Hearing loss can have a major impact on children’s language development, academic success and hearing comprehension. The aim of the present study was to determinate risk factors for severe and profound hearing loss in child candidates for cochlear implantation in southeast of Iran during 2014–2020.This case-control study consisted of 400 children referring to a cochlear implant center (in southeastern Iran) from Bandar Abbas, Zahedan and Kerman during the years 2014–2020 as cases. The subjects were selected using the random sampling method; 200 children hospitalized in Shafa and Afzalipour hospitals were selected as controls.p < 0.05), hospitalization in NICU (OR = 7.21: p < 0.05), family history of hearing loss (OR = 11.47: p < 0.05), Gestational age over 35 (OR = 9.63: p < 0.05), intracranial hemorrhage (OR = 5.18: p < 0.05), consanguineous marriage (OR = 12.48: p < 0.05) and high fever and seizures (OR = 3.02: p < 0.05) were recognized as risk factors for sensorineural deafness in children.Based on the results of the multivariate logistic regression, weight less than 1500 g (OR = 4.40: Most of the risk factors for deafness are preventable, and hereditary factors play an important role in congenital deafness in children. Therefore, genetic counseling before consanguineous marriage, early diagnosis, timely intervention can prevent many cases of hearing loss in children. Hearing is known to be the main sensory access of human at the beginning of life which enables him to identify, pay attention, trace sounds and integrate essential hearing experiences to develop speech and language . The intThis case-control study aimed to elucidate the role of risk factors related to hearing loss and deafness in children. The case sample consisted of 400 children referring to a cochlear implant center (in southeastern Iran) from Bandar Abbas, Zahedan and Kerman during the years 2014–2020. The subjects were selected using the random sampling method; 200 children hospitalized in Shafa and Afzalipour hospitals were selected as hospital controls.Inclusion criteria for the case group were diagnosis of severe (not hearing sounds between 71 and 90 dB) and profound hearing loss (not hearing sounds more than 91 dB) by the physician or undergoing cochlear implantation.P-value of less than 0.05 was considered significant.The controls of this study consisted of children hospitalized in other wards or outpatient clinics of Shafa and Afzalipour hospitals who had no hearing loss or deafness at the time of the study and had no history of this disease in their past. Data was collected through face-to-face, telephone interviews and medical records using a questionnaire. This questionnaire contained information about age, gender, consanguineous marriage and family history that collected from interviews. The other information about medical care gathered from medical records. In order to comply with the principles of medical ethics, oral consent was obtained from patients that interviewed by telephone and written consent was obtained from patients interviewed through face-to-face. Univariate and multivariate logistic regression models were used to analyze the data, and the risk of hearing loss or deafness was expressed as odds ratio calculations and estimates (95% confidence). First, univariate regression was used; the significant variables were then entered into multivariate regression. Data was analyzed using SPSS software (version 24). In this study, 600 children were examined . Out of 400 children, 216 were boys (54.5%) and 184 were girls (46.0%), with the average age of 8.91 ± 4.39. In the control group, 93 were boys (46.5%) and 107 were girls (53.5%), with the average age of 6.73 ± 3.26.p > 0.05)) were significant factors affecting sensory-neurological deafness in children (Table p < 0.05), hospitalization in NICU (OR = 7.21: p < 0.05), family history of hearing loss (OR = 11.47: p < 0.05), Gestational age over 35 (OR = 9.63: p < 0.05), intracranial hemorrhage (OR = 5.18: p < 0.05), consanguineous marriage (OR = 12.48: p < 0.05) and high fever and seizures (OR = 3.02: p < 0.05) were recognized as risk factors for sensorineural deafness in children  . A systeThe Joint Committee on Infant Hearing has noticed family history of hearing loss as risk factor for permanent congenital, delayed onset, or progressive hearing loss since 1973 . In our Consanguinity is common in some regions of Asia and Africa because of socioeconomic, cultural, and religious factors. Historical studies report a strong preference consanguineous marriages in Iranians. Consanguineous marriage is an important risk factor of genetic disorders . Our stuOur study revealed that pregnancy at the age of 35 and higher is associated with increased risk of hearing loss in child. Leslie et al. also reported maternal age as a risk factor for hearing loss . HoweverA pontine intracranial hemorrhage (ICH) evokes several neurological symptoms, due to the various nuclei and nerve fibers; however, hearing loss from a pontine ICH is rare , howeverOur study also revealed that fever and seizures is associated with increased risk of hearing loss in children. This finding was similar to the results of studies by Karanja et al. . Murray Hereditary factors play an important role in congenital deafness in children especially in families with a family history of hearing loss and genetic counseling prior to consanguineous marriage, which is common in Asian countries, are crucial factors to be considered. In addition, early diagnosis, timely intervention, vaccination to prevent meningitis, increased birth care and preventing the use of ototoxic medications can prevent many cases of hearing loss in children and may dramatically reduce the cost of education related to hearing loss.The present study had some limitations. First, Incomplete medical records of some patients. Second Lack of parental responsibility and due to the data were collected retrospectively, which might cause to recall bias. Third, Change patients’ phone number so we couldn’t contact them.


USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas‐mediated gene repair or gene therapy in vitro. AAV‐based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.Usher syndrome (USH) is the most common form of monogenic deaf‐blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin‐encoding  The full phenotypic spectrum of Usher Syndrome is reflected in a pig model carrying a patient‐specific mutation after partial humanization of the porcine USH1C gene. Retinal function in USH1C pigs was improved when harmonin expression was reconstituted by AAV‐mediated gene therapy. Due to the lack of sufficient animal models, the mechanisms of vision loss in Usher syndrome (USH) are incompletely understood and therapies cannot be sufficiently tested. As a consequence, patients inevitably suffer blindness.A pig model for USH reflects the combination of deafness, vestibular dysfunction, and vision loss seen in USH patients. Visual impairment in USH1C pigs, as documented by ophthalmologic examinations and parameterized behavioral tests, is accompanied by morphologic changes in photoreceptor cells. Further, photoreceptor cilia and primary cilia of fibroblasts were consistently elongated in USH1C pigs and facilitated the testing of gene supplementation and gene correction as potential therapeutics. Exploratory gene therapy experiments prove the amelioration of visual function in USH1C pigs.in vivo, at the cellular and the molecular level. Our achievements will promote future research in two relevant ways: (i) reversible changes in primary cilia length make fibroblasts a simple, cheap and robust in vitro evaluation tool for testing treatment options in USH. (ii) The consistent multi‐disciplinary examination of the ocular phenotype paves the way for a comprehensive evaluation of vision in pigs and therefore towards a more reliable judgment of pre‐clinical therapy studies in the USH1C pig model in vivo.The USH1C pig is the first animal model for studying the ocular component of the Usher syndrome et al, et al, et al, Usher syndrome (USH) is the most common form of inherited deaf‐blindness in humans  domain  documented severe hearing deficits in USH1C pigs as early as 3 weeks and up to 2 years of age, indicated by a lack of response even upon click stimulus at sound pressure levels of up to 120 dB sound pressure level (SPL) Figs . The finges Figs E and F. ges Figs . Most ofges Figs . ConsistClinical examination of the eyes in USH1C pigs at 1 year of age revealed changes comparable to early stages of USH in human patients. A reduction of cone photoreceptor function was evident in the central visual streak area analyzed by multifocal electro‐retinography ERG, Fig B and C. USH1C/harmonin expression, particularly in the mouse retina, were inconsistent but indicated localization of harmonin in PRC, MGC, and secondary retinal neurons. , whirlin (encoded by WHRN (USH2D)), and myosin VIIa (encoded by MYO7A (USH1B)) in the retina  for transducing cells of the retina 10 vg/eye) of established AAV8, AAV9, or a modified Anc80 vectors, each of them expressing eGFP under control of a CMV promoter , isolated from USH1C founder animals . Accordingly, the phenotype in USH1C pigs appears to reflect patients well within the published spectrum of disease severity and progression. As a consequence, we would also suppose that translating clinically established endpoints  from human to pig will support the assessment of future pre‐clinical studies in our model. Vice versa, the multi‐disciplinary examination of the model described here defined novel disease hallmarks, which may give rise to alternative endpoints for future clinical trials. The value of additional read‐out parameters such as motion analysis  as primary endpoint . Multi‐species alignments were done by a combination of DiAlign and CHAOS  and a density plot was created. Both, a homology plot at nucleic acid level and a protein conservation plot were calculated in BioEdit on the basis of the nucleotide summary assessment and entropy calculation functions, respectively. Coverage maps of the augmented alignment were plotted by JalView  and purchased from CHORI BACPAC Resources Center . A sequence comprising a 278 bp 5′‐arm, the 1,524 bp humanized fragment including the TGA stop codon in exon 2 and 3 intronic SNPs as well as a 278 bp 3′‐arm were commercially synthesized and provided in a pUC plasmid derivate by GeneArt . A cassette with a PGK/EM7 driven neo gene, providing neomycin resistance in mammalian cells and kanamycin resistance in bacteria . Single cell clones were generated as described  and azaperone 40 mg/ml , according to the manufacturer’s specifications. Fully anesthetized animals were euthanized by intravenous injection of T61® . Pig primary kidney cells were isolated according to standard procedures  and raised for breeding purposes. After reaching fertility, F1 boars were mated to their mothers and sisters for producing USH1C‐null pigs and with wild‐type sows to broaden the genetic background of the future breeding herd. Some litters from hom x het matings were with their mother for suckling, but piglets were kept in a separate box otherwise. Upon calling by the mother, the barrier between the boxes was opened and the time until piglets passed the gate was determined. Non‐responding piglets were nudged by the caretaker after one minute to avoid impaired nurturing.neo selection cassette was detected by endpoint PCR with usharm2f – ush5arm2r and sequencing of the amplicon while Cre‐mediated excision of neo was detected with neoFOR3 – neoREV3 and sequencing of the amplicon.Genomic DNA was isolated from tail biopsies by using the Easy DNA kit (Invitrogen), DNAeasy kit  or Nexttec kit . For verifying the abundance of the modification element, amplicons generated with the primer pair hush_for3 – hush_rev3 were sequenced. The presence of the USH1C_a and USH1C_c variants . Primer pairs ushrt16f – ushrt16r and ushrt18f – ushrt18r