Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10–20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10–65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.The causative agent of ileitis,  Lawsonia intracellularis, an obligate intracellular bacterium [L. intracellularis infection is commonly associated with economic losses [L. intracellularis infections often are exacerbated by co-infecting pathogens [acterium , 2, is tacterium  and infec losses . Clinicac losses –7. L. inathogens . The disathogens , 10.Lawsonia intracellularis is transmitted among pigs by the oral-fecal route. The organism has been shown to survive in the environment for up to 2 weeks in a temperature range of 5–15 °C [4–106 organisms are sufficient to infect a pig [ 5–15 °C . It has ct a pig . Transmict a pig , 12.L. intracellularis infection is mainly accomplished by treatment with antibiotics such as tiamulin, tylosin, chlortetracycline, lincomycin and olaquindox [L. intracellularis shedding and is therefore occasionally used in combination with antibiotics [L. intracellularis infection poses challenges for producers and veterinarians.Control of aquindox , 13, whiaquindox , 14–17. ibiotics . In addiin-feed supplementation [Bacillus spp., are considered to have probiotic potential [Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus on L. intracellularis infection. The results were directly compared to those obtained with a commercial live L. intracellularis vaccine.Probiotic bacteria are widely used in human nutrition. Major proposed probiotic mechanisms include competitive exclusion of pathogenic microorganisms, enhancement of the epithelial barrier, modulation of the immune system and others. Usage of probiotics is considered by many as an alternative to prophylactic antibiotic entation . Severalotential . The objL. intracellularis-free source herd where the dams were regularly tested and found to be negative for L. intracellularis antibodies using a commercial ELISA . The pigs were transported to the Livestock Infectious Disease Isolation Facility at Iowa State University and randomly allocated into six experimental groups housed in four separate BSL-2 rooms with 1–3 pens (approximately 10 m2 each) of 10 pigs each equipped with a nipple waterer and a self-feeder , were supplied feed supplemented with one of the three probiotics , or remained non-treated . At 7 weeks of age all groups except the NEG-CONTROL group were challenged with gut homogenate containing L. intracellularis by gastric gavage. Blood samples were collected from all pigs on a weekly basis. Rectal swabs  were collected at arrival of the pigs at the research facility, prior to inoculation and at day post-inoculation (dpi) 2, 4, 6, 8, 10, 12 and 15.At arrival, the pigs were randomly assigned to six treatment groups with 10–20 pigs each  per metric ton of Bacillus amyloliquefaciens , Bacillus licheniformis  or Bacillus pumilus . Each different feed batch was color coded, labeled with Base, T01, T02 or T03, transported to the Iowa State University research facility and stored at 22 °C in a dark, dry storage room. The room temperature in the storage room was monitored daily. The pigs were fed ad libidum for the duration of the study with their allocated feed  as outlined in Figure The feed used in this study was produced by a local feed mill  without addition of antibiotics or animal proteins. Two different regular base diets, HLN2 and HLN3, were fed to the pigs from 2.5 to 6 weeks (HLN2) and 6 to 9 weeks  at 3 weeks of age. This particular vaccine is a one dose product, contains a live bacterial culture, and is given orally. Prior to vaccination the vaccine was freshly reconstituted according to the manufacturer’s instructions and administered to each VAC-LAW pig by slowly dripping 2 mL of the vaccine with a syringe into the mouth.Pigs in the VAC-LAW group were vaccinated with a commercial L. intracellularis vaccination, using an intestinal homogenate from an experimentally infected pig containing approximately 3 × 107L. intracellularis organisms per mL. The L. intracellularis inoculum was obtained from a commercial supplier . The L. intracellularis concentration in the inoculum was determined by quantitative PCR [Salmonella enterica, enterotoxigenic Escherichia coli, oocysts or parasite eggs. In brief, the inoculum was thawed shortly prior to the inoculation and was transported/stored in several independent smaller containers on ice. Each pig was briefly restrained with a snare and 35 mL of the homogenate was administered by gastric gavage. The overall inoculation took approximately 45 min for all pigs. The order of inoculation was T03-LAW group first followed by T02-LAW, T01-LAW, POS-CONTROL pigs and finally the VAC-LAW pigs.Inoculation was done when the pigs were 7 weeks old, 28 days post tive PCR , 22. TheThe pigs were weighed at arrival (2.5 weeks of age), at inoculation  and at necropsy  blinded to treatment group status. Specifically, ileum mucosal thinking was subjectively assessed using a score ranging from 0 , 1 (mild), 2 (moderate), and 3 (severe). If present, the length of a given lesion was not assessed. However, to assess distribution by histopathology three sections of ileum (3–5 cm apart) and three sections of colon (3–5 cm apart) were collected from each pig. Samples from the ileum, colon and mesenteric lymph node were collected and as per standard research protocols frozen at −80 °C or fixed in 10% buffered formalin for histological analysis.L. intracellularis-specific antibody response was determined using the SVANOVIR®L. intracellularis/Ileitis-Ab antibody test , a competitive ELISA, as per the manufacturer’s instructions. A 40% inhibition of the supplied standard positive serum was set as the criteria for a valid assay. A sample with more than 30% inhibition was considered positive, and a sample with less than 30% inhibition was considered negative.The Lawsonia intracellularis DNA was extracted from the fecal swabs using the KingFisher Flex® magnetic bead-based nucleic acid extraction platform and the MagMAX-96 nucleic acid isolation kit .L. intracellularis was generated to establish the conditions for bacteria quantification initially. The copy numbers of L. intracellularis in the rectal swabs were estimated using a quantitative real-time PCR assay with the following primers and probe: forward primer, 5′-GCGCGCGTAGGTGGTTATAT-3′); reverse primer, 5′-GCCACCCTCTCCGATACTCA-3′; probe, 5′-FAM-CACCGCTTAACGGTGGAACAGCCTT-TAMRA-3′ [A standard curve with a known amount of TAMRA-3′ . The assL. intracellularis infection . While inflammation is not a prominent feature with L. intracellularis infection, the sections were also scored for presence of inflammation .Microscopic lesions were assessed by a veterinary pathologist (TO) blinded to the treatment status. Intestinal sections including several sections of ileum and colon were scored for the presence of crypt epithelial hyperplasia characteristic of Lawsonia intracellularis antigen-specific IHC was performed on the intestinal sections as described [L. intracellularis antigen was scored and ranged from 0 , 1 , 2 , to 3 . To simplify the analysis, a combined ileum lesion score was introduced for which the scores for crypt enterocyte hyperplasia, inflammation and amount of L. intracellularis antigen were combined for a maximum score of 9. Using this combined ileum lesion score, the lesions were classified as absent (score of 0), mild , moderate  and severe .escribed . The preFor the Periodic acid Schiff (PAS) staining, fixed paraffin-embedded ileum sections from pigs were dewaxed and rehydrated according to standard protocols. Following this, sections were immersed in 1% periodic acid for 5 min and in Schiff’s reagent for 15 min as described . Slides ® Red Alkaline Phosphatase Substrate  according to manufacturer instructions. Slides were counterstained with Haemalum Mayer and mounted as described for the PAS staining.Intestinal alkaline phosphatase (IAP) was stained with VectorFor quantitative analysis of PAS and IAP stained slides were scanned using NanoZoomer XR digital slide scanner . For PAS staining, images were captured using brightfield, and for IAP a combination of brightfield and Texas red filter was used. Digital images were acquired at 40× magnification and analyzed using open-access image analysis software ImageJ version 1.49 . Staining was analyzed by quantifying the intensity of the entire sections and a total of three sections per staining were quantified. Briefly, the spectrally opposite color channel was selected, and the intensity threshold was adjusted to highlight goblet cells for PAS staining or the villi for IAP staining .L. intracellularis. Real-time PCR results  were log10 transformed, demonstrated a normal distribution, and were then analyzed. The significance level was P < 0.05 followed by pairwise testing using the Tukey–Kramer adjustments to identify the groups that were different. Non-repeated measures of necropsy and histopathology data were assessed using non-parametric Kruskal–Wallis ANOVA. If a non-parametric ANOVA test was significant (P < 0.05), then Wilcoxon tests were used to assess the differences of pairs of groups. Differences in incidence were evaluated by using Fisher’s exact test. The area under the curve (AUC) of bacterial shedding of each animal and the total AUC for each group was calculated using the log transformed values of the bacterial loads over time. One-way ANOVA and a Bonferroni post hoc test were used to compare groups.The statistical analysis was performed using JMP software . Summary statistics were calculated to assess the overall quality of the data. Analysis of variance (ANOVA) was used for cross-sectional assessment of the average daily weight gain (ADG) and level of fecal shedding of L. intracellularis values of infected  and non-infected pigs (NEG-CONTROL) based on PAS and IAP staining intensity in ileum sections, receiver operating characteristic (ROC) curve analysis SPSS statistics  was used. The cutoff was calculated using the Youden Index. Correlations of PAS or IAP values with overall ileum lesion severity or L. intracellularis DNA shedding length in fecal swabs were also calculated and compared as described [L. intracellularis PCR positive rectal swabs over time in a given pig.To determine a cutoff for escribed . SpecifiL. intracellularis antibodies (data not shown). At the day of inoculation (28 days post-vaccination) all VAC-LAW pigs had seroconverted with a group mean percentage of inhibition value ± SEM of 53.1 ± 1.9 which was significantly (P < 0.05) higher compared to all other groups indicating successful vaccination of this group. The number of pigs that seroconverted within the 15 days between L. intracellularis inoculation and blood collection prior to necropsy was 4/10 in the T01-LAW group , 1/10 in the T02-LAW group (14.7 ± 2.4), 1/10 in the T03-LAW group (12.8 ± 4.2), 10/10 in the VAC-LAW group (44.5 ± 1.8), 0/10 in the NEG-CONTROL group (7.2 ± 1.6) and 13/20 in the POS-CONTROL group (33.9 ± 4.4).At arrival in the research facility, all pigs were negative for L. intracellularis inoculation. Pigs in the NEG-CONTROL group remained free of clinical signs throughout the study period. Individual pigs in all L. intracellularis groups had semisolid or pasty feces on single days throughout the study. Watery or bloody diarrhea (score 3) was present in 4/20 POS-CONTROL pigs and 1/10 VAC-LAW pigs by dpi 15. The average daily gain (ADG) before and after challenge is summarized in Table L. intracellularis infection, all infected groups had significantly less ADG compared to the NEG-CONTROL group without significant differences among