EC-injured muscles showed structural/ultrastructural alterations and changes in electrophysiological sarcolemnic properties. TCs were identified in control and EC-injured muscles by either confocal immunofluorescence (i.e. CD34+CD31− TCs) or transmission electron microscopy (TEM). In EC-injured muscles, an extended interstitial network of CD34+ TCs/telopodes was detected around activated SCs displaying Pax7+ and MyoD+ nuclei. TEM revealed that TCs invaded the SC niche passing with their telopodes through a fragmented basal lamina and contacting the underlying activated SCs. TC-SC interaction after injury was confirmed in vitro by culturing single endomysial sheath-covered myofibers and sprouting TCs and SCs. EC-damaged muscle-derived TCs showed increased expression of the recognized pro-myogenic vascular endothelial growth factor-A, and SCs from the same samples exhibited increased MyoD expression and greater tendency to fuse into myotubes. Here, we provide the essential groundwork for further investigation of TC-SC interactions in the setting of skeletal muscle injury and regenerative medicine.Although telocytes (TCs) have been proposed to play a “nursing” role in resident satellite cell (SC)-mediated skeletal muscle regeneration, currently there is no evidence of TC-SC morpho-functional interaction following tissue injury. Hence, we explored the presence of TCs and their relationship with SCs in an  These peculiar interstitial cells are ultrastructurally characterized by a small nucleated cell body with a scarce cytoplasm that abruptly gives rise to a variable number of extremely long and thin processes, called telopodes, which often exhibit a sinuous trajectory and a dichotomous branching pattern. Telopodes represent the most distinctive feature of TCs and are morphologically recognizable because of their moniliform appearance conferred by the irregular alternation of slender segments, termed podomers, and small cistern-like dilations, referred to as podoms, which accommodate few organelles such as mitochondria, endoplasmic reticulum cisternae and caveolae3. The morphology of the cell body varies depending on the number of telopodes, being typically piriform for one, spindle-shaped for two, triangular for three and stellate for more processes3. Besides the above described ultrastructural traits, it appears that TCs differ from other stromal cells, such as ‘classical’ fibroblasts/fibrocytes and mesenchymal stem cells, for their immunophenotypic profile3. Although TC-specific antigenic markers have yet to be discovered, at present CD34 is considered the most reliable one for light microscopic in situ identification of TCs, which indeed are also commonly referred to as TCs/CD34+ stromal cells4. As in the stromal compartment of vascularized organs the expression of CD34 is shared by TCs and endothelial cells of blood vessels, only the simultaneous detection of CD34 and endothelial cell-specific CD31/platelet-endothelial cell adhesion molecule-1 can allow to unequivocally discriminate between CD34+CD31− TCs and CD34+CD31+ vascular endothelial structures, especially when vessels are captured as profiles with no obvious lumen. Other markers including vimentin, platelet-derived growth factor receptor-α or -β, or c-kit/CD117 in combination with CD34 are also regarded as useful for TC identification3. However, while the expression of CD34 has been definitely demonstrated in stromal cells morphologically identifiable as TCs from a variety of organs, the expression of other markers, such as the above-mentioned, may be weak or inconstant in TCs, possibly being organ/tissue- or animal species-dependent3.Telocytes (TCs) are unique stromal cells newly identified in a variety of organs over the last decade12, including the skeletal muscle interstitium18. Functionally, TCs are thought to contribute to the maintenance of local tissue homeostasis by participating in many physiological processes3. In fact, by means of their telopodes TCs connect either with each other via homocellular connections, or with different cell types via heterocellular connections19, thus building a complex three-dimensional interstitial network, possibly serving as a guidance scaffold to define the correct tissue organization and mediate cell-to-cell signaling. Of note, increasing evidence indicates that TCs may be involved in the modulation of the functionality of other tissue resident cells not only in a juxtracrine manner by the establishment of heterocellular junctions, but also in a paracrine one, via the release and transfer of extracellular vesicles enriched in many bioactive molecules including mRNAs, microRNAs, long non-coding RNAs, proteins and bioactive lipids to neighboring recipient cells23.To date, TCs have been identified in the connective tissue layers of many cavitary organs and in the stromal compartment of non-cavitary organs of humans and various mammals and vertebrate animals15. Interestingly, extracellular vesicles released by telopodes were also detected near SCs13. SCs constitute a small population of mononucleated cells whose name relies on their unique anatomical location at the immediate periphery of skeletal myofibers, just beneath the surrounding basal lamina in intimate association with the myofiber sarcolemma. They are considered the resident adult muscle progenitor/stem cells responsible for the post-natal muscle growth and repair/regeneration24. In healthy skeletal muscle tissue, SCs are mitotically quiescent and transcriptionally inactive, accounting for only ~2–10% of the total myonuclei in adult muscles 25. In this dormant state SCs express the paired box transcription factor Pax7, which is considered the canonical SC-specific marker necessary for their survival and function26, and the myogenic transcription factor Myf5 (detectable in ~90% of quiescent SCs) but not the myogenic regulatory factors, namely MyoD or myogenin25. In damaged muscles, the percentage of SCs may increase up to ~10–15%. In response to signals coming mainly from injured myofibers and infiltrating inflammatory cells, indeed, SCs exit from the quiescent state to re-enter cell cycle and closely recapitulate the steps of embryonic and fetal myogenesis program. Upon tissue damage, at first SCs undergo mitotic division to give rise to a progeny of proliferating adult myoblasts expressing Pax7, Myf5 and MyoD. Subsequently, adult myoblasts downregulate Pax7, acquire myogenin and MRF4/Myf6 expression and differentiate into skeletal myocytes that finally either fuse to one another forming nascent syncytial contractile myofibers or fuse with injured myofibers to repair the damage25. Moreover, there is evidence that only a small percentage of SCs are “true” stem cells, that is cells capable of self-renewal to ensure the replenishment of the basal pool of resident SCs recruitable in case of muscle re-injury27. The recently discovered close spatial relationship of TCs and SCs in the adult skeletal muscle led to the postulation that TCs might communicate with these resident progenitor cells through both juxtacrine and paracrine intercellular signaling, potentially modulating their behavior and influencing their fate to support muscle tissue repair/regeneration13. In line with this assumption, a recent paper investigating the presence and distribution of TCs in human fetal skeletal muscle tissue from lower limbs revealed intriguing changes in these cells during the early steps of the fetal myogenic process28. In particular, it has been demonstrated that TCs increase in number from 9 to 11.5 weeks of gestation and, by means of their telopodes, form an extensive reticular network in close relationship with primary and secondary myotubes undergoing maturation. Strikingly, afterwards the number of TCs appears to be robustly reduced in fetal skeletal muscle tissues at 12 weeks of gestation, where mature myotubes become evident, suggesting that TCs may specifically be implicated in myotube development/formation during the early maturation steps of muscle tissue morphogenesis28. Besides the aforementioned experimental data, the intriguing hypothesis of a morpho-functional interaction between TCs and SCs during muscle repair/regeneration is also based on the increasing evidence that TCs may establish a specialized relationship with putative stem/progenitor cells endowed with regenerative potential in a variety of healthy, injured or pathological tissues/organs34. In this context, TCs may help in recruiting, nursing or guiding such progenitors to proliferate, differentiate and integrate into the tissue architecture. Nevertheless, studies on TCs in the skeletal muscle tissue of mammals are still scanty and, at present, there is no direct experimental evidence to definitely support a morpho-functional TC-SC interaction following skeletal muscle damage. Hence, in the present morphological study we sought to explore for the first time the presence and tissue distribution of TCs in a mouse model of skeletal muscle damage, focusing on their interaction with SCs. The reciprocal in vitro behavior of TCs and SCs isolated from single living endomysial sheath-covered muscle fibers was also assessed.Focusing on the skeletal muscle tissue, within adult muscles TCs have been found widely distribuited throughout the perimysial and endomysial compartments, where their long-distance spreading telopodes are strategically positioned in the close vicinity of striated myofibers with regenerative features, nerve endings, small blood vessels, and often satellite cells (SCs)ex vivo EC-induced extensor digitorum longus (EDL) skeletal muscle damage model by means of both morphological and electrophysiological analyses.We first verified a successful construction of the i.e. swollen myofibers with rounded shape) and the presence of intracellular and interstitial edema as compared to controls  amplitude  resting membrane potential (RMP) depolarization 4 or by transmission electron microscopy (TEM).According to relevant recommendations for the morphological study of TCs and considerable reference data+CD31− immunophenotype18, from CD34+CD31+ vascular endothelial cells . In particular, TCs were located alongside the myofibers and in close vicinity of CD31+ vascular structures , the immunostained TCs were clearly distinguishable from fried egg-shaped endothelial cells that also expressed CD34  TCs have been reported to highly express and secrete this growth factor44. As shown by confocal fluorescence analysis of cells stained with WGA and immunostained for VEGF-A, cultured stromal cells with TC morphological traits from EC-damaged myofibers expressed high levels of VEGF-A, that instead was almost undetectable in TCs from controls -A in a stromal cell-enriched population obtained after myofiber removal Fig.  because ols Fig. . The parols Fig. .Figure 646. Although considerable work has demonstrated that many different cell types residing within the muscle tissue or recruited via the bloodstream may contribute to new myofiber formation owing to their inducible myogenic potential , muscle resident SCs are broadly regarded as the main players in such a regenerative process47.It has been clearly proven that adult skeletal muscle tissue possesses a remarkable ability to regenerate in response to focal injuries. Muscle regeneration represents a highly coordinated