifunctionality in endosymbionts, all of which contribute to the maintenance of endosymbiont metabolic function.A. pisum, lineage LSR1 [Vicia faba cv. The Sutton. All aphids were kept at 20°C in a long day regime of 16 hours of light and 8 hours of dark.Parthenogenetic females of the pea aphid age LSR1 , 5A and age LSR1  were maiE. coli K-12 MG1655 (eco00770) and B. aphidicola from host aphids, including: A. pisum  biosynthesis were analyzed using the KEGG pathway database ,35. Thesbab00770 ), C. cedbcc00770 ), M. perhnera Mp ), S. grabas00770 ) and U. bua00770 ). The coapi00410 ).A. pisum and Buchnera pantothenate biosynthesis gene expression in whole adult female aphids and isolated bacteriocytes using 2CT-ΔΔ methodology [A. pisum RefSeq gene models  and Buchnera gene expression was normalized to atpE ATP synthase F0F1 subunit C . The normalized expression value for each gene was compiled into a heat map /standard deviation). Yellow: z >0, blue: z <0, black: z = 0.Real-time quantitative PCR (QPCR) was used to compare hodology . Methodshodology . Primershodology , and speBuchnera ilvC (BU599) and panC (BU196) coding sequences were amplified from Buchnera LSR1 gDNA, and full-length E. coli ilvC (EG10495), panC (EG11746) and panE (EG13271) coding sequences were amplified from E. coli K-12 MG1655 gDNA; all coding sequences were amplified using Phusion high-fidelity DNA polymerase . Primers were designed to incorporate restriction enzyme sites to allow directional cloning of ilvC, panC and panE coding sequences into pUC19, in frame and downstream of the lacZ start codon; thus, generating pUC19 expression constructs containing ilvC, panC and panE coding sequences under the control of the lacZ promoter . Reaction products were analyzed on 3130xl genetic analyzer (Life Technologies), and sequence data assembled into contiguous sequences using Sequencher v4.9 .Full-length d panC BU96 codingd panC BU96 codingE. coli ilvC/panE double mutant FE8 62, lacY1, tsx-29, glnV44(AS), galK2(Oc), λ-, Rac-0, hisG4(Oc), rfbC1, xylA5, mtl-1, argE3(Oc), thiE1 [E.coli Genetic Stock Center .Functional complement experiments were carried out using the E::tetA; ), obtain), thiE1 ,52), obtE. coli FE8 (ilvC- and panE-) cells were transformed with plasmids pBUC-ilvC, pECO-ilvC, pECO-panE and pUC19 empty vector (negative control) according to [OD600 = 1.0. Functional complementation of E. coli FE8 pantothenate auxotrophs was carried out by plating aliquots (5 μl) of cell suspensions on M63 minimal media agar plates [2PO4 (pH 7.0), 2 g/L (NH4)2SO4, 0.4% (w/v) glycerol, 0.25 g/L MgSO4.7H2O, 50 mg/ml L-isoleucine, 50 mg/ml 3-methyl-2-oxobutanoic acid , 0.5 mg/L FeSO4.7H2O, 0.5 mg/L thiamine, 1.5% (w/v) agar, 50 μg/ml ampicillin, 15 μg/ml gentamicin and 10 μg/ml tetracycline. Aliquots of cell suspensions were also plated on M63 minimal media agar plates supplemented with 50 μg/ml D-pantothenic acid (non-selective) to assess cell viability. E. coli FE8 cell growth on selective plates (without vitamin B5) and non-selective plates (with vitamin B5) was assessed after four days of growth at 26°C.rding to . Transfor plates  containiE. coli AT1371 (panC-) cells were transformed with plasmids pBUC-panC, pECO-panC (positive control) and pUC19 empty vector (negative control) according to [2PO4 (pH 7.0), 2 g/L (NH4)2SO4, 0.4 % (w/v) glycerol, 0.25 g/L MgSO4.7H2O, 0.5 mg/LFeSO4.7H2O, 0.5 mg/L thiamine, 1.5% (w/v) agar and 100 μg/ml ampicillin. Aliquots of cell suspensions were also plated on M63 minimal media agar plates supplemented with 50 μg/ml D-pantothenic acid (non-selective) to assess cell viability. E. coli FE8 cell growth on selective plates (without pantothenate) and non-selective plates (with pantothenate) was assessed after four days of growth at 26°C.rding to . Transfording to  containi


Here, we here present scanning electron microscope data for the existent telocytes (TCs) on the endothelial surface of the wall of pig coronary arteries, internal thoracic arteries and carotid arteries. These cells have a small (8.39 ± 1.97 μm/4.95 ± 0.91 μm) cell body of different shapes  with very long (of about 30 μm) and thin cellular processes called telopodes (Tps), which have uneven calibre. The number of Tps ranges between 2 and 6. Tps typically present the alternation of podoms and podomers, and also have a dichotomic branching pattern. These data could influence the current attempts for elucidating the role(s) of TCs. Telocyte (TC) is a novel type of interstitial cell recently described  in stromThe study was approved by the Institutional Ethics Board of Fudan University, according to the generally accepted international standards. Anaesthetized four pigs were fixed and the thoracic cavity was opened, through a puncture in the left ventricle; 3500 ml of heparin physiological saline and 1000 ml 4% paraformaldehyde solution were perfused at physiological pressure respectively. After the perfusion, different arterial segments of large and middle-sized arteries were removed and prepared for scanning electron microscopy according to routine treatment. Endothelial surface of various arteries was examined and the images were captured by using Philips XL30E SEM.Under SEM, Figure etc.). We showed the presence of a new population of cells (TCs) on the pig endothelial surface with the same ultrastructural features as TCs, previously described by Popescu's group 6], skele's group . We foun


In the past, it was thought that hearing loss patients with residual low-frequency hearing would not be good candidates for cochlear implantation since insertion was expected to induce inner ear trauma. Recent advances in electrode design and surgical techniques have made the preservation of residual low-frequency hearing achievable and desirable. The importance of preserving residual low-frequency hearing cannot be underestimated in light of the added benefit of hearing in noisy atmospheres and in music quality. The concept of electrical and acoustic stimulation involves electrically stimulating the nonfunctional, high-frequency region of the cochlea with a cochlear implant and applying a hearing aid in the low-frequency range. The principle of preserving low-frequency hearing by a “soft surgery” cochlear implantation could also be useful to the population of children who might profit from regenerative hair cell therapy in the future. Main aspects of low-frequency hearing preservation surgery are discussed in this review: its brief history, electrode design, principles and advantages of electric-acoustic stimulation, surgical technique, and further implications of this new treatment possibility for hearing impaired patients.  