Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression.Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues  proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying  MSH6), MutL homologues  and yeast post-meiotic segregation homologues (PMS1 and PMS2) [MLH1, MSH2, MSH6 and PMS2 mutations increase susceptibility to multiple malignancies, most notably colorectal and endometrial cancers [E.coli MutL homologues (MLH) and yeast post-meiotic segregation (PMS) homologue proteins, which have significant amino acid identity and structural similarity to the MLH proteins, among others (reviewed in [DNA mismatch repair (MMR) is pivotal in maintaining genomic stability in both prokaryotes and eukaryotes. There are nine mammalian MMR genes, including E. coli MutS homologues  . In humaogues MLH, MLH3 an cancers . MSH2-MSogues MLH, MLH3 anogues MLH, MLH3 anThe MLH1-PMS2 complex is the primary MutL heterodimer that interacts with both MSH2-MSH6 and MSH2-MSH3 complexes, and is the only heterodimer that participates in repair of single base substitutions. There is partial redundancy in the functions of MLH1-PMS2 and MLH1-MLH3 complexes in IDL repair and DNA damage response , 11. In HR is a critical pathway for repair of DNA double-strand breaks (DSBs). Briefly, in mammals recA-like protein (Rad51) forms complexes with single-stranded DNA (ssDNA) strands. Rad51-ssDNA filaments interact with double stranded DNA (dsDNA) and pair when homologous sequence contacts are made. Subsequently, strand exchange occurs and a hybrid dsDNA/ssDNA complex called a D-loop is formed that is processed by multiple HR sub-pathways, including double-strand break repair (DSBR), synthesis dependent strand annealing (SDSA) and break-induced replication (BIR) . DSBR, SRelatively small degrees of sequence heterogeneity can alter the frequency of DSB-induced HR and SSA in mammalian cells. For example, HR is decreased by >80% for DRs that are 1.2% divergent . When thSaccharomyces cerevisiae, functional mutations of Msh2p, Msh6p, Mlh1p or Pms1p  increase HeR between integrated reporters containing DRs with mismatch heterology [Msh2p, Mlh1p or compound mutations of the three Mlh1p heterodimeric partners, Pms1p/Mlh2p/Mlh3p, increases SSA [MSH2 and MSH6 can suppress HeR [MMR suppression of HeR was first observed in bacteria , 21 wherterology –26, yet ases SSA . In mammress HeR , 28–31, FRA16D and FRA3B have increased DSB rates and are the most common CFS reported in tumors [FRA3B and FRA16D are evolutionarily conserved across eukaryotes [In tumors, DSBs also occur frequently at chromosomal fragile sites (CFS). CFS are AT-rich repetitive sequences that are difficult-to-replicate during mitosis and manifest as gaps flanked by DSBs in different tumors –35. Fragn tumors –39. FRA3karyotes  and encoFhit knockout mice develop gastric carcinomas and skin cancers [FHIT and WWOX, respectively. Fragile histidine triad protein (FHIT) -triphosphatase) spans 10 exons over a 1Mb region at fragile site FRA3B. FHIT is a member of histidine triad subfamily of nucleotide binding proteins. FHIT is a DNA hydrolase that metabolizes substrate AP3A ,P(3)-triphosphate), which promotes DNA replication and inhibits stress response signaling . CRCs an cancers , 44.FRA3B and FRA16D CFS has not been linked to HeR. Here, we report that mammalian MutL homologs MLH1, PMS2 and MLH3 participate in suppression of HeR and SSA. In addition to GFP reporter studies, we computationally identified 6,848 paired DRs with 97%–99.9% sequence identity mapping to unique locations in the mouse genome as substrates for endogenous genomic HeR and SSA. Confirming reporter gene studies in yeast, we demonstrate that mutation of Mlh1, Pms2, Mlh3 or Mlh3/Pms2 increases HeR in MEF DR sequences. Furthermore, for the 1–12 heterologous bases in each paired DR, we show that >94% of base substitution mutations are consistent with gene conversion (where a mismatched base in one direct repeat motif is replaced with its respective paired direct repeat heterologous base). Next, to assess SSA, we used gamma-irradiation to promote endogenous DSBs and show that Mlh1, Pms2 and Mlh3 mutations suppress CNAs, including recurrent CNA hotspots that we previously identified in mouse dMMR intestinal tumors. Finally, we analyze data from dMMR and MMR proficient (pMMR) CRCs with matched normal tissues from The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov). These studies confirm high rates of human endogenous paired DR HeR and reveal that the intervening sequences between paired DRs are deleted more frequently in dMMR vs pMMR CRCs. Specifically, these dMMR recurrent CNA deletions include hotspots at chromosomal fragile sites FRA3B and FRA16D, which have flanking DR and IR sequences and high rates of spontaneous DSBs in CRCs. Overall, these data provide insights into mammalian MutL homolog inhibition of HeR and SSA and are consistent with a potential role for dMMR inhibition of homeologous recombination in tumor suppression.WWOX is a WW domain-containing oxidoreductase at FRA16D. WWOX contains 9 exons and spans 1.2 Mb . Genomicwild-type (Wt), Mlh1−/−, Pms2−/−, Mlh3−/−, and Pms2−/−;Mlh3−/− mouse embryonic fibroblasts (MEFs) matched for passage number.To understand the role of mammalian MutL homologues in HR and HeR, we modified the pDR-GFP homologous recombination reporter by introducing eight closely spaced mutations in GFP to monitor homeologous recombination, similar to neomycin-based reporters previously used to study the role of mammalian MSH2 in HeR  Supplem. These rMLH1−/− MEFs showed only 3 ± 4% suppression . Further experiments in Mlh3−/−, Pms2−/− and Pms2−/−;Mlh3−/− MEFs showed 77.5 ± 0.5%, 60 ± 5% and 13.5 ± 0.5% HeR suppression relative to HR . These studies are consistent with a potential role for mouse Mlh1 in HeR suppression. Similar to previous findings in IDL repair and DNA damage response, these data also are consistent with partially redundant roles for PMS2 and MLH3 in HeR suppression, as results in Mlh1−/− MEFs were not significantly different from those in Mlh3−/−;Pms2−/− MEFs .In Wt MEFs transfected with I-SceI, comparisons of DR-GFP and DR-GFP8mu stable transfectants showed that 1.2% heterology suppressed HeR vs HR by 74 ± 3%, whereas pression  . Targeted DR sequences were then analyzed on an Illumina HiSeq 2500. After mapping sequence reads to mouse genome (mm9), and filtering DR reads for mapping quality score >23 and sequence read depth >10 reads, we successfully obtained sequence data for 5,119 paired DR for further analysis.DSBs that occur between DRs stimulate both HR and SSA . To undeP > 0.15; ANOVA followed by Dunnet's test). Compared with Wt MEF DRs, Mlh3−/−, Pms2−/−, Mlh3−/−;Pms2−/− and Mlh1−/− MEF DRs all had significantly higher mutation rates  . In both Wt and dMMR MEFs, for mutations occurring at HeR-BPs, more than 94% of changes were at the corresponding base in the paired DR   and surrounding bases (non-HeR-BP) in DRs with no CNAs. HeR-BPs in DR sequences had significantly higher mutation rates than surrounding bases Table  ranging P < 0.001, Chi-square). Similarly, for longer gene conversion tracts that include two consecutive heterologous bases with mutations consistent with gene conversion, we observed that Mlh3, Pms2, Mlh1 or combined Mlh3/Pms2 mutations also significantly increased the number of tracts vs Wt MEFs  , as did Mlh3−/−Pms2−/− and Mlh1−/− MEFs (P = 5.91 × 10−13 and 2.22 × 10−115) . Strikingly, shared DR deletion hotspots were observed on mouse chromosomes 6 and 7   Figure . ImportaMlh3, Pms2 or Mlh1 each significantly increased DR CNA rates in γ-irradiated MEFs. Each MutL mutation caused a bias towards genomic deletions, likely due to increased SSA between homologous DR sequences. Mlh1−/− MEFs overall had increased CNA rates vs Mlh3, Pms2 or combined Mlh3;Pms2 mutations. On chromosome 6, an Mlh3 mutation dependent CNA deletion hotspot was observed that was also seen in Mlh1−/− but not Pms2−/− irradiated MEFs. A chromosome 7 CNA deletion hotspot shared by Mlh3, Pms2 and Mlh1 mutant MEFs was also seen that had been previously identified in Mlh3−/−;Pms2−/− deficient intestinal tumors. Furthermore, an Mlh1 (but not other MutL homologue) mutation dependent chromosome 12 CNA amplification hotspot, which had also been previously identified in Mlh3−/−;Pms2−/− intestinal tumors, was observed.In summary, mutation of Using the same methodology and parameters for analysis as in the mouse genome, we identified 12,379 uniquely mappable DRs in the human genome, each carrying 1-12 heterologous bases per paired DR. We then evaluated whether MMR mutations increased paired DR HeR rates in matched normal mucosa-dMMR patient CRC by analyzing genome sequence data from The Cancer Genome Atlas (TCGA-COAD) . We iden−4 vs 3.7 × 10−4p < 0.001, chi-square test) . Secondly, consistent with gene conversion and similar to our findings in mouse MEFs, HeR-BP in TCGA dMMR and pMMR CRCs were found to be significantly enriched for the corresponding base substitution in their matched 5′ or 3′DRs  . Additionally, dMMR CRCs also had higher rates of tracts consisting of two consecutive heterologous bases both with gene conversion consistent mutations  Table . SimilarOverall, these human CRC data are consistent with studies in mouse MEFs and DRs in the mouse genome. In human DRs, both HeR-BPs and surrounding bases have overall elevated mutation rates when dMMR is compared to pMMR CRCs. However, HeR-BPs have higher mutation rates than surrounding DR bases, almost all mutations are consistent with a mechanism of gene conversion in both pMMR and dMMR CRCs, including tracts of gene conversion consistent mutations. Moreover, dMMR CRCs have higher HeR rates than pMMR CRCs.P = 0.00947). Of note, this analysis also revealed two dMMR somatic deletion hotspots that mapped to FRA3B and FRA16D chromosomal fragile sites (CFS) . These dMMR CRC genomic deletions were flanked by DR and IR motifs ranging in size from 46 kb to 338 kb  [Mlh1 and Pms2 are involved in HeR suppression. Furthermore, we extend these findings and provide evidence for mammalian Mlh3 involvement in HeR suppression as well  –26 to mahttp://cancergenome.nih.gov). In