Sphingobium sp. strain TCM1 can degrade tris(2-chloroethyl) phosphate (TCEP) to inorganic phosphate and 2-chloroethanol. A phosphotriesterase (PTE), phosphodiesterase (PDE) and phosphomonoesterase (PME) are believed to be involved in the degradation of TCEP. The PTE and PME that respectively catalyze the first and third steps of TCEP degradation in TCM1 have been identified. However, no information has been reported on a PDE catalyzing the second step. In this study, we identified, purified, and characterized a PDE capable of hydrolyzing haloalkyl phosphate diesters. The final preparation of the enzyme had a specific activity of 29 µmol min−1 mg−1 with bis(p-nitrophenyl) phosphate (BpNPP) as the substrate. It also possessed low PME activity with p-nitrophenyl phosphate (pNPP) as substrate. The catalytic efficiency (kcat/Km) with BpNPP was significantly higher than that with pNPP, indicating that the enzyme prefers the organophosphorus diester to the monoester. The enzyme degraded bis phosphate, bis phosphate and bis(2-chloroethyl) phosphate, suggesting that it is involved in the metabolism of haloalkyl organophosphorus triesters. The primary structure of the PDE from TCM1 is distinct from those of typical PDE family members and the enzyme belongs to the polymerase and histidinol phosphatase superfamily. Their widespread use has led to contamination of various environments, including house dust2, surface water3, and groundwater4. These chemicals are physicochemically and microbiologically stable in the environment and are also reported to be toxic. For example, TCEP was shown to cause adverse effects on various tissues including brain, liver, and kidney5, and is also suspected to be carcinogenic6. In addition, TDCPP induced genotoxicity in several in vitro assays conducted in prokaryotic and eukaryotic cells7. These chlorinated organophosphorus triesters are thus a serious threat to human and ecosystem health.The chlorinated organophosphorus triesters tris(2-chloroethyl) phosphate (TCEP) and tris phosphate (TDCPP) are widely used as flame retardants, mainly in a range of plastic foams, resins, and latexesSphingobium sp. strain TCM1. Subsequently, a phosphotriesterase catalyzing the first step of TCEP and TDCPP degradation, named haloalkylphosphorus hydrolase (HAD), was cloned and purified9. We also recently reported that two alkaline phosphatases, which are metal-dependent phosphomonoesterases (PMEs) named SbPhoA and SbPhoX-II, are involved in the metabolism of TCEP10. However, no information has been reported on a phosphodiesterase (PDE) catalyzing the second step of TCEP degradation. Since the previous studies in our laboratory indicated that the rate-limiting step in the degradation pathway of these triesters in strain TCM1 is the step catalyzed by PDE or PME, identification of the PDE is important to improve the biodegradation of haloalkyl phosphorus compounds using TCM1.We have previously isolated a TCEP and TDCPP-degrading bacterium, 11. The cyclic nucleotide PDEs can be classified into three different groups by their primary structure. Class I PDEs are found exclusively in higher eukaryotes and are further classified into 11 families11. Class II PDEs have been identified in a few organisms, such as yeast, Dictyostelium and Vibrio fischeri13. All class III PDEs are found in prokaryotes, including Escherichia coli14, Mycobacterium tuberculosis15 and Arthrobacter16. Only a few studies have focused on the biodegradation of toxic organophosphate compounds by PDEs. As an example, the glycerophosphodiester phosphodiesterase (GpdQ) from Enterobacter aerogenes is one of the few can hydrolyze a broad range of phosphodiesters, including dimethyl phosphate, diethyl phosphate, bis(p-nitrophenyl) phosphate (BpNPP), cAMP and EA 2192, a degradation product of the nerve agent VX19.PDEs can degrade the phosphodiester bond of various compounds, and include cyclic nucleotide PDEs, phospholipase C, phospholipase D and some nucleases. Cyclic nucleotide PDEs are among the most characterized PDEs; they hydrolyze the