L. intracellularis infected groups.There were no clinical signs in any of the pigs present prior to Lawsonia intracellularis DNA was never detected in rectal swabs of any of the NEG-CONTROL pigs for the duration of the study. L. intracellularis DNA was also not detected in any of the other groups at arrival at the research facility or on the day of inoculation. L. intracellularis shedding was first detected in a POS-CONTROL pig by dpi 2. The prevalence rates of L. intracellularis shedding in fecal swabs are summarized in Figure L. intracellularis for each dpi and the shedding length in weeks is presented in Table L. intracellularis compared to VAC-LAW and POS-CONTROL pigs. The AUC was highest and not different for T01-LAW, VAC-LAW, and POS-CONTROL while it was significantly (P < 0.05) reduced for T02-LAW and T03-LAW  in the T01-LAW, T02-LAW and T03-LAW group compared to the POS-CONTROL pigs. Often the intestinal content was fluid and bloody with necrotic debris adhered to the mucosa. Liquid intestinal content (score 3) was present in 1/10 T01-LAW pigs, 0/10 T02-LAW pigs, 0/10 T03-LAW pigs, 1/10 VAC-LAW pigs and 8/20 POS-CONTROL pigs. In addition, mesenteric lymph nodes were often macroscopically enlarged.Macroscopic lesions were not observed in the NEG-CONTROL group while lesions consistent with L. intracellularis including crypt epithelial hyperplasia, increased numbers of mitotic figures throughout the crypt, depletion of goblet cells and presence of L. intracellularis antigen in enterocytes. The mean microscopic lesions are summarized in Table L. intracellularis associated lesions were identified in 30% of the T01-LAW pigs, 10% of the T02-LAW pigs, 40% of the VAC-LAW pigs and 55% of the POS-CONTROL pigs while severe lesions were not present in NEG-CONTROL or T03-LAW pigs. L. intracellularis antigen was identified in 9/10 T01-LAW pigs (0.7 ± 0.3), 9/10 T02-LAW pigs (0.3 ± 0.2), 9/10 T03-LAW pigs (0.2 ± 0.1), 8/10 VAC-LAW pigs (0.7 ± 0.2), 0/10 NEG-CONTROL pigs, and 18/20 POS-CONTROL pigs (1.2 ± 0.2). The L. intracellularis antigen levels were significantly (P < 0.05) higher in T01-LAW, VAC-LAW and POS-CONTROL pigs compared to NEG-CONTROL pigs, whereas there were significantly (P < 0.05) lower antigen amounts in T02-LAW and T03-LAW compared to POS-CONTROL pigs  lower mean staining intensity compared to NEG-CONTROL pigs for both PAS and IAP.PAS staining reveals the presence of mucopolysaccharides, glycoproteins, and/or glycogen and is traditionally used for the assessment of goblet cells, which are depleted during nfection . The gronfection . The groL. intracellularis infected pigs were considered healthy. For the IAP stain, the AUC was 0.9 ± 0.0. At a cutoff value of 3.6 the sensitivity was 100% and the specificity was 83% and all NEG-CONTROL pigs were considered healthy, while 16.7% (10/60) of the L. intracellularis infected pigs were considered healthy. The correlation of PAS stain intensity with an ileum lesion severity score (a combined histopathology and immunohistochemistry score) was moderate (−0.536) and it was high (−0.752) for L. intracellularis shedding length. The correlation of IAP stain intensity with the ileum severity score was low (−0.467) and it was moderate with L. intracellularis shedding length (−0.672).PAS stain had an AUC of 0.9 ± 0.0. At a cutoff value of 1.6 the sensitivity was 100% and the specificity was 75%. Using this cutoff all NEG-CONTROL pigs were considered healthy while only 25% (15/60) of the L. intracellularis shedding compared to the POS-CONTROL group. The T02-LAW group was significantly different from the POS-CONTROL and the VAC-LAW group on dpi 6 and dpi 8. The T03-LAW group had significantly lower fecal shedding of L. intracellularis throughout the study period (except on dpi 10). In addition, microscopic lesions consistent with severe L. intracellularis infection  were not seen in the T03-LAW pigs. Interestingly, pigs in groups T02-LAW and T03-LAW had a delayed or limited seroconversion against L. intracellularis, with only 10% of the pigs in the T03-LAW group seroconverting by dpi 15. This could reflect the delay in the colonization of the intestinal tissue by L. intracellularis in the T02-LAW and T03-LAW groups and thereby delay in the onset of humoral immune response against the pathogen. This is further corroborated by the observation that no fecal shedding could be observed in the T03-LAW group on dpi 6. Furthermore, on dpi 8, only 2/10 of the T03-LAW pigs were shedding compared to 18/20 POS-CONTROL pigs, indicating the degree of mitigation. Probiotic organisms B. amyloliquefaciens and B. licheniformis, fed to the groups T01-LAW and T02-LAW, respectively, were also effective in mitigating the shedding of the L. intracellularis albeit to a lesser degree than the B. pumilus. While B. amyloliquefaciens and B. licheniformis may also offer a certain level of protection against enteric pathogens in pigs and other livestock species [In this study, T02-LAW and T03-LAW groups showed a delayed  species , their aBacillus spp. mediate their probiotic action have been described [Bacillus spp. has been shown to induce resistance to oxidative damage in host intestinal cells and improve their barrier function [Bacillus spp. are also known to produce cyclic lipo-peptides which have a variety of activities including interactions with biofilms, and anti-fungal, anti-inflammatory, anti-tumor, anti-virus properties [Bacillus spp. The lipopeptides are known to self-assemble and form micelles which have surfactant activity on biological membranes [Bacillus pumilus is known to lyse cells of Vibrio spp. [Bacillus spp. which have potent antibacterial activity, besides other biological activity [Bacillus spp. may have a direct effect on the L. intracellularis and may also prevent the colonization of enterocytes by L. intracellularis by inhibition of the pathogens host cell adhesion or entry.Numerous modes by which the escribed . Secretifunction . Bacilluoperties . Surfactembranes . Bacillurio spp. . Polyketactivity . The com7L. intracellularis organisms and the intragastric inoculation route used in this study, the T03-LAW group had significantly lower fecal shedding of L. intracellularis at dpi 4, 6, 8, 12 and 15 compared to POS-CONTROL and VAC-LAW groups, and fewer and less severe, but not statistically different, gross and histopathological lesions than the VAC-LAW group. The L. intracellularis specific antibodies, especially IgA, induced by vaccines are thought to be important in mediating protection against infection of enterocytes by L. intracellularis and efforts to bolster the IgA in the ileal mucosa by feed supplementation with short-chain fructo-oligosaccharide have shown improved protection against L. intracellularis [B. pumilus may be able to prevent or limit the infection of enterocytes by L. intracellularis due to a yet unknown mechanism. This warrants further investigation especially under field conditions with the natural dynamics of infection, i.e., a lower but constant dose of infectious exposure at play. Identifying the mechanism by which B. pumilus inhibits L. intracellularis, a major primary pathogen in pig production around the world and also a risk factor for other enteric pathogens such as Salmonella enterica serovar Typhimurium [Probiotics may act synergistically when used in combination, as their mode of action may be complementary to one another. Further, in spite of a challenge dose of 3 × 10llularis . In thishimurium , 36, wouB. pumilus efficiently prevented early colonization of L. intracellularis in the pig enteric system as evidenced by reduced fecal shedding with a significantly lower AUC, even in the face of a high-dose experimental inoculation. The two other probiotics, B. amyloliquefaciens and B. licheniformis, displayed any anti-L. intracellularis effect to a lesser extent. This study warrants further detailed exploration of the potential of the B. pumilus to prevent or limit the impact of L. intracellularis infection in growing pigs.The results of this study indicate that


Hearing impairment is a complex medical disorder which has genetic and non-genetic causes. Gap Junction Protein Beta 2 (GJB2) gene variant is a well-known disease-causing gene among patients with hearing impairment. The frequencies of genetic variants in the GJB2 gene are different in each population. This study aimed to discuss the GJB2 gene status in an Iranian population with hearing impairment who referred for prenatal testing. This cross-sectional study was conducted in a genetic laboratory affiliated with Mashhad Jahad Daneshgahi, Mashhad, Iran. A total number of 21 bilateral hearing impaired patients were enrolled in this study. The exons for target GJB2 gene were amplified by polymerase chain reaction after the confirmation of the hearing impairment and the exclusion of the acquired causes of hearing loss. The c.35delG and c.79G>A variants were the first and second most common variants in the study population, respectively. The mean age of the patients was 27.5 (8.7) years and 12 cases were male. There was no significant association between hearing impairment degree and age and heterozygosity status (P=0.376 and P=.074 respectively).The c.35delG and c.79G>A variants were determined as the first and second most common variants in the GJB2 gene, respectively. The mean age of 26 years in this study population indicates the late referral for the evaluation of the hearing difficulty. Furthermore, it highlights the further need to encourage families with a history of hearing impairment to engage in genetic counseling.  The incidence of hearing impairment is about one out of 166 newborns in Iran in which it is much more prevalent than the rest of the world . Syndromic and non-syndromic hearing impairment is a classification, which mostly relies on the genetic basis of the disorder. Syndromic patients show other clinical abnormalities in addition to hearing problems, whereas hearing impairment is only observed among non-syndromic patients .Gap junction-related genes, Ion channel-related genes, and tight junction-related genes are 3 well-known genes which are responsible for most cases of non-syndromic hearing loss . The GJB2 gene, which is a variant of gap junction-related genes, is responsible for the hearing impairment in Iran and many other countries. It has been reported that GJB2 gene variants will result in prelingual hearing loss ,4. AlthoIncomplete population coverage of detection techniques, poor screening tests, and hearing impairment denial of both parents and physicians are reported to be the three main reasons for late diagnosis and the management of patients with hearing loss .Therefore, it is necessary to determine the late referral cases and establish cost-effective genetic testing for hearing loss according to each country’s prevalent variants. Accordingly, it may lead to achieve a successful strategy to reduce the burden of this disorder . The present study was conducted in Iran in order to achieve this goal and also evaluate the genetic diagnostic age for hearing impairment among patients with autosomal recessive non-syndromic hearing loss. This cross-sectional study was conducted in a genetic laboratory affiliated with Mashhad Jahad Daneshgahi, Mashhad , Iran, and was approved by Khorasan Academic Centre for Education, Culture and Research. In total, 21 hearing impaired patients were selected out of 200 individuals referring for prenatal diagnosis between August 2015 and January 2018. With regard to the ethnicity, all the participants were Persian and resident in Mashhad. The hearing impairment was confirmed by an otorhinolaryngologist and required clinical tests, such