In the past three decades, cochlear implantation has evolved from an experimental procedure to represent the standard of care for deaf patients. Advances in processing strategies, implant design, and patient selection criteria have significantly improved implant users' performance. Nowadays, the current frontiers in implantation involve strategies to preserve residual acoustic hearing and the development of algorithms to combine electrical and acoustic hearing. Moreover, it is important to keep in mind that, by preserving apical organ of Corti structures, it is possible to take advantage of new technologies that may lead to regeneration of the inner ear in the future .Cochlear implantation with a standard-length electrode has been a reality in treating patients who have profound deafness. However, the loss of residual acoustic hearing following cochlear implantation is an important clinical consideration when determining the most appropriate options for patients with severe hearing losses . There aHigh frequencies report information about vocal vibration (like the ability to distinguish between “s” and “z”), whereas lower frequencies apprise information regarding the vocal formants and spectral patterns (such as the difference between “b” and “g”) . PatientThe loss of low-frequency hearing during cochlear implantation is the result of the technique used to create the cochleostomy and its size combined with the characteristics of the electrode design  since it may induce substantial damage to the basilar membrane and cochlear hair cells as it advances into the scala tympani , 2.It is known that cochlear implantation with a standard-length electrode and standard surgical technique in patients with some residual hearing results in complete loss of the remaining acoustic hearing , 7. NeveThe idea of acoustic plus electric hearing means a cochlear implant aided by an ipsilateral hearing aid, to benefit from the residual low-frequency hearing of an individual. This idea of electric-acoustic stimulation (EAS) first emerged from the work of two independent groups, one from Iowa, USA, and the other from Frankfurt, Germany. In 1995, the University of Iowa Cochlear Implant research team along with the Cochlear Corporation  developed a shortened electrode array called the Hybrid S. It was designed to be inserted only into the lower basal turn of the cochlea and thus stimulate the missing areas of high frequency for those specific patients with preserved low-frequency thresholds . The HybIn 2003, Gantz and Turner  reportedAnother short electrode named Hybrid L24 has been developed in conjunction with the Cochlear Corporation. It contains 22 electrodes that are 16 mm long and its optimal insertion is 250° of the basal turn of the cochlea. It would still preserve the residual hearing from the apical portions of the cochlea and if the low-frequency hearing is lost, it can be used as a traditional electric device since it has 22 electrodes, similar to a standard one. The FDA trial for the Hybrid L has not been published yet, but the preliminary results from the European clinical trial  demonstrEAS electrode can be used for both round window insertion and cochleostomy techniques [EAS involves mixed cohorts of patients. Gstoettner et al. in 2008 [The Med-El Corporation has also developed a shortened electrode called M, which is 22 mm long with an ideal insertion of 360° from the basal turn of the cochlea. It has a very flexible tip and a significantly reduced diameter in the distal portion. This Flexchniques . Much of in 2008  reportedAn alternative strategy for preserving the low-frequency hearing was developed with standard-length electrodes, but limiting the depth of insertion. Kiefer et al.  implanteThere is controversy in the literature about the preferred electrode length. There is a higher rate of reduced thresholds and anacusis with the long electrodes. Some authors defend this reason for its use, so if residual hearing is lost, the patient can have the full-length electrode to benefit from its electric-only listening mode, which would not happen with a short electrode. Since the short electrode is 10 mm long, it accesses only the 2800 to 4700 Hz range according to the Greenwood frequency placement map of the basilar membrane. Even though this was thought to be a tonotopic mismatch, which could impair discrimination, Hybrid S electrode users in electric-only mode showed similar performance as long-electrode users on consonant recognition tasks . ImproveOne argument in favor of long electrode used to be the likelihood of progressive low-frequency hearing loss. However, Yao et al.  demonstrAnother concern regarding Hybrid or EAS studies is the progression of hearing loss after activation. At the time of implantation, very few patients lose all their residual low-frequency hearing. In the phase 1 Hybrid S10 trial , 2% of tSteroids have been shown to reduce noise-induced cochlear damage and hearing loss and to increase recovery after noise trauma. However, their efficacy has been controversially discussed due to a lack of adequate clinical trials . Also, tIn the initial studies, the indication was closer to standard cochlear implantation for thresholds above 65 dB in the low frequencies between 125 and 500 Hz . After eActual guidelines determine that pure tone audiometry scores for both ears have to be greater than 60 dB between 125 and 500 Hz and below 70 dB at 1500. In addition, monosyllables tested at a 10 dB signal-to-noise ratio (SNR) should not exceed a score of 40% in the best aided condition [mechanical damage during electrode insertion ,shock waves in the perilymph fluid due to implantation,acoustic trauma due to drilling,loss of perilymph and disruption of inner ear fluid homeostasis,potential bacterial infection,secondary intracochlear fibrous tissue formation.Wright and Roland in 2005  first deThe technique used at the University of Iowa for implantation of the cochlear Hybrid S and L devices is described in this review.A standard mastoidectomy is performed through a postauricular incision. A portion of the superior margin of the mastoid cortex is left in place and a suture is passed through the cortex to anchor the electrode before placing it in the cochlea. The objective of this suture is to reduce the spring of the electrode and to prevent movement of the electrode during placement in the scala tympani (ST). A bonny well for the internal processor is also created in the same manner used for a standard cochlear receiver/stimulator. A ridge of bone between the mastoid cavity and the well is left so the implant does not later slide. A subperiosteal pocket is created deeply to the temporalis muscle and pericranium. The facial recess is