phosphodiester bond of cyclic nucleotides and play a critical role in intracellular signaling by selectively hydrolyzing the second messengers cAMP and cGMPSphingobium sp. TCM1, which is named Sb-PDE. This enzyme hydrolyzes dihaloalkyl phosphates such as bis(2-chloroethyl) phosphate (BCEP), bis phosphate (BDCPP) and bis phosphate (BDBPP), which are hydrolysis products of the corresponding trihaloalkyl phosphates, TCEP, TDCPP and tris phosphate (TDBPP). The primary structure of Sb-PDE is distinct from those of typical PDE family members and the enzyme belongs to the polymerase and histidinol phosphatase (PHP) superfamily.In the present paper, we describe the identification, purification and characterization of the PDE from Sb-PDE was purified to homogeneity from Sphingobium sp. TCM1 by chromatography using phenyl Sepharose HP, Q Sepharose HP and Superdex 200 pg columns, with a yield of 14.8%  because these compounds were not available from commercial sources. Sb-PDE could hydrolyze the bis-haloalkyl phosphates BCEP, BDCPP and BDBPP with specific activities of 0.007, 2.02 and 19.5 µmol min−1 mg−1, respectively. The enzyme also displayed PDE activities toward cAMP and cGMP .The substrate specificity of Sb-PDE purified from Sphingobium sp. strain TCM1 were determined to be EFVTQSDQRPP. We performed a Blast search against the draft genome of strain TCM120 using this N-terminal sequence as the query sequence and the putative gene (locus_tag A7Q26_24300) was found. However, no Shine-Dalgarno (SD) sequence was found around the putative translation initiation codon. Moreover, the N-terminal region of the deduced amino acid sequence from the putative Sb-pde gene was 15 residues shorter than that of a hypothetical protein from Novosphingobium resinovorum (accession no. WP_006949123), which otherwise showed 100% amino acid identity with the deduced amino acid sequence of Sb-PDE. We found another initiation codon 45 bp upstream of the previous candidate start codon of the predicted gene (corresponding to 15 amino acid residues), with a SD sequence (5′-aggaga-3′) 4-bp upstream of the codon. From these results, we concluded that the newly found initiation codon could function as the translation initiation site. The putative Sb-pde gene (accession no. LC177422) contained an open reading frame of 1,326 nucleotides encoding a protein of 441 amino acid residues with a predicted molecular weight of 48,702.The N-terminal amino acid residues of Sb-PDE predicted from the gene. An N-terminal signal peptide was predicted by SignalP analysis and the putative cleavage site is consistent with the result of N-terminal amino acid sequence analysis of purified Sb-PDE. Thus, Sb-PDE contains a signal peptide for secretion, and the mature protein consists of 413 amino acids with calculated molecular weight 45,769.The N-terminal amino acid sequence of the purified enzyme was identical to amino acid residues 29–39 of the putative amino acid sequence of N. resinovorum (accession no. EZP79922) and significant similarity with a hypothetical protein from S. japonicum (85% identity)  also revealed that Sb-PDE contains the PHP domain (Pfam accession number PF02811), which is found in bacterial polymerase III α subunits, family X DNA polymerases and histidinol phosphate phosphatases  Fig. . Althougses Fig. .Figure 4Sb-pde gene containing the putative signal peptide coding region of strain TCM1 was expressed in E. coli BL21 (DE3) cells under the control of the T7 promoter. Crude extracts of E. coli cells harboring pET25b showed neither PDE nor PME activity, while those of E. coli cells harboring pET25b-pde showed hydrolase activities of 7.01 µmol min−1 mg−1 for BpNpp and 1.97 µmol min−1 mg−1 for pNpp (PDE activity/PME activity = 3.6), indicating that the expressed gene encodes Sb-PDE. The N-terminal amino acid sequence of recombinant Sb-PDE, EFVTQXDQ (X represents an unidentified amino acid residue), was identical with that of native mature enzyme suggesting that the recombinant Sb-PDE was also processed in E. coli.The Sphingobium sp. strain TCM1, which