as pure-tone audiometry through a diagnostic audiometer.Furthermore, imaging studies were performed where indicated. Pure-tone averages of more than 25 decibels hearing level (dBHL) were defined as hearing loss .The severity of deafness was divided into mild (24-40 dB), moderate (41-55 dB), moderately severe (71-90 dB), severe (71-90 dB), and deep (90 dB). Those couples who had acquired hearing loss because of environmental causes  were excluded from the study. The informed consent and 5ml of venous blood sample were obtained from all participants. Genomic DNA was extracted from peripheral blood by salting out method. Individual exons for target GJB2 gene were amplified by polymerase chain reaction (PCR). Oligonu- cleotide primer pairs for the amplification of each exon were designed online by Primer 3. The PCR was conducted in the 20μL final volume and was performed in the Labcycler  under the following conditions: 94 °C for 5 min followed by 35 cycles of 94 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec. As a final extension, 72 °C was applied for 5 min . Moreover, amplified fragments were separated on 1.5% agarose gel electrophoresis. All PCR products were sequenced  and they were analyzed using Chromas software (Version 2.4.4).The sequence alignment was performed by reference sequences from ENSEMBLE (ENSG00000165474) and the sequence databases for GJB2 gene. The data were analyzed in SPSS software (version 20) through Kruskal Wallis and the Chi-square tests. A total number of 21 patients referred for prenatal diagnosis was enrolled in this cross-sectional study. The mean age of patients was 27.5 (8.7) years and 12 cases were male. According to the results obtained from the Kruskal Wallis test, no significant associations were observed regarding the study variables. There were not any significant associations between the hearing impairment degree and the age and heterozygosity status (P=0.376 and P=.074 respectively). Heterozygote variant of c.487A>G was found in two participants with profound and moderate deafness. Moreover, homozygote variants were identified in one of the healthy participants. Three cases with profound hearing loss had c.457G>A, c.95G>A, and c.88A>G. The results showed that individuals without hearing loss had c.487A>G variant in GJB2 gene. Furthermore, most of the variants were located in the M1 domain of connexin 26 protein . .79G>A were the first and second most common variants among study participants, respectively. Although frameshift variant of 35delG is responsible for 20% of hearing loss in Caucasians, some countries  have reported high prevalence rates. This variant is responsible for one-third of prelingual and sensorineural hearing loss in Greece population  and Ashkenazi Jewish patients (37%), respectively. Approximately, 40% of Iranian population has the genetic background for autosomal recessive non-syndromic hearing loss . This faAlthough there is no significant association between GJB2 variants and hearing loss degree, it has been reported that truncating variants are associated with a greater degree of hearing loss in contrast to non-truncating variants . RegardlMany parents are unaware of their children's hearing impairment in their early life. A recent study conducted in Iran has shown that approximately one-third of parents are unaware of their children hearing loss . Parents A ten-year cohort study has demonstrated the value of hearing screening at birth. Moreover, deafness has been diagnosed in one-quarter of the children at school. Such findings demonstrate the obvious necessity of postneonatal care pathways for the identification of the missed ones. Otherwise, these children will face various difficulties in their later life . This isTraditional intermarriage among deaf people is a common finding in some countries which will increase the number of deaf people in some situations. The genetic testing for GJB2 variant among partners who wish to marry each other and has hearing impairment will predict the possibility of having a hearing impaired child if other genetic and non-genetic causes of hearing loss can be ruled out. However, this mate selection may be forbidden in some cultures and may raise ethical issues . Full awIt is necessary to screen all children with hearing impairment to determine the specific gene variants regardless of the hearing impairment degree. Therefore, further programs for family planning and conducting prenatal diagnosis become easier following the determination of these variants in each population. The mean age of 26 years in this study population indicates the late referral for the evaluation of the hearing difficulty. Moreover, it highlights the further need for public education regarding genetic counseling among families with a history of hearing impairment. Furthermore, a more close relationship between clinicians and geneticists will facilitate further experimental procedures for these families.


Apophysomyces species, Saksenaea species and Lichtheimia (formerly Absidia) species, although uncommon as causes of sinopulmonary mucormycosis, are relatively frequent agents of trauma-related mucormycosis. The pathogenesis of these infections likely involves a complex interaction among organism, impaired innate host defenses, and biofilms related to traumatically implanted foreign materials. Effective management depends upon timely diagnosis, thorough surgical debridement, and early initiation of antifungal therapy.Necrotizing mucormycosis is a devastating complication of wounds incurred in the setting of military (combat) injuries, natural disasters, burns, or other civilian trauma.  Fungi of the order Mucorales are increasingly recognized as important causes of necrotizing wound infections in the setting of military (combat) injuries, burns, natural disaster-related, and other civilian trauma ,4,5,6,7.Rhizopus arrhizus is the most commonly reported cause of pulmonary, sino-orbital, rhinocerebral, and disseminated mucormycosis, less commonly recognized species of Mucorales are reported worldwide in associated with trauma-related disease. Apophysomyces species, Saksenaea species and Lichtheimia (formerly Absidia) corymbifera, albeit uncommon causes of mucormycosis, are relatively frequently reported agents of trauma-related infections  direct injury to soft tissue resulting in local necrosis, as well as impaired blood flow to damaged tissue, (2) traumatic inoculation of foreign material, including soil, rocks, glass, and wood, contaminated with soil-borne Mucorales forming a nidus for possible biofilm formation, (3) establishment of local infection by the Mucorales fungus in deep tissue, and (4) possible synergistic interaction with bacteria and other fungi to establish a necrotizing infection. Since tissue has been injured, either simultaneously or previously from traumatic injury and impaired host inflammatory cells are unable to eradicate the organism, a local infection is able to be established and become self-propagating as hyphal elements further destroy local tissue, prevent capillary influx of white blood cells, and allow further proliferation of organism.Therapeutic intervention follows from this pathogenesis in that successful management would include thorough and meticulous debridement of necrotic tissue, removal of foreign material, as well as antifungal therapy.Combat-associated wounds complicated by invasive fungal infections (IFIs) are associated with serious morbidity and excess mortality . Early oMucorales and other invasive mould pathogens on clinical outcomes of polymicrobial traumatic wound infections [n = 20)), probable (culture + nonvascular tissue invasion (n = 4)), and possible ). The data were collected from records of US military personnel who served in Afghanistan. The following epidemiological and possible risk factors were common to most patients with IFI: Blast injury during foot patrol, injury occurring in southern Afghanistan, lower extremity amputation, and receipt of large volume blood transfusions [Aspergillus spp. in 16, and Fusarium spp. in 9. Reflecting the soil and environmental contamination of severely injured wounds, cultures yielded multiple species of moulds in 10 (28%) of these cases. The median age of the patients with IFIs was 22.9 years and 100% were male. All of the IFIs were associated with injuries sustained from blast injury. Traumatic amputations of the lower extremity above or through the knee were the most common type of initial wounds.Warkentien et al. of the Infectious Disease Clinical Research Program (IDCRP) Trauma Infectious Disease Outcomes Study (TIDOS) group from the Walter Reed National Military Medical Center, Uniformed Services University of the Health Sciences, San Antonio Military Medical Center, and Landstuhl Regional Medical Center  recently reviewed the impact of fections ,9. The isfusions ,23. Amonn = 82) and as case-matched control non-IFIs (n = 136). The authors further evaluated the effect of the type of mould on clinical outcome [Aspergillus spp. (29%), and Fusarium spp. (21%). Among the 29 wound IFIs caused by Mucorales, the most common genera were Mucor spp. (n = 15 (52%), Saksenaea vasiformis (n = 5 (17%)), and Rhizopus spp (n = 1 (3%)). The other species of Mucorales were apparently not cultured or identified to species and may have been diagnosed histologically as mucormycosis. These investigators then compared the potential differences in microbiological features and clinical outcomes between wounds classified as IFIs ). The study also found that the median time to wound-closure was significantly longer (p < 0.001) for IFIs (16 days) than that for the control non-IFIs with or without skin and soft tissue infections . IFI wounds were managed principally by delayed primary closure, full-thickness skin graft, or split-thickness skin graft. Surgical amputations and revisions were more frequently performed in IFI wounds (n = 63 (77%)) than in non-IFI wounds (n = 71 (52%)). Median duration of antifungal therapy for mucormycosis was 31 days . Given the broad range of fungal pathogens causing these infections, when IFI was clinically or microbiologically suspected, a combination of liposomal amphotericin B and voriconazole was empirically initiated for broad antifungal spectrum, pending definitive identification. That voriconazole is added empirically may contribute to the selection of increased virulence of isolates of Mucorales [Wounds infected with a species of the Mucorales required longer median time for wound-closure in comparison to those infected with a non-Mucorales fungal pathogen  rated multiple vortex tornado devastated the community of Joplin, Missouri, USA, resulting in 13 tornado victims with serious necrotizing cutaneous mucormycosis caused by nd glass . ApophysApophysomyces spp. are well described as causes of trauma-related musculoskeletal mucormycosis in immunocompetent hosts. However, Apophysomyces trapeziformis had seldom been reported as an etiological agent. Whole-genome sequencing typing (WGST), which was conducted on four isolates, demonstrated that these four isolates were separate individual strains. Five of these patients died. Apophysomyces trapeziformis, as well as two control isolates of Apophysomyces variabilis [Additional WGST analysis was conducted by Etienne and colleagues from the Centers for Diseases Control (CDC) on 17 outbreak isolates and three control strains of riabilis . While tFusarium spp. Apophysomyces elegans was recovered from one patient. Aspergillus fumigatus was also recovered from one of two patients who later died. Other invasive fungal infections complicating this massive tsunami included Cladophialophora