multi-step process, starting from degeneration and/or necrosis of injured myofibers and ending with formation of new contractile myofibers, aimed to the morpho-functional restoration of damaged muscle tissue49. Specifically, the whole microenvironment surrounding SCs may be subdivided into the so-called “immediate SC niche” and the “microenvironment beyond the immediate niche”. The “immediate SC niche” is represented by the specific anatomical location where SCs reside  and includes different extracellular matrix (ECM) components, signaling molecules diffusing between the myofiber and the SC, cell surface receptors mediating both cell-to-ECM and cell-to-cell interactions or binding regulatory factors, as well as receptors located in the basal lamina sequestering inactive growth factor precursors which serve as a local reservoir to be rapidly activated after damage51. The “microenvironment beyond the immediate niche”, instead, comprises both the local milieu consisting in different interstitial cells and the vascular and neural networks with their associated secretable factors within the stroma between the myofibers of a muscle fascicle, and the systemic milieu including several molecular and cellular signals coming from the entire muscle belly along with neighboring skeletal muscles and bones50. In particular, the juxtacrine and paracrine mutual interactions that SCs establish with the tissue resident or recruited interstitial cells appear crucial in the regulation of the SC quiescent state and even more activation, proliferation and differentiation after injury. In fact, as compared to intact muscles, following tissue damage a high number of cells, putatively supporting SC-mediated regeneration, has been found in the activated microenvironment in the close vicinity of SCs52. These interstitial cells mainly include pro-inflammatory (M1) and anti-inflammatory/pro-regenerative (M2) macrophages54, fibroblasts55, mesenchymal stem/stromal cells such as fibro-adipogenic progenitors56, capillary endothelial cells and perycites58.The behavior and fate specification of SCs are profoundly influenced by the dynamic interplay they establish with the components of the surrounding microenvironment. In healthy intact muscles, the local microenvironment essentially acts for maintaining SCs in a quiescent state; by contrast, during physiological tissue repair after an injury, it releases biochemical and physical signals which promote the activation of SCs and their proper progression into the myogenic differentiation program as well as SC self-renewalin situ identification in the close vicinity of SCs in healthy skeletal muscles17, their possible role during early myogenesis28, and their well-documented interaction and/or cross-talk with stem/progenitor cells in other organs34. Indeed, at present, the effective occurrence of a morpho-functional interplay between TCs and SCs in injured skeletal muscles has yet to be demonstrated with certainty. Hence, we considered that experimental studies aimed at investigating the reciprocal behavior of TCs and SCs in an injured muscle are essential to clarify whether TCs may take part in the regenerative events following muscle tissue damage and, in particular, whether muscle resident SCs and TCs may be privileged partners in such context. In the present study we have described for the first time the distribution of TCs in murine skeletal muscles damaged by forced EC and provided novel experimental evidence supporting their morpho-functional interaction with activated SCs. In particular, confocal immunofluorescence analysis of intact control muscle sections revealed the presence of CD34+CD31− elongated, thin and varicose processes organized in an interstitial network alongside the striated myofibers and around blood vessels, consistent with the previous description of telopodes of TCs in the endomysial and perimysial sheaths of skeletal muscle17. Notably, in EC-damaged muscles this CD34+CD31− telopode network appeared particularly extended and was preferentially arranged around activated SCs that displayed Pax7 and MyoD nuclear positivity and were located in their distinctive site at the periphery of striated myofibers. As expected, and in agreement with a previous report describing the same ex vivo model of muscle damage59, the Pax7+ SCs were more numerous in EC-damaged muscles as compared with controls indicating that these cells presumably left their quiescent state to re-enter cell cycle. In fact, it is noteworthy that SCs positive for MyoD, the myogenic regulatory factor regarded as an early marker of cell activation and myogenic commitment25, were detected in damaged muscles, but not in the intact controls, and were captured in a close spatial relationship with TCs/telopodes. Furthermore, TEM analysis clearly highlighted the close heterocellular interaction between TCs and activated SCs in EC-damaged muscles. Indeed, in these samples, interstitial cells located among striated myofibers and fulfilling the TC ultrastructural identificative criteria were seen to invade the “immediate SC niche”, where, by means of their telopodes, crossed the fragmented basal lamina and contacted the underlying SCs which showed signs of activation, namely cell swelling and lower chromatin density, and appeared incompletely covered by the basal lamina36. In this regard, it is conceivable that a fragmented basal lamina results functional to the prompt migration of activated SCs toward the injury site51, and that activated SCs may contribute themselves to the break down of the basal lamina. In fact, previous studies have shown the capability of SCs to express and secrete functional proteolytic enzymes selectively digesting individual components of the ECM  and their specific tissue inhibitors62. A positive correlation between the expression and activity of matrix metalloproteinases in myoblastic cells (i.e. C2C12 myoblasts and primary murine SCs) and their migratory ability across type-I collagen substrate or basal lamina was also demonstrated62. When considering that TCs have also been reported to express matrix metalloproteinases63, it is tempting to speculate that they might either cooperate with SCs in the digestion of the basal lamina or even mediate the release of tethered pro-myogenic factors from ECM thus contributing to SC activation64. Besides in situ tissue analyses, in the present work the preferential interaction of TCs and SCs from EC-damaged muscles was also confirmed in vitro in a set of experiments performed by culturing single skeletal myofibers surrounded by their endomysial sheath, and the sprouting TCs and SCs. A further intriguing result of our study is the increase in VEGF-A expression found in cultured TCs from EC-damaged muscles. This observation is consistent with previous reports demonstrating an ability of TCs from different tissues, including skeletal muscle, to produce VEGF whose secretion might occur via the release of extracellular vesicles44, and sheds some light on the potential paracrine mechanisms by which TCs may stimulate SC activation and foster myogenic differentiation. In support, it is interesting to note that a prominent role of VEGF-A in modulating the myoblast/SC behavior is being increasingly reported in different healthy and pathological conditions43. Thus, the potential functional role of TC-derived VEGF-A in mediating TC-SC interactions is worth of further investigation. In addition, we believe that future studies aimed at evaluating the possible involvement of juxtacrine mechanisms in mediating the cross-talk between TCs and SCs deserve a particular attention.Only recently, TCs entered this scenario as a new distinctive type of interstitial cells that may potentially behave as nursing cells for SC-mediated skeletal muscle regeneration. However, it must be frankly recognized that such supporting role for TCs has only been supposed on the basis of their i.e. (1) the ex vivo model of muscle damage does not allow to recapitulate all the phases of muscle repair/regeneration process, and (2) the in vitro experimentation obviously eliminates many paracrine/juxtacrine mechanisms that may regulate in situ intercellular interactions and cell functionality), the present findings contribute either to extend our knowledge of TC biology or to shed additional light on the cellular and molecular mechanisms regulating SC functionality, establishing for the first time that SCs and TCs may represent close neighbors and privileged functional partners in a damaged skeletal muscle condition. This study may therefore be considered as a basal groundwork for further investigations devoted to comprehensively decipher the molecular mechanisms underlying the TC-SC interplay during skeletal muscle tissue repair and potentially representing new attractive targets in the field of regenerative medicine.In conclusion, although we recognize some limitations of our study  and exposure to a series of contractures (ten cycles of 1 minute each) in a solution containing a high K+ concentration (mM): 150 K-glutamate, 2.0 MgCl2, 10 KOH, 10 TES e 1.0 K2-EGTA (Sigma-Aldrich). After each cycle of EC, muscles were recovered in physiological Ringer-Krebs solution containing (mM) 140 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 D-glucose and 10 HEPES (Sigma-Aldrich) for 4 minutes. After EDL muscle excision, anesthetized mice were sacrificed by cervical dislocation. All efforts were made to minimize animal suffering and to reduce the number of animals used. A total of 18 mice were used in the experimental procedures. Animals were housed in a Laboratory Animal Facility , fed with standard laboratory diet and tap water ad libitum, kept at 23 ± 1 °C with a 12 hour light/dark cycle. Animal manipulations were carried out in accordance with the Directive 2010/63/EU of the European Parliament and of the European Union council (22 September 2010) on the protection of animals used for scientific purposes. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Florence. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health .Young adult male Swiss mice (25–30 g) were gradually anesthetized with Xylazine , Lidocaine  and Zoletil  from a low dose (20 mg/kg) to a higher dose (40 mg/kg) to avoid a respiratory arrest. The anesthetic mixture was administered intramuscularly. An adequate depth of anesthesia was maintained so that the mice were unresponsive to tail pinch/tactile stimulation. EDL muscles were carefully excised from each limb of the animal: one muscle was subjected to forced EC in isometric condition as previously reportedBoth EC-injured and control EDL muscles (n = 5 each) were fixed with 10% formalin in PBS, dehydrated with a graded alcohol series, cleared in xylene, and embedded in paraffin. Muscles sections (5 μm thick) were deparaffinized and routinely stained with hematoxylin and eosin. Tissue morphology was then observed under a light microscope (Leica DM4000 B) equipped with a DFC310 FX 1.4-megapixel digital color camera and the software application suite LAS V3.8 .59. Briefly, EC-injured and control EDL muscle specimens (n = 5 each) were fixed with 4% cacodylate-buffered glutaraldehyde (pH 7.4) solution for 2 hours, post fixed in 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) for 1 hour at room temperature, dehydrated in a graded acetone series, passed through propylene oxide and embedded in Epon 812 (Sigma-Aldrich). Semithin sections (2 μm thick) were stained with toluidine blue. Ultrathin sections (60 nm thick) were contrasted with UranyLess stain  and alkaline bismuth subnitrate, and then examined using a Jeol 1010 electron microscope  at 80 kV. TCs and SCs detected in electron microscopy images were digitally colored in blue and green, respectively, using Adobe Photoshop CS6 software .Samples were processed for ultrastructural analysis by TEM as previously reported with minor modifications67. The muscle was continuously superfused during recordings at a rate of 1.8 ml/minute  in the experimental chamber with the Ringer-Krebs solution (mM): 120 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5.5 HEPES, and 1 D-glucose (Sigma-Aldrich), at room temperature (22 °C). RMP was recorded in the current clamp mode by inserting a microelectrode into a single skeletal muscle fiber of isolated EC-injured and control EDL muscles (at least 15 myofibers for each muscle). The microelectrodes were pulled by a micropipette vertical puller  from borosilicate glass  and were filled with an internal filling pipette solution containing (mM): 130 KCl, 10 NaH2PO4, 0.2 CaCl2, 1 EGTA, 5 MgATP and 10 HEPES/KOH (pH 7.2) (Sigma-Aldrich). The tip resistance was about 60 MΩ. The junction potential of the electrode was measured prior to make the patch, and then it was subtracted from the recorded membrane potential. The RMP was recorded in physiological solution by using a stimulus waveform I = 0 pA. The membrane passive properties of the fibers  as well as K+ current (IK) were measured in voltage-clamp mode. To estimate the membrane passive properties two 75-ms long voltage step pulses to −80 and −60 mV were usually applied, starting from a holding potential (HP) of −70 mV. To evoke the ionic transmembrane currents, we applied 1-s long voltage pulses ranging from −80 to 50 mV in 10-mV increments (HP = −80 mV). The P/4 procedure allowed us to cancel on line any leakage and voltage-independent ionic currents from the records. The current amplitude (I) was normalized to cell capacitance Cm (in pA/pF) to properly compare the currents recorded from different fibers. The I/Cm values represent the current density (in pA/pF). The Digital-to-analog and analog-to-digital conversions were carried out by using a Digidata 1200 interface . pCLAMP programs  were used for stimulation, recordings and data acquisition. The condition of the fibers was constantly checked by monitoring the RMP and Rm. Fibers remained stable for about 150–240 minutes.Electrophysiological analyses were conducted on EC-injured and control EDL muscles (n = 5 each) essentially as previously reported62. Briefly, single living myofibers were isolated from the digested muscle by a gentle mechanical trituration using a Pasteur pipette, and individually transferred in a 24-well Matrigel-treated plate  or glass coverslips and cultured for 48 hours in SC proliferation medium containing DMEM plus 20% fetal bovine serum , 10% horse serum (Sigma-Aldrich), 0.5% chicken embryo extract  and 100 U/ml penicillin-streptomycin (Sigma-Aldrich) to allow cell sprouting. Then, the myofibers were removed and the derived cells were cultured in proliferation medium until reaching 80% of confluence. The cells were then detached using a solution of 0.05% trypsin–0.03% ethylenediaminetetraacetic acid (Sigma-Aldrich) for 5 minutes at 37 °C and re-seeded in a culture plate for 20 minutes with fresh proliferation medium. After 20 minutes, the non-adherent cells (mostly SCs) were collected and cultured (on gelatin-coated culture plates or glass coverslips) in SC proliferation medium, while the adherent ones 62 were washed and cultured (on culture plates or glass coverslips) in DMEM plus 20% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. The cells were monitored and observed every day under an inverted phase-contrast microscope . Myotube formation from confluent EC and control myofiber-derived SCs cultured for 5 days in their specific proliferation medium was observed under an inverted phase-contrast microscope .Single endomysial sheath-covered living myofibers were isolated from both EC-injured and control EDL muscles  digested in a DMEM solution containing 0.2% collagenase type I (Sigma-Aldrich) and processed according to previously published protocols+ and MyoD+ SCs were performed on 5 random 200 μm2 optical square fields (63×) containing at least 10 muscle fibers, under the confocal Leica TCS SP5 microscope, in each tissue section. At least three tissue sections from each EDL muscle (n = 5) were analyzed. Counting was performed by two independent observers and the count numbers were then averaged.Sections (5 µm thick) of paraffin-embedded EC-injured and control EDL muscle tissue samples were deparaffinized, rehydrated, permeabilized with cold acetone for 10 minutes and then incubated with a solution containing 10% glycine in PBS for 10 minutes to quench autofluorescence. Muscle sections were blocked with 0.5% bovine serum albumin  and 0.2% gelatin in PBS for 30 minutes and incubated at 4 °C overnight with the following primary antibodies: rabbit polyclonal anti-CD34 , rat polyclonal anti-CD31 , mouse monoclonal anti-Pax7 , mouse monoclonal anti-MyoD . Stromal cell/TC-enriched and SC-enriched culture grown on glass coverslips were fixed with 0.5% paraformaldehyde in PBS for 10 minutes at room temperature, permeabilized with cold acetone for 3 minutes and incubated with a blocking solution containing 0.5% BSA (Sigma-Aldrich) and 0.2% gelatin in PBS for 20 minutes. Cell preparations were then incubated overnight at 4 °C with the following antibodies: rabbit polyclonal anti-CD34 , mouse monoclonal anti-Pax7 , mouse monoclonal anti-MyoD  or mouse monoclonal anti-VEGF-A . In some experiments, cell plasma membrane was stained with WGA Tetramethylrhodamine conjugate  for 15 minutes at room temperature in the dark. Primary antibodies were revealed using specific anti-rabbit/anti-mouse/anti-rat Alexa Fluor 488-conjugated IgG  or 568-conjugated IgG  for 1 hour at room temperature. Negative controls were performed by replacing primary antibodies with non-immune serum, while cross reactivity of secondary antibodies was verified by omitting primary antibodies. Immunolabeled samples were rinsed in PBS, mounted using an antifade gel mounting medium  and examined with a Leica TCS SP5 confocal microscope (Leica Microsystems) equipped with a HeNe/Ar laser source, a Leica Plan Apo 63×/1.43NA oil immersion objective, for fluorescence measurements and differential interference contrast (DIC) optics. Series of optical sections  0.4 µm in thickness were taken throughout the depth of the samples at intervals of 0.4 µm and the images projected onto a single ‘extended focus’ image. DIC images were captured to visualize the precise tissue structure and localization of the immunostaining. Counts of Pax7t-test for comparisons between data obtained from EC-damaged and control muscles. P < 0.05 was considered statistically significant.Data are reported as means ± SEM. We used unpaired Student’s


Background: Prior studies have found that rescue and recovery workers exposed to the 9/11 World Trade Center (WTC) disaster have evidence of increased persistent hearing and other ear-related problems. The potential association between WTC disaster exposures and post-9/11 persistent self-reported hearing problems or loss among non-rescue and recovery survivors has not been well studied. Methods: We used responses to the World Trade Center Health Registry (Registry) enrollment survey (2003–2004) and first follow-up survey (2006–2007) to model the association between exposure to the dust cloud and persistent hearing loss . Results: The prevalence of post-9/11 persistent hearing loss among survivors was 2.2%. The adjusted odds ratio (aOR) of hearing loss for those who were in the dust cloud and unable to hear was 3.0 . Survivors with persistent sinus problems, headaches, PTSD and chronic disease histories had an increased prevalence of reported hearing problems compared to those without symptoms or chronic problems. Conclusions: In a longitudinal study, we observed an association between WTC-related exposures and post-9/11 self-reported hearing loss among disaster survivors. A previous publication from the World Trade Center Health Registry (Registry) regarding the cohort of individuals exposed to the events of the WTC disaster of 11 September 2001, found that dust cloud exposure among survivors who evacuated damaged and destroyed buildings was associated with self-reported hearing problems in a cross-sectional analysis . An analThe precise type of hearing loss and mechanism of persistent hearing loss associated with dust cloud exposure are not known. Extremely high noise exposure resulting from the building collapses and their aftermaths, ototoxic chemical exposure, infectious (sinusitis) and physical injury (concussion) all are plausible explanations for this association ,6.Reliable measurements of noise exposure during and following the disaster are mostly lacking. Potential ototoxic exposures that were detected in settled dust at the World Trade Center disaster site included heavy metals, polycyclic aromatic hydrocarbons, and other hydrocarbons . Smoke aThe purpose of this study was to conduct a longitudinal analysis to evaluate the association between exposure to the World Trade Center 11 September 2001 disaster and self-reported persistent hearing loss among residents, passersby, area workers, students and staff from schools in Lower Manhattan (survivors) who enrolled in the World Trade Center Health Registry in 2003–2004, and who responded to a follow-up survey in 2006–2007. The study population has been described in detail elsewhere . BrieflyFor this analysis, we excluded enrollees who were rescue and recovery workers since they were examined separately, those interviewed by proxy and those with pre-9/11 hearing problems. The final analytic sample of survivors was 22,741 . InstituParticipants were asked in the Registry’s Wave 2 survey  whether The enrollment survey (Wave 1  ) includWe performed a descriptive analysis of the proportion of survivors who had reported persistent hearing problems or loss by demographic, health status, and exposure factors. Comparisons were made between those with and without persistent hearing problems using standardized differences that estimate the differences between the means and proportions in each group divided by the standard error . An imbaWe examined the association between WTC-related dust cloud exposure and hearing loss using logistic regression. We adjusted for mode , age at Wave 2 (integer), sex, race/ethnicity, education , smoking history at Wave 2 , persistent sinus symptoms at Wave 2 , persistent headache symptoms at Wave 2 , and chronic disease history . Chronic disease history was a reported physician diagnosis of hypertension, heart disease, angina, heart attack or diabetes at Wave 1 or Wave 2. These conditions have been associated with hearing loss ,14.We ran several secondary analyses to evaluate the robustness of the observed associations. We restricted the sample to those 50 years or younger on 11 September 2001 to determine if the observed effects may be