human genome DRs, HeR-BPs have higher mutation rates than surrounding DR bases. Almost all mutations are consistent with a mechanism of gene conversion in both pMMR and dMMR CRCs, including tracts of two consecutive gene conversion consistent mutations. Overall, these findings are largely consistent with MEF studies, supporting DRs as in vivo endogenous HeR reporters and demonstrating that dMMR CRCs have higher HeR rates than pMMR CRCs.We evaluated DR mutations in human dMMR and pMMR CRCs by using available whole genome sequencing data from the TCGA database  a mouse chromosome 6 deletion hotspot shared by Mlh1−/−, Pms2−/−, Mlh3−/− and Mlh3−/−;Pms2−/− MEFs, (b) mouse chromosome 7 deletion hotspot shared by Mlh1−/−, Pms2−/−, Mlh3−/− and Mlh3−/−;Pms2−/− MEFs that we had been previously identified in adenomas  and (c) rcinomas . Each ofFRA3B  and FRA16D . CFS are long AT-rich repeat genomic sequences that are sites of DNA polymerase stalling during genome replication and have very high rates of spontaneous DSBs in tumors [FRA3B and FRA16D, which encode the tumor suppressor genes FHIT and WWOX respectively, are the two most frequently deleted chromosomal loci in human CRCs. Higher rates of FHIT and WWOX deletions have previously been reported in dMMR vs pMMR CRC cell lines and tumors [FRA3B and FRA16D. Since CFS have high endogenous rates of DSBs, our data are consistent with increased rates of SSA promoting deletions of FHIT and WWOX tumor suppressors in dMMR tumor.In human CRCs, analysis of DRs and IRs revealed two somatic deletion dMMR hotspots at chromosomal fragile sites (CFS) n tumors –61. Whenn tumors –65. FRA3d tumors , 67. MapIn summary, known DNA mismatch repair tumor suppression mechanisms include base-substitution and small in/del mutation repairs as well as DNA damage response initiation. Our data provide the first evidence that homeologous recombination and SSA suppression are also important MMR tumor suppression mechanisms. Poly ADP (Adenosine Diphosphate)-Ribose Polymerase (PARP) inhibitors are an example of a successful mechanistically based therapy that exploits synthetic lethality with HR deficiency in ovarian and breast tumors. Our findings suggest that screens to identify drugs promoting synthetic lethality with increased HeR rates may similarly be effective for targeted treatment of dMMR tumors.2) and washed with PBS. MEFS were then grown in 15% FBS and 100U/ml penicillin, 100 ug/ml streptomycin at 37C, 5% CO2.Mouse embryonic fibroblasts were established from day 12.5 embryos isolated from the uteri of pregnant mice and matched for passage number as previously described . In brie7 cells in 1 ml of phosphate-buffered saline were electroporated with 20 to 25 μg of each uncut plasmid DNA in a 0.4-cm electrode-gap cuvette . Electroporated cells were aliquoted into four or five 10-cm-diameter dishes and kit for primary cells. As I-SceI is expressed and recombinant cells expressing GFP were analyzed using flow cytometry (BD-Biosciences) or colonies were selected in media 24 h after electroporation and were grown in selection G418 (200 μg/ml) media for 14 days before colony counts.We constructed pDR-GFP8mu containing 8 silent mutations in the iGFP of pDR-GFP . PrimaryPrimary MEFS from all cell lines were cultured and irradiated with 5 Grays @ 1.27 Gy/Min. Subsequently the cells were fed fresh media and allowed to grow and passaged twice. The cells were then harvested and genomic DNA was extracted using DNeasy kit (Qiagen) per manufacturer's protocol. All experiments were performed by irradiating exponentially growing cell cultures with 137Cs irradiator .To search for direct repeat (DR) regions in mouse genome (mm9) and human genome (hg19) we used the program Vmatch  using thPaired-end reads sequenced at targeted direct repeat regions were processed at Weill Cornell Genomic Core and then mapped to mouse reference genome (mm9) with BWA method . After rhttp://cancergenome.nih.gov). For each probe, the ratio of normalized probe signal between tumor and the matched normal tissue was used to determine if there was a copy number alteration at the probe site. We considered a corresponding CNA at a site if tumor to normal ratio was >1.5 or <0.5. At each probe site, differential CNA between dMMR and pMMR tumors was calculated with Chi-squared analysis based on the 2 × 2 contingency table. If the expected values in the contingency table were less than 1, we used Fisher exact test following recommendations of Campbell et al. [Level 2 and level 3 SNP array data were downloaded from TCGA data portal . In addition, for 10 pMMR CRC patients, tumor and matching normal whole genome sequence data were also downloaded from TCGA and READ portals. The selection of these patients was based on matching sequence depth in addition to same sequencing center, to reduce potential batch effects. Somatic point mutations for each CRC patient were identified using Mutect [For 10 dMMR CRC patients with available whole genome sequencing tumor and matching normal germline DNA data, pre-processed BAM files were downloaded from TCGA database portal (g Mutect  method b


Hereditary non-polyposis colorectal cancer comprises Lynch syndrome and familial colorectal cancer type X (FCCTX). Differences in genetics, demographics and histopathology have been extensively studied. The purpose of this study is to characterize their immunoprofile of markers other than MMR proteins.We compared the expression patterns of cytokeratins (CK7 and CK20), mucins (MUC2/5 AC/6), CDX2 and β-catenin in Lynch syndrome and FCCTX.p < 0.001), whereas MUC2, MUC5AC and MUC6 were overexpressed in Lynch syndrome tumors compared with FCCTX tumors . We observed no differences in the expression patterns of CK7 and CDX2.Differences were identified for CK20 and nuclear β-catenin, which were significantly more often expressed in FCCTX than in Lynch syndrome (In summary, we identified significant differences in the immunoprofiles of colorectal cancers linked to FCCTX and Lynch syndrome with a more sporadic-like profile in the former group and a more distinct profile with frequent MUC6 positivity in the latter group. Identification of hereditary colorectal cancer provides an unprecedented possibility for cancer prevention through inclusion of family members at increased risk into surveillance programs. Identification and diagnostics of hereditary colorectal cancer requires joint efforts from clinicians, pathologists and geneticists. Hereditary non-polyposis colorectal cancer (HNPCC) represents the most common subset of hereditary colorectal cancer and comprises the major subsets Lynch syndrome and familial colorectal cancer type X (FCCTX). Germline mutations in one of the mismatch repair (MMR) genes and resultant microsatellite instability (MSI) characterize Lynch syndrome, whereas retained MMR function and unknown genetic causes characterize FCCTX –3. LynchThe purpose of this study is to record the immunoprofile of markers well-described in colorectal carcinoma in general but, hitherto, incompletely studied in hereditary colorectal carcinomas. These include cytokeratins, mucin glycoproteins, and CDX2. Specifically, its discriminatory utility in FCCTX- vs Lynch syndrome cases is addressed, as is the feasibility of identifying FCCTX among colorectal carcinomas in general. Additionally, β-catenin is included, to compare the extent of the wnt pathway activation in the two hereditary cohorts, as Wnt-signaling genes are shown to be upregulated in FCCTX tumors.http://www.hnpcc.dk). In Denmark, patients with suspected or verified hereditary colorectal cancer are reported to this register by laboratories and responsible clinicians. In Denmark, colorectal cancer diagnostics includes reflex testing for MMR protein expression using antibodies against MLH1, PSM2, MSH2 and MSH6. Cases with loss of expression are, if applicable, and implying the patient provides consent, referred to genetic counselling. Genetic counselling is performed by clinical genetic counsellors and clinical geneticists at 4 departments countrywide. Following genetic diagnostics, Lynch syndrome was defined as presence of disease-predisposing MMR gene variants (classes 4 and 5) and FCCTX was defined as families that fulfilled the Amsterdam criteria, but had tumors with retained MMR function and for the majority of families also genetic MMR gene testing without mutations. The histopathological profiles of the 2 cohorts have been presented in Klarskov et al. [Patients were identified through the national Danish HNPCC register .Immunohistochemical stainings were performed on fresh 4-μm sections from FFPE tissue that was deparaffinized in Tissue clear. Antigen retrieval was achieved by PT-Link and 3-in-1 buffer, pH 9 (Dako). The sections were processed in a Dako autostainer , applying the antibodies targeting CK7/20, MUC2/5 AC/6, CDX2, and β-catenin , tumor location  and extent of differentiation (54% of Lynch syndrome poorly differentiated/undifferentiated versus 17% of FCCTX tumors (p < 0.001)).Clinical data are summarized in Table p < 0.001). Compared to FCCTX tumors, Lynch syndrome tumors displayed significantly more often expression of the tested MUC glycoproteins. The difference in MUC expression was particularly prominent for MUC6 , less so for MUC2 , and MUC5AC (p < 0.01). FCCTX tumors showed more frequent CK20 expression than did Lynch syndrome tumors (p < 0.001) with an equal distribution in right and left side of the large bowel. In Lynch syndrome tumors, CK20 expression patterns correlated to tumor location with more frequent expression of CK20 in right-sided tumors (77%) than in the left-sided tumors (50%) (p = 0.03).The immunohistochemical profiles for five of the seven markers studied significantly differed between colorectal cancers linked to FCCTX and Lynch syndrome Tables  and 4. AUC6 Fig.  .APC (adenomatous polyposis coli) mutations [Immunohistochemical staining is commonly applied as adjunct diagnostics in colorectal cancer. To this end profiles for the expression of cytokeratins, CDX2 and mucin glycoproteins are well-established markers –11 that utations , which mTumors linked to Lynch syndrome and FCCTX showed significant differences, primarily related to frequent expression of CK20 and nuclear β-catenin in FCCTX and relative over-expression of MUC2, MUC5AC and MUC6 in Lynch syndrome. Sánchez-Tomé et al. likewise reported differences in the immunoprofile of FCCTX carcinomas (27 cases) – and Lynch syndrome carcinomas (18 cases) based on markers selected to analyze colorectal carcinogenesis, including SMAD4, COX2, MUC1, and P53 . DespiteFrequent (70–100%) expression of CK20 has been reported in unselected colorectal cancers , 14–20. CK7 expression did not differ between Lynch syndrome tumors and FCCTX tumors and paralleled the expression levels (10–22%) in unselected and sporadic colorectal cancers reported in the literature , 15, 17.The prevailing CK7−/CK20+ cytokeratin profile is reported in 55–77% of colorectal cancer in general  and was Loss of expression of CDX2 in colorectal cancer has been reported as a negative prognostic marker . CDX2 exOur study on the secreted gel-forming mucins  demonstrated significantly higher values in the Lynch syndrome than in the FCCTX tumors. In this context, the idea that the MSI status may influence mucus production, by altering the genes involved  is notewAPC gene [APC mutation in 62% of their FCCTX cases. Other MMR-mutation negative, familial series  have reported lower frequencies of nuclear β-catenin [β-catenin mutation, another cause of aberrant β-catenin, probably contributes to the occasional aberrant β-catenin expression in Lynch syndrome tumors. Based on the current immunohistochemical study, nuclear β-catenin expression in Lynch syndrome tumors is uncommon compared to its prevalence in FCCTX (16% vs. 52%). In concert, nuclear β-catenin expression was previously recorded in merely 19% of colorectal cancer from 118 Lynch syndrome patients in a study conducted by some of us [Nuclear translocation of β-catenin is a marker of dysregulated Wnt signalling. Diverse mechanisms may induce this event in colorectal carcinoma, the major cause being dysfunction of the APC gene . In totaAPC gene , 39. TheAPC gene  who repo-catenin –42. Give-catenin . β-catenme of us . FurtherDistinct gene expression patterns have been demonstrated in colorectal cancers linked to Lynch syndrome and FCCTX and overall support that FCCTX tumors mimic sporadic MMR-proficient tumors , 45. TheThe strengths of the present study include clinically well-defined and relatively large study populations, immunohistochemical evaluations on whole sections in contraposition to the limited areas available by tissue micro arrays , and evSignificant differences in the immunohistochemical profiles of colorectal cancers linked to Lynch syndrome and FCCTX are not restricted to MMR-proteins. In particular, CK20, MUC2, MUC5AC, MUC6 and β-catenin showed disparate expression patterns that may in part be ascribed to clinicopathologic factors such as tumor location, mucinous components, differentiation, and MSI-status, conceivably reflecting diverse underlying genetic mechanism(s). The chosen antibody panel did not allow differentiation between FCCTX and colorectal carcinoma in general. As knowledge on FCCTX genetic(s) emerges, translation into novel biomarkers, useful in discriminating FCCTX from its sporadic counterpart can be anticipated.


Pleurotus ostreatus is an edible oyster mushroom with medicinal properties and can be cultivated on various agroindustrial biomass, including sawdust containing supplements. Pineapple rind was powdered and used as a supplement of composted sawdust at 2%, 5%, 10%, 12%, 15%, and 20% (w/w) on dry weight basis. A control treatment consisted of composted sawdust supplemented with rice bran at 12% (the most utilized composition in Ghana). P. ostreatus strain EM‐1 was cultivated on these treatments. Factors investigated included the spawn run period, yield, fruiting body weight and size, biological efficiency, and nutritional composition  of fruiting bodies harvested from selected high‐yielding treatments and the control treatment. Full colonization of all treatments occurred by the 34th day of incubation. Enhanced yield, fruiting body weight and size, and biological efficiency were generally recorded with supplementation at lower concentrations (2% and 5%) compared to treatments supplemented at higher concentrations. There was also a supplement concentration‐dependent alteration of the nutritional composition of the mushroom. Powdered pineapple rind can be utilized as an organic supplement at relatively low concentrations in composted sawdust for P. ostreatus strain EM‐1 cultivation. The use of lower concentrations of powdered pineapple rind in composted sawdust is advantageous as relatively less input will be required to produce higher P. ostreatus strain EM‐1 yields. Utilization of pineapple rind for mushroom cultivation will extend the pineapple plant value chain, intensify mushroom production in a sustainable way, and minimize agricultural losses.Pineapple rind is a by‐product of the pineapple processing industry and contains nutrients and other compounds which must be utilized as a bioresource for socio‐economic benefits while preventing the potential problems of improper agroindustrial biomass disposal methods.  Pineapple is mostly consumed fresh within the country, but processed products including packaged fresh‐cut pineapple, cut‐dried pineapple, packaged fruit salads, commercially juiced, and bottled pineapple, and juicing in individual households are also available  Kummer strain EM‐1 originally from Mauritius and maintained on Potato Dextrose Agar slants were used to prepare spawn. Spawn of this mushroom was prepared, using grains of sorghum and millet, obtained from the Nima Market in Accra, combined in a 3:1 (w/w) ratio  was collected from Nature's Best fruit processing incubation factory at CSIR – Food Research Institute, and solar dried for 45 hr to a moisture level of about 18% as determined, using the AOAC method  Kummer strain EM‐1 spawn, incubated till total spawn run and cropped as previously described by Obodai and Johnson  on dry weight basis before bagging. A control treatment supplemented with rice bran (RB) at 12% (w/w) was also set up. Aliquots of quicklime were added to all treatments at 0.5% (w/w). The moisture content of each treatment was also adjusted with tap water to approximately 60%–70% using the squeeze test Buswell, . The mix Johnson . The tre Johnson  with somData collected during incubation included the mycelia growth rate and the spawn run period .Fresh mushrooms were harvested at maturity during cropping. Data collected during the cropping stage included weight and number of fruiting bodies per flush, weight and number of fruiting bodies per bag, and the total yields per bag. Physical characteristics of the fruiting bodies recorded included fruiting body weight, cap and stipe weights, cap diameter and stipe circumference of five randomly selected fruiting bodies of the first flush of mushrooms. Caps and stipes of the selected fruiting bodies were separated by cutting the stipe at the cap attachment with a scalpel. Weights were taken with a Digital Computing Scale . Cap diameters were taken by measuring the diameter of the cap at right angles for each randomly selected fruiting body and averaging the values to represent the cap diameter for that fruiting body. An average of the circumferences of the largest and smallest portion of the stipe was recorded as the stipe circumference of each selected fruiting body.The ratio of cap weight to stipe weight was determined by dividing the average cap weight of the five randomly selected fruiting bodies by the average stipe weight of the same fruiting bodies. Ratio of cap diameter to stipe circumference was also determined by dividing the average cap diameter of the same five randomly selected fruiting bodies by the average stipe circumference of the five fruiting bodies. The total yield per flush as well as the total number of fruiting bodies were also recorded. Mushroom size was the ratio of mean total weight of fresh mushroom harvested per bag and the mean total number of mushrooms harvested per bag as described by Royse, Rhodes, Ohga, and Sanchez . The bio2.4The entire fruiting bodies  from treatments showing the highest biological efficiencies as well as the control treatment were separately sun dried, milled, and kept refrigerated at −10°C until chemical analyses were conducted.2.4.1N × 4.38) was estimated by the Macro‐Kjeldahl method. The crude fat was determined by extracting a known weight of sample with petroleum ether, using a Soxhlet apparatus. Ash content was determined by incineration at 600 ± 15°C for 24 hr. The total carbohydrates were calculated by difference whereas the total energy was calculated according to the equation,Nutritional composition  of the milled samples were determined, using the AOAC procedures AOAC, , 2000. S2.4.2All the minerals; Copper (Cu), Zinc (Zn), and Lead (Pb) were analyzed by AAS  after sample wet digestion according to the method described by the authors   reported Stamets,  for P. oStamets, . Moisturp < .05) by the end of the third week. Spawn run periods for the treatments with varying concentrations of pineapple rind were comparable to the control treatment and differed by 1 or 2 days  than that of the control treatment.Pineapple rind at 2% and 5% supplementation (2% PPR and 5% PPR) both supported the best yield of the fungus in comparison with all the other treatments supplemented with PPR. However, yields obtained from 2% PPR treatment (236.5 ± 37.00) were somewhat comparable to that from the 5% PPR (223.5 ± 14.95) and the control treatment . The yields obtained from 5% PPR were statistically higher  from that of the fruiting bodies obtained from the control treatment (12% RB). As observed for the yields, the highest BE was recorded for the 2% PPR treatment (78.83 ± 12.33%), which was comparable to the BEs of the 5% PPR treatment (74.50 ± 4.98%) and the control treatment (65.23 ± 3.40%). Formulation of sawdust and other substrates have been previously reported  were statistically not significantly different (3.3p < .05) in treatments containing relatively high