bantiana soft tissue infection, Scedosporium apiospermum brain abscess, and Aspergillus fumigatus brain abscess. In addition to these organisms, water-borne bacterial co-infection, including those caused by Aeromonas hydrophila and Pseudomonas aeruginosa, may have also contributed to the pathogenesis of these infections. The devastating Indian Ocean tsunami of December 26, 2004, that killed more than 200,000 estimated victims with yet another estimated 40,000 seriously wounded patients ,12,28, iAmong other cases of mucormycosis associated with natural disasters, Patiño et al. reported the development of 8 cases of necrotizing soft tissue infection caused by Mucorales fungi following the cataclysmic volcanic eruption of Armero, Colombia in 1985 that resulted in more than 23,000 deaths and 4500 wounded, where burns sustained from lava, pyroclastic flows, and other fires may have allowed for inoculation from environmental pathogens ,29. WithRhizopus and Rhizomucor species were recovered in all patients. Bacterial co-infection with Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter baumannii, and Proteus mirabilis was identified. The authors observed that the frequency of mucormycosis in their center from 2005 to 2014 among 477 adult patients was 0.63%, which they further noted was consistent with that of Schaal et al., who reported an incidence of 0.5% in a French military burn center [Absidia corymbifera infection in 2005, according to Christiaens et al. [Mucormycosis of burn wounds has been known for more than one-half century to be associated with a high mortality and severe morbidity. Devauchelle et al. recently reviewed the epidemiology of mucormycosis in burn patients . They idn center ,30. Use s et al. . Kyriopos et al. . In addition to military (combat) injuries, burns, and natural disasters injuries as predisposing factors for necrotizing mucormycosis, injuries associated with civilian industrial, agricultural, and automotive/vehicular accidents also pose a threat for these serious infections. Lelievre, representing the French Mycosis Study Group, published a study of posttraumatic mucormycosis . Cases oApophysomyces elegans complex and Saksenaea vasiformis were recovered more frequently from posttraumatic wounds than from other types of mucormycosis. More patients (94%) underwent surgery for posttraumatic mucormycosis than did those with other forms (48%). Survival at day 90 was greater in posttraumatic mucormycosis (88%) in comparison to that of other types of mucormycosis (48%).Posttraumatic mucormycosis in these patients was seldom associated with underlying diseases  in comparison to other forms of mucormycosis. The preponderance of cutaneous mucormycosis occurred in posttraumatic mucormycosis (87%) vs. other forms of mucormycosis (7%). As these infections were localized to the skin and soft tissue and occurred in a trauma-related clinical setting, an early diagnosis was readily established. Among the causes of mucormycosis, Apophysomyces elegans complex and Lichtheimia (formerly Absidia) spp. were the two most common species recovered from these cases of civilian posttraumatic mucormycosis. Lichtheimia corymbifera has long been associated with post-traumatic necrotizing mucormycosis. Apophysomyces elegans complex was also a common organism recovered from wounds of posttraumatic mucormycosis.Among the 122 cases that were identified from a systematic review of literature, traffic injuries, domestic accidents, natural disasters, and farm accidents constituted the most common events predisposing to civilian posttraumatic mucormycosis. Dissemination from traumatic mucormycosis seldom occurred (9%). Apophysomyces elegans in an otherwise healthy child who sustained a traumatic avulsion injury of her latissimus dorsi [Apophysomyces spp. in trauma associated necrotizing mucormycosis and highlights the need for a high index of awareness of in both pediatric and adult patients.Most patients reported with trauma-associated mucormycosis are adults. Little is known about trauma-associated mucormycosis in pediatric patients. Kordy and colleagues reported the development of severe deep soft tissue mucormycosis caused by us dorsi . The traWhile any of the previously mentioned settings may inflict trauma-related osteoarticular mucormycosis, the preponderance of literature addresses deep soft tissue infections. Little has been written about trauma-related osteoarticular mucormycosis. A systematic review of osteoarticular mucormycosis by the International Osteoarticular Mycoses Consortium from 1978 to 2014  found thStrategies of management of trauma-related mucormycosis follow fundamental principles of diagnosis, empirical antifungal therapy for suspected infection, extensive surgical debridement of necrotic tissue, definitive antifungal therapy for documented disease, topical therapy, and reversal of underlying metabolic or immune-impaired conditions.A heightened clinical suspicion at the time of wound assessment and a rapid laboratory diagnosis are essential in the management of trauma-related necrotizing mucormycosis . Direct Rhizopus, Mucor, Rhizomucor, and Cunninghamella species) within the Mucorales in plasma, bronchoalveolar lavage fluid, and tissue of rabbits with experimental invasive pulmonary mucormycosis [Mucor/Rhizopus, Rhizomucor, and Lichtheimia (formerly Absidia) in a retrospective multicenter study [Deployment of PCR or other molecular diagnostic systems for laboratory diagnosis of wound-associated mucormycosis could complement conventional microbiological methods and guide pathogen-directed antifungal therapy. Rapid molecular diagnostic tools have been developed that may further aid in the diagnosis of necrotizing mucormycosis for those healthcare facilities with clinical laboratories that are resourced with dedicated assays and technologist support. Pioneering work by Kasai and colleagues developed a rapid PCR-based platform that identified several genera  spp. in March 2014 and July 2016, as well as in individual cases in November 2013 and July 2016. Real-time PCR, and Lichtheimia species-specific qPCR detected Lichtheimia ramosa, Lichtheimia ornata, and Lichtheimia corymbifera in crepe bandages and elasticized bandages [Fréalle et al. investigated the possible role of non-sterile bandages used to secure sterile gauze and strips in contact with burn wounds in the Burn Unit of the University Hospital of Lille, France in order to determine their relationship to outbreaks of infections caused by bandages . The autAs trauma-related mucormycosis is an uncommon infection, there are no controlled studies to guide management. Nonetheless, the experience from the medical command caring for servicemen with trauma-related mucormycosis provides the largest body of collective experience in management of this devastating infection . The appAs a guide to resection of tissue, one of the serious challenges is the need to repeatedly resect necrotic tissue resulting in larger wounds. Defining clear margins is essential in limiting resection of viable tissue while receiving infected margins. We have observed that wound margins may appear clinically and histologically intact while still having viable organisms present. We therefore have used a system of intraoperative assessment of resected tissue margins sent by the surgical team to the clinical microbiology laboratory for fluorescent microscopy using calcofluor wet mounts ,41.There are, of course, many variables that must be individualized for each patient. These include the extent, timing, and repeating of debridement, the duration of systemic therapy, use of oral agents, role of adjunct hyperbaric oxygen, repair of major tissue defects, and the timing of skin grafting.Rhizopus species, such as Lichtheimia corymbifera, Saksenaea and Apophysomyces spp. Development of new rapid molecular tools, especially at point of care in a trauma setting would be highly beneficial in guiding therapy [Considerably more work is needed in understanding the pathogenesis, diagnosis and treatment of trauma associated mucormycosis. Appropriate immunocompetent animal models are paramount to understanding the pathogenesis and treatment of these infections. Understanding the environmental microbiology of trauma associated mucormycosis is important to addressing the role of non- therapy ,46,47,48 therapy ,50. Furt therapy . Finally


Human parasitic protozoa cause a large number of diseases worldwide and, for some of these diseases, there are no effective treatments to date, and drug resistance has been observed. For these reasons, the discovery of new etiological treatments is necessary. In this sense, parasitic metabolic pathways that are absent in vertebrate hosts would be interesting research candidates for the identification of new drug targets. Most likely due to the protozoa variability, uncertain phylogenetic origin, endosymbiotic events, and evolutionary pressure for adaptation to adverse environments, a surprising variety of prenylquinones can be found within these organisms. These compounds are involved in essential metabolic reactions in organisms, for example, prevention of lipoperoxidation, participation in the mitochondrial respiratory chain or as enzymatic cofactors. This review will describe several prenylquinones that have been previously characterized in human pathogenic protozoa. Among all existing prenylquinones, this review is focused on ubiquinone, menaquinone, tocopherols, chlorobiumquinone, and thermoplasmaquinone. This review will also discuss the biosynthesis of prenylquinones, starting from the isoprenic side chains to the aromatic head group precursors. The isoprenic side chain biosynthesis maybe come from mevalonate or non-mevalonate pathways as well as leucine dependent pathways for isoprenoid biosynthesis. Finally, the isoprenic chains elongation and prenylquinone aromatic precursors origins from amino acid degradation or the shikimate pathway is reviewed. The phylogenetic distribution and what is known about the biological functions of these compounds among species will be described, as will the therapeutic strategies associated with prenylquinone metabolism in protozoan parasites. Trypanosoma cruzi), African human trypanosomiasis (Trypanosoma brucei), severe leishmaniasis (Leishmania spp.), malaria (Plasmodium spp.), and severe coccidiosis  mevalonate. Similarly, Leishmania tropica and T. brucei synthesize the polyprenylated side chain of UQ via the MEV pathway -GGPP in phytylated prenylquinone strongly suggests that both parasites possess an active pathway for phytol biosynthesis. Moreover, the incorporation of [3H]-phytol into phytylated quinones in both parasites suggests an active phytol phosphorylation process for the salvage of phytol, which is typical of some photosynthetic organisms  aminobenzoic acid (pABA) into the prenylquinone [13C6] pABA incorporation into UQ occurs, the intermediate that undergoes deamination remains unknown. This discovery suggests that the mechanisms of action of sulfonamide and other classic antifolates could be involved with inhibition of UQ biosynthesis  4-hidroxybenzoate and radiolabeled isoprenic moieties incorporation into UQ -GGPP in phytylated prenylquinone strongly suggests that both parasites possess an active pathway for phytol biosynthesis. Moreover, the incorporation of [3H]phytol into tocopherol in Leishmania suggests an active phytol phosphorylation process for the salvage of phytol, which is typical for some photosynthetic organisms [P. falciparum and L. amazonensis and weakly by nitisinone in L. amazonensis. Inhibition with usnic acid showed a dose-response effect on the growth of both parasites, causing