were used to amplify USH_b splice variants (pig specific primers binding on exons according the splice pattern of human GenBank entry NM_153676.4). Afterward, Sanger sequencing was performed on PCR amplicons.RNA was isolated from powdered tissue samples using TRIzol (Invitrogen). Approximately 100 mg of powdered tissue was grinded up in 1 ml of TRIzol by Polytron PT2500E  in a pulsatile manner to avoid overheating. Further steps were carried out as suggested by the TRIzol protocol and RNA was stored at −80°C. For cDNA synthesis, samples were treated first with DNase I (Invitrogen) to remove a possible contamination with genomic DNA. Then, RNA was reversely transcribed into cDNA with SuperScriptTM III Reverse Transcriptase (Invitrogen). For determining USH1C transcripts, cDNA was used in RT–PCRs with the primer pair ushrt2f – ushrt2r or ushrt2f – urt4r, located in the consistently transcribed exons 1 and 8 or 6, for detecting all splice variants of the gene. Primer pairs ushrt11f – ushrt11r and ushrt13f – ushrt13r were used to amplify et al, Tissue was powdered and then processed as previously described ;run("Smooth");run;run.The script for measuring synapse width is:run("8‐bit");run;run;//setThreshold;run;run(“Measure”).The script for measuring connecting cilium length is:The temporal bones of the pigs were dissected to expose the middle and inner ears. Their cochleae were dissected immediately and the organ of Corti was exposed. The tectorial membrane was removed prior to fixation. Dissected cochleae were fixated in 2,5% glutaraldehyde, 4% PFA in 0.1 M Sörensen’s phosphate buffer. After several washing steps with 0.1 M Sörensen’s phosphate buffer, cochleae were dehydrated in ethanol, critical‐point dried, and gold sputtered in an argon atmosphere. Specimen were imaged with a Philips ESEM XL30 scanning electron microscope .et al, ® M‐1 and polymerized at 60°C. For the pre‐embedding labeling, we followed a previously established protocol .For conventional TEM dissected eyeballs were pre‐fixated for 2 h in buffered 2.5% glutaraldehyde containing sucrose and post‐fixated in buffered 2% OsO4 as previously described  in Renlam® M‐1 resin. Flatmount samples were heat‐polymerized and glued on the top of empty Araldit blocks. Ultrathin sections of the embedded specimens were prepared with an Ultracut S ultramicrotome (Leica) and collected on Formvar‐coated copper or nickel grids. Sections were counter‐stained with heavy metals before imaging and analyzing in a Tecnai 12 BioTwin TEM , equipped with a SIS Mega‐View3 CCD camera  was used. The images were processed using Adobe Photoshop CS (Adobe Systems). Connecting cilia length or synapse width were analyzed directly in the TEM using the measuring tool of the Olympus SIS image analysis system . Quantification steps have been performed with Fiji/ImageJ.Retina sections were first post‐fixated in 2.5% glutaraldehyde buffer and second in 0.5% OsO4 buffer. After dehydration, sections were flatmounted between ACLARet al, 2. 5 days later 10% FCS were added. Outgrown cells were transferred to new complete medium for further culturing. For ciliogenesis experiments, 5 × 105 cells were seeded per well (in medium containing 10% FCS and 5% PS) and 24 h later cultured in OPTI‐MEM reduced‐serum medium (Invitrogen by Thermo Fisher Scientific). After 48 h of starvation, cells were washed in PBS and fixed with 2% PFA in PBS. After washing and permeabilization with 0.1% Triton‐X in PBS, cells were blocked with 0.5% cold‐water fish gelatin, 0.1% ovalbumin in PBS followed by incubation with primary antibodies overnight at 4°C. After washing, coverslips were incubated with secondary antibodies for 1 h at room temperature. Samples were washed and then mounted in with Mowiol (Roth). The primary ciliary length and number of ciliated fibroblasts were quantified by using Fiji. Lengths were measured directly in immunostained images taken with a 63‐x objective. For this purpose, the Arl13b‐positive axoneme was measured from the tip to the cilia base, which was labeled for pericentrin, a common marker for the ciliary base.Pig primary kidney cells and primary dermal fibroblasts were isolated according to standard procedures , 0.02 mg/kg atropine sulfate  and 20 mg/kg ketamine . Anesthesia was continued by intravenous injection of propofol  according to effect. After endotracheal intubation, pigs were mechanically ventilated and cardiovascular function was monitored throughout the procedures. To exclude eyeball movement during examinations, a peripheral muscle relaxant  was applied. For ABR on older animals, animals were starved overnight before intramuscular application of TKX , ketamine 5 mg/kg , and xylazine 1 mg/kg .For examination with ERG, mfERG, OCT, AF, FA, and ABR on 3‐week old animals were anesthetized by intramuscular injection of 2 mg/kg azaperone  and Natus® TIP‐300 insert earphones (AF). Recording stainless steel needle electrodes were positioned subcutaneously ipsilateral to the stimulated ear over the mastoid at the base of the ear (−) and at the vertex in the midline of the skull corresponding to Cz (+). A ground electrode was positioned in the neck. Electrode impedance was < 2 kOhm. Click stimuli  were delivered at supramaximal stimulation intensity (100 dBSPL) to one ear and masking noise was applied to the contralateral ear (−40 dBSPL). Filter settings were 100 Hz for high pass and 3 kHz for low pass filters. The ABR represented the averaged signal of 1,000–1,500 10 ms recordings. ABR was recorded twice for each ear to ensure reproducibility of the ABR peaks in WT animals (n = 3) or absence of recognizable peaks in USH1C‐null animals (n = 3), respectively. If there was no recordable ABR at 100 dBSPL in USH1C‐null animals, ABR was also conducted at 120 dBSPL (twice for each ear). Additional testing comprised ABR at decreasing steps of 10 dB in WT piglets. Hearing threshold was determined as the minimal click intensity that still evoked a noticeable potential negative peak after peak V. ABR for other time points were conducted with four subcutaneous needles placed in vertex, forehead, and mastoids with specialized audiometric equipment. 8 weeks and 2 years old animals were examined with eABRUSB  and headphones  were used on the 8 weeks old piglets. Click stimuli  and tone pips  were applied. The evoked signal was filtered by band‐pass filter of range from 300 Hz to 3 kHz. All the responses were averaged 200–400 times for each intensity and analyzed with eAudio software. On 3 months, 6 months and 2 years old pigs, acoustic stimuli were generated with an evostar 2 system  and presented to the animal via headphones . Click stimuli (duration of electric pulse 150 µs) and the tone pips  were applied. The signal from an electrode was filtered by band‐pass filter over the range of 100 Hz to 2.5 kHz. The response was averaged 200–500 times on each intensity. This signal was processed with and analyzed using Evostar software. The threshold response to each frequency and click was determined as the minimal tone/click intensity that still evoked a noticeable potential peak in the expected time window of the recorded signal. This evaluation process was the same for both systems.In 3‐week‐old USH1C and WT piglets, ABR was recorded with standard electrodiagnostic equipment  using a 61‐hexagon stimulus and according to the standard of the International Society for Clinical Electrophysiology of Vision  were applied. Amplifier gain was set to 50,000 and the band‐pass from 5 to 100 Hz. There were eight recording cycles of 47 s each. The 61 traces are displayed separately in a false‐color‐coded amplitude map identifying P1 amplitude . All traces were also grouped into one response value and the averaged first‐order kernel function displayed as single trace. Luminance levels in the pig eye were calculated from the light intensity measured at the floor. Given the complex physical correlation between light source, reflection, and sensing in an indoor 3D space, it is challenging to determine corresponding luminance levels in the pig eye, but considering the general reflexion parameter ρ as 0.2 seems a fair approximation.Full‐field ERG was recorded with a corneal Kooijman electrode . Platinum needles were placed subcutaneously at the temple and top of the head between the ears as reference and ground electrodes respectively. Stimuli were brief white flashes (4 ms) delivered from a light source within the Kooijman electrode  under general anesthesia and during full pupil dilation. During the acquisition, the corneal surface was protected with methylcellulose eye drops while lids were held open using lid specula. After 30deg and 55deg infrared and red‐free images were recorded using scanning laser ophthalmoscopy (SLO), animal received a flush of approximately 5 mg/kg fluorescein and the angiography was recorded using the 55 degree lens at multiple time points .et al, The design was adapted from a previous publication , STATISTICA_13 , MATHEMATICA_12 , Champaign, IL (2018) and PASW 24 , and Prism_5 .Pigs were trained until they walked straight through the obstacle course to the food trough without stopping or hesitating. After a training period of three weeks, the pigs were performing the course once a week. The pigs entered the course individually in random order, they had to pass the obstacles and at the end they were rewarded with a cookie in a food trough. The test was conducted under light and dim conditions. Each run was documented by video. The pigs were marked with numbers to facilitate recognition. The times the pigs needed to pass the course were measured. All contacts with the obstacles or the wall were counted. When a pig nudged one obstacle more than once in a playful way in the same area, this was counted as one touch. When a pig touched one obstacle at different sites, each contact was counted separately. When a pig swept along an obstacle with its snout, this was counted as two touches. When a pig turned around before reaching the end or when a pig was lame, the pig was excluded for the test day. For analyzing the trajectory, the dimensions of the course and the gaps of the slatted floor were measured and scaled at 1:100 . Using the slatted floor as a ruler, each footstep of each animal in each qualified run were plotted in the map. X and Y coordinates of footsteps as well as the corresponding times when the foot touched the ground were transferred to an excel file. Trajectories were analyzed from these data sets with the R package trajr. Briefly, we first imported and smoothed all trajectories to avoid any source of noise by applying a Savitzky‐Golay smoothing filter ) were applied in the subretinal space with Extendible 41G subretinal injection needle (23 gauge/0.6 mm) . After 5 weeks injection sites were evaluated by OCT (see above) and funduscopy, confirming the concise injection sites. Following enucleation, the eyecup was dissected and fixed with 4% PFA in PBS. Flattened whole mount preparations of the eyecup were analyzed by epifluorescence using a handhold device . Samples were processed for cryosectioning and analysis as described above.Distinct AAVs  were generated by the viral core facility at Boston Children’s Hospital Viral Core at the Massachusetts Eye and Ear Infirmary. AAVs were injected subretinally into the eyes of WT pig at an age of 30 months using an ophthalmologic surgery microscope . Using inhalation anesthesia , morphology of the retina was examined via OCT iVue  on both eyes before the injection. 