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Summary Maintenance of a healthy photoreceptor-retinal pigment epithelium (RPE) interface is essential for vision. At the center of this interface, apical membrane protrusions stemming from the RPE ensheath photoreceptor outer segments (POS), and are possibly involved in the recycling of POS through phagocytosis. The molecules that regulate POS ensheathment and its relationship to phagocytosis remain to be deciphered. By means of ultrastructural analysis, we revealed that Mer receptor tyrosine kinase (MERTK) ligands, GAS6 and PROS1, rather than αVβ5 integrin receptor ligands, triggered POS ensheathment by human embryonic stem cell (hESC)-derived RPE. Furthermore, we found that ensheathment is required for POS fragmentation before internalization. Consistently, POS ensheathment, fragmentation, and internalization were abolished in MERTK mutant RPE, and rescue of MERTK expression in retinitis pigmentosa (RP38) patient RPE counteracted these defects. Our results suggest that loss of ensheathment due to MERTK dysfunction might contribute to vision impairment in RP38 patients., Graphical Abstract, Highlights • POS are ensheathed in vitro by human embryonic stem cell-derived RPE • POS ensheathment is upregulated by MERTK ligands: GAS6 and PROS1 • αVβ5 integrin receptor ligands do not stimulate POS ensheathment • MERTK is essential for POS ensheathment and fragmentation before internalization, Using ultrastructural scanning electron microscopy (SEM)-based analysis, Almedawar et al. show that MERTK ligands, GAS6 and PROS1, upregulate photoreceptor outer segments (POS) ensheathment in human embryonic stem cell-derived retinal pigment epithelial cells. The authors also demonstrate that MERTK is essential for POS ensheathment and fragmentation before internalization, and suggest that ensheathment defects contribute to the pathology of retinitis pigmentosa type 38.

Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.

Background and Aims Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of intrahepatic interferon-γ- (IFNγ) producing lymphocytes have been documented in patients with PSC, yet their functional role remains to be determined. Methods Liver tissue samples were collected from patients with PSC. The contribution of lymphocytes to liver pathology was assessed in Mdr2−/− x Rag1−/− mice, which lack T and B cells, and following depletion of CD90.2+ or natural killer (NK)p46+ cells in Mdr2−/− mice. Liver pathology was also determined in Mdr2−/− x Ifng−/− mice and following anti-IFNγ antibody treatment of Mdr2−/− mice. Immune cell composition was analysed by multi-colour flow cytometry. Liver injury and fibrosis were determined by standard assays. Results Patients with PSC showed increased IFNγ serum levels and elevated numbers of hepatic CD56bright NK cells. In Mdr2−/− mice, hepatic CD8+ T cells and NK cells were the primary source of IFNγ. Depletion of CD90.2+ cells reduced hepatic Ifng expression, NK cell cytotoxicity and liver injury similar to Mdr2−/− x Rag1−/− mice. Depletion of NK cells resulted in reduced CD8+ T cell cytotoxicity and liver fibrosis. The complete absence of IFNγ in Mdr2−/−x Ifng−/− mice reduced NK cell and CD8+ T cell frequencies expressing the cytotoxic effector molecules granzyme B and TRAIL and prevented liver fibrosis. The antifibrotic effect of IFNγ was also observed upon antibody-dependent neutralisation in Mdr2−/− mice. Conclusion IFNγ changed the phenotype of hepatic CD8+ T cells and NK cells towards increased cytotoxicity and its absence attenuated liver fibrosis in chronic sclerosing cholangitis. Therefore, unravelling the immunopathogenesis of PSC with a particular focus on IFNγ might help to develop novel treatment options. Lay summary Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and fibrosis, whose current medical treatment is hardly effective. We observed an increased interferon (IFN)-γ response in patients with PSC and in a mouse model of sclerosing cholangitis. IFNγ changed the phenotype of hepatic CD8+ T lymphocytes and NK cells towards increased cytotoxicity, and its absence decreased liver cell death, reduced frequencies of inflammatory macrophages in the liver and attenuated liver fibrosis. Therefore, IFNγ-dependent immune responses may disclose checkpoints for future therapeutic intervention strategies in sclerosing cholangitis.