opened widely, and the round window niche is totally exposed by removing the bone overlying and anterior to the facial nerve. With a 1 mm diamond burr, the bony overhang of the round window niche is drilled away to expose the entire round window membrane.Before creating the cochleostomy, the surgical field must be extensively irrigated and meticulous hemostasis must be done in order to prevent bone chips and blood debris from entering into the cochlea upon opening. The cochleostomy is created in the inferior-posterior quadrant of a box created by drawing a line at the superior margin of the round window and a perpendicular line at the inferior margin of the round window . EnterinThe electrode and processor must be seated in position before opening the endosteum of the cochleostomy. Two holes are drilled into the tegmen tympani, which allow a 2-0 or 4-0 nylon suture to be passed through the bone in order to secure the Hybrid S and L electrode array (not the ground electrode). Once the processor and electrode are seated and secured, a temporalis fascia “washer” with a 1.5 mm × 1.5 mm diameter is harvested and flattened in a fascial tissue press. It is then punctured with a straight needle and the electrode is inserted through the fascia up to the Dacron collar in order to seal the scala tympani at the cochleostomy. Opening the endosteum into the scala tympani with a 0.2 mm footplate hook must be the final act of the procedure to reduce the time the cochlea is open. The electrode is advanced slowly into the scala tympani (over 1 to 2 minutes) in order to minimize insertional trauma and to allow perilymph displacement.There are some intraoperative tests described in order to further improve the safety of hearing preservation surgery, such as the measure of cochlear microphonics  and audiThe middle ear is not packed with muscle or fascia. The periosteum should then be closed completely over the receiver/stimulator and the electrode. Soft tissues are then closed in the standard way.Helbig et al.  recentlyCurrent literature has already reported several discrete advantages to both modalities, but the full benefits of an EAS of the ipsilateral cochlea are still under investigation.Patients with preserved low-frequency hearing show significant improvements in their discrimination scores. Hybrid S electrode users continue to improve on CNC scores over 1 to 2 years after activation. In the Hybrid S trial , 48% of EAS users had preoperatively open-set sentence recognition of 24% and after 12 months of use, scores averaged 71%. Monosyllable recognition also improved from 16% to 44% on average.Exceptional improvement in speech understanding is possible for patients in the combined mode with all electrodes activated: some patients score more than 90% on the CNC monosyllabic word test. FlexImprovements in discrimination tasks have also been observed in long-electrode users with preserved acoustic hearing. Patients who received the Med-El Combi40+ with 19 to 24 mm of insertion scored 75% on monosyllabic tests after 1 year, an important improvement from the 9% preoperative scores.Distinguishing the correct words in a background of competing talkers is a challenging test for traditional cochlear implant users. Normal-hearing listeners have a signal-to-noise ratio (SNR) of −30 dB and −15 dB for competing talkers . The aveHybrid S users do better than traditional cochlear implant patients, but not as well as normal-hearing listeners in noisy backgrounds. In a subgroup of 27 Hybrid S patients with at least 12 months of activation, SNRs ranged from −12 to +17 dB (average −9 dB) . PatientEAS electrode changed their preoperative open-set sentence scores with a SNR of +10 dB from 14% to 60% after 1 year of activation [Hybrid L electrode patients also improved their SNR preoperatively from 12.1 dB to 2.1 dB postoperatively . Those wtivation .Long-electrode users with preserved hearing in low frequencies also benefit when listening in noise. Med-El Combi40+ users with a low-frequency threshold of <80 dB scored better than patients with a cochlear implant and no residual hearing with a SNR of +5 dB . GstoettIt is known that traditional cochlear implant users have difficulties identifying and enjoying music because of the extremely complex encoded spectral information. They can usually distinguish lyrics to a certain degree but have significant trouble with pitch, timbre, and melody recognition , 36.Music appreciation has been a part of the research protocol for the Hybrid S/L trials. Patients with preserved acoustic hearing have a distinct advantage over traditional cochlear implant users regarding pitch, lyrics, melody, and timbre of the instruments. When Hybrid users were provided with excerpts of easily recognizable American songs, they were able to correctly identify the songs 65%–100% of the time, similar to normal-hearing listeners. In tasks regarding pitch recognition and melody without lyrics, the Hybrid users still scored better than the traditional cochlear implant patients, but not as well as normal-hearing listeners did , 37.Brockmeier et al.  studied Apical cochlear preservation may become a significant issue in the future. It is possible that treatments will emerge that require naïve cochlear tissue . This concern is relevant for families weighing the risks and benefits of bilateral cochlear implantation for deaf children. The Iowa CI research team has recently reported a series of 9 children with bilateral implants: one ear with a standard long electrode and the other with Hybrid S12 research electrode . The chiAlthough hearing preservation in cochlear implantation is technically challenging, the importance of preserving residual low-frequency hearing cannot be underestimated in light of the added benefit of hearing in noisy atmospheres and music quality for EAS users. Electrode designs and processor technology are constantly being improved. Nowadays, patients undergoing hearing preservation surgery can expect a long-term low-frequency hearing preservation rate of 50–70%. Although surgeons have largely modified the initially described “soft surgery” technique over the years, there is not a unique protocol to be followed.Few would have imagined the progress that has been made in the past 30 years regarding aiding deafened patients, and the future promises to be equally exciting. The potential EAS advantages highlight the value of the endeavors to preserve low-frequency hearing in the implanted ear and to continue amplification where appropriate. Further research is also needed to maximize outcomes for recipients with different degrees of hearing loss who use devices that combine electric and acoustic stimulation.