200 µl of AAVs . For examination of specific allelic constellations, cells nucleofected with urg1 and urt1.3 were seeded onto 96‐well plates and single cell clones were produced. DNA was isolated from independent clones and PCR amplicons were generated by primer pair ushHS1f – huUSH1r, sequenced with primer ush1s, and analyzed by Sanger sequencing.Different constellations of the gRNAs urg1 and urg2 with correcting ssODN urt1, urt2, urt1.1, urt1.2, urt1.3, urt1.4, and urt1.5 were tested in primary fibroblasts. For this, plasmids encoding Cas9 and the respective gRNA as well as correcting oligonucleotides were nucleofected under standard conditions (Richter 5 primary porcine fibroblasts were transfected with 0.4 µg of an endotoxin‐free pDest_Harm_a1_S/F plasmid (Invitrogen), expressing splice form a1 of the human USH1C gene under the control of the CMV promoter. Transfection was performed with the 4D‐NucleofectorTM X Unit (Lonza), using the P2 Primary Cell 4D‐NucleofectorTM X Kit S and program FS113, according to the manufacturer’s protocol. After transfection, cells were seeded in 10% FCS and analyzed for ciliogenesis as described above.For restoring gene expression, 2.5 × 1011 vg Anc80.CMV.harmonin_a1 in 200 µl PBS  was injected subretinally into the right eye, while the left eye was sham‐treated with 200 µl PBS vehicle, according to the procedures described for the AAV transduction experiment (see above). Animal #10056 was injected at an age of 2.5 years and experiment was terminated after 12 months after gfERG examination. Eyes were collected from the vesicular and non‐vesicular areas of the retina for immunofluorescence and Western blot analyses. Animal #10054 was injected at an age of 3.5 years. After intermediate gfERG examination 3 months after injection, the experiment is ongoing.1 × 10Sophia Grotz: Validation; Investigation; Visualization; Methodology; Writing—original draft; Writing—review and editing. Jessica Schäfer: Validation; Investigation; Visualization; Methodology; Writing—original draft; Writing—review and editing. Kirsten A Wunderlich: Investigation; Methodology. Zdenka Ellederova: Methodology; Project administration. Hannah Auch: Validation; Investigation; Methodology. Andrea Bähr: Supervision; Investigation; Methodology. Petra Runa‐Vochozkova: Supervision; Investigation; Methodology. Janet Fadl: Investigation. Vanessa Arnold: Investigation. Taras Ardan: Investigation; Methodology. Miroslav Veith: Investigation; Methodology. Gianluca Santamaria: Software; Formal analysis; Methodology. Georg Dhom: Investigation. Wolfgang Hitzl: Formal analysis; Methodology. Barbara Kessler: Methodology. Christian Eckardt: Investigation. Joshua Klein: Investigation. Anna Brymova: Investigation. Joshua Linnert: Investigation. Mayuko Kurome: Methodology. Valeri Zakharchenko: Methodology. Andrea Fischer: Supervision; Investigation; Methodology; Writing—review and editing. Andreas Blutke: Investigation; Methodology; Writing—review and editing. Anna Döring: Investigation. Stepanka Suchankova: Investigation; Methodology. Jiri Popelar: Supervision. Eduardo Rodríguez‐Bocanegra: Validation. Julia Dlugaiczyk: Supervision. Hans Straka: Formal analysis. Helen May‐Simera: Conceptualization; Investigation; Methodology; Writing—review and editing. Weiwei Wang: Resources. Karl‐Ludwig Laugwitz: Conceptualization; Methodology; Writing—review and editing. Luk H Vandenberghe: Resources. Eckhard Wolf: Conceptualization; Methodology; Writing—review and editing. Kerstin Nagel‐Wolfrum: Conceptualization; Validation; Investigation; Visualization; Methodology; Writing—original draft; Writing—review and editing. Tobias Peters: Formal analysis. Jan Motlik: Supervision; Methodology; Project administration; Writing—review and editing. M Dominik Fischer: Supervision; Validation; Visualization; Methodology; Writing—original draft; Writing—review and editing. Uwe Wolfrum: Conceptualization; Supervision; Funding acquisition; Validation; Investigation; Visualization; Methodology; Writing—original draft; Writing—review and editing. Nikolai Klymiuk: Conceptualization; Supervision; Funding acquisition; Validation; Investigation; Visualization; Methodology; Writing—original draft; Project administration; Writing—review and editing.CRediT author contributions listed above, the contributions in detail are:In addition to the SG contributed to molecular analysis and breeding of animals, designed and performed behavior tests and contributed to writing of the manuscript. JS contributed to molecular and morphological analysis, and ciliation analysis in primary fibroblasts. KAW contributed to tissue sampling and processing, imaging (IF and SEM), data analysis and interpretation. ZE contributed to breeding attempts sub‐retinal intervention and ABR measurements. HA evaluated Gene Repair. ABä managed intervention on pigs and anesthesia. PR‐V contributed to Gene Repair study. JF contributed to genetic modification. VA contributed to quantitative analysis of the synaptic phenotype in cones. TA managed intervention on pigs. MV performed subretinal injection. GS performed trajectory analysis. GD contributed to animal management and tissue sampling. WH performed data analysis. BK contributed to Somatic Cell Nuclear Transfer and performed Embryo Transfer. CE performed RT–PCR on USH1C transcripts. JK participated in the molecular and morphologic analysis of eyes after USH1C gene therapy. ABr contributed to ERG measurements. JL performed ciliation analysis in primary fibroblasts. MK and VZ performed Somatic Cell Nuclear Transfer. AF, SS, and JP performed ABR measurement. ER‐B participated in ophthalmologic analysis. ABl conducted pathological examinations and tissue preparation. AD contributed to ERG measurement. JD and HS contributed to the analysis of the vestibular phenotype. HM‐S assisted with tissue dissection and cochlear preparation. K‐LL and EW contributed to the design of the study and the writing of the manuscript. WW and LHV provided AAV vectors. KN‐W contributed to the design of the study, interpretation of data and writing of the manuscript. TP performed ERG and OCT measurements. JM contributed to the design of the study, orchestrated breeding attempts, sub‐retinal intervention and ABR measurements. MDF led the ophthalmological characterization of animals, performed the vitreoretinal surgery and contributed writing of the manuscript. UW designed and coordinated the study, performed molecular and morphological analysis, interpreted data and wrote the manuscript. NK designed and coordinated the study, performed bio‐informatic and molecular analysis, interpreted data and wrote the manuscript. ER‐B contributed to analysis of ERG and OCT measurements.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Movie EV1Click here for additional data file.Movie EV2Click here for additional data file.Movie EV3Click here for additional data file.Movie EV4Click here for additional data file.Movie EV5Click here for additional data file.Movie EV6Click here for additional data file.Movie EV7Click here for additional data file.Source Data for Figure 1Click here for additional data file.


Speech intelligibility in multitalker settings is challenging for mostcochlear implant (CI) users. One possibility for this limitation isthe suboptimal representation of vocal cues in implant processing,such as the fundamental frequency (F0), and the vocal tract length(VTL). Previous studies suggested that while F0 perception depends onspectrotemporal cues, VTL perception relies largely on spectral cues.To investigate how spectral smearing in CIs affects vocal cueperception in speech-on-speech (SoS) settings, adjacent electrodeswere simultaneously stimulated using current steering in 12 AdvancedBionics users to simulate channel interaction. In current steering,two adjacent electrodes are simultaneously stimulated forming achannel of parallel stimulation. Three such stimulation patterns wereused: Sequential (one current steering channel), Paired (twochannels), and Triplet stimulation (three channels). F0 and VTLjust-noticeable differences , in addition to SoSintelligibility (Task 2) and comprehension (Task 3), were measured foreach stimulation strategy. In Tasks 2 and 3, four maskers were used:the same female talker, a male voice obtained by manipulating both F0and VTL (F0+VTL) of the original female speaker, a voice where only F0was manipulated, and a voice where only VTL was manipulated. JNDs weremeasured relative to the original voice for the F0, VTL, and F0+VTLmanipulations. When spectral smearing was increased from Sequential toTriplet, a significant deterioration in performance was observed forTasks 1 and 2, with no differences between Sequential and Pairedstimulation. Data from Task 3 were inconclusive. These results implythat CI users may tolerate certain amounts of channel interactionwithout significant reduction in performance on tasks relying on voiceperception. This points to possibilities for using parallelstimulation in CIs for reducing power consumption. Cochlear implants (CIs) are devices that can restore hearing in patientssuffering from profound hearing loss. Although many CI users obtain goodspeech performance in quiet, their speech intelligibility dropssignificantly in the presence of a background interfering signal known asthe cocktail-party scenario e.g., . There alistening in the dips” of the masker; however, CIusers usually find this situation more favorable in comparison to scenariosinvolving a single-talker masker  listeners, who have been shown in the literature tobenefit from spectral dips or temporal modulations in a fluctuating maskerto obtain release-from-masking , CI userr masker .A possible explanation for these reported perceptual differences between NH andCI listeners may be attributed to the ability of the former group to usevoice cue differences that exist between multiple simultaneous talkers insuch speech-on-speech (SoS) scenarios e.g., . On the linear frequency scale, while elongating VTL resultsin the compression of the spectral envelope toward lower frequencies. On alogarithmic frequency scale, shortening VTL leads toa translation of the formant peaks in the spectrum toward higherfrequencies, while elongating VTL leads to a translation of the formantstoward lower frequencies. This effect directly influences the formantfrequency space defining vowel boundaries  on SoS and voice cue perception in CIlisteners by using simultaneous stimulation of multiple channels to inducevarious degrees of channel interaction. The effect of this channelinteraction on the representation of F0 and VTL cues in the speech signal isalso investigated using a 3D model to illustrate the increase in channelinteraction caused by various stimulation strategies that differ in thenumber of simultaneously stimulated channels used. Beyond the purpose ofevaluating the importance of spectrotemporal cues for F0, VTL, and SoSperception, there is also a potential benefit in using parallel stimulation,as it was originally proposed in the literature as a method of reducingpower consumption e.g., . One wayintelligibility and comprehension.The participants were also asked to do different activities in each of thesetasks as follows. In Task 2, SoS intelligibility was measured in a mannersimilar to previous literature (Three research questions were addressed in this study: (a) whether increasingthe number of parallel stimulated channels , thereby increasing channel interaction, decreases thesensitivity to F0 and VTL differences in CI users, which was measured usingJNDs in Task 1; (b) whether this effect is also reflected as a reduction inSoS perception (Tasks 2 and 3); and (c) whether some parallel channelstimulation could be deployed for reducing power consumption withoutsignificantly impairing voice cue and SoS perception. In Tasks 2 and 3, SoSperception was measured as a function of systematically increasing ΔF0 andΔVTL between target and masker speakers. The setup was designed such that asingle-talker target speaker was embedded in a single-talker masker to modelone of the more challenging background interference scenarios for CI usersas previously mentioned . The tarterature , 2007. Pterature . In thisterature . Anadvaterature . Forexaterature .The hypotheses were as follows for each research question, respectively: (a)Reductions in sensitivity are expected to increase as a function ofincreased channel interaction, and should be larger for VTL compared to F0,because