age-related. We did a stratified analysis for those with and without PTSD (defined as a Posttraumatic Stress Checklist—Civilian Version score of 44 or greater), and a separate stratified analysis by sex. We also used a more restrictive case definition based on responses to the Wave 2 question whether or not they had a medically diagnosed condition or disability that currently affected their hearing. Statistical analyses were performed using SAS 9.4 .p < 0.0001). The prevalence of self-reported post-9/11 hearing loss or problem among survivors at the enrollment interview (2003–2004) was 1275/22,544 (5.7%). The overall prevalence of self-reported persistent hearing problems or loss (at both enrollment and follow-up surveys) among survivors in the Registry was 2.2%. Women reported a slightly higher proportion (2.4%) compared with men (2.0%) . The meaThe prevalence of hearing loss increased with higher levels of dust cloud exposure, from 1.3% among those not in the dust cloud to 2.2% for those in the dust cloud but able to hear, then to 5.7% for those in the dust cloud but unable to hear. Survivors with persistent sinus problems, headaches, PTSD and chronic disease histories had an increased prevalence of reported hearing problems compared to those without symptoms or chronic problems. In a fully adjusted model , the adjIn secondary analyses (data not shown), restricting the age at 9/11 of survivors to 50 years or less did not substantively change the overall pattern of the results. When the logistic regression analysis was stratified by PTSD at Wave 2, the magnitude of effect was reduced when limited to those with PTSD, and this was further reduced when limited to those without PTSD, although the dust cloud exposure measure remained statistically significant in both cases. When a separate case definition based on reported disability due to hearing loss was used, the overall pattern of results did not change substantively. Similarly to our findings among 9/11 rescue and recovery workers, we observed an association between World Trade Center dust cloud exposure and reports of post 9/11 persistent hearing loss among World Trade Center disaster survivors, including Lower Manhattan residents, passersby, area workers, and school staff. In a fully adjusted model, those who could not hear while in the dust cloud on 9/11 had a three-fold increased odds ratio for persistent hearing loss compared to those not in the dust cloud. This relationship did not change significantly when secondary analyses were conducted limiting age to less than 50 years and stratifying by PTSD. The WTC dust, based on analyses of settled dust, was 80%–90% made up of a highly re-suspendable alkaline mixture of crushed concrete, gypsum  and synthetic vitreous fibers, such as slag wool or fiberglass  ,16. MuchThe reason why Asian and Hispanic survivors had a significantly increased adjusted odds ratio in this study is unclear. Prior studies of hearing loss after blast events rarely include information on race/ethnicity of the affected populations. A higher prevalence of hearing impairment has previously been found among US Spanish and Hispanic adults compared with the overall population, associated with noise exposure, socioeconomic factors and diabetes/pre-diabetes . The prePTSD without traumatic brain injury has been shown to be associated with hearing loss among Iraq and Afghanistan veterans . The reaHigh-level noise exposure was likely at the same time periods and locations where the dust cloud exposure occurred, although actual environmental measurements of both noise and airborne dust are lacking. Exposures with sound energy equivalent to 85 dBA for 8 hours exceed the recommended limits ,21. ExceThe role of exposure to traumatic events such as 9/11 and their prolonged impact on physical and mental health outcomes is the subject of increasing focus by investigators ,29,30,31Hearing loss is a major public health problem as it is the third most common chronic physical condition following hypertension and arthritis among United States adults . The conStrengths: This study is longitudinal and represents responses from a relatively large cohort of survivors. Simultaneous reporting of both exposure and outcome at two time points allowed a more detailed analysis of hearing loss among this disaster-exposed population of survivors than had been previously published.Limitations: Loss to follow-up between surveys may have resulted in selection bias. Those who participated in Wave 2 tended to have fewer PTSD symptoms. This suggests that the prevalence of hearing loss at Wave 2 may be underestimated in this study. The self-reported nature of the survey data means that misclassification of exposure and outcome may have occurred. The type of hearing loss could not be determined based on the survey answers. We had no information about post-9/11 noise exposure from hobbies or other sources. The public health impact of hearing loss among survivors of the WTC disaster is an ongoing issue. The specific nature and causes of hearing loss among World Trade Center survivors remain to be determined. Analysis of pre- and post-9/11 audiograms among New York City Fire Department workers involved in health surveillance has been recently published . HoweverHearing loss is not currently a certifiable WTC-related condition under the World Trade Center Health Program, making access to affordable treatment options potentially difficult. Health insurance typically does not cover hearing aids. Public health officials should consider including questions about hearing trouble in surveys following future disasters involving chemical, dust, or blast exposures, and audiometric screening is recommended as part of ongoing surveillance for subsequent health effects.


Hearing is important for achieving a good quality of life, because a partial or total inability to hear is a barrier to communication and language development. Hearing loss, especially disabling hearing loss, is associated with delayed cognitive development in children and early cognitive decline in older adults.Unaddressed hearing loss is one of the leading causes of morbidity; it is ranked fourteenth in terms of disability adjusted life years and second in years lived with disability globally.Hearing loss was highlighted at the World Health Assembly in 2017, when Member States unanimously adopted a resolution on the prevention of deafness and hearing loss.Bulletin of the World Health Organization will publish a theme issue on the public health approach of hearing loss.Against this background, the We welcome papers focusing on identifying and filling the gaps in evidence across comprehensive hearing-care services, from promotion of ear and hearing care, to screening, hearing devices and rehabilitation. In particular, the papers should report on unmet needs, outcomes of services, and effective and sustainable initiatives to reach underserved groups. Submission of papers reporting on both the magnitude of diseases and conditions, such as ear infections, meningitis and rubella, that can affect hearing, are encouraged. As well, papers addressing health system issues and promoting an intersectoral approach to ear and hearing care, such as looking beyond health. As much as possible, papers should seek to integrate examples from low- and middle-income countries across life course.Bulletin’s guidelines for contributors (available at: http://www.who.int/bulletin/volumes/96/1/18-990118/en/), and the cover letter should mention this call for papers.The deadline for submission is 30 November 2018. Manuscripts should be submitted in accordance with the


Varicose veins are a common problem worldwide and can cause significant impairments in health-related quality of life, but the etiology and pathogenesis remain not well defined. This study aims to elucidate transcriptomic regulations of varicose veins by detecting differentially expressed genes, pathways and regulator genes.We harvested great saphenous veins (GSV) from patients who underwent coronary artery bypass grafting (CABG) and varicose veins from conventional stripping surgery. RNA-Sequencing (RNA-Seq) technique was used to obtain the complete transcriptomic data of both GSVs from CABG patients and varicose veins. Weighted Gene Co-expression network analysis (WGCNA) and further analyses were then carried out with the aim to elucidate transcriptomic regulations of varicose veins by detecting differentially expressed genes, pathways and regulator genes.From January 2015 to December 2016, 7 GSVs from CABG patients and 13 varicose veins were obtained. WGCNA identified 4 modules. In the brown module, gene ontology (GO) analysis showed that the biological processes were focused on response to stimulus, immune response and inflammatory response, etc. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the biological processes were focused on cytokine-cytokine receptor interaction and TNF signaling pathway, etc. In the gray module, GO analysis showed that the biological processes were skeletal myofibril assembly related. The immunohistochemistry staining showed that the expression of ASC, Caspase-1 and NLRP3 were increased in GSVs from CABG patients compared with varicose veins. Histopathological analysis showed that in the varicose veins group, the thickness of vascular wall, tunica intima, tunica media and collagen/smooth muscle ratio were significantly increased, and that the elastic fiber/internal elastic lamina ratio was decreased.This study shows that there are clear differences in transcriptomic information between varicose veins and GSVs from CABG patients. Some inflammatory RNAs are down-regulated in varicose veins compared with GSVs from CABG patients. Skeletal myofibril assembly pathway may play a crucial role in the pathogenesis of varicose veins. Characterization of these RNAs may provide new targets for understanding varicose veins diagnosis, progression, and treatment. Varicose veins are a common problem worldwide, affecting approximately 25% of the population  and causTranscriptomic alteration of great saphenous vein can lead to vascular wall structure and morphological changes, which may contribute to the pathogenesis of varicose veins. RNA-Sequencing (RNA-Seq) is a relatively novel approach to transcriptome profiling, which can obtain the complete transcriptomic information and allow for more extensive analysis compared with microarray platforms . Gene exIn this study, we harvested GSVs from CABG patients and varicose great saphenous veins (GSV) from conventional stripping surgery. RNA-Seq technique was used to obtain the complete transcriptomic data of both GSVs from CABG patients and varicose GSV. WGCNA and further analyses were then carried out with the aim to elucidate transcriptomic regulations of varicose veins by detecting differentially expressed genes, pathways and regulatory genes. We integrated RNA-Seq to associated gene networks and pathways with pathological process in varicose veins.The study population consists of patients who underwent CABG because of coronary artery disease and patients who underwent conventional great saphenous vein stripping surgery because of varicose veins. The GSVs from CABG patients were harvested during the CABG surgery and varicose great saphenous vein samples were harvested during the conventional GSV stripping surgery. The GSV phlebectomy was carried out about 5 cm above the knee and the specimens were 2 cm in length. When harvested, the surrounding tissue was removed and each specimen was divided into 2 segments, one stored in liquid nitrogen with a frozen tube and the other one in 10% formalin solution, both immediately. This study was carried out in accordance with the recommendations of the ethics committee of China-Japan Friendship Hospital with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of China-Japan Friendship Hospital.The GSVs from CABG patients and varicose veins were fixed in 10% formalin for at least 24 h. Specimens were dehydrated, embedded with paraffin and sectioned  for further investigations. Hematoxylin-Eosin (HE), Masson trichrome and elastic fiber staining for morphologic analysis were carried out for both GSVs from CABG patients and varicose veins. We measured the thickness at 4 points around the vessel circumference, including 12-o’clock, 3-o’clock, 6-o’clock and 9-o’clock. Then, the histopathological analysis was carried out under an optical microscope  with Image J software (Version 1.48). The person who performed the histopathological examination was blinded to whether the specimens were GSVs from CABG patients or varicose veins. The immunohistochemistry staining for ASC, Caspase-1 and NLRP3  was carried out with antibodies for human ASC , Caspase-1  and NLRP3  using EnVison method. Brown or brown-black particles in the cytoplasm were considered as positive expressions.