an increase in ROS levels [Tocopherol biosynthesis was detected in asexual stages of rganisms . In thisS levels ,190. It S levels .Plasmodium and Leishmania [P. falciparum, which is indicative of the antioxidant function [Plasmodium is unclear. Nevertheless, tocopherol biosynthesis in P. falciparum appears stimulated in vitro when the parasites are grown under 20% oxygen [P. falciparum, there remain many uncertainties regarding the mechanism of the biosynthetic process. Specifically, there are no putative sequences for tocopherol biosynthesis annotated in the P. falciparum genome. Furthermore, metabolic labeling using isoprenic precursors only proved the polyprenylation process. The aromatic biosynthesis or external acquisition remains unexplored. Even before the characterization of tocopherol biosynthesis in apicomplexan parasites, it was shown that the infected red blood cell membranes contained 5 fold more vitamin E and C than uninfected membranes. However, α-tocopherolquinone was not detectable in infected red blood cell membranes. Thus, some authors suggested that a parasite-mediated vitamin redox modification had occurred [The effect of usnic acid on parasite viability and oxidative stress was reversed by the addition of exogenous α-tocopherol in ishmania ,190. Simfunction . Howeverfunction . Consequoccurred . In vivooccurred . In thisoccurred .T. cruzi and P. berghei transmission have reported HPPD inhibition effects in hematophagous vectors [Plasmodium, whereas nitisinone has been shown to be extremely toxic for several hematophagous vectors, even at clinically achievable blood concentrations. Some authors have suggested HPPD inhibitors as selective insecticides, but these inhibitors should also be considered to control the spread of hematophagous vector-transmitted diseases [Leishmania parasites as an excellent drug target for further exploration [Studies focused on  vectors ,203. Usndiseases ,203. Tyrloration .P. falciparum to be inhibited by norfluorazon, a PDS enzyme inhibitor in plants and algae [P. falciparum a putative PDS or a cofactor that could be used for these purposes. However, treatment of the parasite with norfluorazon apparently led to increased phytoene levels and inhibition of carotenoid biosynthesis [Plasmodium could use prenylquinones for the biosynthesis of carotenoids.In several plants and algae, the enzyme phytoene synthase provides phytoene, which is then desaturated by the enzyme phytoene desaturase (PDS) for carotenoid biosynthesis. In plants, PDS is dependent on PQ as a hydrogen acceptor, whereas some fungi and bacteria use another cofactor ,205. Fornd algae ,207. It T. acidophilum and subsequently from Wolinella succinogenes and Bacteroides gracilis [Chlorobium thiosulphatophilum and Chloropseudomonas ethylicum [Thermoplasma acidophilum [C. thiosulphatophilum and C. ethylicum, and the structures of these compounds correspond to 1-oxomenaquinone-7 [TpQ-7 or methylmenaquinone-7 was initially isolated from gracilis . ChQ wasthylicum ,210,211.dophilum . Nucleardophilum . On the uinone-7 ,209,211.E. histolytica, suggesting that this compound could be one of the main quinones involved in TPMET. The reduction of 1,2-naphthoquinone-4-sulphonic acid (NQSA), ferricyanide and α-lipoic acid (ALA) is evident, which suggests that TPMET redox chains are present in E. histolytica. Due to these quinone-mediated redox chains, it is possible to transfer electrons from the intracellular NADH or NADPH oxidation reactions to other compounds located in the extracellular space [E. histolytica amoebic liver abscesses, this redox chain could be essential for parasitic survival [L. donovani, ChQ was found in fractions containing purified plasma membranes. In addition, in this case, prenylquinone was suggested to be involved in ALA, NQSA and ferricyanide reduction [Nandi et al.  in 2011 ar space . In E. hsurvival . Similareduction .E. histolytica and L. donovani, the two prenylquinones can be destroyed by UV light irradiation, which leads to TPMET loss [E. histolytica most likely acquired genes responsible for MQ biosynthesis [L. donovani and green sulfur bacteria, which is consistent with the endosymbiosis theory for trypanosomatid ancestors [In both MET loss ,67. HoweMET loss ,67. Bothynthesis ,67. On tncestors . Both stncestors ,67. Regancestors ,67.Prenylquinones are molecules involved in essential metabolic reactions in all organisms, for example, prevention of lipoperoxidation, mitochondrial respiratory chain or as enzymatic cofactors.Surprisingly, only few studies involving prenylquinones as drug targets have been conducted so far, most of them about UQ-related metabolism ,54. The L. amazonensis and Plasmodium falciparum) and MQ  [An important fact is that the aromatic and isoprenic precursors of the prenylquinones were more studied then their specific biosynthesis pathways. In fact, biosynthesis inhibition studies have only been performed in vitro for tocopherol (in arasite) ,64. SeveLeishmania and those enzymatic inhibitors of tyrosine degradation and tocopherol biosynthesis affects Leishmania proliferation and P. falciparum [Trypanosomatidae species, Apicomplexa pathogens seems to require the shikimate pathway for the synthesis of aromatic precursors and corroborating this statement, glyphosate and shikimic acid analogs demonstrated effect against apicomplexan pathogens however there is an evidence that the last enzyme of shikimate pathway is not essential for the P. berghei viability [In regard to the aromatic precursor formation in parasitic protozoa, tyrosine degradation has been demonstrated to be essential in lciparum ,63,64,89iability .G. lamblia is sensitive to the statins mevastatin and mevinolin, two classical inhibitors of the MVA pathway and for consequence the isoprenic precursors synthesis [Blastocystis, Acanthamoeba and some apicomplexan parasites, including Toxoplasma, Cryptosporidium and Plasmodium, it was demonstrated the susceptibility to several other statins and there is an evidence of enzymatic capacity to metabolize MVA in P. falciparum extracts [E. histolytica.Regarding to the isoprenic precursor formation in parasitic protozoa drug targets are concentrated in MEP and MVA pathways. First of all, it was previously demonstrated that ynthesis ,106. In extracts ,108. Curd-xylulose-5-phosphate reductoisomerase) enzyme [Plasmodium spp., Babesia spp. and Theileria spp. proliferation, however fosmidomycin has a poor effect on Toxoplasma, Theileria and Eimeria viability although the MEP pathway is a proven isoprenoid source for apicomplexan parasites, [After the identification of MEP pathway in apicomplexan parasites a vast number of researchers concentrated their efforts around the compound fosmidomycin and analogs. Fosmidomycin is a specific inhibitor of the DXR (1-deoxy-) enzyme ,112,113.rasites, ,111,112.The elongation of isoprenoid chain is also a promisor chemotherapeutic target against parasitic protozoa. For example, in trypanosomatids and apicomplexan parasites the risedronate and its analogs have shown to be effective FPP/GGPP synthase inhibitors ,125,126.Plasmodium was explored and the MQ biosynthesis inhibitor (RO 48-8071) affected the parasite proliferation and seems to increase in the UQ pool while decreasing MQ biosynthesis [Plasmodium falciparum and L. amazonensis and Nitisinone in L. amazonensis, two classical inhibitors of HPPD [It is important to recapitulate here that the apparent function of MQ-4 in ynthesis . In the  of HPPD ,190.Amoebozoa, Apicomplexa, and Euglenozoa philas. As seen in this review, all prenylquinones have a correlation with the endosymbiotic events that occur within these organism’s evolutionary history. These metabolites could be involved in essential physiological functions, for example, prevention of lipoperoxidation, participation in the mitochondrial respiratory chain, TPMET or as enzymatic cofactors. In order to better understand parasite physiology and to discover new drug targets it is recommended to continue the research on the protozoan prenylquinone profile, biosynthesis pathway and biological functions.Human parasitic protozoa cause a large number of diseases worldwide. For some of these diseases, no effective treatments to has been identified to date and drug resistance has been observed. For all these reasons, the discovery of new etiological treatments is necessary. Most likely due to the protozoa variability, uncertain phylogenetic origin, endosymbiotic events and evolutionary pressure for adaptation to adverse environments a surprising variety of prenylquinones can be found in these organisms. In this review were discussed prenylquinones that have been previously characterized in human pathogenic protozoa. UQ is the most widespread but also MQ, tocopherols, ChQ and t TpQ can be found


Remarkably, its role in human cognitive architecture makes it a stage of crucial importance for the study of socio-affective cognition, also in relation with psychopathology such as anxiety. Among socio-affective stimuli, faces occupy a place of first importance. How faces and facial expressions are encoded and maintained in VWM is the focus of this review. Within the main theoretical VWM models, we will review research comparing VWM representations of faces and of other classes of stimuli. We will further present previous work investigating if and how both static  and changeable  facial features are represented in VWM. Finally, we will examine research showing qualitative differences in VWM for face representations as a function of psychopathology and personality traits. The findings that we will review are not always coherent with each other, and for this reason we will highlight the main methodological differences as the main source of inconsistency. Finally, we will provide some suggestions for future research in this field in order to foster our understanding of representation of faces in VWM and its potential role in supporting socio-affective cognition.Visual working memory (VWM) is one of the most investigated cognitive systems functioning as a  Faces are processed in a unique fashion starting from initial perceptual stages . The domain-specific approach sustains that face processing is carried out in specialized modules . ContrarVWM is a core cognitive system defined by a limited-space in terms of capacity in which visual information is temporarily stored and manipulated for further processing  and in tFlexible resource models  and lowest VWM capacity compared to other stimuli . When dealing with visually complex items  a particular class of models seems relevant. n models  suggest n models  has been stimuli .Traditionally, VWM has been studied for simple and abstract stimuli  . NeverthFor a better comprehension of the studies reviewed in the subsequent sections, this section provides a brief overview on methodological aspects related to VWM research.change detection task (CDT)  . Basicaletrieval . AlthougGiven the extensive use of this paradigm, this has led to a great proliferation of CDT variants, sometimes at the expense of the interpretation of the results. The most common CDT manipulations regard the amount and/or type of the memory array and test display items, the duration of both the memory array (with a significant impact on the amount of available encoding time for each displayed item) and the retention interval, and the type of test display presented after the retention interval  or also sustained posterior contralateral negativity (SPCN)  and it has been suggested that the intraparietal sulcus (IPS) is the main neural generator  called enerator . It is cenerator  between enerator  and resoenerator  of storeenerator .Given the great variability in the methods employed and results obtained in the context of VWM studies, we selected those investigations that used a comparable methodology in order to facilitate comparison between results. In some cases, the results of the different studies discussed here are not directly comparable because of differences in the stimuli used  and/or participants’ task . For this reason, we have tried to indicate details useful to the readers for a critical analysis of the results. In the following sections, we focus on studies using CDT with faces with particular attention to studies that measured the CDA. In the last section of this review, we also discuss studies that considered the relationship between face representations and individual differences . This review does not aim to be exhaustive but rather to identify and present selected examples of evidence that may help clarify the critical link between VWM functioning and the complexity of social cognition focusing on the main source of social information, that is others’ faces.K) compared to inverted ones, and, according to the face inversion effect  and capacity (Cowan’s K) for other-race faces. These findings suggest that, similar to inverted faces, other-race faces are processed, at both configural and featural levels of processing, less efficiently  and sensitivity (d’) for negative facial expressions, especially angry, compared to happy and neutral expressions  and on the environment  could be extracted from facial expressions . Using sressions .Furthermore, this benefit is observed only when angry faces are presented in the memory array but not in the test display . In addiK). Opposite to the angry benefit, a cost for angry faces has been also observed (d’) emerges , observed  when comobserved . Interes emerges .K vs. d’) seems to influence this effect , using real vs. schematic faces, different encoding times and/or dependent variables (Cowan s effect . Similars effect . Importas effect  and thisd’) for angry faces (compared to happy) (threat tagging) in which identity is coupled with valence and congruent stimuli  can interact with this representation (d’) for all emotional faces. Similarly, inducing a feeling of social exclusion  seem to enhance performance (o happy) . An angro happy) . Authorsentation . Maran exclusion  or incluxclusion  improvedxclusion . Overalln task-relevant stimuli should be greater than amplitude for n stimuli, some of which are task-irrelevant. Including emotional face distractors in the memory array  and using an identity CDT (1 or 2 to-be-remembered faces), Dealing with task-relevant and irrelevant (distractors) information is another important VWM facet. Filtering efficiency interacts with individual VWM capacity  and withK) for all facial expressions in individuals with higher self-reported anxiety, without affecting precision.Psychopathology is another critical factor in social cognition. Anxiety, in particular, has been widely studied in relation to WM and generally correlates with lower WM capacity  toward sad faces compared to individuals with non-melancholic depression  on the representation of faces in VWM. Negative faces, in particular angry, are associated with better VWM performances. However, a great methodological variability in stimuli choice and CDT parameters makes it difficult to compare findings. As previously shown, results could drastically change using schematic vs. real faces or different probing methods. Future research in this field, if not of interest, should keep paradigms’ parameters fixed, only varying socially relevant information. Otherwise, an orthogonal variation of CDT parameters within the same study could be useful .VWM is defined a hub of cognition  where inPsychopathology is another important aspect in social environment and often related to changes in basic cognitive functions. Again, different methods and different psychopathological conditions are difficult to integrate. However, it is interesting noting that psychopathology and VWM functioning are related. Alexithymic individuals have the worst VWM performance for happy faces  and indiflexible resource models and the neural object-file theory (At the neural level, the CDA seems to be influenced by facial information. It has been demonstrated that the CDA is modulated according to the amount  and alsoe theory , 2009, oFG wrote the first draft of the manuscript. PS provided critical revision. Both authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PrPres deposits in the neural retina were identified in the outer and inner plexiform layers (OPL and IPL) in a limited number of sporadic Creutzfeldt-Jakob diseases (sCJD) and two variant CJDs [res in the neural retina remains unknown in other types of CJDs. Therefore, we analyzed 16 prion cases from our brain bank, including sporadic, familial, and iatrogenic CJDs by using retinal sections [Creutzfeldt-Jakob disease (CJD) is clinically characterized by progressive dementia and neuropathologically characterized by deposits of a protease-resistant isoform of the prion protein   and 12F10 (142–160) . The retinal sections were processed by using a Ventana Discovery automated immunostainer. Evaluations of 3F4 and 12F10-immunoreactive deposits (PrPres-irs) of the outer and inner plexiform layers were performed by using both antibodies in a semiquantitative manner: 0 = none, 1 = positive and scattered, 2 = positive , as well as in the ganglion cell and nerve fiber layers (GCL and NFL); thus, we calculated the frequency of cases with PrPres-irs staining, separated on the basis of each anatomical region of the neural retina.At the time of autopsy, full permission was obtained from each patient’s next-of-kin. The posterior portion of the eye ball was removed with a scalpel, leaving the cornea and lens for funereal purposes. The following cases were available: nine cases of sCJD MM1), two cases of sCJD , one case of sCJD (MM2), three cases of familial CJD (fCJD) (two of V180I and one of M232R), and one case of iatrogenic CJD . We classified sCJDs based on the Parchi’s methodology , 5. We a, two casres-irs staining exhibited granular and fine synaptic patterns in the OPL and IPL, respectively. Although PrPres-irs staining was always present in both OPL and IPL, PrPres-irs staining was stronger in sCJD (MM2), fCJD, and dCJD cases than in sCJD (MM1) cases , fCJD, and dCJD cases , p-tau (AT8), p-synuclein, or TDP-43-irs staining in the retina. No PrPres-irs staining was observed in retinas from control cases. There was no clear PrPres-irs staining in the optic nerves.In some instances, fine-dot PrPses Fig. . No PrPrres-irs in the retina. Our methodology analyzing the posterior portion of the eye ball accurately reflected the pathologic condition of the neural retina in prion diseases. Indeed, a previous study showed that PrPres-irs were not prominent in the anterior portion of the neural retina [Clinical characteristics, such as age at onset, duration, gender, and initial presentation were not associated with the presence or absence of PrPl retina .res-irs within the retina in a series of cases of sCJD (MM1), sCJD , fCJD, and dCJD. Although we did not quantitatively evaluate the amount of PrPres in each case, PrPres-irs in the OPL and IPL may be more prominent in fCJD and dCJD cases. In addition, PrPres-irs were occasionally observed within layers of neural retina other than the OPL and IPL.Our study is the first to describe PrPres accumulation. Head et al. performed a detailed analysis of PrPres in the neural retina [res-irs were present in the OPL (granular pattern) and IPL (synaptic pattern) in one case of sCJD (MM1) and two cases of variant CJD. PrPres was reported as less detectable in the neural retina of sCJD (MM1) [res in the neural retina in vCJD, but not in sCJD, by Western blotting analysis [res-irs in the neural retina. There was no clear association between the clinical duration and PrPres-irs in the neural retina.Protease-sensitive normal cellular PrP was identified in the neural retina of healthy controls . Becausel retina , 2; theyanalysis . In Gersanalysis . We suspres staining in the OPL and IPL; thus, PrPres must be analyzed in other layers of the retina with another methodology, such as laser micro-dissection or biochemical analysis, because PrPres-irs staining in other layers was very weak. In the future, specific eye examinations may become a potential biomarker for the clinical diagnosis of prion diseases, similar to potential clinical diagnosis of AD by detection of amyloid-beta deposits in the retina [res accumulation in the neural retina is common findings even in atypical clinical form of sCJD  as well as fCJD and dCJD.The present study has some limitations. The sample sizes were small, except for cases of sCJD (MM1). This study was able to describe PrPe retina . In term


Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina  The retina is the main visual sensory tissue in mammals. During retinal development, the optic cup derived from the rostral diencephalon is composed of inner and outer walls that differentiate into the neural retina (NR) and retinal pigment epithelium (RPE), respectively. NR progenitors are widely present in the developing NR epithelium and function to expand to generate photoreceptors and other types of retinal neurons.in vitro differentiation of retinal progenitors and their derivatives from pluripotent stem cells (PSCs)23. Moreover, mouse and human embryonic stem cell (ESC) aggregates have the ability to self-organize optic cups in three-dimensional (3D) culture5. The ESC-derived NR self-organizes the formation of multiple retinal layers reminiscent of the postnatal retina. NR progenitors in this culture system have a radial glia-like epithelial morphology, and expand to give rise to photoreceptors and other retinal neurons in a stage-dependent manner, resembling the process in vivo5. The self-organizing stem cell culture technique developed in these studies, named SFEBq (serum-free floating culture of embryoid body-like aggregates with quick aggregation), is now used to generate 3D-organoids as an attractive model for recapitulating mouse and human organogenesis in vitro26.Recent advances in stem cell biology have enabled 6. In our method, designated the BMP method, timed addition of bone morphogenetic protein 4 (BMP4) to the 3D-differentiation culture selectively induces human retinal progenitors at the expense of telencephalic progenitors. Furthermore, we showed that hESC-derived retinal progenitors can be induced to differentiate into NR-RPE-conjugated two-domain 3D-retina, another type of retinal cup, using a fate-biasing culture technique named the induction-reversal culture method. The two-domain 3D-retina contains two key features: a ciliary margin-like stem cell niche and a centro-peripherally polarized NR. The human ciliary margin-like stem cell niche, self-formed at the margin of the NR and RPE, fosters a retinal stem-like cell population and functions to expand the NR by de novo progenitor generation. The centro-peripherally polarized NR tends to maintain a stratified continuous epithelium structure, reminiscent of the developing NR epithelium in vivo. Therefore, using the BMP method combined with the induction-reversal culture method, hESC-aggregates can be robustly differentiated into two-domain 3D-retina that self-forms a high-quality stratified continuous NR epithelium6.Toward future applications in cell therapy, we previously reported a modified SFEBq culture method for robust generation of 3D-retina31. Indeed, transplantation of retinal tissues and cells from primary embryonic tissues has the potential to improve