concentrations of PPR (15 and 20%) compared to all the other treatments, including the control treatment. Stipe weights followed similar trends and ranged from a mean weight of 1.0 to 6.0 g across the treatments. Mean cap diameters ranged from 5.3 cm to 7.0 cm whereas mean stipe circumferences ranged from 2.4 cm to 3.2 cm across the treatments. The increasing order of the ratio of cap weight to stipe weight was 1.3, 3.0, 3.1, 4.1, 5.7, 5.8, and 5.9 for treatments 5% PPR, 10% PPR, 2% PPR, 12% PPR, 12% RB, 20% PPR, and 15% PPR, respectively. The dimensional ratio of the cap to the stipe ranged from 2.0 to 2.4 across the treatments.Whole fruiting body, cap, and stipe weights, cap diameter, and stipe circumference of five fruiting bodies from the first flush of mushroom are presented in Table P. ostreatus cultivated on composted sawdust and cropped using a variety of cropping techniques. It is, however, noteworthy that supplementation of composted sawdust with PPR at relatively high concentrations (≥15% w/w) has a negative influence on the cap and stipe weights as well as the cap diameters of fruiting bodies obtained from the substrate.The fruiting body parameters reported herein are comparable to that reported by Baka, Narh, Obodai, Dzomeku, and Takli  for the 3.4p > .05), whereas there were statistically significant differences (p < .05) in the values for the other nutritional parameters. For instance, the significantly highest and lowest protein contents were recorded in the fruiting bodies obtained from treatments 2% PPR and 5% PPR respectively. Significantly lower values of copper, zinc, and lead were recorded for fruiting bodies obtained from treatment 2% PPR while the values recorded for the fruiting bodies obtained from treatments 5% PPR and 12% RB did not differ significantly.Moisture, ash, fat, protein, carbohydrate, and energetic values of the powdered mushroom fruiting bodies ranged from 10.56 to 10.84 g/100 g.dw, 8.34 to 8.40 g/100 g.dw, 1.29 to 1.45 g/100 g.dw, 20.4 to 21.65 g/100 g.dw, 57.85 to 59.08 g/100 g.dw and 329.5 to 334.34 Kcal/100 g.dw, respectively (Table P. ostreatus cultivated on composted sawdust. Consumption of P. ostreatus and other mushrooms is usually based on the nutritional composition, medicinal value, and organoleptic properties, which sometimes differ in the caps and stipes of the fruiting bodies (Oboh & Shodehinde, Ash, carbohydrate, copper, zinc, and lead contents of this study were relatively higher while all the other nutritional values were relatively lower than that reported by the authors (Obodai, Ferreira, et al., P. ostreatus. In addition, the concentration of PPR in composted sawdust is relevant for altering the nutritional characteristics of the mushroom's fruiting bodies obtained from composted sawdust.These results demonstrate that substrate formulation influences the nutritional and mineral composition of fruiting bodies of 4P. ostreatus strain EM‐1 comparable to the mushroom cultivated on composted sawdust supplemented with rice bran at 12%. PPR at low concentrations also significantly enhances the biological efficiency of composted sawdust in comparison with supplementation with rice bran at 12%. The study also demonstrates the supplement concentration‐dependent alteration of the nutritional composition of the mushroom. Therefore, PPR can be considered to be a good organic supplement of composted sawdust for P. ostreatus strain EM‐1 cultivation when used at low concentrations.This study shows that supplementation of composted sawdust with PPR at low concentrations results in yields and mushroom size of The authors declare that they have no known conflict of interest.


Childhood hearing impairment is a significant cause of disability in developing countries, where infectious diseases are the leading cause of avoidable pediatric hearing loss . The WorThe provision of hearing services in the Pacific Islands is currently limited . AlthougThe WHO reports that 60% of global childhood hearing loss is preventable: prenatal and postnatal infections 31%), birth-related causes (17%), ototoxic medicines (4%), and other causes such as substance abuse (8%) [%, birth-The integration of new pediatric audiology services into existing public health care programs should have a positive outcome if these programs are accepted and well-attended by the community. Given the success of the WHO Expanded Program on Immunization in developing countries, immunization clinics have been identified as an alternative platform for Infant Hearing Screening (IHS) programs in nations where a hospital-based Universal Newborn Hearing Screening Program may not be feasible . The curSchool-screening programs are another well-known public health approach to managing significant health conditions and improving educational outcomes. The development of school-based ear and hearing assessments in the Pacific Islands may collaborate with the WHO Global School Health Initiative to optimize successful implementation of the program . This inThere are a number of challenges  that may prevent attendance at a hospital or health clinic. School hearing screening programs offer an acceptable community-based alternative, and would enable early access to primary health care services for ear/hearing disorders. A key aim of these programs would be to facilitate medical and, possibly, rehabilitative and educational interventions for children identified with a hearing impairment.Hospital-based Ear, Nose & Throat (ENT) Clinics are among the best candidates for the development of public health hearing services for children in the Pacific Islands. Given their existing commitment to otological health care, ENT Clinic staff members are the most likely to appreciate the additional contribution of pediatric audiology services in the overall diagnosis and management of a child with OM-induced hearing loss. ENT Clinic staff members may also have the greatest motivation for acquiring audiometry skills, satisfying the principle of capacity-development in the implementation of locally sustainable services. An opportunity for formal qualifications would be desirable as it provides local staff members with recognition for their skills.Where ENT medical services are accepted and well-attended by the community, the integration of audiometry services into the ear health care framework has been successful. At the time of writing, ENT Registered Nurses in the Solomon Islands and Vanuatu are able to provide audiometry testing as part of their ENT outpatient service. Given that these ENT nurses include a school outreach service as part of their clinical program, the development of an ear and hearing screening program performed by the ENT nurses is a clear possibility. The ENT Clinic may also extend their community-based services to Infant Ear and Hearing Screening programs, targeting early-onset otitis media, and monitoring infants at risk of sensorineural hearing impairment.In addition to community-based public health programs, a hospital-based audiology service should consider the implementation of a hospital-based hearing screening program for survivors of meningitis. Given the estimated burden of post-meningitic hearing impairment in the Pacific Region, the results of a routine hearing screening program for all meningitis survivors could lend extra support to the implementation of immunizations against the multiple causal pathogens of meningitis. A hospital-based hearing screening program may eventually expand to assess other patients at risk of hearing loss .Although the focus of the present review has been a public health approach to reducing the burden of preventable and treatable causes of childhood hearing impairment in the Pacific Islands, some comments about (re)habilitation audiology services are warranted as pilot Infant Hearing Screening programs will identify children with permanent congenital and early-onset hearing loss. In accordance with the principles of international health development, the implementation of (re)habilitation audiology services must consider sustainability. Indeed, the WHO recommends the provision of amplification devices  only where support and maintenance services are also available . The addAlthough many Pacific Island nations have a School for the Deaf or special schools for children with disabilities, the provision of hearing aids for students is limited . While tShould the provision of rehabilitation audiology services be sustainable, there may be more community acceptance of hearing aid services if they are delivered at a School for the Deaf or special school, since a hospital-based service may inadvertently lead to negative perceptions of permanent hearing loss primarily as a disease or illness. Referral pathways should, however, continue to exist between such centres and the ENT Clinic to ensure any additional ear disease is managed as required. Given the tropical climate of many Pacific Island nations, the use of hearing aids increases the risk of otitis externa, as well as cerumen impaction.Another benefit of a school-based service would be that hearing aid care and maintenance are provided by staff in regular contact with the hearing-impaired child. It must be noted, however, that the Pacific Island region is currently moving towards Inclusive Education strategies rather than encouraging separate schools for children with a disability. The Inclusive Education policies are largely based on the fact that attendance at special schools is difficult, and that Inclusive Education is more appropriate given the economic and geographic challenges of the Pacific Island context.The development of pediatric audiology services in the Pacific Islands should consider a public health approach. The integration of childhood hearing services with existing public health initiatives by international organizations such as the WHO and UNICEF, should reduce the burden of preventable childhood hearing loss in the Pacific Islands. Collaboration with the local ENT Clinic, including training and capacity-development of ENT Clinic staff members, is an attractive option in the development of local audiology services for children.