The present study examined differences in modality use during episodes of joint attention between hearing parent-hearing child dyads and hearing parent-deaf child dyads. Hearing children were age-matched to deaf children. Dyads were video recorded in a free play session with analyses focused on uni- and multimodality use during joint attention episodes. Results revealed that adults in hearing parent-deaf child dyads spent a significantly greater proportion of time interacting with their children using multiple communicative modalities than adults in hearing parent-hearing child dyads, who tended to use the auditory modality  most often. While these findings demonstrate that hearing parents accommodate their children’s hearing status, we observed greater overall time spent in joint attention in hearing parent-hearing child dyads than hearing parent-deaf child dyads. Our results point to important avenues for future research on how parents can better accommodate their child’s hearing status through the use of multimodal communication strategies. Imagine a world in which the way that people communicate is inherently different from how you communicate: You use visual information and they insist on using auditory information. The result is confusion and miscommunication. For many children who are born deaf, this is the reality they initially face. This is because 90 percent of deaf children are born to hearing parents , meaningLanguage development is often delayed in deaf children of hearing parents  because This mismatch may pose difficulties for parents as well. Hearing parents of deaf children may experience stress specifically with regard to their child’s deafness , and thoResearch that is informative on the issue of communicative accommodation involves use of the Still Face Paradigm, in which a mother is instructed to maintain a neutral, unemotive face at prescribed intervals during normal interaction with her infant . AlthougAnother way to examine whether and how parents accommodate their children communicatively is through their efforts to establish joint attention. Joint attention is the ability to focus simultaneously on an object or event and another person, sometimes described as “shared intentionality” . Joint aWhile the comparison of hearing parent-deaf child dyads to hearing parent-hearing child dyads is helpful, it is just as important to compare modality-matched dyads . A hearing parent’s use of the auditory modality with a deaf child highlights one of the primary difficulties in hearing parent-deaf child communication. Hearing parents rely on oral language in the rest of their lives but cannot use it to communicate with their children effectively. While this may seem obvious, the instinctive use of oral communication by hearing parents affects important basic interactions, such as when parents direct their children’s attention to objects and events in their surroundings. In a study on children’s visual perception of Manual Coded English (MCE), a communication method which involves a hearing mother speaking while signing to her deaf child, mothers who used more deaf-friendly means of communication had children who saw more complete versions of the mothers’ signed utterances . For exaClearly, which modalities are used in communication between hearing parents and deaf children is a topic that merits further research. While previous research e.g.,  has examn = 4 females) aged 18.2–36.7 months  and their hearing parents , participated in the study. While all children were candidates for cochlear implantation, none of them had received an implant; the children were being instructed predominantly using the oral method. None of the children produced any spoken or signed language during videotaping in our sample. Each child was receiving at least 1 h per week of speech therapy, as well as some basic instruction in ASL. In addition, four hearing children  ages 18.3–36.7 months  and their hearing parents  took part in the study. Participants were aged-matched, and were from the Southwestern and Northeastern United States. Each was recruited via the National Institute of Health website or local recruitment. The sample was primarily Caucasian , and all but one parent had completed at least high school. This study was carried out in accordance with the recommendations of the University of Connecticut Institutional Review Board and the Stanford University School of Medicine Institutional Review Board with written informed consent from all subjects. For participants who were young children, parents provided written informed consent. All subjects gave written informed consent in accordance with the Declaration of Helsinki.Four severely to profoundly deaf children . Videos of hearing parent-deaf child dyads were then transmitted from collaborators at Stanford University to researchers at University of Connecticut using Research Electronic Data Capture (REDCap) electronic data capture tools hosted at Stanford University  were used during a free-play session between the child and his/her primary caregiver, which occurred as part of a visit with a speech language pathologist (deaf children) or to the Husky Pup Language Lab at the University of Connecticut (hearing children). The speech language pathologist or experimenter instructed the caregiver to play with the child as she would at home; play sessions were video recorded for approximately five minutes (iversity . REDCap http://tla.mpi.nl/tools/tla-tools/elan/), and is available free of charge. We use coding criteria for joint attention based on the work of The videos were coded for joint attention using ELAN , languagVideos were reviewed for visual clarity and Adobe Premiere Pro (CS6) was used to cut the video to the start and end time of the play session. The start time of the play session was at the first frame in which the testing room’s door was closed, leaving the child and parent alone. The end of the play session was at the first frame in which the experimenter opened the door to end the play session. These two values were subtracted to give a baseline length of time for the play session. Next, intervals in which the video was uncodeable were marked. An uncodeable interval was defined as an interval of at least 5 s in which at least one participant’s face was not visible. The amount of uncodeable time was subtracted from the baseline length of time to yield a total length of play session for each participant.during its use within a joint attention episode. Most instances of use of the auditory modality with deaf children were brief and the hearing parent would proceed to a different modality of engagement.Very few instances of child-initiated joint attention were observed; thus, this construct was not included for analysis in the paper. Moreover, in the present study, only successful bids for joint attention  were coded and quantified. A successful joint attention episode involved the adult making a bid for the child’s attention using pointing, gaze switching between the object and the child, tapping or touching the child, deliberate waving in the child’s visual field, changing affect, and/or language; this bid was then responded to by the child using pointing, gaze switching between the object and the parent, tapping or touching the parent, grasping the object of interest, deliberate waving in the parent’s visual field, changing affect, and/or language. This type of episode could also occur if a parent shifted the child’s attention from one object to another