visual function34. To supply adequate amounts of retinal tissues and cells for cell therapy, 3D-retina generated from hPSCs is a promising graft source. Photoreceptor precursors purified from ESC-derived retinal tissue can survive and make contact with the host retina36. Retinal sheet dissected from mouse ESC-3D-retina can engraft into even severely degenerated mouse retina, and graft-derived photoreceptors form direct synaptic connections with the host bipolar cells37. In addition, mouse iPSC-derived 3D-retina transplanted into end-stage retinal degeneration model mice (rd1) improved visual function29. Human 3D-retina, generated from hESCs using the BMP method and the induction-reversal culture method, differentiated into rod and cone photoreceptors that developed a structured outer nuclear layer in a primate model of severe retinal degeneration38. Importantly, we recently published another paper about functional maturation of human 3D-retina transplantation in the end-stage retinal degeneration model mice39. These studies demonstrate the potential of hPSC-derived 3D-retina as a graft source for retinal transplantation therapy.Retinitis pigmentosa is a group of hereditary diseases characterized by a loss of photoreceptors and is the major cause of untreatable blindness in developed countries. Transplantation of photoreceptors and/or retinal progenitors is a promising therapeutic option6, based on published protocols41. However, coculture with feeder cells is not a preferred procedure for generating clinical-grade human 3D-retina, partly because of the high costs. Therefore, we aimed to differentiate 3D-retina from feeder-free hESCs and hiPSCs. Application of Rho-associated kinase (ROCK) inhibitor to hPSCs markedly prevents dissociation-induced apoptosis and enhances survival to expand dissociated hPSCs42. Recombinant laminin-511 E8 fragments (LM511-E8), containing minimal integrin-binding sites, have strong adhesive activity for hPSCs43. Nakagawa et al. developed a feeder-free culture method to reprogram and expand hPSCs using a culture medium named StemFit combined with ROCK inhibitor and LM511-E8 matrix44.In our previous studies, hESCs used for retinal differentiation were routinely maintained on a feeder layer of mitotically-inactivated mouse embryonic fibroblasts (MEFs)45. Shh signaling promotes ventralization and posteriorization in neural patterning and retinal tissue formation in hESC-differentiation culture in some context5. Using this new culture method, we demonstrated that several hESC and hiPSC lines robustly differentiated into 3D-retina. Furthermore, we transplanted human iPSC-derived 3D-retina to examine whether feeder-free iPSC-derived 3D-retina can engraft and undergo maturation in vivo.Toward retinal cell therapy, we aimed to generate 3D-retina from hPSCs maintained in a feeder-free (Ff-) culture condition. Here we established an efficient differentiation culture technique to direct Ff-hPSCs toward self-organizing 3D-retina formation by modulating TGF-beta and Shh signaling pathways. The TGF-beta superfamily signaling pathway, including TGF-beta, Nodal/Activin and BMP signaling pathways, has negative effects on neural differentiation in early development; therefore, inhibition of TGF-beta signaling pathways utilizes to promotes neural differentiation in PSC-differentiation cultureRx::Venus line) was used according to the hESC research guidelines of the Japanese government and cultured at RIKEN. hiPSC-1231A3 line was established by Kyoto University and derived from ePBMC(R) purchased from Cellular Technology Limited (http://www.immunospot.com/), and gifted from Kyoto University44. hiPSC-C3 and hiPSC-LPF11 lines were a gift from N. Takamura, M. Higuchi, and A. Fujiki (Sumitomo Dainippon Pharma). hPSCs were maintained on LM511-E8 matrix (Nippi) in StemFit medium (Ajinomoto) according to a published protocol44 with slight modifications. Prior to passaging, culture plates (Iwaki) were coated with LM511-E8 matrix in PBS at 37 °C for at least 1 h. hPSC colonies were treated with TrypLE Select Enzyme (Gibco) at 37 °C for 4–7 min, and dissociated into single cells by gentle pipetting. The dissociated PSCs were suspended in StemFit medium and counted. The single-cell PSC suspension was plated on LM511-E8 matrix-coated plates at a density of 700–1700 cells/cm2 and cultured in StemFit medium with 10 µM Y-2763242. The medium was changed to StemFit medium without Y-27632 on day 1 and further changed on days 3, 5, and 6 at least. Passages were performed every sixth or seventh day.hESC-KhES-1 line  in differentiation medium  supplemented with 20 µM Y-27632 and 30 nM SAG (d0-SAG method) under 5% CO2 at 37 °C. The differentiation medium was growth factor-free CDM (gfCDM) supplemented with 10% KSR, while gfCDM alone comprised 45% Iscove’s modified Dulbecco’s medium , 45% Hams F12 , Glutamax, 1% chemically defined lipid concentrate (Gibco), and 450 µM monothioglycerol (Sigma)46. The day of SFEBq culture initiation was defined as day 0, and recombinant human BMP4 (R&D Systems) was added at 1.5 nM (55 ng/ml) on day 3. Thereafter, the concentration of BMP4 was diluted by half with a half-medium change every third day. For the Ff-hiPSC without preconditioning, treatment with 300 nM on day 0 efficiently improved aggregate growth , 100 nM LDN193189 (LDN), and/or 300 nM smoothened agonist (SAG) for 18–30 h prior to differentiation. The SFEBq culture method was then performed as described6. The NR epithelium generated from hPSCs was subjected to two-step induction-reversal culture under the following conditions. For the induction culture, NR-containing aggregates generated from hPSCs on days 14–23 were transferred from 96-well plates to 9-cm non-cell-adhesive petri dishes , and further cultured in suspension for 3 days under 5% CO2 in DMEM/F12-Glutamax medium (Gibco) containing 1% N2 supplement (Gibco), 3 µM CHIR99021 , and 5 µM SU5402 . For the reversal culture, the floating aggregates with an RPE-like thin epithelium were cultured in suspension under 5% CO2 in NR-differentiation medium containing DMEM/F12-Glutamax medium, 1% N2 supplement, 10% FBS , 0.5 µM retinoic acid , 0.1 mM taurine (Sigma), 0.25 µg/ml Fungizone (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin. The medium was changed every third or fourth day until day 178. Floating aggregates were analyzed using inverted microscopes .Induction-reversal culture and long-term NR culture were performed as described47. SD-Foxn1 Tg(S334ter)3Lav nude rats (RD-nude rats) were obtained from the Rat Resource and Research Center49. Ff-hiPSC-derived 3D-retina (1231A3) on day 58 were transplanted to 6–8-week-old RD-nude rats. Similar results were obtained in four eyes.In all animal experiments, the animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. All animal experiments were conducted with approval from the Animal Research Committee at RIKEN Center for Developmental Biology Institute and only performed at RIKEN. Transplantation into the subretinal space of rats were performed as described6. Aggregates were fixed with 4% PFA and sectioned with a cryostat (Leica). Frozen sections were treated with or without heat-based antigen retrieval in Target Retrieval solution (Dako) at 105 °C for 15 min. The primary antibodies used in this study were as follows: Chx10 , Rx , Pax6 , Mitf , Crx , Recoverin , NRL , RXR-gamma , Rhodopsin , S-opsin , L/M-opsin , PKCalpha , HuNu , Calbindin , N-cadherin , FoxG1 , Ctip2 , Tbr1 . Nuclear counter-staining was performed with DAPI . Stained sections were analyzed with a fluorescence microscope  and a confocal microscope . Image analyses were performed with ImageJ 1.48 v software (NIH).Immunohistochemistry was performed as described6 cells) in control or preconditioning conditions (n = 4) and lysed with RLT buffer (QIAGEN). After differentiation, 16–32 aggregates per each time points were lysed with RLT buffer (n = 4). Total RNA was extracted with an RNeasy micro kit (QIAGEN) according to manufacturer’s instructions. Quantitative PCR reactions (qPCR) were performed by using TaqMan gene expression assays (Thermo Fisher Scientific). Relative mRNA expression was determined by the delta-delta Ct method with gapdh as an endogenous control. The standard error of the mean (SEM) were calculated using Excel software (Microsoft). The qPCR primers used in this study were as follows : gapdh (Hs02758991_g1), ACTB (Hs01060665-g1), Rx , Chx10 , Pax6 (Hs00240871_m1), Sox1 (Hs01057642-s1), Sox2 (Hs01053049_s1), Oct3/4 , Nodal (Hs00415443-m1), ID1 (Hs03676575-s1), Gli1 (Hs00171790_m1), Patched-1 , Gata4 (Hs00171403-m1), Gata6 (Hs00232018-m1), Hand1 (Hs02330376-s1).Feeder-free hiPSCs were cultured in 6 well plate (~2 × 10t-test (parametric) for two-group comparisons and with ANOVA followed by post-hoc Tukey’s test for multi-group comparisons. Values of p < 0.05 were considered to indicate statistical significance. *p < 0.05; **p < 0.01 and ***p < 0.001 for all figures.Statistical analyses were performed with Excel software (Microsoft) and R software (ver 3.6.1). Statistical significance was tested with Student’s 44. hiPSC-1231A3 cells expanded stably in the feeder-free maintenance culture, and their doubling time was approximately 12–18 h. 3D-retinal differentiation culture was then carried out using the SFEBq-BMP method6. Ff-hiPSC-1231A3 cells were dissociated into single cells, plated in V-bottom 96-well plates, and cultured in differentiation medium (gfCDM + KSR) in the presence of Y-27632. Unexpectedly, we observed that Ff-hiPSC-derived aggregates reproducibly collapsed at around day 6 in 3D-differentiation culture . The 1231A3 hiPSC line, established in Kyoto University using episomal vectors expressing Yamanaka factors, was maintained on LM511-E8 matrix in StemFit medium according to published protocolure Fig. . When, bure Fig. . We thenTo test our hypothesis, we added several candidate signaling molecules around day 0 of 3D-differentiation culture and examined their effects. First, we observed that transient treatment of Ff-hiPSC-aggregates with SAG  promoted aggregate growth during 3D-differentiation culture Fig. , d0-SAG.Second, we examined the effects of preconditioning before starting 3D-differentiation culture. Ff-hiPSCs were cultured in the Ff-maintenance culture condition with FGF2 in the presence or absence of various signaling molecules for 1 day Fig. . Oct3/4 We optimized the method by combining the d0-SAG treatment Fig.  and the 52. The preconditioned-iPSC-derived aggregates in each condition were found to contain a neuroepithelium on their surface, and the majority of the cells in the neuroepithelium were positive for Chx10 and Rx  or left untreated as control and differentiated in the presence of SAG and Y-27632 Fig. . The Ff- Rx Fig. . Thus, h6.Importantly, we observed that the morphology of the NR epithelium differed depending on the preconditioning conditions Fig. . SpecifiNext, we investigated whether the preconditioning method is a generally useful methodology for the generation of 3D-neuroepithelium from feeder-free hESCs and hiPSCs. For this, we examined the effects of preconditioning in: 1) multiple ESC and iPSC lines; 2) another commonly used feeder-free culture medium; 3) various types of well-plates in 3D-differentiation culture; and 4) formation of non-retinal neuroepithelium.We cultured multiple ESC and iPSC lines in the preconditioning or control conditions, and induced their differentiation with the SFEBq-BMP method Fig. . As anot5. First, we checked the mRNA expression of ID1 , Nodal , Gli1 and Patched-1 56, and confirmed that SB, LDN, SB + SAG and LDN + SAG treatment decreased the mRNA level of ID1, while SAG, SB + SAG and LDN + SAG treatment increased the mRNA levels of Gli1 and Patched-1  cells maintained in StemFit medium on LM511-E8 matrix were treated with SB, LDN, SAG, SB + SAG or LDN + SAG for 24 h or left untreated as controls. Total RNA extracted from the iPSCs was subjected to qPCR analysis Figs. , n = 4. d-1 Fig. . These rn = 4, 16–32 aggregates per each conditions). We tested the mRNA expression of PSCs (Oct3/4 and Sox2), neural , retinal  and mesoendodermal  markers and normalized with gapdh mRNA expression  preconditioning of undifferentiated hiPSCs affects the proportions of NR and RPE in 3D-retina; 2) LDN-preconditioned hiPSCs and SB-preconditioned hiPSCs tend to differentiate into NR-dominant aggregates and NR-RPE-conjugated two-domain aggregates, respectively; and 3) SB-type aggregates and LDN-type aggregates can both self-form a multilayered stratified NR epithelium with an NR progenitor layer and a photoreceptor layer.in vitro for 178 days as reported previously6. SB-type 3D-retina was fixed and immunostained for photoreceptor markers. We found that the NR epithelium in the 3D-retina self-formed a multilayered structure comprising an outer photoreceptor layer with Crx+ and Recoverin+ cells, a middle layer with Chx10+ cells, and an inner layer with Calbindin+ horizontal cells and Chx10−/Pax6++ cells  carrying a mutated human rhodopsin transgene (SD-Foxn1 Tg(S334ter)3-LavRrrc line)49. Ff-hiPSC-derived SB-type 3D-retina (1231A3) on day 58 was dissected and transplanted into the subretinal space of RD-nude rats, as described previously38. At 259 days after transplantation (317 days after initiation of differentiation), the eye balls were fixed and immunostained. We observed that the hiPSC-derived 3D-retina became engrafted between the host RPE and host NR , human PSCs have been routinely cultured on feeder cells in many laboratories60. We previously developed a 3D-retina differentiation culture method using hESCs on feeder cells6. Mass production and quality control of feeder cell banks are associated with high costs, approximately similar to those of hPSC banks. For example, feeder cells such as MEFs are xeno-materials and must undergo virus negativity tests. Furthermore, culture of hPSCs on feeder cells requires good manipulation skills. In contrast, feeder-free culture on LM511-E8 matrix has a low cost and is easy to manipulate for hPSC expansion44. Therefore, we developed a preconditioning method to generate 3D-retina from Ff-PSCs. The combination of feeder-free PSC-culture and the modified SFEBq method is a technically simple method for generating 3D-retina, compared with our previous method.Since human ESC lines were established by Thomson et al. developed the SFEBq culture method to regulate the size and quality of individual PSC-aggregates24. In the modified method developed in the present study, the variety of organoid quality, such as morphologies of individual organoids and proportions of NR and RPE, may be smaller than those in our previous method6 . Possible mechanisms for this are that: 1) feeder-free culture may reduce the variety of PSC states compared with on-feeder culture; and 2) the preconditioning method modify the levels of signaling pathways to reduce the variety of 3D-retina. By using a combination of the Ff-PSC culture, SFEBq, preconditioning, BMP, and induction-reversal methods, it is now possible to robustly generate 3D-retina with controlled NR-RPE proportions.One of the methodological challenges in 3D-differentiation culture is how to reduce the variety of organoids at both the intra-batch level and inter-batch level. Eiraku 61. Ff-hiPSC maintenance culture and/or initial steps of differentiation culture are critical steps to generate 3D-retina robustly.The reason why feeder-free hPSC-derived aggregates in SFEBq culture tend to collapse still remain elusive. Ff-hiPSCs without preconditioning might have a possibility to prone to mesoendodermal fate instead of ectodermal fate Fig. . Interes7, Wnt signaling inhibitor7, IGF116, ROCK inhibitor in hPSC suspension culture42, SFEBq method6, 2D/3D-combination culture method of hPSC aggregates12, low concentration KSR4, extracellular matrix addition on PSC aggregates5, 2D-conflent culture method18, COCO62 and BMP signaling agonist6. In the present study, we identified Shh signaling agonist in the early step promoted aggregate growth and neural tissue formation (d0-SAG method). Although Wnt priming method in mouse ESC maintenance culture was reported63, major improvements in human PSC retinal differentiation culture protocols were made after the removal of FGF2 to start differentiation. The removal of FGF2 greatly changes the hPSC state, therefore we developed preconditioning method in the presence of FGF2. The preconditioning method a minor modification in the maintenance culture is useful culture methodology to adjust differentiation protocol for each hPSC cell lines without major changes after starting differentiation.Previous studies reported changes made in the early steps of retinal differentiation culture: TGF-beta/Nodal signaling inhibitor25. Indeed, control Ff-iPSC (without preconditioning) tended to differentiate into the mesoendoderm-biased state, which the mRNA levels of Sox2, Sox1, Pax6 and Chx10 were not sufficiently induced and the mRNA levels of Gata4, Gata6 and Hand1 were increased. It is therefore possible that preconditioning with the TGF-beta/Nodal/BMP signaling inhibitor and/or Shh signaling agonist may bias Ff-hPSC state to neural fate instead of mesoendodermal fate.The mechanisms how preconditioning (before differentiation) affected 3D-neuroepithelium formation (after differentiation) still remain unclear. Preconditioning method might consist of two steps: first, preconditioning in the presence of FGF2 changed the initial state of Ff-hPSCs in maintenance culture; then, preconditioned Ff-hPSCs efficiently self-formed 3D-neuroepithelium in differentiation culture.Multiple preconditioning methods, namely short-term treatment with SB, LDN, SAG, SB + SAG and LDN + SAG, promoted self-formation of neuroepithelium, respectively. To elucidate the state of preconditioned Ff-hPSCs, we examined mRNA expression of preconditioned Ff-hiPSCs. We found that SB- and LDN-treatments reduced the mRNA level of TGF-beta signaling target ID1 and SAG-treatment increased that of Shh signaling targets Gli1 and Patched-1. These results raise a possibility that preconditioning might bias from highly stable pluripotency-maintained state to a state suitable for 3D-differentiation culture in the levels of signaling pathways. Preconditioning with SB, LDN, SB + SAG and LDN + SAG increased the mRNA level of early neural differentiation marker Sox1. The Nodal/Activin signaling pathway promotes induction of the mesoendodermal fate, while the TGF-beta and BMP signaling pathways promote induction of the mesoendodermal fate and extraembryonic fate in the early stage and epidermal fate at the later ectodermal stageImportantly, preconditioning of Ff-hPSCs can control the proportions of NR and RPE within 3D-retina: LDN-preconditioned hiPSCs and SB-preconditioned hiPSCs tended to differentiate into NR-dominant aggregates and NR-RPE-conjugated two-domain aggregates, respectively. We reasoned that preconditioning might affect the positional information and local patterning of self-formed neuroepithelium along the centro-peripheral axis in the eye field.Taken together, we believe that preconditioning might bias the initial PSC state to affect differentiation in several steps: 1) biasing from highly stable pluripotency-maintained state toward a state suitable for 3D-differentiation culture in the levels of signaling pathways, 2) promoting 3D-neuroepithelium formation and growth after differentiation and 3) affecting the local patterning within the 3D-neuroepithelium. We therefore proposed that optimization of preconditioning stimuli is the useful methodology to direct initial Ff-PSC state toward self-organizing 3D-neuroepithelium with controlled positional information.26. A series of studies by Sasai, Eiraku and colleagues revealed the mechanism for optic cup formation65. Self-organizing hPSC culture also applied to analyze the mechanism of de novo neurogenesis from the ciliary margin-like retinal stem cell niche6. Canto-Soler and colleagues demonstrated that hiPSC-derived 3D-retina can respond to light stimuli12. The feeder-free culture and modified SFEBq method in the present study, in combination with genome editing technology, will provide a technically easy method to induce genetically-modified 3D-retina for research on organogenesis. Important issues, such as species differences in retinal development and features of human retina, key determinants for differentiation of human rods and cones, and generation of fovea structures, may be resolved in future studies.Self-organizing culture of 3D-organoids is a powerful tool to study organogenesis66. Modeling of inherited retinal diseases is an important theme for developing therapeutic strategies67. Ff-hiPSC-derived 3D-retina provides a platform for research on retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), and glaucoma11. Toward the development of therapies for glaucoma, PSC-derived retinal ganglion cells from 3D-retina can be cultured in vitro for examination of neurite outgrowth70. Primary retinal ganglion cells purified from the eye were engrafted into the retina in vivo71, raising the possibility that hPSC-3D-retina may contribute to cell therapies for glaucoma.iPSC technology brings a new dimension to drug discovery and regenerative medicine72. Two different teams published important studies related with RPE transplantation in patients74. In retinal degeneration diseases, such as severe AMD and RP, progressive photoreceptor loss leads to irreversible vision loss. Transplantation of human PSC-derived photoreceptors is a promising therapeutic option76. Nevertheless, numerous points remain to be determined, such as transplantation of immature cells or mature cells, retinal progenitors or photoreceptors, cell suspension or retinal sheet, unfrozen or frozen cells, contributions of material transfer, retinal state of patients (hosts), immunosuppression method, surgical devices, and manufacturing procedures compliant with cGMP. Among these, a series of studies by Takahashi, Mandai and colleagues have indicated that PSC-derived 3D-retina has the potential to engraft, undergo maturation in vivo, and provide 3D-retina-derived photoreceptors in direct contact with the host bipolar cell layer in retinal degeneration model animals39. In the present study, we observed that Ff-hiPSC-derived 3D-retina can engraft into retina in vivo, similar to the case with 3D-retina derived from hESCs on MEF feeder cells. We recently published another manuscript about the positive effects of human Ff-hPSC-derived 3D-retina transplantation in model animals47. Toward photoreceptor transplantation therapy, we are now addressing the manufacturing process and a new methodology to control the quality of 3D-retina using SFEBq technology.Clinical scientists and stem cell biologists have been developing new strategies for cell therapy. Indeed, removal of neovascular lesion followed by transplantation of an autologous iPSC-derived RPE sheet improved foveal thickness in a patient with neovascular AMD• Optimized self-organizing culture can generate 3D-retina from feeder-free hPSCs• hPSC preconditioning by modulating TGF-beta and Shh promotes 3D-retina formation• hPSC preconditioning affects the proportions of neural retina and RPEin vitro and in vivo• hPSC-3D-retina differentiates into rod and cone photoreceptors Supplementary Figures