was isolated previously in our laboratory from soil8, can degrade TCEP to inorganic phosphate and 2-chroloethanol. It is considered that a phosphotriesterase, PDE and PME are involved in the degradation of TCEP. While phosphoesterases catalyzing the first and third steps of TCEP degradation in TCM1 have been identified in our previous studies as a HAD9 and an alkaline phosphatase10 respectively, no information has been reported on a PDE from this microorganism. In the present study, Sb-PDE from Sphingobium sp. TCM1 was purified and characterized and the gene was cloned.Sb-PDE, whereas the enzyme showed only weak activity toward BCEP, cAMP and cGMP. TCM1 can degrade both TCEP and TDCPP, but the generation of 2-CE from TCEP was slower than that of 1,3-DCP from TDCPP8. BCEP may be a relatively poor substrate for Sb-PDE. These results suggest a possible involvement of Sb-PDE in the metabolism of haloalkyl phosphates in TCM1, although involvement of the enzyme in cyclic nucleotide(s) metabolism was not established.BDCPP and BDBPP were efficiently degraded by Sb-PDE was inhibited by dithiothreitol and 2-mercaptoethanol. Since Sb-PDE is possibly secreted extracellularly and has two cysteine residues, disulfide bond essential for enzyme activity or protein folding may be present in the enzyme.The enzyme contained a predicted signal peptide for secretion and the mature protein is a homodimer with a subunit molecular mass of 45.8 kDa. No consensus twin-arginine-transport (TAT) motif is found in this signal peptide, suggesting that the enzyme is probably secreted in a Sec-dependent manner. Enterobacter aerogenes19 and PdeA from Delftia acidovorans21 exhibit PME activity. Furthermore, alkaline phosphomonoesterases, such as PhoA from Escherichia coli22 and PhoD from Aphanothece halophytica23, exhibit both PME and PDE activity. Therefore, we determined the PME activity of Sb-PDE. The purified enzyme had PME activity in addition to PDE activity, but the PDE activity was 3.9-fold lower. Kinetic analysis revealed that the catalytic efficiency (kcat/Km) with BpNPP was significantly higher than that with pNPP. These results indicate that this enzyme is a PDE.It was reported that PDEs such as GpdQ from Sb-PDE hydrolyzed both a phosphodiester and a phosphomonoester. However, it has already been reported that two alkaline phosphatases, SbPhoA and SbPhoX-II, are involved in the utilization of TCEP by Sphingobium sp. strain TCM1 and the double gene mutant did not grow on TCEP medium10. These results suggest that Sb-PDE catalyzes only the second step in the degradation of organophosphorus triesters, and not the third (phosphomonoester hydrolysis) step, although the PME activity of the enzyme toward monohaloalkyl phosphate was not determined in this study.Sb-PDE from TCM1 is distinct from those of typical PDE family members. Sb-PDE contained a PHP domain, which is part of the amidohydrolase superfamily, while no consensus sequences of class I, class II or class III PDEs were found. The PHP domain is found in bacterial polymerase III α subunits, family X DNA polymerases and histidinol phosphate phosphatases and the biochemical functions of this domain in these enzymes are well characterized. The C-terminal PHP domain of DNA polymerase X shows Mn2+-dependent 3′, 5′-exonuclease activity25 and histidinol phosphatase catalyzes the dephosphorylation of l-histidinol phosphate to histidinol26. The PHP superfamily also includes numerous proteins of unknown function24. In fact, many proteins with sequence similarity to Sb-PDE are annotated as hypothetical proteins in databases. Although it was reported that Elen0235 from Eggerthella lenta can hydrolyze a cyclic phosphodiester to a vicinal diol and inorganic phosphate27, little is known about whether proteins belonging to the PHP superfamily display PDE activity.The primary structure of 24, and structurally characterized enzymes of the PHP superfamily contain a trinuclear metal center in the active site31. Sb-PDE and several homologous proteins have the four conserved motifs24 that have been