VTL is a primarily spectral cue, while F0 cues could still bepreserved in the temporal aspect of the signal even if the spectralcomponent is compromised; (b) These reductions in sensitivity for F0 and VTLcues are expected to be reflected as a reduction in both SoS intelligibilityand comprehension performance; (c) Some degree of parallel stimulation, suchas Paired, may not lead to a significant reduction in voice cue and SoSperception compared to Sequential, which could warrant the use of Pairedstimulation as a potential low-power stimulation strategy.Twelve native German CI users with Advanced Bionics (AB) devices wererecruited from the clinical database of the Medizinische HochschuleHannover (MHH) based on their clinical speech intelligibility scoresin quiet and in noise. To ensure that participants could perform theSoS tasks, the inclusion criteria were to have a speechintelligibility score higher than 70% in quiet and 20% in noise at a+10 dB signal-to-noise ratio on the Hochmair–Schulz–Moser (HSM)sentence test . It is wSpeechTransformation and Representation based on AdaptiveInterpolation of weiGHTed spectrogram  in AB devices is a sound coding strategythat processes the audio signal through an automatic gaincontrol. Next, a spectral analysis is performed using ashort-time fast Fourier transform to compute the slow varyingenvelopes in each analysis band. In parallel stimulation, thespectrum is analyzed using a spectral peak locator to estimatethe most dominant frequency component in each analysis band.Finally, the slowly varying envelopes are logarithmicallycompressed into the electric dynamic range of each participantbetween the threshold and the most comfortable level. Eachanalysis band is then assigned to two simultaneously stimulatedelectrodes . The curThe effect of spectral smearing using the F120 Sequential, Paired,and Triplet strategies was first analyzed in simulation using a3D finite element model of the electrically stimulated cochlea(see The spread of electric current in the cochlea is simulated in a 3Dfinite element method (FEM) from the geometry of the cochleacontaining the Scala tympani, Scala vestibuli, Reisner membrane,basilar membrane, the modiolus, and the nerve. The left panel inGerman tokens were processed with the Sequential, Paired, andTriplet sound coding strategies using the same levels ofstimulation (threshold and most comfortable levels). Thestimulation patterns served as input to the computational modelthat estimated the excitation patterns. For each participant in the study, the F120 Sequential was fittedby adjusting the threshold and most comfortable levels of eachelectrode individually. Next, the strategy was activated, andthe participant was asked to perform a loudness scaling taskcontaining the presentation of a Consultative Committee forInternational Telephony and Telegraphy (CCITT) noise at 65 dBSPL free field. The threshold and most comfortable levels forthe strategy were then adjusted globally until the participantstated a comfortable loudness percept. Afterward, the Paired andTriplet sound coding strategies were fitted by globallyadjusting the most comfortable level across all electrodes bythe same amount starting from the Sequential map fitting whilepresenting the same noise signal.In a typical AB device, power consumption is a function of thesupply voltage required to power the CI. This supply voltage isdetermined based on the maximum current the CI can deliverwithout causing damage to the electrodes. The power consumed bythe device is then estimated as the multiplication of the supplyvoltage with the sum of the leakage current and averagestimulation current. The leakage current, reaching around 1 mAin a typical CI, is used to power the implant’s internalcircuitry and contributes the largest share of the powerconsumed. This type of current depends on the technology(transistor sizing) used to manufacture the CI hardware andincreases with decreasing transistor sizing. The averagestimulation current, however, is bounded by the maximum currentlevel fitted for each CI user. Thus, the most effective methodto reduce power consumption is to reduce the supply voltage.NeverthFrom this analysis, it can be concluded that adding parallelchannels causes current smearing which, in turn, causes areduction in the current levels required to achieve the sameloudness percept, thus achieving the proposed currentsavings.The methods for this experiment are largely similar to the onesdescribed in Recordings were made in a sound-isolated anechoic chamber at theUniversity Medical Center Groningen, NL, using a RØDE NT1-Amicrophone mounted on a RØDE SM6 with a pop-shield . The microphone was connected to aPreSonus TubePre v2 amplifier  with noise filtering below 80 Hz. The amplifier outputwas recorded through the left channel of a DR-100 MKII TASCAMrecorder  at a samplingrate of 44.1 kHz. Seventy-five consonant–vowel (CV) syllableswere manually extracted from the recorded words in the corpus,resulting in a list of combinations of the consonants  and vowels .A single trial consisted of concatenating three random CVsyllables, with a 50-ms silence in between, to form a triplet ofsyllables. Within the trial, the same triplet of syllables waspresented three times, with a 250 ms silence gap between eachpresentation. One of these three presentations was processed tohave a different voice , asindicated by the vectors from the origin of the plane to the red crosses shown in Following the paradigm used in a number of previous studies , 2018, JThe JND measurement for each of the three voice vectors wasrepeated three times per strategy, resulting in a total of 27experimental conditions (3 voice vectors × 3 repetitionseach × 3 coding strategies). Experimental conditions wereblocked per strategy, meaning that a participant would performall conditions for a given strategy before switching to the nextone, and the order of the strategies was randomized perparticipant. Participants were blinded to the strategiestested.Training was administered before the beginning of each strategyblock with two voice vectors different than those used for datacollection:  and . Each training condition was terminated aftersix trials, whether the algorithm had converged or not. Visualfeedback was always provided.Stimuli taken from the German HSM sentence test  were useSameTalker, F0,VTL, and F0+VTL, respectively,in the rest of this article. The parameters for F0 and VTL werechosen based on the findings of an earlier study, in which CIusers showed reduced SoS intelligibility and comprehension whenthe voice of the masker was manipulated with parameters takenfrom the top-right quadrant in Four different masking voices were created as shown in Test sentences were taken from Lists 1–8 and 16–19, while maskerswere constructed from Lists 9 and 10. Training sentences wereobtained from Lists 11, 12, and 13, with one list randomlyassigned per strategy. All sentences assigned for constructingthe maskers were processed offline before data collection usingSTRAIGHT, with all combinations of ΔF0 and ΔVTL highlightedearlier. For the Same Talker condition, the masker sentenceswere also processed with STRAIGHT, without changing F0 or VTL.All target sentences were kept as the natural, unprocessedversion (not processed with STRAIGHT).Within a trial, the masker sequence started 500 ms before the onsetof the target sentence and ended 250 ms after the offset of thetarget. For the specific ΔF0 and ΔVTL combination within thetrial, the masker was constructed from random 1-s-long segmentsselected from the masker sentences previously processed withSTRAIGHT. A raised cosine ramp of 2 ms was applied to thebeginning and end of each segment before concatenating them toform the masker sequence. Finally, both the beginning and end ofthe entire masker sequence were ramped using a 50-ms raisedcosine ramp.Target sentences were calibrated at 65 dB SPL, and the intensity ofthe masker sequence was adjusted relative to that of the targetto obtain the required TMR. The TMRs used for training and datacollection in this task were set to +8 dB and +12 dB,respectively, following the protocol of The SoS paradigm for this experiment was based on that used byA short training was provided for each strategy block, with bothauditory and visual feedback. During the training phase of agiven strategy, 12 sentences were randomly selected from theassigned training list: Six sentences were presented in quiet,while the remaining six were presented with a competing masker.The masker voice used for training was assigned different valuesfor ΔF0 and ΔVTL than those used during data collection .Data collection was composed of a total of 240 trials for all threestrategy blocks (20 sentences per list × 4 voice conditions × 3strategies) generated offline prior to the beginning of theexperiment. The trials within a strategy block were randomized.No feedback was provided, and the stimulus was presented once.The participants’ responses were scored on a word-by-word basisusing a graphical user interface programmed in MATLAB. Inaddition, the verbal responses were recorded and stored as datafiles for later offline inspection.Response words were scored in the following fashion: The German HSMsentences include words that are hyphenated in the corpus, suchas “Wochen-ende” (weekend). These words, although writtenwithout the hyphen, are hyphenated in the HSM corpus to bescored separately. Only the part repeated by the participant wasmarked as correct. In addition, the response word was alsoconsidered correct if a participant changed the order of thewords in the sentence.A response word was considered incorrect if only a part of the wordwas repeated for words that are not hyphenated in the HSMcorpus, such as saying “füllt” (fills) when the word was“überfüllt” (crowded). In addition, confusion of adjective form,for example, saying “keiner” (“not any” as used with a masculinenoun) instead of “keine” (“not any” as used with a femininenoun), or confusing the Dativ with the Akkusativ article, forexample, confusing “der” with “dem” or “den,” was alsoconsidered incorrect. Confusion of verb tenses or incorrect verbconjugation was considered incorrect. A total of four scheduledbreaks were programmed into the experiment script; however,participants were encouraged to ask for additional breakswhenever they felt necessary. The whole procedure lasted forabout 1–1.5 hr, including breaks.Bevers bouwen dammen in de rivier[Beavers build dams in the river]) and false version . All sentences are grammatically andsyntactically correct.The voice conditions for the masker in this experiment were thesame as those defined in Experiment 1. The masker sequence wascreated as described in Experiment 1 from Lists 9 and 10 fromthe HSM material. Target sentences were based on Germantranslations of the Dutch SVT developed by Translation from Dutch to German and the evaluation was performedthoroughly by three independent native German speakers: Two ofthose speakers were also fluent in Dutch, while the third hadsufficient knowledge of the language , 4 true and 4 falsesentences were randomly picked and assigned to the trainingblock of each strategy. No true–false pair was assigned to thesame training block.In each training block, two true and two false sentences were firstpresented without a competing masker, followed by the remainingtwo true and two false sentences, which were presented with acompeting masker. This masker also had the same voice parametersas those of the training masker voice used in Task 2 and at thesame training TMR of +12 dB. Both audio and visual feedback wasprovided: Participants were shown if the sentence was true orfalse, and the sentence was shown on the screen while the wholestimulus was replayed through the loudspeaker.The remaining sentences  not usedfor training were used for data collection. These sentences weredistributed among the number of conditions tested (4 maskervoice conditions × 3 strategies), and no true–false