Bombyx mori is an economically important species. White muscardine caused by Beauveria bassiana is the main fungal disease in sericulture, and understanding the silkworm responses to B. bassiana infection is of particular interest. Herein, we investigated the molecular mechanisms underlying these responses in two silkworm strains Haoyue  and Kang 8  using an RNA-seq approach. For each strain, three biological replicates for immersion treatment, two replicates for injection treatment and three untreated controls were collected to generate 16 libraries for sequencing. Differentially expressed genes (DEGs) between treated samples and untreated controls, and between the two silkworm strains, were identified. DEGs and the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the two strains exhibited an obvious difference. Several genes encoding cuticle proteins, serine proteinase inhibitors (SPI) and antimicrobial peptides (AMP) and the drug metabolism pathway involved in toxin detoxification were considered to be related to the resistance of K8 to B. bassiana. These results revealed insight into the resistance and susceptibility of two silkworm strains against B. bassiana infection and provided a roadmap for silkworm molecular breeding to enhance its resistance to B. bassiana.The silkworm  Bombyx mori, is an important economic insect for silk production in many developing countries [Beauveria bassiana is a major pathogenic fungus in sericulture. White muscardine disease caused by B. bassiana tends to break out in moist climate and leads to enormous damage [The silkworm, ountries . It is aountries ,3,4. Beas damage .B. bassiana infecting silkworms can be separated into three different stages: surface adhesion, cuticle penetration, and replication and host killing [B. bassiana infection remains poorly understood. Investigating the silkworm response to B. bassiana infection is of particular interest since it provides a roadmap for silkworm molecular breeding to enhance its resistance against B. bassiana. In addition, it gives useful information for genetic modification of the fungus to enhance its efficiency in pest control [B. bassiana using the suppression subtractive hybridization method [B. bassiana infection, and identified 1430 DEGs [B. bassiana. However, these studies only focused on one silkworm strain without considering differences between resistant and susceptible strains.In the natural environment, the process of  killing . After c killing , but the control . Hou et n method . In 2014430 DEGs . The resB. bassiana infection, in contrast to the Kang 8 (K8) strain that is highly resistant. K8 is a valuable germplasm resource preserved in Guangdong Sericultural Germplasm Bank and is well known for good healthiness. The molecular mechanisms underlying their different responses to B. bassiana infection remain unknown at present, and investigating differences in gene expression during B. bassiana infection may help us to understand the genes responsible for resistance in K8.Haoyue (HY) is a bivoltine silkworm strain originating from Japan  and has B. bassiana infection. In this study, the molecular mechanisms underlying the different responses to B. bassiana infection in HY and K8 were investigated via transcriptome analysis with the Illumina HiSeq 2500 platform. DEGs and their associated pathways were identified, and the results provide insight into the genes responsible for B. bassiana resistance and susceptibility.RNA-Seq is a revolutionary technology ,13 proviB. bassiana infection. As shown in 5 conidia/mL), almost all HY silkworms were dead  at 168 hpi, whereas nearly 70% of the K8 silkworms were alive and able to spin cocoons (5 conidia/mL) showed similar result  analysis. The results  essentiaTo better understand the identified DEGs, we classified them according to their biological functions . In the In the injection experiment, a greater number of genes encoding cuticle protein, AMP, lipoprotein (30K protein), and cytochrome P450 were up-regulated in K8, while more genes encoding peptidases and MFS proteins were up-regulated in HY A. A largp < 0.05), and no common pathway was detected in these two strains. For the injection experiment, 40 and 32 pathways were significantly enriched with DEGs in K8 and HY, respectively (p < 0.05), and 19 pathways were enriched with DEGs in both strains. In K8, the pancreatic secretion pathway, which was related to digestion and abortion of nutrition, was clearly enriched in both immersion and injection experiments. In HY, “phagosome”, “lysosome”, “glycerolipid metabolism” and “ECM-receptor interaction” pathways were enriched in both immersion and injection experiments.Pathway enrichment analysis was performed on DEGs identified in each group . For theB. bassiana is distinct in the two silkworm strains, and the genes that are differentially expressed between HY and K8 strains may be responsible for the dissimilar responses to the fungal pathogen. We therefore compared DEGs between HY and K8 strains. HY and K8 samples consisted of three control samples, three immersion samples and two injection samples, respectively. A total of 260 DEGs were detected, including 116 up-regulated genes and 144 down-regulated genes in K8 (q < 0.05 and log2 (Fold change) ≥ 1, deSeq). As shown in 2 (Fold change) ≥ 3. Under the standards, six genes could be considered as K8-specific genes  were selected for interrogation by qPCR, the results of which confirmed their specific expression in the K8 strain. Cytochrome P450 proteins function as a major class of detoxification enzymes [B. bassiana infection.Furthermore, DEGs of eight HY samples and eight K8 samples were also compared. Six genes were specifically expressed in K8, including three encoding cytochrome P450 family proteins. Two of these , carboxylesterase (BGIBMGA012122), and UDP-glucuronosyltransferase , were all up-regulated in the K8 strain. Similar to cytochrome P450, these enzymes are essential components of insect toxin detoxification pathway . We propll types  and thisll types . A previll types .B. bassiana infection in highly susceptible (HY) and highly resistant (K8) strains using an RNA-seq approach. The results revealed that a larger number of genes encoding cuticle protein, SPI, AMP and 30K protein were up-regulated in the K8 strain and down-regulated in the HY strain. Moreover, a series of detoxification-related genes were specifically up-regulated in the K8 strain. It could be speculated further that K8 silkworms could increase level of cuticle protein and SPI, which thereby inhibited the germination and penetration of B. bassiana conidia, and then activate the drug metabolism pathway to remove the toxins secreted by B. bassiana, ultimately resulted in high resistance to B. bassiana. Further research on the exact mechanisms of these genes is needed.The insect-fungus interactions have been studied for several years. However, the mechanisms of resistance remain poorly understood, and many of the key genes and proteins involved have not been established. In the present study, we investigated host response to B. bassiana) and K8 (resistant to B. bassiana) were provided by the Guangdong Sericultural Germplasm Bank . Larvae were reared on fresh mulberry leaves at 27 °C, and newly exuviated larvae of the fifth instar were used in all experiments.The trial was approved by the Experimental Animal Ethics Committee of Sericulture and Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences . The silkworm strains HY (sensitive to B. bassiana (Genbank ID: KM205065), isolated from silkworm larvae, was preserved at 4 °C on slants of potato dextrose agar. Conidia, harvested from cultures grown for 7 days at 26 °C and 95% relative humidity, were suspended in sterile distilled water for experiments.The fungal strain 5 and 5 × 105 conidia/mL) for 15 s, respectively [B. bassiana. For the control group, the silkworms were immersed in sterile distilled water for the same period. Each assay was repeated four times.For each silkworm strain, newly exuviated larvae of fifth instar were immersed in the conidial suspension (1 × 105 conidia/mL) as described above. At 96 h post infection (hpi), three independent larvae of each strain were collected respectively. For eliminating the effect of the body wall on B. bassiana defenses in K8 and HY strains, injection experiments were also performed. Since 10 h post injection has been shown as an appropriate time point for investigating the insect responses to B. bassiana conidia in previous reports [7 conidia/mL) were injected into the hemocoel of Bombyx mori fifth instar 86 h larvae, and 10 h later, two independent larvae of each strain were collected for subsequent sequencing experiments. Untreated fifth-instar larvae were used as the control group at 96 h. All silkworm samples were therefore collected at the same developmental stage (96 h post exuviation) to eliminate the influence of development on gene expression [Newly exuviated larvae of the fifth instar were immersed in the conidial suspension  according to the manufacturer’s protocol. RNA purity was assessed by using an ND-1000 NanoDrop . Each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 Tape Station , and all samples had an RNA integrity value above 7.0. Briefly, mRNAs were isolated from total RNA, fragmented to approximately 200 bp, and subjected to first strand and second strand cDNA synthesis followed by adaptor ligation and low-cycle enrichment according to the instructions of the TruSeq RNA LT/HT Sample Prep Kit .Purified library products were evaluated using the Agilent 2200 Tape Station and Qubit 2.0 software (Life Technologies). RNA-Seq was performed at RiboBio Co., Ltd.  using the HiSeq 2500 platform . Prior to sequencing, raw data were filtered to produce high-quality clean data, and all subsequent analyses were performed using clean data.2 ratio ≥1. Pathways significantly enriched with DEGs were identified based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and hypergeometric test (q < 0.05).For annotation, clean data were mapped to the silkworm transcriptome reference database using Soap2 . Only reBmactinA3 gene was used as the endogenous control [Ct−ΔΔ method [To validate the results of DEG analysis, qPCR experiments were performed on a Roche LightCycler 480 system using SYBR-GREEN1 fluorescent reagents . All designed primers were synthesized at Beijing Liuhe Huada Genomics Technology Co., Ltd.  . The Bma control . qPCR ret method .B. bassiana) and HY (sensitive to B. bassiana) strains to investigate the reasons for their distinct susceptibility to B. bassiana. Different responses against B. bassiana infection were observed. Several genes encoding cuticle proteins, SPI and AMP and the drug metabolism pathway involved in toxin detoxification were considered to be related to the resistance of K8 to B. bassiana. Further molecular mechanism-based studies of host resistance to fungus are still needed.In this study, we carried out RNA-Seq on silkworm K8 (resistant to