® 3.0 Fluorometer . RNA integrity and concentration were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system .RNA was isolated with TRIzol Reagent . RNA degradation and contamination were detected by 1% agarose gels electrophoresis. RNA purity was checked using the Qubit® UltraTM RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations. Index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QIAquick PCR kits and elution with EB buffer, subsequently, terminal repair, A-tailing and adapter were implemented. The aimed products were retrieved by agarose gel electrophoresis and PCR was performed. Then the library was completed.A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextThe main procedure for library construction included mRNA concentration, fragmentation, cDNA synthesis, end repairing, add A-tailing and adapter, fragments selection and library purification. The reagent used included NEBNext super speed RNA Library Prep Kit for Illumina; Beckman AM Pure XP beads, Beckman; 80% Ethanol; Promega magnetic frame PCR instrument, ABI 9700.® RNA Assay Kit in Qubit® 3.0 to preliminary quantify and then dilute to 1 ng/μl. Insert size was assessed using the Agilent Bioanalyzer 2100 system , and qualified insert size was accurately quantized using StepOnePlusTM Real-Time PCR System .RNA concentration of library was measured using QubitThe clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end reads were generated. The Sequencing quality was controlled by MultiQC tool .P-value. The threshold of DEGs was set as FDR < 0.05.The number of reads mapped to each gene was counted using HTSeq v0.6.0. Fragments per kilobase of exon per million reads mapped (FPKM) was then calculated based on length of the gene and reads mapped to the gene. Then, raw RNA-Seq data of GSVs from CABG patients and varicose veins was subjected to DEGs analysis. DEGs were identified by DESeq2  package SD > 0.25) and built the co-expression network using WGCNA package in R language , including the biological process, cellular components and molecular functions using clusterProfiler packages .t-test. For non-normally distributed variables, we performed a non-parametric test. For discrete variables, the chi-square test or Fisher’s exact test were performed. Data analysis was performed using SPSS version 22  and R software version 3.2.3 (package “WGCNA” “DESeq2”). A P-value of <0.05 was considered statistically significant.Continuous variables were present as mean and standard deviation. Discrete variables were present as percentages. For continuous variables, we carried out the normality test. For normally distributed variables, we performed independent https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA523048).The RNA-Seq data in the study can be found in NCBI’s Sequence Read Archive (SRA) accession: PRJNA523048  aligner. This resulted in an average of 19033962 uniquely mapped reads per sample, of which an average of 97.56% were mapped in the intragenic region (within introns or exons). On average 19572270 reads were detected among the mapped reads. Read counts were estimated at the gene level using HTSeq. Finally we identified 333 differentially expressed genes, including 87 up-regulated and 246 down-regulated . The volWeight gene co-expression network analysis approach was applied to the data count obtained from RNA-Seq of 7 GSVs from CABG patients and 13 varicose vein samples. We used the differentially expressed genes to construct the co-expression network. Gene modules (clusters of highly co-expressed genes) were detected and assigned different colors. Finally, we identified 4 modules containing 285 genes, labeled by yellow, brown, blue and gray . Among tr = 0.92, P = 1 × 10-8). Therefore, for further analyses, we selected the gray module as a module of interest. The gray module was also associated with CEAP  categories, the presence of coronary artery disease, side of the great saphenous vein, vascular wall thickness and collagen/smooth muscle ratio.In these identified modules, an eigengene was calculated. Then, we correlated the selected traits and modules’ eigengene to calculate the module-traits relationships . The resGenes in the modules were subjected to GO functional analysis and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. Gene functional analysis of DE genes can be found in The immunohistochemistry staining showed that the NLRP3 expression in varicose veins was decreased compared with GSVs from CABG patients . The expThe histopathological analysis showed that in varicose veins, the thickness of vascular wall, tunica intima, tunica media and the collagen/smooth muscle ratio were significantly increased, and the ratio of elastic fiber/internal elastic lamina was decreased . The detIn the present study, we carried out WGCNA based on RNA-Seq data, clinical information and the histopathologic characteristics of GSVs from CABG patients and varicose veins from conventional surgery and identified several candidate genes and biological processes which may contribute to the pathogenesis of varicose veins.It is commonly considered that weakness of venous wall, venous valve dysfunction, and increased intravenous pressure are the main causes of varicose veins . Though Gomez reported  that decSome studies have investigated the differentially expressed genes in primary varicose veins. NLRP3 inflammasome is a key component mediating sterile inflammation. This is a complex consisting of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1. It has been reported to be closely related to vascular dysfunction. NLRP3 inflammasome can be activated by cholesterol crystal and is essential for atherogenesis , and hasIt has been reported that insulin-like growth factor I stimulates myofibril development and decreases smooth muscle α-actin . In variOur study has some limitations. First, the sample size is relatively small. GSVs from CABG patients are not strictly normal, which may cause a bias. Increased cardiovascular risk in CABG patients might increase the expression of NLRP3 inflammasome markers, since those patients often presented hypertension, diabetes and obesity, which have been associated with NLRP3 inflammasome activation. Second, though WGCNA allows for more extensive analysis, over analysis of RNA-Seq data may lead to type I error. Third, this is a preliminary study, and further studies, such as RT-qPCR, western blotting, gene knocking down/out and overexpression should be carried out to shade light of the underlying mechanisms.This study shows that there are clear differences in transcriptomic information between varicose veins and GSVs from CABG patients. Some inflammatory RNAs are down-regulated in varicose veins compared with GSVs from CABG patients. Skeletal myofibril assembly pathway may play a crucial role in the pathogenesis of varicose veins. Characterization of these RNAs may provide new targets for understanding varicose veins diagnosis, progression, and treatment.JZ, JW, and PL designed the study. XF, CW, LP, JG, and WS collected the data. CS, YaC, JK, YiC, FW, and QN analyzed the data. QN, JZ, XF, ZY, and JW 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.


Approximately 25% of colorectal cancer patients in sub-Saharan Africa are younger than 40 years, and hereditary factors may contribute. We investigated the frequency and patterns of inherited colorectal cancer among black Zimbabweans.MLH1, MSH2 and EPCAM deletions using multiplex ligation-dependent probe amplification. Next generation sequencing using a 16-gene panel was performed for cases with phenotypic features consistent with familial colorectal cancer. Variants were assessed for pathogenicity using the mean allele frequency, phenotypic features and searching online databases.A population-based cross-sectional study of ninety individuals with a new diagnosis of colorectal cancer was carried out in Harare, Zimbabwe between November 2012 and December 2015. Phenotypic data was obtained using interviewer administered questionnaires, and reviewing clinical and pathology data. Cases were screened for mismatch repair deficiency by immunohistochemistry and/or microsatellite instability testing, and for MSH2 c.2634G>A pathogenic mutation, c.