pair wasassigned to the same condition. This resulted in seven true andseven false sentences per voice condition per strategy. A testTMR of +10 dB was used in this task because pilot measurementswith P01-P03 revealed that a test TMR of +10 dB for this taskwas expected to yield performance in the middle of thepsychometric function compared to a test TMR of +8 dB as wasused in Task 2. All stimuli were generated offline for all threestrategy blocks and pseudorandomized within each block. Duringdata collection, no feedback was given.lme4 package = 4.70,p = .03, but no significantdifferences in JNDs between the different voice cues,F = 1.98,p = .18. The interactioneffect between strategy and voice cue was also found to benonsignificant, F = 0.91,p = .47.p values were then adjusted formultiple comparisons using the FDR method  = 4.59,p = .03, and F0+VTL JNDs,F = 4.56,p = .03, but not on VTL JNDs,F = 1.23,p = .31.A similar LMM (including only a random intercept per participant as therandom effect) was applied to each type of JND separately  to study how stimulation strategy (channel interaction)affects each individual voice cue. A similar ANOVA to the one appliedon the previous general model was also applied here for each modelseparately, and R method . These ASE = 0.13,t(22) = 2.87,p = .03—but did not seem to beaffected by Paired stimulation—β = 0.07, SE = 0.13,t(22) = 0.59,p = .56. On the contrary, VTLJNDs were neither affected by Paired—β = 0.12,SE = 0.12, t(22) = 1.01,p = .39—nor by Tripletstimulation—β = 0.19, SE = 0.12,t(22) = 1.54,p = .21—compared to Sequential.Finally, the participants’ JNDs to differences along both F0 and VTL(F0+VTL condition) also significantly increased (worsened) by about1.35 st when the stimulation strategy was changed from Sequential toTriplet—β = 0.37, SE = 0.12,t(22) = 3.02,p = .03—but not from Sequential toPaired—β = 0.19, SE = 0.12,t(22) = 1.57,p = .21.The post hoc analyses of these tests revealed that F0 JNDs increased byabout 1.44 st as the strategy changed from Sequential toTriplet—β = 0.36, The binary per-word scores  were modeled usinglogistic regression as implemented by a generalized linearmixed-effects model (GLMM) with a logit link function. The logisticregression model was fitted to the binary per-word score with strategyand masker voice, along with their interaction, as the fixed-effects.The interaction between stimulation strategy and masker voice wasincluded to test for the significance of the effect observed in theright panel of 2) tests performed on the fixed-effects of themodel instead of the traditional F-test statistics.The ANOVA revealed a significant effect of stimulation strategy,χ2(2) = 27.29,p < .0001; masker voice,χ2(3) = 36.32,p < .0001; and theirinteraction, χ2(6) = 37.34,p < .0001.As with the analyses of the JND task, an ANOVA  = 9.34,p = .01; F0 condition,χ2(2) = 8.99, p = .01;VTL condition, χ2(2) = 26.39,p < .0001; and F0+VTLcondition, χ2(2) = 34.69,p < .0001  = 58.27,p < .0001; and Tripletstimulation, χ2(3) = 26.99,p < .0001, but not forPaired stimulation, χ2(3) = 7.08,p = .07. Under Sequentialstimulation, participants were found to gain a significant improvementin SoS intelligibility under the VTL, β = 0.66,SE = 0.24,z = 2.76,p = .02, and F0+VTLconditions, β = 0.97, SE = 0.15,z = 6.47,p < .0001, compared to theSame Talker condition; however, there was no difference in SoSintelligibility between the Same Talker and F0 conditions, β = 0.17,SE = 0.18,z = 0.93,p = .39. The voice benefitobserved under the Triplet strategy arose from the significantimprovement in SoS intelligibility for the F0+VTL condition comparedto the Same Talker condition, β = 0.57, SE = 0.13,z = 4.36,p < .0001, but there was nosignificant difference between SoS intelligibility for either the F0or VTL conditions compared to the Same Talker conditions(p > .08). There was novoice benefit observed under the Paired strategy for F0, VTL, orF0+VTL compared to the Same Talker condition(p > .08). Together withthe data representation in the right panel of The significant interaction effect from the global ANOVA indicates thatthe benefit in SoS intelligibility obtained from changing the maskervoice cues relative to those of the target was affected by the amountof channel interaction: As the channel interaction increased , the benefitobtained from the voice differences between masker and target speakers decreasedsignificantly  = 15.58,p = .74, and was thus notincluded in the final LMM. An ANOVA similar to that applied to the LMMin the JND task was also applied to the LMM modeling thed’ data and revealed no effect of strategy,F = 2.68,p = .07; masker voice,F = 1.82,p = .15; or their interaction,F = 1.20,p = .31; on thed’ accuracy scores.arm rate .The d’ 2(2) = 0.006,p = .997; masker voice,χ2(3) = 0.049,p = .997; or their interaction,χ2(6) = 0.167,p = .9999, on RTs.The right panel of d’ accuracy scores and RT data for theVTL condition. It appears that as participants give less accuratescores as the channel interaction increases, they also give theseresponses faster. However, this response strategy seems to change forthe condition F0+VTL. In that condition, as channel interactionincreases, participants also give less accurate responses, but they doso at slower speeds. Because the analyses yielded nonsignificanteffects, no further conclusions could be drawn from this task.In this task, no effect of strategy could be observed either for SoScomprehension accuracy or RTs. Qualitatively, this implies thatparticipants may be compromising accuracy for speed or vice versa andthat these response strategies differ per condition. Consider, forexample, the This study investigated whether increasing channel interaction as a result ofsimultaneously stimulating multiple channels in the CI would lead to areduced sensitivity to F0 and VTL cues (Task 1) and, correspondingly,reduced SoS intelligibility and comprehension performance (Tasks 2 and3).The data from the JND task revealed that, in line with what was expected,increasing channel interaction significantly reduced CI users’sensitivity to voice cues , asdemonstrated by the main effect of stimulation strategy in addition toa lack of interaction effect between voice cue and stimulationstrategy. When mild channel interaction exists, as was the case whenPaired stimulation was compared to Sequential, sensitivity to voicecue differences was not significantly affected. However, as thechannel interaction increased, as was the case with Tripletstimulation, sensitivity to voice cue differences was reduced. Becauseno significant interaction effect between stimulation strategy andvoice cue was observed in the overall model, the effect of strategyshould not be expected to differ for each voice cue. The fact thatpost hoc analyses revealed no significant effect of strategy on VTLJNDs may have arose from the relatively smaller differences in VTLJNDs across all three strategies compared to the F0 differences, eventhough a trend for worsening VTL JNDs could be observed. In an earlierstudy with vocoders, A second observation concerns the effect of channel interaction on F0JNDs. Because F0 information is encoded in both spectral and temporalcues , it was The data from the SoS intelligibility task demonstrated an effect ofchannel interaction, a benefit from voice differences between targetand masker speakers, and a significant interaction effect betweenthese two factors. Compared to Sequential stimulation, increasing thechannel interaction was shown to impair SoS intelligibility scoresonly for Triplet stimulation but not for Paired stimulation. Thisindicates that for mild cases of channel interaction, baseline SoSintelligibility could still be maintained. However, for more extremecases of channel interaction, as in the case of Triplet stimulation,SoS intelligibility scores become severely degraded.The significance of the voice benefit under Triplet stimulation iscounterintuitive, as it was expected that the severity of the channelinteraction would impair the benefit from voice differences comparedto less severe cases of channel interaction . This is because the hypothesis is that channelinteraction is expected to impair the transmission of voice cues.However, in the Triplet case, SoS intelligibility not only becomescapped but is also severely reduced compared to Sequential yielded a benefit in SoS intelligibility. In a previous study , the autTaken together, these data indicate that CI users may benefit differentlyfrom voice cue differences depending on which speaker space theycover. However, this benefit from voice differences between target andmasker is reduced as the amount of channel interaction increases, aswas demonstrated by the significance of the interaction effectobserved between stimulation strategy and voice cue. This means thatas channel interaction becomes substantial, CI listeners may not beable to benefit from voice cue differences between competing talkersin SoS scenarios.It is important to mention that CI users may use different acoustic cuesthat contribute to phonetic perception than those used by NH listenersto obtain word recognition. It should also be noted that the results presented in this study shouldbe treated as the best-case-scenario when comparing performance of CIusers under Sequential, Paired, and Triplet stimulation. This isbecause higher performing CI users had to be recruited for this studyto avoid potential floor effects, as was explained in the Methodssection. A larger, more representative sample of CI users should betested in a follow-up study to better assess the effects ofsimultaneous stimulation on CI users performing at all levels so thatthe findings would be more generalizable to a wider range of CIusers.The data from the SoS comprehension were nonconclusive regarding theeffect of channel interaction: Comprehension accuracy and RT measuresrevealed no effect of either channel interaction or voice cue. Theobservations from this task seem to highlight the different responsestrategies between listeners. In addition, a large amount ofintersubject variability was also observed within this small sample ofCI participants, which also limits the scope of conclusions that canbe drawn from this task. In order to obtain performance results thatcan be more generalizable to the larger CI population, a larger samplecontaining of CI participants should also be tested in upcomingfollow-up studies.The findings from this study highlight the importance of spectrotemporalresolution when performing tasks that depend on voice-cue perception. Thisraises the question of whether CIs could be fitted with the goal ofmitigating the effect of decreased spectrotemporal resolution that may arisefrom channel interaction. Several studies e.g.,  have proThe second goal of this study was to determine the amount of parallel channelstimulation that would not contribute to a significant reduction inperformance. While Paired stimulation yielded performance results that werenot significantly different from Sequential stimulation on all three tasks,it still remains an open question as to whether Paired stimulation mayimpact other aspects of sound perception, such as sound quality. Forexample, This study showed that increasing channel interaction by increasing the numberof simultaneously stimulated channels significantly reduced CI users’sensitivity to voice cues . Compared toSequential stimulation, increasing the channel interaction was shown toimpair SoS intelligibility scores only for Triplet stimulation but not forPaired stimulation. SoS comprehension accuracy and RT measures revealed noeffect of either channel interaction or voice cue, although some evidencefor a change in participant response strategy could be observed. The lack ofa detrimental effect of Paired stimulation on voice cue sensitivity and SoSintelligibility provides evidence that parallel stimulation could be used asa method for reducing power in CIs without impairing performance on tasksrelying on voice cue perception.