using the previously mentioned techniques. Any indication of the auditory modality being used is To record joint attention in ELAN, a 5 s “rule of engagement” was followed . Similarly, there was a 5 s rule of disengagement, i.e., a joint attention episode was deemed to be terminated after neither participant engaged in joint attention behavior for 5 s. If either participant re-engaged within the 5-s window, the length of the episode was extended; the episode ended at the start of the first period of 5 s that displayed no joint attention behaviors.All successful, adult-initiated joint attention episodes were then coded separately for both the parent’s and the child’s uses of the following modalities: auditory, visual, tactile, auditory-visual, auditory-tactile, visual-tactile, and auditory-visual tactile. The criteria are as follows. One episode could have multiple modalities used within it, as specified by the following categories:Behaviors in the auditory modality involved using sound to gain the attention of the other member of the dyad. These included language, humming, other vocal sounds , hitting an object to make noise, clapping (if the other member of the dyad was unable to see the clap), and causing a toy to produce noise  This modality was coded for when there was no possible way for the other dyad member to have received visual input with the auditory input.The visual modality included behaviors that somehow incorporated the visual field in getting the other member’s attention. These included waving, gesturing, pointing, making eye contact, holding an object directly in the other member’s visual field, causing a toy to light up (but not produce sound), demonstrating play with toys, offering a toy to the other partner (without using any of the behaviors described in the auditory section), making faces, and changing affect. As no ASL was produced in any of the dyads, it was subsequently excluded from the coding criteria.The tactile modality involved using touch, either direct or indirect. Examples included tapping/touching the other person, tickling, hugging, holding, grabbing on to the other person’s clothing, tapping the ground to create vibrations, and touching the other person with a toy .This multimodal classification involved criteria for both the auditory and visual modalities occurring simultaneously. Examples included gesturing while talking, presenting a toy while describing it, reacting to a visual event , and demonstrating affect while producing any sort of sound.This multimodal classification included criteria for both the auditory and tactile modalities. This included running a toy over the other partner while making appropriate noises , holding/grasping hands while signing , and touching the other person with a toy that made noise.This multimodal classification included criteria for visual and tactile modalities. It included behaviors such as taking a toy and making it “hop” up the other person’s arm (without making noise), making eye contact with the other person while also touching them, grabbing the other person’s arm while pointing, and touching the other person with a toy within their visual field while not producing any auditory output.This multimodal classification included simultaneously occurring behaviors encompassed by the criteria for the auditory, visual, and tactile modalities. It included holding a child while pointing and talking to them, making eye contact while singing and touching the other person, and both people playing a clapping game that involves auditory output of some sort while making eye contact.To record modality use in ELAN, the start of the production of a modality was coded in real time . However, there was a two second rule of discontinuing the modality, i.e., a participant could pause in production of the modality for up to 2 s and have the subsequent production be part of the same episode. Modality episodes were deemed to be terminated after neither participant engaged in any of the modality criteria behaviors for over 2 s, with the end time of the episode being the end time of the last modality production. Abrupt changes in modality type  were coded in real time, with no rule of engagement or disengagement.n = 3) for these measures was calculated at >90% agreement.Data were extracted from individual videos using the “View Annotation Statistics” function in ELAN. Total times were extracted for length of time spent in joint attention. In addition, modality times were extracted, after having been coded as a controlled vocabulary in ELAN (and a dependent tier of joint attention). These data were then analyzed as described in Section “Analyses.” Inter-observer reliability (Table 1 for proportions and lengths of time spent in joint attention for each dyad). With regards to modality, seven modality metrics were computed for both parents and children. This was done by extracting the total amount of time spent in each of the seven modalities, and dividing each in turn by the total amount of time spent in joint attention in the free play session. Mann–Whitney U analyses were conducted not only to compare joint attention behavior between the hearing parent-hearing child and hearing parent-deaf child groups, but also to compare modality use by both parents and children in the two dyad types.In order to account for differences in the lengths of free play sessions, the metric of proportion of total session length spent in joint attention was computed. To compute this metric, the total amount of time spent in this episode type was extracted from ELAN for each participant. These times were divided by the total session length (excluding uncodeable time) for each participant, i.e., total time spent in adult-initiated, successful bids for joint attention was divided by total session length .We then evaluated modality use by adults across dyad types during periods of joint attention. Because no instances of tactile-only or auditory-tactile modality combinations were produced by adults in either dyad type, these modalities were excluded from further analysis. First, a comparison of the differences in proportion of time spent in the auditory modality reveals that adult in hearing parent-hearing child dyads spent significantly more time in the auditory modality than adults in hearing parent-deaf child dyads, U = 4, p < 0.05, such that adults in hearing parent-deaf child dyads spent a greater proportion of time using this combination than hearing parents of hearing children. In contrast, analysis of the visual-tactile modality demonstrated that adults in hearing parent-hearing child dyads spent a significantly greater proportion of time using this combination than adults in hearing parent-deaf child dyads, U = 9, p < 0.05. Finally, the only case in which adults used three modalities simultaneously involved auditory-visual-tactile communication. In this case, adults in hearing parent-deaf child dyads spent significantly more time in the auditory-visual-tactile modality than adults in hearing parent-hearing child dyads, U = 1, p < 0.05.What about instances in which two modalities were used during joint attention episodes? A comparison of the proportion of time adults in the two dyad types spent communicating in the auditory-visual modality revealed a significant difference, U = 14, p < 0.05. Likewise, deaf children spent a significantly higher proportion of time using the visual modality than hearing children, U = 2, p < 0.05. These differences in unimodal communication channel make