(1896+1_1897–1)_(*193_?)del , and one fulfilling the Amsterdam criteria, with MLH1 and PMS2 deficiency, but no identifiable pathogenic mutation. Two other cases had a strong family history of cancers, but the exact syndrome was not identified. The prevalence of Lynch syndrome was 3·3% (95% CI 0·7–9·4), and that of familial colorectal cancer was 5·6% .Three Lynch syndrome cases were identified: Identifying cases of inherited colorectal cancer in sub-Saharan Africa is feasible, and our findings can inform screening guidelines appropriate to this setting. The incidence of colorectal cancer is gradually rising in some countries in sub-Saharan Africa, including Kenya, Nigeria, and Zimbabwe. In ZimbaApproximately 1 in 4 individuals with colorectal cancer in sub-Saharan Africa is under the age of 40 years., 6 Thus,While this theory is plausible, there is lack of solid supporting evidence. Previous studies reported the frequency of mismatch repair deficiency in archived tissue, without further, comprehensive analysis., 9 PhenoNewly diagnosed cases of colorectal cancer were prospectively recruited in Harare, Zimbabwe, between November 2012 and December 2014, as previously described. They werThe participants were recruited within six months of a histologically confirmed diagnosis of colorectal cancer. After informed consent, phenotypic data was obtained using interviewer-administered questionnaires covering demographic variables, and detailed personal, and family history of cancer. Data on tumour pathology  were obtained from pathology and endoscopic reports.MLH1, MSH2 and EPCAM deletions was performed regardless of the mismatch repair status using multiplex ligation-dependent probe amplification (MLPA). A phenotype-guided strategy was then used to select cases for next generation germline sequencing. The phenotypic features considered included family history of cancer and colorectal cancer, prior history of cancer, age < 40 years, mucinous or signet ring morphology, presence of synchronous tumours, polyposis, mismatch repair-protein status and BRAF V600E result.Venous blood was drawn for germline DNA, and paraffin-embedded tumour blocks were obtained from pathology laboratories. Initial screening for Lynch syndrome was performed using immunohistochemistry and/or microsatellite instability testing. Immunohistochemistry was used as a parallel method of assessing mismatch repair deficiency, and to possibly indicate the mutated gene. Screening for germline Microsatellite instability (MSI) testing was performed on paired germline and tumour DNA samples using the Promega MSI Analysis System version 1.2 . Germline DNA was obtained from peripheral blood, or normal tissue on tumour slides using standard techniques. Fluorescently-labelled primers were used for co-amplification of seven markers; five mononucleotide , and two pentanucleotide (Penta C and Penta D) repeat markers. The PCR products were separated by capillary electrophoresis , and the output analysed with GeneMapper version 4.0 software . The results were interpreted as follows: MSI at 2 or more mononucleotide loci, MSI-high (MSI-H); MSI at a single mononucleotide locus, MSI-Low (MSI-L) and no instability at any of the loci, microsatellite stable (MSS).MSH2 and anti-PMS2 mouse monoclonal antibodies , and anti-MLH1 and anti-MSH6 mouse monoclonal antibodies  on formalin-fixed, paraffin-embedded tissue sections. Tissue sections with normal colonic mucosa adjacent to the tumour were selected for immunohistochemistry. The normal areas acted as internal positive controls, while external control positive controls were also included . A Ventana Benchmark XT autostainer and the Opti-view detection system  were used, following the manufacturers’ protocol. The stained slides were interpreted by an experienced pathologist (DG).Immunohistochemistry was performed using anti-MLH1 protein deficient, with a high level of microsatellite instability (MSI-H) were sequenced for the BRAF V600E mutation. The following primers were used: Forward, 5'-CTACTGTTTTCCTTTACTTACTACACCTCAGA-3'; and reverse, 5'-ATCCAGACAACTGTTCAAACTGATG-3'. Sequencing was performed using BigDye terminator v3.1 cycle sequencing kit  following standard protocols.All cases that were MLH1, MSH2 and EPCAM germline deletions was performed using the SALSA MLPA kit  according to the manufacturer’s protocol. Briefly, germline DNA was denatured, followed by hybridisation with MLPA probes, ligation and amplification. The amplified products were visualised using capillary electrophoresis, and the output analysed using the Coffalyser.Net software (https://www.mlpa.com).MLPA for MUTYH, EPCAM, MSH2, MSH6, MLH1, PMS2, FBX011, APC, BMPRIA, KLLN, PTEN, POLE, POLD1, TP53, SMAD4, and STK11). DNA quality assurance was performed using agarose gel electrophoresis and spectrophotometry (Nano-Drop 1000) . DNA quantification was performed using real time PCR with the RNase P assay. DNA dilutions and library preparation were performed on the Ion Chef using the DL8 kit , followed by template preparation and sequencing on the Ion Chef personal genome machine as per manufacturer’s protocol. The output was analysed using the Ion Reporter . Variants with a mean allele frequency< 0.01 were considered potentially pathogenic, and cross-referenced with online databases, including Clin-Var (https://www.ncbi.nlm.nih.gov/clinvar/), InSiGHT (https://www.insight-group.org/variants/databases/), and LOVD (http://www.lovd.nl/3.0/home). Phenotypic characteristics, particularly family history, immunohistochemistry and MSI status were considered in evaluating pathogenicity.Next generation sequencing of germline DNA from peripheral blood was performed on selected cases using a custom panel of 16 colorectal cancer genes , mean (with standard deviations), and median (with inter-quartile range) were used to summarise data. The statistical analysis was performed using Stata MP Version 12.0® .A total of 101 black Zimbabwean patients with newly diagnosed colorectal cancer were recruited in Harare, between November 2012 and December 2015. Complete analysis for hereditary colorectal syndromes was performed in 90 cases . The meaMLH1 and PMS2 deficient (and BRAF V600E negative), one was MSH2 and MSH6 deficient, and one had isolated MSH2 deficiency. In the 53 cases where both immunohistochemistry and MSI results were available, there was concordance in all, but one case (98·1% concordance)  .st degree relatives, one of whom met the Amsterdam criteria for Lynch syndrome. One case of Lynch syndrome was identified on next generation sequencing, a 48-year-old woman with a rectal tumour, and a pathogenic variant in MSH2, , but no obvious family history of colorectal cancer _(*193_?)del, in a 41-year-old man with a rectal adenocarcinoma. The tumour was mismatch repair protein proficient and there was a history of unspecified intestinal cancer in his brother, and maternal aunt _(*193_?)del , and the case fulfilling the Amsterdam criteria. The individual fulfilling the Amsterdam criteria was a 34-year-old woman with a well-differentiated adenocarcinoma in the transverse colon, and a history of colorectal cancers in both 1st and 2nd degree relatives . [There were 3 Lynch syndrome cases  giving aelatives . The tumentified . They weo42Ser). Therefore, it can be reasonably concluded that, the minimum prevalence of inherited colorectal cancer syndromes in our population ranges from 3·3% (95% CI 0·7–9·4) to 5·6% .This study aimed to establish the role of the established hereditary syndromes in colorectal cancer in sub-Saharan Africa, where the frequency of early-onset disease is disproportionately high. We concluded that up to 1 in 18 patients with colorectal cancer have an underlying high penetrance genetic predisposition. However, we found no evidence to support the hypothesis that the high frequency of early onset colorectal cancer in our population is associated with frequent germline mutations in the established high penetrance genes, particularly Lynch syndrome. This correlates with the axiom that, while most hereditary colorectal cancers occur at an early age, most early onset colorectal cancers are not hereditary.In our study, 3·3–5·6% of patients had hereditary colorectal cancers, which is similar to estimates from the traditional high incidence countries. The prevOur findings have implications for screening for Lynch syndrome in clinical practice in low resource settings. International guidelines recommend universal screening for mismatch repair deficiency of all colorectal cancers occurring below the age of 70 years., 20 UnivBRAF V600E screening may not be useful in sub-Saharan Africa. The 3 cases that were MSI-H, MLH1 deficient and BRAF V600E negative were all found to be sporadic after sequencing. The 4th case had phenotypic features of Lynch syndrome, and the BRAF V600E result would have been inconsequential in clinical practice. A retrospective study in Ghana found no BRAF mutations in 88 unselected cases, despite a high proportion of MSI-H tumours.[MLH1 hypermethylation promoter testing in African populations require further confirmation in a hypothesis driven study. An analogy can be made drawn with the Chinese population, where a low frequency of BRAF mutations was noted, and analysis for MLH1 hypermethylation was shown to be more efficient for triaging cases for further genetic analysis.[MLH1 hypermethylation in our study because the number of cases was too small to justify the added cost. Moreover, next generation sequencing was performed in all the cases that may have required MLH1 hypermethylation analysis.In contrast, our findings suggest that  tumours. Taken toanalysis. We did nATM, SMAD3, or high penetrance syndromes primarily associated with other cancers such as BRCA1 and BRCA2, which were not part of our panel. For example, one of our participants, who had a prior history of breast cancer, and a family history of breast and gastrointestinal cancers could have a BRCA 1 or 2 mutation. It is increasingly recognised that BRCA 1 or 2 mutations are associated with colorectal cancer, although this may not be causal.[Our study had limitations which must be taken into account when interpreting the findings. First, we used a phenotype guided strategy to select cases for sequencing to reduce costs. Consequently, we may have missed some inherited colorectal cancer syndromes, and our findings must be regarded as the minimum possible prevalence. However, it is unlikely that we underestimated the prevalence of Lynch syndrome, which we screened for comprehensively using a combination of immunohistochemistry, MSI testing, MLPA, and next generation sequencing. Second, it is possible that we missed pathogenic germline variants in moderate penetrance colorectal cancer genes such as e causal. Third, nHowever, this is the first prospective study to comprehensively evaluate the contribution, and patterns of inherited colorectal cancer syndromes in the poorly researched populations of sub-Saharan Africa. This study had comprehensive phenotypic data, and was based on an unselected population from both rural and urban areas. Thus, it can be considered representative of patients with colorectal cancer in sub-Saharan Africa. It forms a solid basis for larger studies, covering an even wider array of colorectal cancer susceptibility genes, and for context-specific clinical guideline development. As the immediate next step, it would be reasonable to consider performing whole exome sequencing for both tumour and germline tissue in individuals under the age of 50 years to more fully explore the involvement of other genes in early onset of colorectal cancers in our population.To conclude, at least 1 in 30 patients with colorectal cancer in our population have Lynch syndrome. Identifying cases of inherited colorectal cancer and instituting screening among family members can have a quantifiable impact on the incidence in low prevalence countries. The use of a detailed family history, complemented by screening for Lynch syndrome using immunohistochemistry should be encouraged in these countries, where genomic technologies are unavailable in clinical practice. Ultimately, the goal should be to screen all young patients for Lynch syndrome in these countries using immunohistochemistry, and to intensify research into the causes of colorectal cancer in this demographic group. Our study demonstrates the feasibility of next generation sequencing in low- and medium-income countries, and hopefully these will eventually cascade into clinical practice as the costs continue to fall. Lastly, the majority of colorectal cancers in young people in Zimbabwe, and probably sub-Saharan Africa are not due to Lynch syndrome. This provides a unique opportunity for collaborative research into early onset colorectal cancer, which is an emerging global challenge.S1 File(XLSX)Click here for additional data file.