Purpose Speech in persons who stutter (PWS) is associated with disturbed prosody (speech melody and intonation), which may impact communication. The neural correlates of PWS’ altered prosody during speaking are not known, neither is how a speech-restructuring therapy affects prosody at both a behavioral and a cerebral level. Methods In this fMRI study, we explored group differences in brain activation associated with the production of different kinds of prosody in 13 male adults who stutter (AWS) before, directly after, and at least 1 year after an effective intensive fluency-shaping treatment, in 13 typically fluent-speaking control participants (CP), and in 13 males who had spontaneously recovered from stuttering during adulthood (RAWS), while sentences were read aloud with ‘neutral’, instructed emotional (happy), and linguistically driven (questioning) prosody. These activations were related to speech production acoustics. Results During pre-treatment prosody generation, the pars orbitalis of the left inferior frontal gyrus and the left anterior insula were activated less in AWS than in CP. The degree of hypo-activation correlated with acoustic measures of dysprosody. Paralleling the near-normalization of free speech melody following fluency-shaping therapy, AWS normalized the inferior frontal hypo-activation, sooner after treatment for generating emotional than linguistic prosody. Unassisted recovery was associated with an additional recruitment of cerebellar resources. Conclusions Fluency shaping therapy may restructure prosody, which approaches that of typically fluent-speaking people. Such a process may benefit from additional training of instructed emotional and linguistic prosody by inducing plasticity in the inferior frontal region which has developed abnormally during childhood in PWS.

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.

For children who stutter (CWS), there is good evidence of the benefits of treatment for pre-school age, but an evidence gap for elementary school age. Here we report on the effectiveness of a fluency shaping treatment for 6- to 9-year-old children. The main treatment component is the reinforcement of soft voice onsets. An intensive in-patient group treatment phase lasts 6 days, followed by a 6-month maintenance phase with 3 in-patient weekend group refresher courses. Child and a parent participate together in various treatment activities. In this controlled intervention study (waitlist control, intention-to-treat design) assessments were performed before treatment (T1), 4 weeks after the intensive phase (T2), at the end of the maintenance phase (T3), and 1 year later (T4). Participants were 119 children (108 boys, 11 girls, age 5.5‑10.4 years). Control conditions included a subgroup with delayed treatment (N=25) as well as the assessment of complexity of utterances, inter-rater reliability, and speech naturalness. From before treatment to 1-year follow-up, percent stuttered syllables and OASES-S (Overall Assessment of the Speaker's Experience with Stuttering - School-age) scores decreased with large effect size. Speech naturalness improved during this period but did not reach the level of non-stuttering children. Complexity of utterances increased during the intensive phase, but only temporarily. Twenty children (16.8 %, including dropouts) showed no demonstrable treatment benefit. Fluency shaping treatment can be effectively applied to young school children. It is assumed that parental support, group therapy, intensive treatment, and regular exercises at home are essential.

Unfavourable climatic conditions force Iranian beekeepers to translocate over large distances in the course of the year. However, irrespective of the main place of production, the honey is always labeled with the name of the beekeepers' hometown, which leads consequently to mislabeled products. The present study investigates the capability of DNA metabarcoding to locate the geographical origin of honey. The molecular markers (ITS2 and rbcL) allowed identification of 926 plant species in studied samples. A comprehensive review of floristic reference books specified 34 key species that could be used to successfully determine the geographical origin in 91.4% of samples. These key species were usually present in honey with tiny amounts and thus, conventional palynology might not be able to detect them. The present investigation indicates that although ITS2 is able to detect more species than rbcL, utilizing a combination of both markers provides more robust evidence of geographical origin., Food science, Food analysis, Food technology, Molecular biology, Honey, Geographical origin, Metabarcoding, ITS2, rbcL

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5′ homology arm (HA) of 8 kb and 3′HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3′HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

Purpose Persons who stutter (PWS) should be referred to the most effective treatments available, locally or regionally. A prospective comparison of the effects of the most common stuttering treatments in Germany is not available. Therefore, a retrospective evaluation by clients of stuttering treatments was carried out. Method The five most common German stuttering treatments (231 single treatment cases) were rated as to their perceived effectiveness, using a structured questionnaire, by 88 PWS recruited through various sources. The participants had received between 1 and 7 treatments for stuttering. Results Two stuttering treatments (stuttering modification, fluency shaping) showed favorable and three treatments (breathing therapy, hypnosis, unspecified logopedic treatment) showed unsatisfactory effectiveness ratings. The effectiveness ratings of stuttering modification and fluency shaping did not differ significantly. The three other treatments were equally ineffective. The differences between the effective and ineffective treatments were of large effect sizes. The typical therapy biography begins in childhood with an unspecified logopedic treatment administered extensively in single and individual sessions. Available comparisons showed intensive or interval treatments to be superior to extensive treatments, and group treatments to be superior to single client treatments. Conclusion The stuttering treatment most often prescribed in Germany, namely a weekly session of individual treatment by a speech-language pathologist, usually with an assorted package of mostly unknown components, is of limited effectiveness. Better effectiveness can be expected from fluency shaping or stuttering modification approaches, preferably with an intensive time schedule and with group sessions. Educational objectives: Readers will be able to: (a) discuss the five most prevalent stuttering treatments in Germany; (b) summarize the effectiveness of these treatments; and (c) describe structural treatment components that seem to be preferable across different kinds of treatments.