To the date, many drugs are developed to treat this condition, in particular biological treatments are the most important therapy that are used to control the disease.We read with very interesting the paper published on January 16 by Sin et al2We reviewed data of 39 Caucasian patients genotyped for HLA‐C*06, 18 out of 39 patients were naive to biologic treatment, all patients were evaluated by both rheumatologists and dermatologists for the diagnosis. In our cohort, 19 patients were male and 20 females, the mean age was 56.0 ± 12.1 years (mean ± SD) and the duration of the PsA disease was 6.7 ± 5.4 years . The HLA‐C*06 allele was detected by standard polymerase chain reaction using allele specific primers . Ten out of 39 (26%) were HLA‐C*06 positive (six in heterozygosis and four in homozygosis).The severity of psoriasis and response to treatment were evaluated using the Psoriasis Area and Severity Index (PASI) score at baseline and then at follow‐up visits on weeks 12, 24, and 52. We evaluated the proportion of patients achieving ≥50% reduction in PASI score (PASI 50), a ≥75% reduction in PASI score (PASI 75), a ≥90% reduction in the PASI score (PASI 90), and a ≥100% reduction in the PASI score after 12, 24, and 52 weeks. We evaluated also the following variables: Disease Activity Score (DAS‐44), Patients Visual Analogue Scale (VAS), Modified Nail Psoriasis Severity Index (mNAPSI), Dermatology Life Quality Index (DLQI), Tender Joint Count (TJC), Swollen Joint Count (SJC), C‐Reactive Protein (CRP), and Erythrocyte Sedimentation Rate (ESR) at each time point.3P‐value = .048), PASI‐100 , DAS‐44 , and at week 52, defined by DAS‐44 , was correlated with the number of biological treatments used. Furthermore, a correlation between body weight and clinical outcome at week 12, defined by DAS‐44 , and ESR level at week 24  were observed (Table An ANOVA analysis was realized and no differences statistically significant were found between HLA‐C*06 positive and negative patients at baseline. Additionally, no correlations were found between clinical response and presence of allele C*06 at univariate logistical regression analysis; although a better clinical response at week 12, defined by PASI‐50 (coef [CI] = −1.98 [0.03‐0.99] 4Our data suggest that only 26% of patients affected by psoriatic arthritis are carriers of the HLA‐C*06 allele, in line with the data reported by Sin et al. Furthermore, HLA‐C*06 allele seems not correlated with a higher disease severity or a better clinical outcome. On the other hand, a multiple biological treatment failure and a high heavy weight seem to be connected with a poor response to certolizumab pegol.The authors declare no conflicts of interest.Conceptualization: Annunziata Dattola, Elisabetta BottiWriting ‐ original draft preparation: Annunziata Dattola, Dante Raffaele Caro CaposienaWriting ‐ review and editing: Marco Galluzzo, Sara Mazzilli, Elena Campione, Gennaro Melino, Eleonora Candi, Luca Bianchi, Annunziata Dattola


Vitis vinifera L. to fungal diseases is the integration of genetic resistance by the interspecific crossing between V. vinifera varieties and resistant species. However, the products of such hybrids are still not accepted by customers, particularly due to their organoleptic characteristics, not least influenced by their polyphenolic profile.A promising way to overcome the susceptibility of P > 0.05) within the two harvests, with the exceptions of epicatechin and caftaric acid. Anthocyanin diglucosides were found in ten of the red breeding lines, malvidin‐3,5‐O‐diglucoside being predominant in nine of them. Total polyphenol content of the unknown progeny individuals and international breeding lines was comparable, with the exception of significantly increased amounts of gallic acid and some flavonoids.A total of 58 resistant breeding lines, 41 from international programs and 17 new progeny individuals, were grown in one untreated vineyard to exclude any variances by climatic and pedologic conditions or vineyard practice. A total of 60 polyphenols  were determined in grapevine berries by ultrahigh‐performance liquid chromatography–mass spectrometry in two consecutive years. The overall profiles were rather consistent (variation The comprehensive study reported herein of the polyphenolic profile of hybrids from international breeding programs, but also of new breeds from private initiatives, all cultivated in the same vineyard, will support the selection of promising candidates for further breeding programs to overcome impairment due to undesired sensory characteristics of new highly resistant varieties. The history of cultivation of the predominant domesticated species Vitis vinifera L. can be traced back more than 6000 years.Daktulosphaira vitifoliae) and downy mildew (caused by the oomycete Plasmopara viticola) were introduced from North America into Europe, a sophisticated pest and pathogen control has been mandatory for successful viticulture. Whereas breeders managed to gain control over grape phylloxera, the susceptibility of V. vinifera against P. viticola, as well as Erysiphe necator (syn. Uncinula necator), the ascomycete causal agent of powdery mildew, remains problematic in viticulture. A widely accepted and eco‐friendly strategy for reduction of necessary treatments with fungicides is the integration of genetic resistance by interspecific breeding between V. vinifera and North American or Asian resistant species.Grapevine is not only one of the most important horticultural crop species, reflected by ~8 × 10A big disadvantage of conventional breeding is the tremendous amount of time required to produce results. Detection of genetic markers and genetic engineering can be used to assist in speeding up the selection of promising highly resistant breeds, and profiling approaches can provide insight into the composition of secondary metabolites in grapes, or products thereof. One class of constituents with an enormous impact on the overall properties of grapes, and particularly of wine, the most important product thereof, are polyphenolic compounds. Polyphenols are a structurally heterogenous class of aromatic compounds, with usually two or more phenolic hydroxyl functions. Exceeding a certain chromophore size, their color ranges from yellow to red or dark blue. The phenolic functions make these compounds participators in redox reactions, hence facilitating the chemical interaction with other organic compounds. In addition, owing to their aromaticity, polyphenols are very effective radical scavengers. Polyphenols are responsible for the coloration of wine, and to some extent participate in the wine odor, either directly or indirectly in the aging process of wine. Besides their influence on the appearance of wine, their contribution to the taste sensations of the consumer is of importance. Polyphenol congeners contribute to taste sensations such as sweet, acidic, and bitter, and they attribute to impressions such as velvety, smooth, or astringent in our mouth.The overall polyphenol metabolite profiles, however, vary considerably among different cultivars and are under strict genetic control. Thus, they have been shown to represent useful markers for the authentification of grapes and wines.V. vinifera grapes was intensively studied during the last few decades.V. viniferaThe polyphenolic composition of About a decade ago, an ambitious breeding and cultivation project aiming at the production of new mildew‐resistant varieties lacking negative quality traits was initiated. A total of 58 genotypes, 41 highly resistant breeds and 17 new and uncharacterized progeny individuals (PIs), were grown in an untreated vineyard located in Marlengo, South Tyrol. The aim of the VITISANA project was to comprehensively characterize these breeds independent of influences by macroclimatic and pedologic conditions or vineyard practice. The analytical strategy chosen was to utilize an already well established and validated liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analytical procedure capable of quantifying dozens of polyphenolic congeners in combination with a strict harvesting plan aiming at the exclusion of biological confounding factors. Harvests of two consecutive years were investigated to address the need for biological replicates, since only single plant (vine) analysis was mandatory to ensure genetic uniformity of the novel accessions. For comparability between the different breeds, ripeness of the grapes was monitored carefully, and only ripe clusters were processed for further analysis. To represent the biological range of berry growth on a vine, three individual clusters were harvested and berries from all clusters were processed.Vitis berlandieri × Vitis riparia), grown in loamy sand (see Supporting Information Table http://meteobrowser.eurac.edu/app_direct/meteobrowser) for the Meran/Merano station (distance to the vineyard about 2 km). In 2017 and 2018, at least three clusters per single accession (about 1000 g) were harvested from one plant at maturity level (18 °Bx). The plant material was frozen in liquid nitrogen, transported on ice, and stored at −80 °C until analysis.Fifty‐eight grapevine accessions were grown in an untreated vineyard located in a private winery in Marlengo  . Forty‐one thereof were breeding lines (BLs) originating from various European breeding programs, and 17 lines were new PIs from a private breeder , Italy). Detailed information for all accessions, including variety number VIVC, age of vine, medium yield, species involved, and origin of mildew resistance is provided in Supporting Information Table Rosé‐colored accessions were considered as white‐skinned grapes in statistical evaluation and figures; all red accessions were of skin color ‘noir’  analysis was carried out on an Acquity UPLC system  coupled to a Xevo TQMS System (Waters) with an electrospray ionization source. Phenolic compounds (except anthocyanins) were analyzed according to Vrhovsek et al.