Purpose: We assessed the safety and efficacy of a technically advanced subretinal electronic implant, RETINA IMPLANT Alpha AMS, in end stage retinal degeneration in an interim analysis of two ongoing prospective clinical trials. The purpose of this article is to describe the interim functional results (efficacy).Methods: The subretinal visual prosthesis RETINA IMPLANT Alpha AMS  was implanted in 15 blind patients with hereditary retinal degenerations at four study sites with a follow-up period of 12 months . Functional outcome measures included (1) screen-based standardized 2- or 4-alternative forced-choice (AFC) tests of light perception, light localization, grating detection (basic grating acuity (BaGA) test), and Landolt C-rings; (2) gray level discrimination; (3) performance during activities of daily living (ADL-table tasks).Results: Implant-mediated light perception was observed in 13/15 patients. During the observation period implant mediated localization of visual targets was possible in 13/15 patients. Correct grating detection was achieved for spatial frequencies of 0.1 cpd (cycles per degree) in 4/15; 0.33 cpd in 3/15; 0.66 cpd in 2/15; 1.0 cpd in 2/15 and 3.3 cpd in 1/15 patients. In two patients visual acuity (VA) assessed with Landolt C- rings was 20/546 and 20/1111. Of 6 possible gray levels on average 4.6 ± 0.8  were discerned. Improvements (power ON vs. OFF) of ADL table tasks were measured in 13/15 patients. Overall, results were stable during the observation period. Serious adverse events (SAEs) were reported in 4 patients: 2 movements of the implant, readjusted in a second surgery; 4 conjunctival erosion/dehiscence, successfully treated; 1 pain event around the coil, successfully treated; 1 partial reduction of silicone oil tamponade leading to distorted vision (silicon oil successfully refilled). The majority of adverse events (AEs) were transient and mostly of mild to moderate intensity.Conclusions: Psychophysical and subjective data show that RETINA IMPLANT Alpha AMS is reliable, well tolerated and can restore limited visual functions in blind patients with degenerations of the outer retina. Compared with the previous implant Alpha IMS, longevity of the new implant Alpha AMS has been considerably improved. Alpha AMS has meanwhile been certified as a commercially available medical device, reimbursed in Germany by the public health system. Inherited retina degenerations (IRD) are a broad group of genetic retinal disorders of varying severity with retinitis pigmentosa being the most common form. Its estimated prevalence is between 1 in 3,000–7,000  and IRIS® II  and the subretinal device RETINA IMPLANT Alpha AMS . It should be noted that the epiretinal prosthesis devices are transmitting visual information via electrical stimulation of the ganglion cells, whereas the subretinal approach aims to replace the degenerated photoreceptors and is stimulating the bipolar cells of the remaining inner retina. Thus, patients blind because of damage of the inner retina  or other diseases which cause more than degeneration of photoreceptors cannot benefit from the RETINA IMPLANT Alpha AMS. Efficacy results from 29 blind patients wearing the RETINA IMPLANT Alpha IMS device—a precursor of Alpha AMS—have been published recently  and one site in Great Britain  patients with end-stage of a hereditary retinal degeneration were recruited for the participation in the clinical trial. Patients with rod-cone and cone-rod degenerations, as well as choroideremia, who were not able to use their remaining vision for localization of objects, self-sustained navigation and orientation  were eligible for participation.In three study sites in Germany  and ability to read normal print in earlier life, optically corrected without magnifying glass.Ophthalmologic exclusion criteria were a period of appropriate visual functions <12 years, Optical Coherence Tomography (OCT) findings of retinal edema and/or scar tissue within target region for implant as well as absent layering of the inner retina, heavy clumped pigmentation at posterior pole or any other ophthalmologic disease with relevant effect upon visual function . Additionally, systemic diseases that might imply considerable risks with regard to the surgical interventions and anesthesia, neurological and/or psychiatric diseases, hyperthyroidism or hypersensitivity to iodine and pregnancy/nursing, and participation in another interventional clinical trial within the past 30 days were exclusion criteria.The subretinal implant RETINA IMPLANT Alpha AMS Figure  attachedThe RETINA IMPLANT Alpha AMS is an advanced version of the previous Alpha IMS device; the main changes are summarized in Table Inclusion and exclusion criteria were essentially analogous in both studies (see Section Patient Recruitment).Details of the surgical implantation procedure have been described previously .Treatment duration was not specified in either study, as the implant is intended to remain in the eye as long as it provides benefit to the patient. The follow up duration was 12 months post-implantation surgery for both studies. The device was activated for the first time ~1 month after surgery. Subsequent follow-up visits were done 2, 3, 6, 9, and 12 months after surgery. As both studies are not completed at the time of submission of this manuscript, some patients have not yet completed the study  with implant-ON vs. OFF, as assessed via ADL tasks and recognition tasks. Secondary efficacy endpoints in both studies were significant improvement of visual acuity/light perception and/or object recognition. All tests have been described previously in detail . The patient was asked how many objects could be seen, where they were and what they were. Correct responses were documented as scores (from 0 to 4 for each of the three questions).The second task used four dining objects  which were placed around a large white plate serving as reference for the patient. The patient was asked how many objects could be seen, where they were and what they were. Correct responses were documented as scores (from 0 to 4 for each of the three questions).To test the eye-hand coordination, a large  white ring or square frame was placed on the table and the patient was given either a chess piece or a small white egg cup. The patient was asked to localize the ring/square visually and place the chess piece/egg cup inside the frame. The test was counted as passed if the patient managed to place the object inside the frame without it standing on the frame itself.2, respectively. Each of the combinations was tested three times in a random order and the patient had to determine whether there was a difference in brightness and if so, which side was brighter. For the subsequent analysis, the 50/50%  was not included. For the other six combinations a grayscale was counted as correctly discerned if 2 out of 3 repetitions were correctly distinguished.For assessment of grayscales, the screen  was divided in half with one side always showing a gray area with 50% of the screen's brightness. The other side showed a gray value of 0% (white), 25, 37.5, 50, 62.5, 75, or 100% (black) with minimal and maximal brightness of typically 8 and 200 cd/mLight threshold perception, light source localization, and motion detection of dot patterns were tested on a screen, viewed at 60 cm, as either 2- or 4-alternative forced-choice (AFC) tests of 8 trials each  was not implied. Statistical analyses were performed with the software JMP, version 13.0.0 (SAS Institute Inc.). All tests performed were non-parametric, given the small sample size and thus the inadequacy to test for normal distribution. For assessments which were graded (ADL tasks shapes and table set-up) the Wilcoxon test with paired data was used, analyzing the difference between scores with implant ON vs. implant OFF. Patient data where only one condition was present  was omitted from the analysis. For analyses of assessments which were graded as “passed” or “failed,” the Fisher's exact test was used. An adjustment for multiple testing was not done for any of the analyses performed.In total, 21 patients were screened for RETINA IMPLANT Alpha AMS. The reasons for exclusion after screening were as follows: pre-existing glaucoma, suspected non-functionality of the inner retina, remaining visual function allowing shape detection, macular edema, and heavy clumped pigmentation at posterior eye pole. One patient withdrew consent prior to the implantation.www.clinicaltrials.gov NCT01024803) and five participants  from Great Britain  with a total mean age (± SD) of 55.2 ± 10.2 years between 34 and 70 years received the RETINA IMPLANT Alpha AMS in one eye. Fourteen of the participants had retinitis pigmentosa and one had cone-rod dystrophy. Visual function prior to implantation was light perception without projection (fourteen participants) or no light perception (one participant). None of the participants had any other eye diseases that might have affected the visual pathway.Ten participants  from three sites in Germany  the implant was probably damaged slightly during implantation and did not function correctly. In another patient (OX-RI-04) a combination of damage to the connecting foil and incorrect implantation procedure resulted in a non-functional chip. The chip was subsequently replaced successfully in an exchange procedure, although this was an “off-study” procedure as the patient was no longer part of the formal clinical trial. The results of the remaining 13 patients for each of the particular tests are given in the following paragraphs. A summary of the best results achieved with the implant switched ON for each individual patient is shown in Table  the implResults for the ADL task are summarized in Figure The pair-wise comparison of scores with implant ON vs. implant OFF showed that detection (“how many”) and localization (“where”) of geometric shapes was significantly better at all-time points, when the implant was switched on  and localization (“where”) of table objects was significantly better at all-time points when the implant was switched ON .Detection of grating orientation was significantly better with the implant switched ON compared to OFF at month 2, 3, and 12 . Landolt C-ring measurements under regular ophthalmological conditions have only been reported for one other retinal prosthesis, namely the supra-choroidal device from Bionic Vision Australia, where one patient was able to achieve a maximum value of 20/4451 visual acuity  achieved in our cohort was 3.3 cpd by one patient. This patient was not able to identify Landolt C-rings; supporting the wide-spread notion that correct recognition of orientation of lines in a four alternative forced choice test that involves sampling of many lines over a large area is not related to visual acuity determined by simple optotypes in very low vision. The result of 3.3 cpd has been achieved by one patient with the RETINA IMPLANT Alpha IMS as well , and as the data of all assessments is comparable to the later time-points the authors conclude that re-learning vision is not, or only to a very small extent, necessary for this subset of patients. The authors' empirical experience confirms that mostly within days up to 2 weeks after the first switch-on of the device the best achievable vision for the individual patient could be estimated based on the present results. The authors believe that this is one of the most important differences between bionic vision with subretinal and epiretinal devices; while a subretinal device only tries to replace the photoreceptor function, stimulating the remaining bipolar cell network, the vision is perceived more “naturally” because the whole remaining visual pathway is used. In contrary, in epiretinal visual devices, the third visual neuron is stimulated, bypassing the preprocessing of the bipolar cell layer. As the second visual neuron already performs important signal processing, a vision circumventing this natural step might require more learning and adaptation of the higher visual areas. The improved durability compared