The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic–nuclear distribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they participate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA. Using both green fluorescent protein (GFP) fusion- and immunology-based strategies, we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal–nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression.

Background and Aims Newborn hearing screening and early intervention for congenital hearing loss have created a need for tools assessing the hearing development of very young children. A multidisciplinary evaluation of children's development is now becoming standard in clinical practice, though not many reliable diagnostic instruments exist. For this reason, the LittlEARS ® Auditory Questionnaire (LEAQ) was created to assess the auditory skills of a growing population of infants and toddlers who receive hearing instruments. The LEAQ relies on parent report, which has been shown to be a reliable way of assessing child development. Results with this tool in a group of children who received very early cochlear implantation are presented. Methods The LEAQ is the first module of the LittlEARS ® comprehensive test battery for children under the age of two who have normal hearing (NH), cochlear implants (CIs) or hearing aids (HAs). The LEAQ is a parent questionnaire comprised of 35 “yes/no” questions which can be completed by parents in less than 10 min. Sixty-three children who received unilateral CIs at a young age were assessed longitudinally and their performance was compared to that of a NH group. Results All CI children reached the maximum possible score on the LEAQ on average by 22 months of hearing age, i.e. 38 months of chronological age. In comparison, the NH group reached the maximum score by 24 months of age demonstrating that auditory skills of CI children often develop quicker than those of NH children. In the two comparison groups of children aged (a) younger and older than 12 months, and (b) between 6–9 and 21–24 months at first fitting, the early implanted children reached the highest scores faster than the later implanted children. Furthermore, three children with additional needs were tested. They showed slower growth over time but also received benefits from early implantation. Conclusions The LEAQ is a quick and effective tool for assessing auditory skills of very young children with or without hearing loss. In our study, the auditory skills of children with CI progressed very quickly after implantation and were comparable with those of NH peers.

In order to investigate whether the Lidcombe Program effects a short-term reduction of stuttered speech beyond natural recovery, 46 German preschool children were randomly assigned to a wait-contrast group or to an experimental group which received the Lidcombe Program for 16 weeks. The children were between 3;0 and 5;11 years old, their and both of their parents’ native language was German, stuttering onset had been at least 6 months before, and their stuttering frequency was higher than 3% stuttered syllables. Spontaneous speech samples were recorded at home and in the clinic prior to treatment and after 4 months. Compared to the wait-contrast group, the treatment group showed a significantly higher decrease in stuttered syllables in home-measurements (6.9%SS vs. 1.6%SS) and clinic-measurements (6.8%SS vs. 3.6%SS), and the same increase in articulation rate. The program is considered an enrichment of currently applied early stuttering interventions in Germany. Educational objectives: Readers will discuss and evaluate: (1) the short-term effects of the Lidcombe Program in comparison to natural recovery on stuttering; (2) the impact of the Lidcombe Program on early stuttering in German-speaking preschool children.