sense and, we would argue, validate our measurement system .We now turn to analyses of children’s use of different modalities during joint attention. No instances of the tactile modality were observed, so this was excluded from further analysis. Beginning with the auditory modality, results demonstrated—not surprisingly—that children in hearing parent-hearing child dyads  spent a significantly greater proportion of time using the auditory modality than children in hearing parent-deaf child dyads , U = 13.5, p < 0.05. A comparison of use of the auditory-tactile combination of modalities revealed that hearing children spent significantly more time using it than deaf children, U = 10, p < 0.05, as was the case for the visual-tactile combination as well, U = 13, p < 0.05. Finally, we observed that deaf children spent significantly less time using the auditory-visual-tactile combination than hearing children, U = 10.5, p < 0.05.Turning to multimodal comparisons, analyses revealed that hearing children of hearing parents spent a greater proportion of time in the auditory-visual modality than deaf children of hearing parents, Our results highlight interesting differences in both unimodal and multimodal communication used during episodes of joint attention by parents and children in hearing–hearing and hearing-deaf dyads. Some of the results make sense; others are more surprising and, perhaps, concerning. At the very least, these data demonstrate the variability in accommodation made by parents across different parent–child dyads.First, we found that hearing parents of hearing children spent a significantly greater proportion of time communicating with their children in both the auditory-only modality and the visual-only modality than hearing parents of deaf children. In other words, hearing parents of hearing children used more unimodal communication during joint attention episodes than hearing parents of deaf children. Of course, the shared use of oral language in hearing parent-hearing child dyads produced a richer body of linguistic interactions overall and, because rich linguistic interactions beget rich attentional interactions, joint attention is no doubt easier for these dyads to establish. The lack of complex, language-based interactions between hearing parents and their deaf children could explain some of the discrepancies in modality use between the two dyad types.Second, in contrast to previous research showing that hearing parents tend to use the auditory modality most often when trying to engage their deaf children , our finin joint attention relative to hearing parent-deaf child dyads. Of course, the overall amounts of joint attention were small and so we do not wish to make too much of this difference. However, while the present study extends the body of research on this topic by further detailing modality use between the two dyad types, it raises several questions about the nature of communication between hearing parents and their deaf children that merit further investigation. Thus, the preliminary findings of the present study should serve to motivate future research on this issue.With regard to children’s use of uni- and multimodal communication, we observed that deaf children spent a greater proportion of time than hearing children using only the visual modality. While this is not surprising given that the visual modality is accessible to a deaf child while the auditory is not, this raises the question of whether children are aware that their parents communicate differently than they do. Another item of note is that hearing children produced the only instances of the auditory-tactile combinations that we observed. When considering the parent and the child data, the overall pattern suggests that hearing parent-hearing child dyads were communicating more in general, an interpretation that is consistent with our finding that hearing parent-hearing child dyads spent a greater proportion of time There are several additional factors that necessarily constrain interpretation of our results. First, it is important to note that the sample size is quite small. More observations are needed from more dyads of both types. Another shortcoming is that, although the deaf and hearing children were age-matched, the children are quite varied in age across the dyads. Free play with an 18-month-old is quite different from that with a 36-month-old. Thus, this variability undoubtedly influenced the findings of the present study. Moreover, the hearing parent-deaf child dyad with the oldest child produced the least he amount of joint attention. Why? We can only speculate that this older child found the new toys provided in the study of great interest and willfully chose to focus on the toys rather than the parent. An examination of how parent–child interaction changes over time and relates across time would help clarify some of these questions, as well as facilitate more sophisticated and detailed understanding of the dynamics of age and interaction.how communication is facilitated in joint attention in the two types of dyads, but what is going on during these different types of engagement and how it affects the children’s subsequent development.Nonetheless, while the present study has raised more questions than it has answered with regard to modality use in joint attention between parents and their children, it demonstrates that detailed coding of modality use in parent–child communication can provide important insights into how parents accommodate their children’s particular communicative needs, whether they are hearing or deaf. This should motivate additional research of this type. Future studies will be needed to address not only The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Desmodus rotundus and four other group-living bats, Artibeus jamaicensis, Carollia perspicillata, Eidolon helvum and Rousettus aegyptiacus, under the same captive conditions of fixed association and no ectoparasites. We conducted 13 focal sampling sessions for each combination of sex and species, for a total of 1560 presence/absence observations per species. We observed evidence for social grooming in all species, but social grooming rates were on average 14 times higher in vampire bats than in other species. Self-grooming rates did not differ. Vampire bats spent 3.7% of their awake time social grooming (95% CI = 1.5–6.3%), whereas bats of the other species spent 0.1–0.5% of their awake time social grooming. Together with past data, this result supports the hypothesis that the elevated social grooming rate in the vampire bat is an adaptive trait, linked to their social bonding and unique regurgitated food sharing behavior.Evidence for long-term cooperative relationships comes from several social birds and mammals. Vampire bats demonstrate cooperative social bonds, and like primates, they maintain these bonds through social grooming. It is unclear, however, to what extent vampires are special among bats in this regard. We compared social grooming rates of common vampire bats  Long-term cooperative relationships are most evident in primates –6, but eDesmodus rotundus) [Myotis bechsteinii). These two species both have long lifespans and demonstrate fission-fusion social dynamics, where individuals maintain long-term social associations while moving between several roost trees [In bats, adult social grooming is female-biased in species with female philopatry –58, and otundus) , 59–60. otundus)  comparedst trees –63. In bst trees , 57. Becst trees .Patterns of social grooming among categories of individuals also differed between the two species. In the Bechstein’s bat, adult female social grooming was not detectably symmetrical, and was predicted by kinship, occurring mostly between adult mothers and daughters, sometimes between sisters, and only rarely between non-kin . In vampIt is not entirely clear if vampire bat social grooming is typical or exceptional when compared to other bats or non-primate mammals. One hypothesis is that social grooming in vampire bats is exceptional in quantity and quality, because it is related to their uniquely cooperative food sharing behavior . Like math and 6th place out of 53 neotropical bats for average number of parasitic streblid flies per bat [Alternatively, the apparent distinctiveness of vampire bat social grooming might stem from purely ecological factors. Social grooming may be more obvious in vampire bats due to higher levels of ectoparasite infestation. Bat fly density has been linked to species-level grooming rates  and the  per bat . A sampl per bat  and a laDesmodus rotundus, two frugivorous bats Carollia perspicallata and Artibeus jamaicensis (Family: Phyllostomidae) and two Paleotropical fruit bats, Rousettus aegyptiacus and Eidolon helvum (Family: Pteropodidae). Importantly, the adult bats we compared have fixed levels of social association (stable group composition) and no insect ectoparasites.Comparing social grooming data across studies can be difficult due to study differences in ectoparasite density, temperature, sampling method, visibility, and level of human disturbance. In this study, we took advantage of an opportunity to compare captive vampire bats with four other captive group-living bat species housed at the same facility under the same light/dark schedule, temperature, humidity, and levels of human disturbance. We compared adult-to-adult social grooming in vampire bats Artibeus, Carollia, Eidolon, and Rousettus were housed together (4.5 x 3 x 2 m), while females and a few castrated males (see below) of these species were housed together in a different cage (same dimensions). All Desmodus were housed together (3 x 1.5 x 2 m), but sex could still be assigned with certainty because sexes tended to segregate  and bats were individually marked. In all cages, bats were free to hang at any place in the cage. In all species, we only observed interactions between adult bats, and only one dependent offspring was present (in Desmodus). Work was approved by the University of Maryland Institutional Animal Care and Use Committee (Protocol R-13-30).All bats were cared for by the Organization for Bat Conservation , and housed at 25–28 degrees Celsius with >33% humidity on a half-reversed 12 h light/dim light cycle in flight cages that allowed free association among cagemates. Male Artibeus, Desmodus, and Carollia, we obtained 13 samples from both males and females  that attempts to sample a female bat actually sampled a castrated male, because 6 female Eidolon and 9 female Rousettus were housed with two castrated male Eidolon and two castrated male Rousettus. In To compare social grooming and other behaviors across species, we conducted focal sampling. We took instantaneous (“on the beep”) focal samples of a randomly chosen bat. For During each of 26 focal sampling sessions, observers took one observation every 10 s for 10 min . During each observation, observers reported the presence or absence of social grooming (chewing or licking another bat’s body), self-grooming (scratching or licking its own body), feeding, and aggression. We measured self-grooming to see if social grooming differed merely due to differences in grooming, we measured feeding because feeding rates could limit and hence explain social grooming rates, and we measured aggression to see if this behavior was positively or negatively linked to social grooming. Focal sampling sessions occurred at haphazard times when bats were active. However, using logistic fit in JMP 12 we detected no effect of sample time on presence of social grooming either overall or within each species. Because observers also switched to a different species and sex after each observation, there was no reason to expect biased sampling of sexes or species.Desmodus as the control species. This method computes ranks on all observations then compares Desmodus to all other species in a pairwise manner, and provides p-values with a Bonferroni adjustment. We repeated this analysis for self-grooming. To test for an effect of sex on social grooming rates, we compared social grooming in males and ‘no testes’ bats across all species using a Wilcoxon test. To compute means and 95% confidence intervals of social grooming rates, we used bootstrapping with 1000 permutations in R [To calculate percent time spent social grooming, self grooming, fighting, or feeding, we divided the number of observations of these behaviors by 60 (the number of samples per session). We compared social grooming rates across both species and sexes. To test if vampire bats performed social grooming more than other species, we conducted nonparametric comparisons using the Dunn Method for joint ranking in JMP 12 with ons in R .To check for potential inter-observer bias, we ran a permuted linear model in R (lmPerm package) testing for effects of both ‘species’ and ‘observer’ on social grooming rates. We also repeated this procedure with ‘feeding rates’ and ‘aggression’ instead of ‘observer’ to see if observed differences in these factors could help explain social grooming rates after accounting for species differences.Desmodus were higher than for the other species: Artibeus , Carollia , Eidolon , and Rousettus , Carollia , Eidolon , and Rousettus  is elevated beyond the rates found in four other group-living bat species (Under similar conditions of captivity and in the absence of external parasites, social grooming in vampire bats ( species . Vampire species . Vampire species , and is Desmodus rotundus forms stable female social groups of 8–12 adults with offspring, living in a roost site that is defended by a single dominant male against invasions by other males, i.e. resource defense polygyny [Carollia perspicillata female groups vary in size from 2–18 bats, and are based around resource defense polygyny with males performing courtship displays and defending territories [Artibeus jamaicensis also demonstrates resource defense polygyny, and both males and females appear to form cooperative relationships in the wild [Rousettus aegyptiacus and Eidolon helvum in the wild, but both species form aggregations of hundreds to thousands of both male and female individuals [Rousettus pups converge on adult repertoire of calls through vocal learning [Are social grooming differences explained by general differences in social structure? The bat species in this study are all group-living in the wild, but vary in their social structures and degree of sociality. polygyny , 64. Indpolygyny , and maypolygyny , which apolygyny –75. Caroritories , 77. Vocritories  and betwritories . In contritories . Artibeuthe wild , 80–82. the wild . These mthe wild . Unlike ividuals . These alearning . Both spVampire bats are the only bats that perform regurgitated food sharing , and theWilkinson  suggeste