predicted to be involved in the formation of the metal binding sites . p-Nitrophenyl phosphate sodium salt (pNPP) was from Nacalai Tesque . Restriction enzymes and other DNA-modifying enzymes were obtained from Takara Shuzo (Kyoto), Toyobo (Osaka) and New England Biolabs . DNA purification kits were from Qiagen . Phenyl Sepharose HP, Q Sepharose HP and Superdex 200 pg columns were from GE Healthcare UK Ltd. . All other chemicals were of analytical purity and were purchased from Wako Pure Chemical Industries and Nacalai Tesque. The LC-10Ai Bio-Inert high-performance liquid chromatography (HPLC) system was from Shimadzu (Kyoto).BDBPP and TDCPP were purchased from Wako Pure Chemical Industries . BE. coli BL21 (DE3) was used as the host for the expression of Sb-PDE. Sphingobium sp. TCM1 was aerobically cultivated in MAY medium containing 20 µM TCEP as the sole phosphorus source32. E. coli BL21 (DE3) were grown in Luria-Bertani (LB) medium containing 100 µg/ml ampicillin.Sphingobium sp. TCM1 was cultivated aerobically at 30 °C for 48–50 h and harvested by centrifugation . After washing cells twice with 50 mM MOPS buffer (pH 7.4), the cells were resuspended in the same buffer and homogenized by sonication, followed by a centrifugation at 20,000 × g for 30 min. The cell free extract thus obtained was fractionated by ammonium sulfate precipitation (30–60% saturation). The resulting precipitate was dissolved in M/G buffer (50 mM MOPS (pH 7.4) containing 10% (v/v) glycerol) containing ammonium sulfate at 30% saturation and loaded onto a phenyl Sepharose HP column (2.6 × 11 cm) equilibrated with same buffer. After the column was washed with M/G buffer containing ammonium sulfate at 30% saturation, the enzyme was eluted by using a linear gradient (540 ml) from 30% to 0% ammonium sulfate at a flow rate of 0.7 ml/min. The fractions with PDE activity were collected, combined and concentrated by using an Amicon stirred cell apparatus with a YM-10 membrane . After buffer exchange into 50 mM Tris-HCl (pH 8.0) containing 10% (v/v) glycerol (T/G buffer), the concentrate was subjected to a Q Sepharose HP column (2.6 × 11 cm) pre-equilibrated with T/G buffer and the Sb-PDE was then eluted by using a linear gradient (360 ml) from 0 to 600 mM NaCl in the T/G buffer at a flow rate of 1.0 ml/min. The active fractions were collected, concentrated and loaded onto a Superdex 200 pg column (1.6 × 60 cm) equilibrated with M/G buffer containing 150 mM NaCl. The enzyme was eluted with the same buffer at a flow rate of 0.5 ml/min and the fractions with the enzyme activity were pooled, concentrated and stored at −80 °C until use.p-nitrophenol from BpNpp and pNpp. A 1.5-ml standard assay mixture contained 50 mM Tris-HCl buffer (pH 8.8), 15 mM BpNpp for PDE assay or 15 mM pNPP for PME assay, and an appropriate amount of the enzyme, which was added to the mixture to start the assay. A mixture without enzyme was used as the blank. The activity of the enzyme was determined as the increase in absorbance at 410 nm using a UV-2500PC spectrophotometer (Shimadzu). The kinetic parameters of the enzymes for BpNPP and pNPP were estimated by fitting the initial reaction rates to the Michaelis-Menten equation with the program SigmaPlot 12.5 . Data are expressed as mean ± S.E. of the three independent experiments.PDE and PME activities were respectively assayed at 30 °C by monitoring spectrophotometrically the liberation of Sb-PDE toward BCEP, BDCPP and BDBPP were determined by the increase in the amount of 2-chloroethanol, 1,3-dichloro-2-propanol and 2,3-dibromopropanol, respectively, as described below. BCEP was produced by an incubation of TCEP  with a recombinant His-tagged TCM1-HAD (0.87 μg/ml) with specific activity of 1.86 µmol min−1 mg−1  in 50 mM Tris-HCl buffer (pH 8.0) at 30 °C for 24 h. BDCPP was produced from TDCPP in the same way. Complete hydrolysis of TCEP and TDCPP by His-tagged HAD were analyzed by gas chromatography (GC), and the stoichiometric productions of the corresponding halo alcohols were also confirmed by analyses with a gas