to the earlier version Alpha IMS is also contributing to the stable nature of the observations. As can be seen in Figure The results from the ADL tasks show comparable mean scores as in the previously published cohort of 29 patients with RETINA IMPLANT Alpha IMS  compared to two out of eight who could while the device was turned OFF (5 patients failed the test and one patient did not perform this test). Similar results were obtained in the BaLM (Basic Light and Motion) tests, where eight out of eight patients completing the study had light perception with the implant ON compared to only one patient with the implant OFF. Seven out of these eight patients could also locate the light source correctly with the implant ON, but none of them was able to pass the motion test at month 12. Interestingly, seven patients were able to pass the basic grating acuity test at this time-point. This might be due to the fact that the standardized motion test applied in this study can be regarded as more challenging to the visual system for these patients as it also relies heavily on the setting of stimulation frequency, motion speed range and eye movement. However, even if not passing the formal limited motion speed test range, many patients reported spontaneously about perceiving moving objects such as car lights, animals, persons etc. As the working frequency of the implant can differ from patient to patient and from visit to visit (between 1 and 10 Hz), the perception of motion is different for each patient and difficult to assess over the entire physiological range of motion perception.The safety profile of the RETINA IMPLANT Alpha AMS shows four patients experiencing a total of eight Serious Adverse Events (SAEs). Postoperative movement of the implant was detected in two patients and was treated by repositioning the implant in a second surgery. Two patients experienced conjunctival dehiscence (one patient once and the other patient three times); all of them were treated by surgical intervention. One patient presented with a partial loss of silicone oil tamponade which was refilled in a second surgery. The same patient described pain in the region of the implanted ceramic housing in case of pressure a couple of months later. Initially, a surgical intervention was planned, but the patient declined and stated that the pain subsided. A surgical intervention for revision of the retroauricular cable positioning was finally done 10 months after the trial close-out visit (22 months after implantation). Adverse events were comparable to the safety profile described for the previous version Alpha IMS  discussing interim results of the clinical trial NCT01024803 was submitted to the notified body (ID 0197) in Germany. Based on this documentation, RETINA IMPLANT Alpha AMS received the CE-mark in March 2016 from the notified body while the clinical trial was still continued for long term evaluation (Registration No. II 60119225 0001), according to European rules for medical devices. The Retina Implant Alpha AMS is now commercially available, with reimbursement by public health insurances at selected ophthalmological centers in Germany.These studies were carried out in accordance with the recommendations of ICH Guidelines for Good Clinical Practice (CPMP/ICH/135/95) July 1996 with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the Medical Faculty and University Hospital of Tuebingen, located in Gartenstrasse 47, 72074 Tuebingen, Germany and the National Research Ethics Service Committee London—Dulwich, Skipton House, 80 London Road, London SE1 6LH.Substantial contributions to the conception or design of the work: KS, KB, DB, FG, UG, RM, JRa, JRo, HS, NT, AR, and EZ. Substantial contributions to the acquisition of data for the work: KS, CC, TE, KK, LK, GS, JT, and EZ. Substantial contributions to the analysis and interpretation of data for the work: KS, RS, TE, NT, and EZ. Drafting the work or revising it critically for important intellectual content: All Authors. Final approval of the version to be published: All Authors. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: All Authors.KS, LK: Employed by University of Tuebingen by means provided by Retina Implant AG, Reutlingen for the clinical trial, travel support. RS, UG, NT: Employees from Retina Implant AG, Reutlingen. FG, EZ: Stock ownership in Retina Implant AG, Reutlingen, paid consultant, holder of patents as inventor/developer, travel support from Retina Implant AG, Reutlingen. AR: Paid consultant, holder of patents as inventor/developer, travel support from Retina Implant AG, Reutlingen. HS: Stock ownership in Retina Implant AG, Reutlingen, paid consultants. JT: travel support from Retina Implant AG, Reutlingen. The other 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.


This study was to investigate some new pathological genes in colorectal adenocarcinoma of human.Human colorectal adenocarcinoma tissues and normal colorectal tissues were taken and suppression subtractive hybridization (SSH) and cDNA microarray techniques were employed. From differentially expressed 86 expressed sequence tags (EST), 10 EST of the SSH were selected as seed sequence for bioinformatics analyses, semi-quantitative RT-PCR and PCR-sequencing. Each lane of semi-quantitative RT-PCR was analyzed by Q1 software.Among these 10 EST, it has been found that ES274070, ES274071, ES274076 and ES274081 may play role in the onset of colorectal adenocarcinoma in human.The ES274070, ES274071, ES274076 and ES274081 are related to the onset of human colorectal adenocarcinoma. The initiation and development of colorectal adenocarcinoma are multistep process that is involved multi-gene changes. During this process, cell division and differentiation are abnormally regulated by the many genes . It willThis study aims to screen and clone the genes related to genesis of human colorectal carcinoma and further study their function and relationship with the colorectal adenocarcinoma. We selected 10 expressed sequence tags (EST) from suppression subtractive hybridization (SSH) combining cDNA microarray as seed sequences to extend them in order to acquire full-length cDNA and validate expressiong by RT-PCR. It will be helpful for us to further study their function; moreover, this will make us find early diagnostic and curative targets.The normal human colorectal tissues and the human colorectal adenocarcinoma (HCRAC) tissue samples from HCRAC patients in West China Hospital of Sichuan University were snap-frozen in liquid nitrogen immediately after surgery and stored at -80°C. Nine pairs of HCRAC and normal colorectal tissues had been used, isolation of RNA was performed by the TRIzol method (Invitrogen) according to manufacturer’s instructions. The SSH combined with cDNA microarray was performed . The 10 We obtained 1260 differentially expressed clones by SSH. The cDNA microarray showed total 86 EST being identified were more than half past one fold or two-fold differentially expressed genes between two tissues. After they had been sequenced and analyzed by bioinformatics analysis, the sequence data were accepted by Genebank and were identified as EST and got genebank accession number and dbEST number. The 1721bp length cDNA sequence for ES274070 was formed by DNAStar Software and it located in human chromosome 17q21.2. The open reading frame (ORF) was predicted and its product contains 111 amino acids, its molecular weight is 12.8KD, PI is 8.79. On NCBI website, it is found that it has the homology 1649bp sequence (BC042012). The Serial analysis of gene expression (SAGE) indicates that there is expression in oligodendroglioma, astrocytoma, prostatic carcinoma, ovarian cancer, breast cancer, and carcinoma of testis, sarcoenchondroma, and high expression in brain tumor tissue. Semi-quantitative RT-PCR results for its’ ORF were showed that its mRNA in colorectal cancer tissue was significantly higher than that of normal colorectal tissues (P = 0.011), .The 2790bp length cDNA sequence for ES274071 was also formed. The sequence extends 1550bp from 5’end and 820bp from 3’end. It is located in human chromosome 9q34, ORF’s length is 834bp. Its coded protein is SET translocation, we found the registered sequence NM003011 which is homologous with the assembled sequence, the length is 2577bp. We assembled the full-length cDNA by extending 309bp compared with NM003011 by RT-PCR. The result of ES274071 ORF was shown by sequencing and bioinformatics analysis, it was extended 16bp compared with NM003011 and we got accession number EF534308 .We also found that ES274073 corresponding full-length sequence were human mitochondrial genome (NM183047). Semi-quantitative RT-PCR results of NM183047 showed there was no significant difference in normal tissues and colorectal cancer tissues (P > 0.05). The ES274076 corresponding full-length sequence was human zinc finger protein (NM002644), semi-quantitative RT-PCR results of its ORF showed that its ORF in colorectal cancer tissue was lower than that of normal organizations (P = 0.044), .In fact we found that two EST (ES274075 and ES274084) are the same sequence. Its coded protein may be human collagen protein type. ES274077 located in human chromosome 3q24.3. Using the software DNAStar to assemble the matching sequences together to form a 4503bp cDNA sequence. The ORF was predicted and its product contains 268 amino acids. Its product is ELL associated factor-1 (EAF-1). The SAGE indicates that there is expression in oligodendroglioma, astrocytoma, prostatic carcinoma, ovarian cancer, breast cancer, carcinoma of testis, sarcoenchondroma, and high expression in brain tumor tissue. ES274081 located in human chromosome 20q13.1. The ORF was predicted and its product contains 432 amino acids. Its product is hypothetical protein LOC388969. The SAGE indicates that there is expression in cervical tumor, gastrointestinal tumor, glioma, head and neck tumor, leukemia, lung tumor, ovarian tumor, pancreatic tumor, and high expression in head and neck tumor tissue. ES274083 is located in human chromosome 2q11.2. The ORF was predicted and its product contains 268 amino acids. Its product is S-adenosylhomocysteine hydrolase (SAHH). The SAGE indicates that there is expression in breast (mammary gland) tumor, cervical tumor, colorectal tumor, leukemia, liver tumor, lung tumor, lymphoma, retinoblastoma, and high expression in retinoblastoma tissue. ES274085 is located in 13 chrosome (103-313bp) and 14 chrosome (1-103bp).The ORF of ES274070 located in BC042012 code the kind of hypothetical protein LOC90110, until now, there is its function report , 6. ES27ES274077encoded protein is ELL associated factor-1 (EAF-1), which is a positive regulation protein for ELL and RNA polymerase II. EAF-1 has been found to have a dominant effect in leukemogenesis -15. The In conclusion, with the development of molecular biology, more and more data need to be analyzed, this is possible because of the development of computer and the other sciences and techniques, so the bioinformatics have became very useful tool and new approaches. The colorectal adenocarcinoma is a common malignant and a multi-genes and multi-steps disease. Through the bioinformatics analyses, we are able to screen the data from cDNA microarray, thus we avoid wasting of time and manpower. It is very helpful for us to search the pathologic genes associated with the development of human colorectal adenocarcinoma. Furthermore, ES274070, ES274071 and ES274076 and ES274081 were shown related to onset of human colorectal adenocarcinoma.