Retinal ganglion cells (RGCs) extend axons out of the retina to transmit visual information to the brain. These connections are established during development through the navigation of RGC axons along a relatively long, stereotypical pathway. RGC axons exit the eye at the optic disc and extend along the optic nerves to the ventral midline of the brain, where the two nerves meet to form the optic chiasm. In animals with binocular vision, the axons face a choice at the optic chiasm—to cross the midline and project to targets on the contralateral side of the brain, or avoid crossing the midline and project to ipsilateral brain targets. Ipsilaterally and contralaterally projecting RGCs originate in disparate regions of the retina that relate to the extent of binocular overlap in the visual field. In humans virtually all RGC axons originating in temporal retina project ipsilaterally, whereas in mice, ipsilaterally projecting RGCs are confined to the peripheral ventrotemporal retina. This review will discuss recent advances in our understanding of the mechanisms regulating specification of ipsilateral versus contralateral RGCs, and the differential guidance of their axons at the optic chiasm. Recent insights into the establishment of congruent topographic maps in both brain hemispheres also will be discussed. The vertebrate retina is composed of six major neuronal cells types: the photoreceptors (rods and cones), located in the outer nuclear layer, that respond to light, and the retinal interneurons , located predominately in the inner nuclear layer that modify and relay the visual information from the photoreceptors to the retinal ganglion cells (RGCs) located mainly in the RGC layer at the inner surface of the retina A. RGCs, En route to their targets, all RGC axons first have to navigate into the optic fibre layer at the inner surface of the retina where they extend in a highly directed fashion towards the optic disc, their exit point from the eye. The axons then extend in the optic nerves towards the ventral midline of the diencephalon , where the two nerves meet to form the optic chiasm. In species such as chicken and zebrafish where the two eyes have no visual overlap, all axons cross the midline at the optic chiasm to project to targets on the opposite  side of the brain. However, in species with binocular overlap in the visual field, such as mice and ferrets, distinct subsets of axons project to targets on both the same  and contralateral side of the brain . From thThe proportion of RGC axons that project ipsilaterally at the optic chiasm varies widely between species and is related directly to the position of the eyes in the head, and consequently the degree of binocular overlap in the visual field . In humaIn this manuscript we review our current understanding of the mechanisms controlling the specification of ipsilaterally and contralaterally projecting RGCs and the molecular mechanisms that guide the axons of these cells to their main targets in the brain. Recent work providing insights into the co-ordination of visual map formation on both sides of the brain also will be discussed.Retinal progenitors are multipotent and give rise to all of the neuronal cell types within the vertebrate retina. The different cell types are generated in overlapping waves, with RGCs the first cell type generated B 14,15,,15,16,17Sfrp1 and Sfrp2 the retina is abnormally thick and contains an overabundance of early generated retinal cell types and fewer later born neurons ,3029,30] neurons . Conversll types . Togethell types ,35.dicer mutants markers of late generated progenitors are not expressed, late generated cells types are not produced and generation of early cell types, such as RGCs, is increased due to a prolonged period of genesis. Key miRNAs important for the early-to-late switch in retinal progenitor cell competence are let-7, microRNA-125 and micro-RNA-9. Ectopic early expression of these microRNAs accelerates the generation of late-born retinal cell types, whereas blocking function of these miRNAs inhibits production late generated cell types [Temporal differences in retinal progenitor cell competence are driven by intrinsic changes in the progenitor cells, with progenitor cells present at early embryonic stages expressing distinct sets of transcription factors from those present during late embryonic/postnatal development ,27. Micrll types ,27,28,36Atoh7 (Math5) is necessary but not sufficient for commitment to a RGC fate. Atoh7-expressing cells give rise to multiple retinal cell types, and only a small percentage of Atoh7-positive progenitors (~11%) differentiate into RGCs. Moreover, RGC can be generated by Atoh7-negative progenitors, particularly at early stages of development [Atoh7 (Math5) lack over 80% of RGCs, with cells instead adopting an amacrine or cone photoreceptor fate [Atoh7 is required both cell autonomously and in neighbouring cells to establish RGC competence, with other intrinsic and extrinsic factors required for commitment to the RGC fate. Nearly 1500 transcripts that are expressed differentially between Atoh7-positive and -negative progenitors have been identified, and encode for factors involved in a diverse range of processes including neuronal differentiation and development, cell migration, neuron projection, and neurotransmission [Atoh7 include the transcription factors Brn3b (Pouf42), and Islet-1 (Isl1) which act in parallel pathways to regulate morphological differentiation and survival of RGCs , a receptor for Sonic hedgehog (Shh). antially . Loss ofantially ,78. Howeing RGCs .Ten-M2 fewer RGC axons than normal project ipsilaterally. The number of Zic-2-positive RGCs is not altered obviously in the absence of Ten-M2, but expression of EphB1 is decreased substantially within peripheral ventral retina. These finding suggest that Ten-M2 controls axon laterality at the chiasm by regulating EphB1 expression in peripheral ventral retina [EphB1 expression.Another potential downstream effector of Zic2 is Ten-M2, a member of the highly conserved Ten-m/Odz/Teneurin family of transmembrane glycoproteins. In mice Ten-M2 is expressed throughout the RGC layer, and in mice lacking l retina . HoweverIsl2 results in an increase in ipsilateral projections that originate specifically in ventrotemporal retina. Associated with this, the number of Zic2-positive RGCs in ventrotemporal retina is increased in Isl2 mutants. Based on these findings it has been proposed that Isl2 acts to repress ipsilateral identity in Isl2-expressing RGCs located in ventrotemporal retina rather than actively specifying contralateral identity [Sox4, Sox11 and Sox12, have been identified as key regulators of contralateral RGC differentiation and specification -binding isoforms of vascular endothelial growth factor (VEGF-A) also help promote contralateral growth at the mouse optic chiasm. Through alternative splicing, the ugh NRP1 ,92. VEGFc chiasm . In contc tracts . FurtherNrp1 mutants [Ephb1 mutants in which many ipsilaterally-specified RGC axons are misrouted into the contralateral optic tract [Interestingly, although NRP1 is not expressed by ipsilaterally projecting RGCs , both th mutants . The lac mutants , and in  mutants ,95. Moreic tract . Accordiic tract ,10,96.Nrp1a is expressed by zebrafish RGC axons as they extend towards and through the chiasm midline, whereas Sema3D is expressed in the ventral diencephalon. Morpholino knockdown of Nrp1a or Sema3d results in some axons aberrantly projecting ipsilaterally [Sema3d also results in aberrant ipsilateral projections, suggesting that the precise level of Sema3D is important for mediating midline crossing [Islr2, which is expressed normally by RGCs, develop ectopic ipsilateral projections. However, mice lacking Islr2 do not display obvious defects in axon crossing at the optic chiasm, but exhibit defects in axon fasciculation and an increase in RGC axons projecting into the contralateral optic nerve [NRP1 also is required for contralateral growth of RGC axons in zebrafish which normally have a purely crossed chiasmatic projection. Interestingly, in this species the identified NRP1 ligand is a member of the class 3 semaphorin family. aterally ,98. Overic nerve .Dicer in the retina and ventral forebrain, the proportion of axons projecting ipsilaterally is increased substantially. RGC axons also make other guidance errors in conditional Dicer mutants, including increased projections into the contralateral optic nerve, and wandering away from the optic pathway into aberrant regions of the brain. Zic2 localisation is not altered obviously in the retina of conditional Dicer mutants, indicating that miRNAs are likely involved in regulating the guidance of RGC axons rather than specification of ipsilaterally or contralaterally projecting RGCs [miRNAs also are important for regulating contralateral growth at the mouse optic chiasm. In conditional mouse mutants lacking ing RGCs . HoweverThe main RGC recipient nuclei in the brain are the dorsal lateral geniculate nucleus and the superior colliculus A. The suThe superior colliculus receives projections from 85% to 90% of RGCs in mice  and is oEphB1 and EphB2 are expressed in a high ventral to low dorsal gradient in the retina and ephrinB1 in a high medial to low lateral gradient in the superior colliculus. In mice lacking EphBs or ephrinBs ectopic termination zones are formed, lateral to the appropriate position [The most studied mechanisms by which RGC axons sort to form a topographic map in the superior colliculus is the combination of Eph/ephrin signaling and patterned spontaneous retinal activity ,114. Forposition ,128. Depposition . Moreovein vivo does not affect neuronal identity specification or axon pathfinding during the development of topographic maps, but impairs axon branching and pruning once axonal RGCs growth cones reach their correct topographic position in the target tissues [RGC axon terminals undergo an extensive refinement at postnatal stages in a process depending on spontaneous activity generated in the retina before eye opening to form appropriate topographic and eye-specific maps . Ipsilat tissues . In addi tissues .Spontaneous activity originating in the retina is propagated throughout the visual system and results in corresponding waves of activity in the superior colliculus. Although thought to originate autonomously in each eye, a subset of waves propagates in a highly temporally and spatially correlated manner in both hemispheres of the superior colliculus. One potential function proposed for this synchronisation of activity is to ensure bilateral congruent refinement of RGC arbors within the colliculus . A direcUnc5c or following specific knockdown of Unc5c in the retina, the number of axons projecting into the contralateral optic nerve is decreased substantially. Conversely, ectopic expression of Unc5c in RGCs is sufficient to drive axon growth towards the opposite eye. Unc5c is expressed specifically by contralaterally projecting RGCs and expression of Unc5c appears to be negatively regulated by Zic2. In Zic2 hypomorphic mutants the Unc5c expression domain is expanded, whereas overexpressing Zic2 in the retina decreases Unc5c expression. Moreover, RGCs that express Zic2 do not project into the contralateral optic nerve [Slit and dicer mutants or mice lacking specific heparan sulphate synthetic enzymes, the proportion of RGC axons projecting to the opposite eye is increased substantially [Active guidance mediated by Netrin-1 expressed ventrally at the chiasm and Unc5c expressed in ventronasal RGCs prevents a subset of RGC axons from entering the optic tracts, and instead drives them into the contralateral optic nerve. In mice lacking ic nerve . Other gantially ,148,149.Unc5c expression, the presence of retina-retina projections and the extent of axonal refinement in visual targets supports this idea. In zebrafish, in which RGC axons project directly to their final location in the tectum, independent of activity, a retina-retina projection is not evident and Unc5c is not detected in the retina. In contrast, mice, chicken and ferrets, in which activity-dependent axonal refinement is crucial for establishing RGC axon termination zones, all have axons that project to the opposite retina, and the size of this projection correlates with the extent of Unc5c expression in the retina [In silico modelling supports the idea that retina-retina projections are important for the bilateral co-ordination of retinal waves in species where refinement depends upon spontaneous activity. When the size of the target tissue is larger than that of the retina the models indicate that bilateral symmetry of retinotopic mapping depends on synchronisation of activity in both maps C. It reme retina . Taken aConsiderable progress has been made in understanding the mechanisms that specify ipsilaterally versus contralaterally projecting RGCs, and that guide growth of their axons to targets on both sides of the brain. However, it is clear that much remains to be discovered. For example, recent microarray gene profiling of ipsilaterally versus contralaterally projecting RGCs identified over 300 genes that are differentially expressed between these two cell populations , the fun