Dictyostelium discoideum, unicellular amoebae aggregate when starved and form multicellular fruiting bodies in which stress-resistant spores are held aloft by dead stalk cells. Fruiting bodies are thought to be adaptations for dispersing spores to new feeding sites, but this has not been directly tested. Here we experimentally test whether fruiting bodies increase the rate at which spores are acquired by passing invertebrates.Many microbial phenotypes are the product of cooperative interactions among cells, but their putative fitness benefits are often not well understood. In the cellular slime mold Drosophila melanogaster accumulate spores on their surfaces more quickly when exposed to intact fruiting bodies than when exposed to fruiting bodies physically disrupted to dislodge spore masses from stalks. Flies also ingest and excrete spores that still express a red fluorescent protein marker.D. discoideum increase the likelihood that invertebrates acquire spores that can then be transported to new feeding sites. These results thus support the long-hypothesized dispersal benefits of altruism in a model system for microbial cooperation.Multicellular fruiting bodies created by  Microbes appear to cooperate to acquire resources, fend off competitors, survive harsh conditions, and disperse to new habitats. We say Dictyostelium discoideum[Dictyostelium aggregates migrate to soil surfaces using temperature, light, and gas cues[D. discoideum[One model system for microbial cooperation is the cellular slime mold iscoideum. These uiscoideum. This hyiscoideum. Dictyosgas cues, presumagas cues. The laciscoideum, suggestDictyostelium is thus a model system for microbial cooperation in which the benefits of cooperation are poorly understood. Here, we attempt to remedy this awkward situation by experimentally testing whether fruiting bodies increase the likelihood that spores are picked up and carried by passing invertebrates—the first step in dispersal. The primary vectors of D. discoideum dispersal are unknown, so we exposed fruiting bodies to Drosophila melanogaster as an experimentally tractable stand-in and tested whether flies acquire spores more readily from intact than from disrupted fruiting bodies.There is no direct evidence that fruiting bodies actually increase dispersal, however. Invertebrate grooming might remove spores before they get dispersed. Or perhaps spores are just as easily acquired when invertebrates step on them. The biological function of fruiting bodies might instead be to provide protection from nematodes and fungi,9. DictyD. melanogaster either to intact fruiting bodies or to fruiting bodies physically disrupted to dislodge spore masses and collapse stalks . After 7 hrs of exposure to intact fruiting bodies, ~1% of the original cell population could be recovered from flies. Flies acquired spores more readily from intact than from disrupted fruiting bodies , though they did accumulate spores in both treatments.We exposed  Figure A. DuringD. discoideum. We observed fluorescence on fly legs, wings, eyes, and mouthparts . We did not test whether excreted spores were viable, but we believe it likely. Viable Dictyostelids have been found in the guts of wild-caught pillbugs, earthworms, and ground-feeding songbirds[Drosophila guts[In addition to the spores on fly surfaces, we also observed flies ingest and excrete spores. Flies may have ingested spores by grooming after contacting fruiting bodies or by drinking from fluid droplets in spore masses [D. discoideum on a strain of Klebsiella pneumoniae bacteria (obtained from N. Buttery) with spontaneous resistance to the antimicrobial G418. We stored D. discoideum and K. pneumoniae strains at -80°C in 20% (v/v) glycerol. We obtained wild-type (Canton-S) Drosophila melanogaster from Y. Ben-Shahar (Washington University) and maintained them at 22°C in 200 ml vials containing 50 ml Formula 4–24 Instant Drosophila Medium  with constant light.Carolina. NC28.1 in (rfp). We grewK. pneumoniae lawns on 2.0% (weight/volume) agar plates of SM medium  at 22°C. We dislodged spore masses from fruiting bodies by banging plates upside down, harvested spores from the plate lid with 1.0 ml KK2 buffer , and resuspended them in KK2 to 1 × 107 cells/ml using spore counts in a hemacytometer.We tested how fruiting bodies affect dispersal by exposing flies to either intact fruiting bodies or to fruiting bodies disrupted so that most spores rested on the substrate instead of at the end of upright stalks Figure . To obtaD. discoideum fruiting bodies, we first plated 106 D. discoideum spores onto SM plates with 100 μl of stationary phase K. pneumoniae culture grown for 2 days in SM broth at 22°C without shaking. We incubated plates for 2 days at 22°C with passive humidity and overhead light. We harvested log-phase D. discoideum cells off these plates with an ethanol-sterilized plastic spatula and resuspended them in 12 ml cold KK2. We centrifuged cells for 3 min at 300 × g, washed them three times in 12 ml cold KK2, and resuspended them to 1.0 × 108 cells/ml using spore counts in a hemacytometer. We deposited 107 cells (100 μl) onto 10 ml KK2 agar (2.0% w/v) in the bottom of a 50 ml conical tube. We incubated these tubes 4 days at 22°C with ambient humidity and overhead light, during which time D. discoideum cells created fruiting bodies.To obtain We left fruiting bodies intact in control tubes. For the experimental treatment, we disrupted fruiting bodies by banging conical tubes onto a hard surface several times, causing fruiting bodies to fall over and/or spore masses to fall onto the surface of the agar. We anesthetized adult flies with FlyNap , introduced 5 males and 5 females into each tube, and let the tubes sit sideways without further disturbance. We kept conical tubes sideways so that flies would need to walk into or fly into fruiting bodies, rather than just fall onto them. To sample the flies in a tube, we anesthetized flies, collected them together into 250 μl KK2 buffer supplemented with 0.1% NP-40 detergent, vortexed the tube for ~20 seconds, and determined the density of recovered spores recovered using counts in a hemacytometer. We replicated these experiments using bacteria and amoebae independently grown from frozen stocks on different days.R v2.15.3 using the glm command. Data were overdispersed, so we used quasipoisson errors with a log link function. We accounted for variation in the number of flies and hemacytometer area counted using the offset command. Because each data point came from a separate tube (destructive sampling), we modeled time as a fixed effect. We tested the significance of model terms by performing F tests on models fit with and without the term of interest.We analyzed data by fitting generalized linear models to spore count data in rfp for 24 hr as described above, except to maximize fluorescence signal we supplemented SM media with 5 μg/ml of the aminoglycoside antibiotic G418 to select against nonfluorescent mutants that appear during cell culture. After exposure, we anaesthetized flies and incubated them 20 min at -20°C. We acquired reflected light and red fluorescent images of flies using a Zeiss SV11 stereomicroscope  and assembled composite images using Adobe Photoshop .To visualize spores on flies, we exposed 10 flies to fruiting bodies of NC28.1 The data set supporting the results of this article is available in the Dryad repository at doi:10.5061/dryad.9ht88.The authors declare that they have no competing interests.js conceived the study. All authors contributed to experimental design. js performed the experiments, analyzed the data, created the figures, and wrote the paper. DCQ and JES provided feedback on manuscript drafts. All authors read and approved the final manuscript.