Page 2: Lines 11–14 should read as follows: “…Unlike the genomes of other gammaproteobacterial methanotrophs, the USCγ draft genome contains nearly all essential genes for a complete serine biosynthesis pathway for formaldehyde assimilation. However, the enzymes unique to the typical RUMP pathway, hexulose-6-phosphate synthase and hexulose phosphate isomerase, were not detected….”Volume 5, no. 17, e00047-17, 2017,


Newborn hearing screening programs aim to lower the ages at audiological intervention among hearing-impaired children. In Wallonia and Brussels (Belgium), audiological intervention data are not collected in the screening program, and the ages at initiating audiological care have never been assessed. This study aimed to assess the evolution in the ages at initiating audiological intervention in the context of a newborn hearing screening program implementation.This population-based descriptive study used data from the Belgian healthcare billing database. The main outcomes were the children’s ages at the initial audiological assessment, hearing-aid fitting, and cochlear implantation. Results were compared to the same outcomes from another Belgian regional program (Flanders) that was implemented one decade earlier. Annual birth cohorts from 2006 to 2011 were included in the study.In Wallonia-Brussels, the median ages for all outcomes tended to decrease over time but remained higher than in Flanders for each birth cohort. For all outcomes except the hearing-aid fitting, differences in median ages between the two regions became less pronounced during the study period. In 2006, < 23% of the children from Wallonia-Brussels received any audiological care before the age of 12 months and these proportions were approximately 2-fold greater in the subsequent birth cohorts. For all outcomes, early care (< 12 months) was typically delivered less frequently in Wallonia-Brussels, compared to the delivery in Flanders. These region-specific differences exhibited a decreasing trend over time, and statistically significant differences were less common in the later birth cohorts.We conclude that the hearing screening program in Wallonia and Brussels promoted earlier audiological intervention among hearing-impaired children. However, milestones recommended by experts for an early intervention were not totally encountered. We also recommend collecting audiological intervention data as part of this program, which can facilitate more accurate and regular program evaluation. Hearing impairment affects 1–3 children per 1000 newborns in the well-baby nursery population and 2–4% of infants who are admitted to neonatal intensive care units –7, whichEarly audiological intervention provides remarkable benefits for hearing-impaired children, as early identified hearing-impaired children show better language outcomes, compared to late-identified children –13. ThesIn Belgium, the French- and Flemish-speaking areas are the political authorities for preventive medicine and they exhibit differences in their implementation of preventive healthcare programs, including newborn hearing screening. In the French-speaking area , the hearing screening program was implemented at the end of 2006, and is only routinely monitored for its screening process. The program does not collect data regarding audiological care outcomes, such as enrolment in intervention services, hearing-aid fitting, or cochlear implantation , and conIntermutualiste Agency), and includes data regarding billed healthcare and administrative information from all seven public health insurers in Belgium. The healthcare data are identified using Belgian billing codes, and the Intermutualiste Agency extracted all relevant data for 2006–2011 in June 2015, which allowed us to analyse annual birth cohorts and ages at initiating audiological intervention.As the Wallonia-Brussels screening program did not collect data regarding audiological care outcomes, this population-based descriptive study used data from the Belgian healthcare billing database . This database is managed by a public agency . The billing database does not include medical diagnosis or hearing screening-related outcomes, which made it impossible to examine outcomes that were directly related to the hearing impairment diagnosis and/or screening outcome.We selected all relevant billing codes that were in place in 2006 or during the following years. The codes for an initial audiological assessment were widely reported in the database because they may be used for children with other disabilities that are associated with a transiently elevated hearing threshold . Therefore, to only include children with permanent hearing impairment in this outcome, we only considered cases with both an initial assessment code and a code for hearing-aid fitting and/or cochlear implantation. Consequently, children who were not provided with hearing technology were not included in the initial assessment outcome .The children’s age for each outcome was calculated in months , based on their date of care and date of birth. If several codes were used for a single event in the same child (e.g. two cochlear implants were placed at different times), we only considered the first code. We considered the first event in the ‘any audiological care’ outcome.This study evaluated annual birth cohorts from 2006 (the first available year in the billing database) to 2011. Children were included if they underwent audiological care  at < 3 years of age to ensure a standardised comparison, as the different birth cohorts had unique follow-up periods. This cut-off was selected because it was reported as the age at diagnosis without a hearing screening program , 8. The Children were grouped according to their birth region  using the ZIP code for their residence at birth, in order to associate them to the relevant regional screening program. The outcomes according to birth cohort and region were calculated as median ages and percentiles (25th and 75th), and trend lines for median values by region were fitted to these results using Microsoft Excel. Next, we analysed the outcomes according to the proportions of children who received the four types of audiological care  before the age of 12 months (“early” care) or between the ages of 12 and 36 months (“late” care). The Joint Committee on Infant Hearing recommends initiating intervention before the age of 6 months  but veryAgence Intermutualiste (Program Act 24/12/2002), and received its approval; the agency performed the analyses for each individual’s data and aggregated the data for this study.This study was performed based on the legal mandate of the The median ages for any audiological care, initial assessment, and cochlear implantation showed a large initial decrease in Wallonia-Brussels, with some increases in intervention age in the 2009–2010 cohorts, and a resumption of the downward trend in 2011. In particular, the median ages at the initial assessment exhibited a large decrease from 21.0 months (P25–P75: 12.0–27.0 months) in 2006 to 9.0 months (P25–P75: 5.0–22.0 months) in 2008. The any audiological care outcome exhibited a similar trend, and the age at cochlear implantation initiated a large decrease 1 year later, from 24.0 months (P25–P75: 16.0–29.0 months) in 2007 to 14.5 months (P25–P75: 12.0–18.0 months) in 2009. In contrast, outcomes were more constant over time for the longer-standing program in Flanders, but the Flanders cohort also showed some increased ages at intervention in 2009–2010 . For example, in Wallonia-Brussels, half of the hearing-impaired children underwent any audiological care before approximately 21 months in 2006, and although this improved to approximately 12 months in the last cohorts, children in Flanders benefitted from an earlier intervention  , and these differences became less pronounced over time. The exception to this trend was the hearing-aid fitting outcome. For the same outcomes, the distributions (P25–P75) typically overlapped between Brussels-Wallonia and Flanders, with the exception of cochlear implantation in 2007. Results were based on small size groups Fig. .Considering the median ages, delay between initial assessment and hearing-aid fitting was shorter in Wallonia-Brussels than in Flanders in most birth cohorts, while delay between initial assessment and cochlear implantation was shorter in Flanders Fig. .Less than 23% of the children in the 2006 cohort from Wallonia-Brussels received any audiological care or underwent an initial assessment before the age of 12 months. These proportions evolved greatly in the subsequent birth cohorts (approximately 2-fold greater). A significant increase was also observed after the 2008 cohort in the proportions of children undergoing early (< 12 months) cochlear implantation. In contrast, the proportions of early initial assessment were approximately 50–70% in Flanders, and the proportions of early hearing-aid fitting were approximately 2-fold greater than those in Wallonia-Brussels, with the exception of the 2009 cohort. In Flanders, the proportions of early care did not exhibit any major changes over time, even if the proportions of early cochlear implantation exhibited variability over time . Nevertheless, these region-specific differences exhibited a decreasing trend over time, and statistically significant differences were less common in the later birth cohorts  of these delays in Belgium, but previous studies in other contexts have found that delayed interventions may be related to parental or professional factors, administration of healthcare and hearing services, additional disabilities, or financial barriers  –28. MostWe encountered methodological limits due to our study design. First, as most studies are designed using the Joint Committee on Infant Hearing’s age cut-off (< 6 months) , it is dThis study design also included other important strengths. First, our data were population-based and representative of care that was delivered, as Belgium provides universal access to healthcare. Second, our study included hearing-impaired newborns who were missed by the screening program at birth but subsequently identified during the 3-year follow-up period . This is not typical of other studies that evaluated hearing screening programs, as they typically evaluated data from the screening program itself, with sometimes insufficient follow-up period. Third, our design used population-based data that can be routinely collected via healthcare billing, which indicates that this approach is a potentially low-cost option for monitoring audiological intervention among hearing-impaired children.We recommend accurately assessing the long-term success of the screening program. To that end, we recommend enhancing the data collection in the Wallonia-Brussels screening program with information regarding basic outcomes at the enrolment in intervention services, hearing-aid fitting, and cochlear implantation. If enhancement of data collection is not possible due to legal or organizational issues, we recommend using at least named and medical data from the intervention services or other sources, in accordance with professional secrecy or privacy concerns, to monitor the long-term effects of the screening program.Early detection and intervention among hearing-impaired children is the main purpose of newborn hearing screening programs. Given the absence of data on audiological intervention in the Wallonia-Brussels screening program in Belgium, we used data from a healthcare billing database to evaluate the success of this program. Our findings indicate that the median ages for initiating audiological intervention, and the proportions of children who received early audiological care, improved after the implementation of the screening program. In Wallonia-Brussels audiological intervention was typically initiated at a later age than in Flanders, where the screening program was implemented a decade earlier. The database that we used was not designed for analysing audiological outcomes among hearing-impaired children, which raises several methodological questions. Therefore, we strongly recommend developing a method or database for collecting accurate data regarding screening and audiological data in hearing-impaired children, as these data are needed to perform accurate and ongoing evaluations of the Wallonia-Brussels hearing screening program.