Adults with poorer peripheral hearing have slower phonological processing speed measured using visual rhyme tasks, and it has been suggested that this is due to fading of phonological representations stored in long-term memory. Representations of both vowels and consonants are likely to be important for determining whether or not two printed words rhyme. However, it is not known whether the relation between phonological processing speed and hearing loss is specific to the lower frequency ranges which characterize vowels or higher frequency ranges that characterize consonants. We tested the visual rhyme ability of 212 adults with hearing loss. As in previous studies, we found that rhyme judgments were slower and less accurate when there was a mismatch between phonological and orthographic information. A substantial portion of the variance in the speed of making correct rhyme judgment decisions was explained by lexical access speed. Reading span, a measure of working memory, explained further variance in match but not mismatch conditions, but no additional variance was explained by auditory variables. This pattern of findings suggests possible reliance on a lexico-semantic word-matching strategy for solving the rhyme judgment task. Future work should investigate the relation between adoption of a lexico-semantic strategy during phonological processing tasks and hearing aid outcome. Hearing impairment is a highly prevalent neurological condition that is associated with changes in brain organization, i.e., neural plasticity . This apPlasticity associated with hearing loss is found not only in the auditory pathway, including primary auditory cortex  and audiLong-term memory underpins language processing. To achieve understanding of a spoken message, the incoming speech signal has to be mapped onto knowledge representations stored in long-term memory , 2019. PHearing impairment contributes to adverse listening conditions by making the speech signal weaker and reducing its spectral and temporal fidelity . AlthougConsidering the key role of phonology in speech understanding, it is not surprising that hearing impairment is associated with changes in phonological processing ability. This applies both to individuals with severe hearing impairment . and to Phonological processing ability is typically measured using a rhyme judgment task in which participants are asked to compare the sound of different words . Visual Using a visual rhyme judgment task, Rhyme judgment requires the activation of phonological representations relating to the final syllables of the relevant words. These representations reflect the sublexical patterning of phonemes including consonants and vowels. A double dissociation of the processing of consonants and vowels in neuropsychological patients shows that these are distinct categories of speech sounds . Vowels The purpose of the present study is to investigate the relationship between phonological processing skills and low and high frequency hearing loss in adults with mild-to-severe hearing impairment. We predict that better phonological processing ability will be associated with better thresholds in both low and high frequency ranges reflecting the relative preservation of both vowel and consonant representations. Different impact of consonant and vowel cues during phonological processing will be indicated by a differential contribution of low and high frequency ranges. We also predict that speed of access to representations in long-term memory will account for additional variance in phonological processing skills along with working memory capacity, especially under mismatch conditions.The data for the current study were obtained from the N200 database  under a SD = 8.6 years) were included in the present study. They were all experienced hearing aids users with bilateral, symmetrical sensorineural hearing loss in the range mild-to-severe and were recruited to the longitudinal N200 study from the patient population at the Linköping University Hospital. They all had normal or corrected-to-normal vision and were native Swedish speakers. Data for all participants who had completed the rhyme judgment task at the first time point of the longitudinal N200 study are included in the present study.212 individuals  with a mean age of 60.7 years  and the better ear PTA dB across 2000, 3000, and 4000 Hz for consonant frequency . We acknowledge that the chosen vowel frequencies do not include the higher formant frequencies for front or rounded vowels. As the frequencies 6000 and 8000 may also contribute to the ability to distinguish consonants, we calculated a third better ear PTA dB across these two high frequencies . Mean better ear PTA in the three frequency ranges is reported in Pure tone audiometry (air-conduction thresholds) was performed in accordance with ISO/IEC 8253 (2010) on all participants in both ears at the frequencies 250, 500, 1000, 2000, 3000, 4000, 6000, and 8000 Hz. We were interested in the contribution of hearing acuity in the vowel and consonant frequency ranges. Vowels are identified most accurately by their first two formant frequencies, with F2 being the more important. Back vowels are likely to have second formant frequencies below 1,000 Hz while front vowels have second formant frequencies above 1,000 Hz, with some above 2,000 Hz. Swedish also has some rounded vowels which rely on third formant frequencies above 2,000 Hz. This means that there is an overlap between the lower frequencies that distinguish vowels and the higher frequencies in the range of 2,000 to 4,000 Hz which distinguish consonants. In order to optimize separation of frequencies distinguishing vowels and consonants while keeping constant the number of data points used, we calculated the better ear pure tone average (PTA) dB across 250, 500 and 1000 Hz for vowel frequency  and how many years they had used hearing aids . See Participants also reported number of years with hearing problems R+O+. Words that rhyme and are orthographically similar, e.g., FRITT/VITT .(2)R+O−. Words that rhyme but are not orthographically similar, e.g., KURS/DUSCH .(3)R-O+. Words that do not rhyme but are orthographically similar, e.g., TAGG/TUGG .(4)R−O−. Words that neither rhyme nor are orthographically similar, e.g., HÄST/TORN .There are eight trials in each category. Button-press responses are given and the dependent variable is the mean response time for correct decisions for each of the four different trial types. Previous work has shown that it is harder to solve mismatching  than matching  trials  and thusIn the physical matching test, two printed letters are presented simultaneously on a computer screen and the participant’s task is to determine whether or not they are identical. Button press responses are given. There are 16 trials equally divided between true and false, and the dependent variable is the mean response time for correct trials. This is a measure of cognitive processing speed.In the lexical decision task, strings of three letters (two consonants and one vowel) are presented on a computer screen and the participant’s task is to determine whether or not the string constitutes a Swedish word. The strings are Swedish words, Swedish pseudowords  or Swedish non-words . Button press responses are given. There are forty trials evenly distributed between true and false with pseudowords and non-words occurring equally often across false trials. The dependent variable is the mean response time for correct trials. This is a measure of lexical access speed.The reading span test  is a tesThe data for the present study were collected at one and the same session lasting between 2 and 3 h. This was the first of three testing sessions, constituting the first time point of the longitudinal N200 study see .All analyses were done using R  = 498.53, MSE = 41,119.68, p < 0.001, η2 = 0.221, where accuracy, as expected, was greater under match compared to mismatch conditions . As rhyme judgment accuracy approached 100% in the match conditions, meaning that variance was skewed, response time was used as an index of phonological processing ability in the correlation and regression analyses. The response time ANOVA also showed a main effect of condition F = 236.12, MSE = 0.02, p < 0.001, η2 = 0.332, where responses, again as expected, were faster under match compared to mismatch conditions . Together, these two ANOVAs confirm that the rhyme judgment task was solved more accurately and faster under match compared to mismatch conditions.The ANOVA of rhyme judgment accuracy (proportion correct) scores showed a main effect of condition, r = 0.12, p = 0.09; Match r = 0.13, p = 0.06. To determine the relative contribution of age, auditory and cognitive variables to variance in rhyme judgment performance under match and mismatch conditions regression analyses were performed.Correlations are shown in 2 of 0.45. For the match condition, lexical access speed and reading span both contributed to the model with adjusted R2 of 0.55. The final models are presented in Robust regression analysis with stepwise elimination was performed separately for rhyme judgment performance under match and mismatch conditions. Mean response time was the dependent variable. The variables included in the regression were age; hearing thresholds at vowel, consonant and high frequencies, duration of hearing problems and hearing aid use as well as cognitive variables including reading span, lexical access speed and cognitive processing speed. The same remaining variables were found on all five imputed datasets. Therefore, pooled estimates over all datasets are presented. For the mismatch condition, only lexical access speed was a significant predictor, and that model had an adjusted RIn the present study, we tested the phonological processing abilities of 212 individuals with varying degrees of hearing impairment in the range mild-to-severe taking part in the N200 study . We did The visual rhyme judgment task used in the present study included four different types of trials that fall into two categories: mismatch  and match . There were 16 trials in each category. This is fewer than in some previous studies. For example, the study by In line with previous work studying individuals with severe hearing impairment , resultsIn the present study, the associations between phonological processing speed and variables of interest including lexical access speed, hearing thresholds and reading span were tested separately for match and mismatch conditions using Pearson’s correlations. Phonological processing speed was strongly associated with lexical access speed and cognitive processing speed. Both these associations were positive showing that individuals with faster lexical access and cognitive processing speed also have faster phonological processing. Associations between phonological processing speed and hearing thresholds were surprisingly weak, considering our predictions, suggesting that phonological representations do not contribute to performance on the rhyme judgment task above and beyond lexical access speed. This is only likely to be the case if a non-phonological strategy is adopted. There was a moderate negative association with reading span (a measure of working memory capacity) showing that faster phonological processing is associated with greater working memory capacity, in line with our prediction.Regression analysis resulted in two models explaining variance in phonological processing speed. One model related to mismatch conditions and showed that variance was largely explained by lexical access speed, but that there was no contribution of auditory measures; this was not surprising, considering the pattern of correlations. Importantly, although both lexical access speed and cognitive speed correlated with phonological processing speed, cognitive speed was not retained in the final regression model relating to mismatch. This suggests that lexical access speed specifically, rather than cognitive processing speed in general, is important for phonological processing in the sample studied here, in line with our prediction. Surprisingly, reading span did not contribute above and beyond lexical access speed to the model pertaining to mismatch conditions. The other model related to match conditions and showed that variance was, again, largely explained by lexical access speed, but that reading span also contributed. Recently, Brain plasticity in connection with hearing impairment is both a threat and an opportunity, and auditory rehabilitation should target neural organization associated with good hearing health . ActivatOne way of maintaining a phonological strategy in preference to a lexico-semantic strategy during word processing may be targeted phonological training . FergusoIn conclusion, in a group of 212 adults with hearing impairment, we found evidence that auditory measures tested were only very weakly correlated with performance on a visual rhyme judgment task and once lexical access speed was accounted for, auditory measures did not explain additional variance in visual rhyme judgment performance. We tentatively propose that this finding may be explained by reliance on a lexico-semantic word-matching strategy during visual rhyme judgment, making phonological representations redundant or even a source of confusion. Future work should investigate possible neural plasticity in left-hemisphere phonological processing networks of individuals with early-stage hearing impairment and how it can be shaped to promote good hearing health. It would also be beneficial to retest the original prediction that auditory factors would correlate with better performance in rhyme judgment tasks by using a more appropriate set of frequencies to test for consonant and vowel perception.This study was carried out in accordance with Swedish legislation. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Regional ethics review board, Linköping, Sweden.MR conceived the article and wrote the first draft. MR, HD, BL, TL, and JR designed the study and contributed to the final version of the article. HD performed the statistical analyses.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.