Previous studies suggest that anatomical anomalies [Foundas, A. L., Bollich, A. M., Corey, D. M., Hurley, M., & Heilman, K. M. (2001). Anomalous anatomy of speech-language areas in adults with persistent developmental stuttering. Neurology, 57, 207-215; Foundas, A. L., Corey, D. M., Angeles, V., Bollich, A. M., Crabtree-Hartman, E., & Heilman, K. M. (2003). Atypical cerebral laterality in adults with persistent developmental stuttering. Neurology, 61, 1378-1385; Foundas, A. L., Bollich, A. M., Feldman, J., Corey, D. M., Hurley, M., & Lemen, L. C. et al., (2004). Aberrant auditory processing and atypical planum temporale in developmental stuttering. Neurology, 63, 1640-1646; Jancke, L., Hanggi, J., & Steinmetz, H. (2004). Morphological brain differences between adult stutterers and non-stutterers. BMC Neurology, 4, 23], in particular a reduction of the white matter anisotropy underlying the left sensorimotor cortex [Sommer, M., Koch, M. A., Paulus, W., Weiller, C., & Buchel, C. (2002). Disconnection of speech-relevant brain areas in persistent developmental stuttering. Lancet, 360, 380-383] could be at the origin of persistent developmental stuttering (PDS). Because neural connections between the motor cortex and basal ganglia are implicated in speech motor functions, PDS could also be associated with a dysfunction in basal ganglia activity [Alm, P. (2004). Stuttering and the basal ganglia circuits: a critical review of possible relations. Journal of Communication Disorders, 37, 325-369]. This fMRI study reports a correlation between severity of stuttering and activity in the basal ganglia and shows that this activity is modified by fluency shaping therapy through long-term therapy effects that reflect speech production improvement. A model of dysfunction in stuttering and possible repair modes is proposed that accommodates the data presented here and observations previously made by us and by others.

Neuroimaging studies have indicated that persistent developmental stuttering (PDS) may be associated both with an abnormality in white matter of left-hemispheric speech areas and a right-hemispheric hyperactivity. The latter may compensate for the deficient structural connectivity in the left hemisphere. To investigate the effects of stuttering therapy on brain activity nine male adults with PDS underwent functional magnetic resonance imaging (fMRI) before and within 12 weeks after fluency shaping therapy. Brain response differences during overt sentence reading before and after therapy were assessed by utilizing random effects analyses. After therapy, a more widespread activation was observed in frontal speech and language regions and temporal areas of both hemispheres, particularly and more pronounced on the left side. Interestingly, distinct posttreatment left-sided activation increases were located directly adjacent to a recently detected area of white matter anomaly [M. Sommer, M.A. Koch, W. Paulus, C. Weiller, C. Buchel (2002). Disconnection of speech-relevant brain areas in persistent developmental stuttering. The Lancet, 360, 380-383] suggesting that fluency shaping techniques reorganize neuronal communication between left-sided speech motor planning, motor execution, and temporal areas. Hence, a therapeutic mechanism can be assumed to remodel brain circuitry close to the source of the dysfunction instead of reinforcing compensation via homologous contralateral brain networks.The reader will learn about and be able to: (1) describe brain activation changes detected shortly after fluency-shaping therapy; (2) identify left-hemispheric regions where a (re)functionalization after fluency-shaping therapy seems to occur adjacent to a recently described abnormal white matter region in PDS subjects; and (3) discuss how an effective cerebral compensation mechanism for stuttering could work.

This article reviews some of our recent functional magnetic resonance imaging (fMRI) studies of stuttering. Using event-related fMRI experiments, we investigated brain activation during speech production. Results of three studies comparing persons who stutter (PWS) and persons who do not stutter (PWNS) are outlined. Their findings point to a region in the right frontal operculum (RFO) that was consistently implicated in stuttering. During overt reading and before fluency shaping therapy, PWS showed higher and more distributed neuronal activation than PWNS. Immediately after therapy differential activations were even more distributed and left sided. They extended to frontal, temporal, and parietal regions, anterior cingulate, insula, and putamen. These over-activations were slightly reduced and again more right sided two years after therapy. Left frontal deactivations remained stable over two years of observation, and therefore possibly indicate a dysfunction. After therapy, we noted higher activations in persons who stutter moderately than in those who stutter

Summary After 10 days of cultivation at 37 °C, the herbicide resistance encoded by the chimaeric pat 4l gene (coding region from Streptomyces viridochromogenes fused to the 823 bp CaMV35S promoter) was strongly reduced in all of the 27 independent transgenic Nicotiana tabacum SRI lines analyzed. This reversible reduction occurred in sterile and unsterile culture in the first and second generation and even when the overnight temperature was reduced to 24 °C. Neither the enzyme activity, the protein nor the pat 4l specific RNA could be detected in the heat treated plants, regardless of the number of copies and the hemior homozygous state. In contrast to this, the expression of the synthetic pat S coding region fused to the 534bp CaMV35S promoter and coding for essentially the same protein, was stable in heat treated plants. The exchange of the GC rich coding region of the pat 4l gene by the AT rich synthetic DNA fragment carrying the pat S coding region led to the stabilization of the specific RNA steady state level. However, the presence of the transgene-encoded protein at 37 °C could only be achieved by using specific 5′ and 3′ untranslated regions of the synthetic patS gene.