−1, and desolvation gas flow was 1000 L h−1.Anthocyanins were analyzed according to Arapitsas et al.,O‐galactosides of quercetin and syringetin were calculated using the calibration of their respective 3‐O‐glucosides, and procyanidin B3 was calculated with the calibration of procyanidin B1. All data reported did exceed the limit of detection figures of the assay.Peak identification, optimization of multiple reaction monitoring events, and quantitation were performed with standards as described by Vrhovsek Data were processed using Waters MassLynx (version 4.1) and TargetLynx software (Waters). Data analysis and production of graphs, including one‐way analysis of variance, box‐plots, and heatmaps, were performed using R software , Excel 2016 , and SPSS Statistics software, version 24.0 . For principal component analysis (PCA), SIMCA 13.0 software was used . Unit‐variance scaling and mean centering were applied for heatmaps and PCA, and compounds with more than 50% missing values were excluded in the heat maps. Dendrograms were constructed by hierarchical clustering analysis using ‘Canberra’ as the distance function and Ward.D2 as the hierarchical clustering algorithm.Following the strategy to lower agro‐chemical input by promoting mildew‐resistant varieties, almost 100 novel plants derived from crossing resistant genotypes and quality varieties were cultivated in an untreated vineyard in South Tyrol. Fifty‐eight accessions (41 highly resistant breeds and 17 new progeny lines – for details see Supporting Information Table −1) a median RSD of 9.2% was found, with 80% of all RSD values below 14.5%. For P‐type analytes (median concentration 15.7 mg kg−1) a median RSD of 17.2% was found, with 80% of all RSD values below 28%. These data are in agreement with the data presented in the validation study,Since the general figures such as limit of quantitation and analytical repeatability of merit of the applied assay were already published as part of the method validation, repeated sampling was not performed in that investigation.O‐rhamnoside in red grapes contributed to the moderate differentiation between white and red grapes , whereas the white grapes of the cultivar ‘IV065’ did show >90% elevated levels of the stilbenoids trans‑ and cis‐piceide and of arbutin in 2017. Cultivar ‘IV063’ was close to the 95% confidence interval in 2017 and was a clear outlier in 2018 due to approximately twofold concentrations of the same stilbenoids and a moderate increase in kaempferol glycosides. The cultivar ‘IV029’ was observed as an outlier in 2018 due to increased amounts of flavan‐3‐ols.The red accession ‘Cerason’ was an outlier in 2018 due to a tenfold increase in stilbenoids  differences in the phenolic profiles between the harvests in 2017 and 2018 were observed with a very few exceptions (see Supporting Information Tables −1 FW to 74.0 mg kg−1 FW in red grapes (P = 0.01) and from 136.8 mg kg−1 FW to 72.1 mg kg−1 FW in white grapes (P = 0.002). Levels of caftaric acid were comparable in 2017 in red and white grapes (28.3 mg kg−1 FW and 28.7 mg kg−1 FW respectively) but were markedly (P = 0.01) elevated to 51.0 mg kg−1 FW in 2018 in white grapes. Although a great variance was observed between years for most accessions when looking at single compounds, no significant differences were detected considering the overall phenolic profile (P > 0.1).As already shown qualitatively in the PCA Fig. , no stat−1 FW in red berries and 31.4 mg kg−1 FW in white grapes. When Liang et al.V. vinifera grapes, vanillic acid accounted for about 70% of all acids, with amounts up to 43 mg kg−1 FW in wine grapes and up to 76 mg kg−1 FW in table grapes. In our study, vanillic acid could only be detected in a few red accessions (<15 mg kg−1 FW). The dominating benzoic acids in all samples were gallic acid and its condensed dimer ellagic acid, the former with amounts of 1.5–36.6 mg kg−1 FW in red grapes and 0.9–41.9 mg kg−1 FW in white grapes and the latter with amounts of 0.8–63.3 mg kg−1 FW in red grapes and 5.4–74.1 mg kg−1 FW in white grapes. In recent years, ellagic acid in particular has been a focus of interest owing to its various pharmacological properties. In a recently published review, ellagic acid was even discussed as a potential lead for novel therapeutics.et al.V. vinifera.Seven phenolic acids were quantified, the contents and distribution are shown in Fig. −1 FW in white grapes and 3.7 mg kg−1 FW in red grapes). Notable higher concentrations were detected in three white breeds only . Levels of caftaric acid were, as already discussed, comparable in 2017 in red and white grapes but significantly elevated in white grapes in 2018.Hydroxycinnamic acids and their esters are the dominant phenolics in white wines and, besides flavonoids, also in red ones.trans‐Resveratrol and its cis‐isomer, its glucosides trans‑ and cis‐piceide, the hydroxylated derivative piceatannol, and isorhapontin were quantified in this study.Stilbenoids are well‐known constituents in grapes, and also in several other edible berries, due to their role in plant defense, as allelochemicals, and for their interesting health‐related characteristics.−1 FW) than white accessions did (3.1 mg kg−1 FW), and new BLs had a higher mean level of stilbenoids (10.5 mg kg−1 FW) than progeny lines (5.4 mg kg−1 FW) (see Supporting Information Tables −1 FW and 57.7 mg kg−1 FW of trans‑ and cis‐piceide respectively (2017: 12.5 mg kg−1 FW and 23.1 mg kg−1 FW); in ‘IV065’, on the other hand, the trans‑ and cis‐piceide contents decreased from 20.0 mg kg−1 FW and 52.5 mg kg−1 FW respectively in 2017 to 0.9 mg kg−1 FW and 3.7 mg kg−1 FW respectively in 2018. The red grapes of ‘Cerason’ contained the highest amount of total stilbenoids in 2018 (47.9 mg kg−1 FW) and an extraordinary high level of trans‐resveratrol (8.9 mg kg−1 FW) and piceatannol (7.8 mg kg−1 FW) compared with all other breeds. Although those increased stilbenoid levels correlate with observed levels of mildew infection in leaves and clusters of ‘Cerason’  and could be discussed as host response to the fungal attack, utmost care must be taken not to overlook possible other triggers contributing to stilbenoid formation. In contrast to the report of Pedneault and Provost,Red accessions showed a higher median content and wider distribution of total stilbenoids ‐catechin, (−)‐epicatechin, (+)‐gallocatechin) and dimeric forms  were quantified in the present study.P = 0.12; see Supporting Information Table −1 FW in 2017 and 711.6 mg kg−1 FW in 2018, compared with red accessions with median values of 519.4 mg kg−1 FW in 2017 and 483.3 mg kg−1 FW in 2018  were found between the median content in white grapes (327.9 mg kg−1 FW) and red grapes (223.5 mg kg−1 FW), with insignificant variation between years. The situation was different for (−)‐epicatechin, however, where there was a noteworthy drop in the median amount to about half, as discussed earlier.As reported in other studies,Condensed catechin and/or epicatechin derivatives represent a further prominent group of flavanols which are considered as the main oxidants in grapes.P < 0.001; see Supporting Information Table −1 FW versus 70.5 mg kg−1 FW). The flavonol‐richest accession was ‘IV029’, with a total of 331.0 mg kg−1 FW in 2018  but only in traces in white grapes in a very few cases .Red‐colored grapes, on the other hand, were characterized by the presence of myricetin, myricetrin, and syringetin glycosides, which were found in all red accessions , to some extent also in grapes classified as rosé colored (29.3–84.4 mg kg−1 FW), and, surprisingly, in two white accessions, namely ‘IV067’ (24.8 mg kg−1 FW) and ‘Souvignier gris’ (45.5 mg kg−1 FW). Differences between 2017 and 2018 harvests were not significant. Malvidin‐3‐O‐glucoside (oenin) was the most prominent congener. About 15% of all anthocyanins were acetylated, and 15% were present as coumaric acid esters. ‘Cerason’, a variety from the Czech Republic, contained not only the highest total amounts of anthocyanins (5955.5 mg kg−1 FW) but also of acetyl esters (>30%). The dominating representative in the rosé‑ and white‐colored breeds was cyanidin‐3‐O‐glucoside (>97%).As to be expected, anthocyanins were found in all 23 red accessions in varying amounts . Cultivar ‘LU2’ was outstanding not only because of its highest content of diglucosides (average of 2295.5 mg kg−1 FW) but also because of their high proportion of about 90% of total anthocyanins in this plant. Further BLs with extraordinary high amounts were ‘LU1’ and ‘Leon Millot’ (45.3–61.6%). In ‘Coarna N. × Pierelle × SV 20366’ peonidin diglucoside was the most abundant derivative, whereas malvidin‐3,5‐O diglucoside was predominant in the other nine lines. Taking into account that the acceptable limit of diglucosides in wine is 15 mg L−1 . The 17 new PIs were from a private breeder in South Tyrol (Supporting Information Table versus 4.2 mg kg−1 FW). White PIs also contained more flavonols than the white BLs did . The same tendency was observed for rutin and quercetin‐3‐O‐glucuronide (6.9 versus 4.2 mg kg−1 FW and 59.0 versus 29.5 mg kg−1 FW respectively). Luteolin‐7‐O‐glucoside was higher in white and red PIs. Anthocyanin diglucosides, particularly the legally problematic malvidin‐3,5‐O‐diglucoside, were not detectable in any of the PIs.Comparing the results between the international BLs and the PIs that had never been investigated before, it became evident that the differences in the total amount of phenolics was insignificant. However, significant differences were found for several subclasses  or as absolutes (PIs) from private initiatives, were investigated. Variations between genotypes due to macroclimatic and pedologic conditions were excluded as all the grapevines were grown in one and the same untreated vineyard in northern Italy. This allowed the unbiased comparison and differentiation of the hybrids; differences found in the polyphenol pattern originate in the different genotypes of the hybrids solely. Variations of the polyphenolic profiles between two vintages were relatively limited, confirming the stability of the observed polyphenol patterns in the accessions investigated. Since this study was limited to single individuals, it is a must that these findings are reproduced in follow‐up studies in larger experimental cultivation plots. It was also observed that ten of the BLs investigated producing red‐colored grapes contained anthocyanin diglucosides. In nine of these the undesired malvidin‐3,5‐Table S1. List of studied accessions with names, internal (InnoVitis) code, VIVC  variety number, berry skin color, utilization, origin, agronomical and botanical informationTable S2. Soil parameters at growing site at Marlengo, South Tyrol, Italy.Table S3. Concentration of phenolic classes in grape berries in mg kg‐1 FW (fresh weight) in harvest of 2017 and 2018Table S5: Statistical analysis for repeated sampling (n = 3) in selected cultivars. Mean concentrations, standard deviations (SDs) and relative standard deviations (RSDs) of 20 key metabolites.Table S6. Significance of variance for concentrations of phenolic classes between the harvest in 2017 and 2018Table S7. Significance of variance for concentrations of phenolic classes between the red and white grape accessionsTable S8. Statistical significance of variance of concentrations of phenolic metabolites between 2017 and 2018 and between white and red grape accessionsTable S9. Concentrations of phenolic classes found in grapevine hybrids of different breeding origins harvested in 2017 and 2018 (in mg kg‐1 FW)Fig. S1. PCA contribution plot of white grapes vs. average (anthocyanins excluded)Fig. S2. PCA contribution plot of red grapes vs. average (anthocyanins excluded)Fig. S3. Box plot illustrating the total phenolic content in all accessions (red and white grapes) in 2017 and 2018Fig. S4. Box plot illustrating the total phenolic content in red (R) and white (W) accessions in 2017 and 2018Fig. S5. Box plot illustrating the content of epicatechin in red (R) and white (W) accessions in 2017 and 2018Fig. S6. Box plot illustrating the content of caftaric acid in red (R) and white (W) accessions in 2017 and 2018Fig. S7. Heatmap of white grape accessions and identified compounds in the two harvests 2017 and 2018Fig. S8. Heatmap of red grape accessions and identified compounds in the two harvests 2017 and 2018Click here for additional data file.Table S4. Concentration of phenolic compounds in grape berries in mg kg−1 FW (fresh weight) in harvest of 2017 and 2018 (provided as separate Excel file)Click here for additional data file.


We report two patients with PMM2‐CDG who developed end stage renal disease (ESRD). Renal abnormalities of clinical significance have only been reported in about 6% of patients with PMM2‐CDG and have rarely been reported as the cause of death. Given the recurrent episodes of acute kidney injury associated with hospital admissions and the accelerated development of ESRD thereafter in our two patients, we recommend proactively involving Nephrology early in the care of these patients. We report two patients with PMM2‐CDG who developed ESRD, and hope to bring attention to the potential importance of renal decline in PMM2‐CDG.1PMM2 gene located on the 16p13.3 locus.The congenital disorders of glycosylation (CDG) are defects in glycan synthesis and in the proper attachment of glycans to proteins and lipids that results in multiple organ system involvement.Individuals with PMM2‐CDG have a host of clinical and laboratory abnormalities .In this report, we present two patients with PMM2‐CDG with normal renal function at birth and early infancy, who subsequently developed lethal end stage renal disease (ESRD). To the best of our knowledge, death associated with renal failure has only been reported once before in an infant with PMM2‐CDG and congenital nephrotic syndrome Table .52PMM2 gene sequencing confirmed the following compound heterozygote variants: p.Gly15Arg and p.Arg141His.Patient 1 was a female with PMM2‐CDG who died at 4 years and 11 months of age, due to ESRD. Her renal manifestations included cystic renal disease and proteinuria. Her other medical issues at time of death included global developmental delay, hypotonia, abnormal liver function, protein‐losing enteropathy, hypoalbuminemia, pancytopenia, congenital hypothyroidism, hypoglycemia secondary to hyperinsulinism, retinal dystrophy, seizure disorder, bilateral moderate‐to‐severe hearing loss, coagulopathy, pericardial effusion, and eczema. She was born at 37 weeks' gestation to a 27‐year‐old primigravida mother of European ancestry, who had an overall healthy pregnancy, and a father of African‐American ancestry. At 8 weeks of age, she was hospitalized due to failure to thrive and emesis. She was subsequently found to have elevated serum transaminases and hypoalbuminemia, resulting in a diagnosis of a congenital disorder of glycosylation. At 4 months, her renal ultrasound demonstrated increased echogenicity. Furthermore, she exhibited both tubular and glomerular proteinuria as evidenced by increased urine beta‐2 microglobulin and microalbumin, respectively.Clostridioides difficile infection and hypoglycemia. Her serum creatinine was normal at this time. A random urine protein to creatinine ratio  and urine microalbumin  were significantly elevated and serum albumin was decreased  with associated edema. She was provided supportive care with periodic albumin infusions to optimize volume status.At 7 months, she was hospitalized for a Between 12 and 14 months, her random urine protein to creatinine ratio, urine microalbumin, and urine beta 2‐microglobulin levels showed improvement.At 24 months, she underwent extensive hospitalization for hypoglycemia due to hyperinsulinism and also experienced repeated episodes of pericardial effusion. Renal ultrasound at this time showed poor corticomedullary differentiation. At 26 months, she demonstrated decline in renal function with a serum creatinine of 0.7 mg/dl. Her glomerular proteinuria had mostly resolved at this time, however her tubular proteinuria was further exacerbated.At 32 months, she was hospitalized for a febrile illness, presumed to be meningitis, and her serum creatinine increased to 1.1–1.2 mg/dl during this admission. At 36 months, she continued exhibiting tubular proteinuria, persistently creatinine, secondary hyperparathyroidism, and anemia, all believed to be related to her moderate‐to‐severe chronic kidney disease (CKD). A renal ultrasound indicated the development of numerous new bilateral renal cysts, consistent with a diagnosis of cystic dysplasia.At 46 months, she developed pancytopenia and persistent deficiencies in Factor IX, Factor XI, antithrombin‐3, and protein C during a hospitalization for gastroenteritis, sinusitis, pulmonary nodular amyloidosis, and confirmed parvovirus infection. Consistent with an acute kidney injury, her serum creatinine level increased dramatically to 3.2 mg/dl with subsequent improvement. Despite a promising transient increase in all cell counts, she ultimately continued to exhibit trilinear pancytopenia.In the final months of her life, she underwent multiple hospital admissions for various illnesses and gastrointestinal bleeding. Her serum creatinine continued to rise. Her progressive, multifactorial anemia and thrombocytopenia led to a greater susceptibility to GI bleeds that required repeated, regular transfusions of packed blood cells and platelets.In her final days, treatment options of chronic dialysis and renal transplant were discussed with her parents given the severity of her CKD. After careful deliberation, her parents decided against pursuing renal replacement therapy and to focus on her quality of life. She was admitted to the hospital for pain control at 59 months for comfort care and passed away.PMM2 gene sequencing confirmed the following compound heterozygote variants: p. Gly42Arg and p. Pro113Leu.Patient 2 was a male with PMM2‐CDG who died at 11 years and 6 months of age due to ESRD. This patient was the subject of a previous report emphasizing perturbations in the vascular barrier that led to anasarca.He was born at full term to a 36‐year‐old primigravida mother of European ancestry, who had an overall healthy pregnancy, and a father also of European ancestry. During his first year of life, he presented low levels of protein C, protein S, anti‐thrombin, factor V, factor VII, factor IX, and factor XI—putting him at increased risk for clotting and bleeding. Furthermore, he presented an ongoing history of easy bruising, purpura and petechiae, a prominent genitalia fat pad, occasional epistaxis, osteopenia, and abnormal endocrine function including thyroid binding globulin deficiency.He showed evidence of increased renal echogenicity and renal cysts at 3 years of age. He also demonstrated mild persistent proteinuria, though his serum creatinine was maintained within the normal range until age 8  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients and/or guardians for being included in the study. Additional informed consent was obtained from all patients for which identifying information is included in this article.Data S1: Supporting Information.Click here for additional data file.


Several SARS‐CoV‐2 variants have emerged since the pandemic, bringing about a renewed threat to the public. Delta variant (B.1.617.2) was first detected in October 2020 in India and was characterized as variants of concern (VOC) by WHO on May 11, 2021. Delta variant rapidly outcompeted other variants to become the dominant circulating lineages due to its clear competitive advantage. There is emerging evidence of enhanced transmissibility and reduced vaccine effectiveness (VE) against Delta variant. Therefore, it is crucial to understand the features and phenotypic effects of this variant. Herein, we comprehensively described the evaluation and features of Delta variant, summarized the effects of mutations in spike on the infectivity, transmission ability, immune evasion, and provided a perspective on efficient approaches for preventing and overcoming COVID‐19. SARS‐CoV Delta (B.1.617.2) variant has been classified as variants of concern (VOC) by World Health Organization (WHO). The reproductive number (R0) of SARS‐CoV‐2 wild type and Delta variant is 2.3‐5.7 and 5‐8, respectively. Patients infected by Delta variant exhibit shorter mean generation time and mean serial interval, higher virus load and hospitalization rate compared to those infected by wild‐type SARS‐CoV‐2. With the joint efforts of the global community, vaccine protection and effective supervision of SARS‐CoV‐2 positive individuals greatly reduced COVID‐19 hospitalizations and deaths. However, in recent months when B.1.617 lineages, especially B.1.617.2 variant  rapidly spread, even though more than five billion vaccine doses have been administered globally (https://www.who.int/), thousands of new cases are diagnosed every day. Therefore, it is necessary to have a more detailed understanding of the SARS‐CoV‐2 Delta variant.Coronavirus disease 2019 (COVID‐19) is a highly contagious viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2).−4 replacement per site for every year.https://www.who.int/en/activities/tracking‐SARS‐CoV‐2‐variants). By August 24, 2021, cases infected by Alpha, Beta, Gamma, and Delta variants have been reported in 192, 141, 86, and 163 countries, respectively (https://apps.who.int/iris/bitstream/handle/10665/344560/CoV‐weekly‐itrep24Aug21‐eng.pdf?sequence=1&isAllowed=y).As a typical RNA virus, the pace of mutation is about 10www.gisaid.org). The WHO classified the Delta variant as a VOI on April 4, 2021. Later, because of its astonishing transmissibility and infectivity, the Delta variant displaced other pre‐existing lineages and its percentage in all circulating viruses significantly raised to 89.8% on May 10, 2021 among sequences in India (www.gisaid.org). Delta variant caused 17 million COVID‐19 patients including reinfections during March‐May 2021.In early 2021, the variant responsible for the outbreak of COVID‐19 in India was initially called “double mutant” due to E484Q and L452R mutations in spike.2www.gisaid.org), which triggers a new wave of global infection.The reproductive number (R0) of SARS‐CoV‐2 wild type has been estimated to be as high as 2.3‐5.7 Figure 11, 12The life cycle of the Delta variant in host cells is shown in Figure Several properties of the Delta variant have been analyzed to explain why Delta broke through a variety of variants. On the one hand, the Delta variant spike fuses more efficiently with target cells with low human angiotensin‐converting enzyme 2 (hACE2) level than other variants, and its pseudovirus infects target cells significantly faster than other variants.3Ct value below 20.The double mutations of E484Q and L452R in the spike of B.1.617 lineages had caused global concerns since E484 mutation appeared in the rapidly spreading variants Beta and Gamma and was associated with immune evasion.Meanwhile, the Delta variant exerts resistance to some anti‐N‐terminal domain and anti‐RBD therapeutic monoclonal antibodies for COVID‐19 including Bamlanivimab.The RBM epitopes in RBD that overlap with ACE2 are immunodominant. T478, E484, and L452 residues are located in immunodominant RBM epitopes and could influence the binding ability by antibodies from serum or some therapeutic antibodies.44.1WHO has advocated some precautions to protect us from COVID‐19, such as cleaning hands, wearing a mask, keeping rooms well ventilated, etc., especially for healthcare workers (HCWs) who are not only in close contact with COVID‑19 patients, but also in a poorly ventilated environment. Previous studies have shown that the infection rates of SARS‐CoV‐2 among HCWs range 2.2–44% which is significantly higher than that of the general population.4.2Studies have demonstrated that pre‐existing cellular immunity in people infected by other coronaviruses might contribute to the different disease severity between COVID‐19 patients,Moreover, oral and nasal mucosae serve as the primary defense to prevent pathogens from entry and infection.5Mutations will occur in the future due to natural selection, random genetic drift, or influence of the environment.The authors declare no conflict of interest.Xiawei Wei and Kang Zhang conceived the study and revised the manuscript. Xuemei He, Cai He, and Weiqi Hong wrote the article.Not applicable.