chromatograph mass spectrometer (GCMS).The hydrolytic activities of Sb-PDE (570 μg/ml) was added to 1.95 ml of the hydrolytic mixture of TCEP described above, followed by incubation at 30 °C. A mixture without enzyme was used as the blank. An aliquot (200 μl) of the incubation mixture was removed after 0, 15, 30, 60, 90, 120 and 180 min. 2-Chloroethanol in the aliquot was extracted with 1 ml of ethyl acetate and analyzed by GC-MS.To assay PDE activity toward BCEP, 50 µl Sb-PDE (2.85 μg/ml) was added to 1.95 ml of the hydrolytic mixture of TDCPP. An aliquot (200 μl) of the incubation mixture was removed after 0, 15, 30 and 60 min. 1,3-Dichloro-2-propanol in the aliquot was extracted with 1 ml of ethyl acetate and analyzed by GC-MS.PDE activity toward BDCPP was measured in the same way, but 50 µl of a much lower concentration of Sb-PDE enzyme (2.85 μg/ml). An aliquot (200 μl) of the reaction mixture was removed after 0, 15 and 30 min. 2,3-Dibromopropanol in the aliquot was extracted with 1 ml of ethyl acetate and analyzed by GC-MS.The assay of PDE activity toward BDBPP was carried out at 30 °C, using a mixture (2 ml) containing 50 mM Tris-HCl buffer (pH 8.0), 66 μM BDBPP and 50 µl of PDE activity toward cAMP and cGMP was measured using a colorimetric cyclic nucleotide phosphodiesterase assay kit .8. Concentrations of 2-chloroethanol, 1,3-dichloro-2-propanol and 2,3-dibromopropyl phosphate were determined using a GCMS-QP2010 (Shimadzu), as described8.Concentrations of TCEP and TDCPP were analyzed using a GC-17A equipped with a flame photometric detector with a phosphorus optical filter (Shimadzu), as described in a previous reportThe native molecular mass of the enzyme was analyzed by gel filtration with a HPLC equipped with the Superdex 200 pg column. The gel filtration chromatography was performed at a flow rate of 0.5 ml/min with M/G buffer containing 150 mM NaCl. The molecular mass marker proteins  consisting thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) were used as the standard.Metal ion content was determined by inductively coupled plasma mass spectrometry using an Agilent7700x . The purified enzyme (10 nmol) was digested in 5% nitric acid for 24 h to release the metal ions. The sample was then diluted with 5 volume of ultrapure water and was directly subjected to ICP-MS measurement.E. coli cells harboring pET25b-pde was resolved by SDS-PAGE. The resolved proteins were transferred to a Sequi-Blot polyvinylidene difluoride (PVDF) membrane  by electroblotting and the PVDF membrane stained by Coomassie Brilliant Blue R-250. The protein bands on the PVDF membrane were cut out from the membrane, and the N-terminal amino acid sequence of the protein was determined by automated Edman degradation with a Shimadzu model PPSQ-21 protein sequencer.Purified enzyme or crude extract of Sb-pde gene was amplified from genomic DNA of Sphingobium sp. TCM1 by PCR with primers TCM Pde Ex F1 (5′-AAGGAGATATACATATGGTTAAGCATAGAAACTTCCGCGC-3′) and TCM Pde Ex R1 (5′-AGCAGGTATTTCATATTACATCCAGCCGTCGAGATCG-3′). The resulting PCR product was ligated into NdeI-digested pET25b using the In-Fusion HD Cloning Kit . The plasmid for the expression of Sb-PDE was named pET25b-pde. The pET25b-pde was introduced into E. coli BL21 (DE3), and the cells were grown in LB medium containing 100 µg/ml ampicillin at 30 °C for 3 h. The culture was supplemented with 1 mM isopropyl-1-thio-β-d-galactopyranoside and grown for further 14 h at 20 °C. The cells were harvested, resuspended in 50 mM MOPS buffer (pH 7.4) and homogenized by sonication.The 33 using a protein assay kit (Bio-Rad) with bovine serum albumin as the standard. SDS-PAGE in reducing conditions was performed as described by Laemmli34.Protein concentration was determined by the Bradford methodSupplementary information


Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32  and Mlh1-mlh3-45  displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new  During meiosis, diploid germ cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. The segregation of chromosomes at the first meiotic division is dependent in most organisms on at least one genetic exchange, or crossover event, between chromosome homologs. Homologs that do not receive a crossover frequently undergo nondisjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such events have been linked to human disease and infertility. Recent studies suggest that the Mlh1-Mlh3 complex is an endonuclease that resolves recombination intermediates into crossovers. Interestingly, this complex also acts as a matchmaker in DNA mismatch repair (MMR) to remove DNA replication errors. How does one complex act in two different processes? We investigated this question by performing a mutational analysis of the baker’s yeast Mlh3 protein. Five mutations were identified that disrupted MMR but not crossing over, and one mutation disrupted crossing over while maintaining MMR. Using a combination of biochemical and genetic analyses to further characterize these mutants we illustrate the importance of protein-protein interactions for Mlh1-Mlh3’s activity. Importantly, our data illustrate how defective meiotic components can alter the outcome of meiotic recombination events. They also provide new insights for the basis of infertility syndromes. MutShomolog (MSH) proteins, which in turn recruit MutLhomolog (MLH) proteins to form ternary complexes containing mismatched DNA, MSH factors, and MLH factors. These interactions result in the recruitment of downstream excision and resynthesis proteins to remove the error [S. cerevisiae repair of insertion deletion loops greater than one nucleotide in size primarily involves the MSH heterodimer Msh2-Msh3 and the MLH heterodimer Mlh1-Pms1 [During mismatch repair (MMR), insertion/deletion and base-base mismatches that form as the result of DNA replication errors are recognized by he error . In S. clh1-Pms1 . Howeverlh1-Pms1 –8. Accurlh1-Pms1 –12. Faillh1-Pms1 .2E(X)4E) metal binding motif also found in Mlh3. Previous work demonstrated that a point mutation within this motif (mlh3-D523N) conferred mlh3Δ-like defects in MMR and crossing over. These included a mutator phenotype, a decrease in spore viability to 70% (from 97% in wild-type), and a two-fold reduction in genetic map distances [in vitro, and Mlh1-mlh3-D523N is defective in endonuclease activity [Yeast Mlh1-Pms1 and its human ortholog MLH1-PMS2 both exhibit an endonuclease activity that is essential for MMR –15. Thisactivity –17.S. cerevisiae cell in meiotic prophase, of which ~90 are repaired to form COs between homologous chromosomes, with the rest repaired to form noncrossovers  are induced throughout the genome in a s NCOs; –23). In . In S. cs  were observed, suggesting that Mlh1-Mlh3 is the primary factor required for CO resolution in the interference-dependent CO pathway [mlh3-/- mice are sterile with an 85–94% reduction in the number of COs; germ cells in these mice fail to maintain homologous pairing at metaphase and undergo apoptosis [Genetic and physical studies summarized below support a major role for Mlh1-Mlh3 in promoting meiotic CO formation in the interference-dependent CO pathway. 1. Genetic studies performed in yeast showed that ing over , 35, 36.ing over , 8, with pathway , 38. Forpoptosis , 39.msh4 mutants. Based on these findings and other observations they propose that Msh4-Msh5 is required at the invading end of the DSB to stabilize recombination intermediates such as SEIs, while Zip3 acts to promote second-end capture steps at the ligating end of the DSB [in vitro [Much remains to be understood on how biased resolution of dHJs in the interference-dependent pathway is achieved. A working model, supported by genetic and molecular studies outlined below, is that the STR complex and a subset of ZMM proteins process and interact with DSB repair and SEI intermediates to create a dHJ substrate that can be resolved by the Mlh1-Mlh3 endonuclease and Exo1 to form primarily COs , 40–46.  the DSB . In suppin vitro , 43. Furin vitro , 47, 48.mms4, slx4, yen1) results in a similar reduction of CO levels (~10% of wild-type levels) suggesting a pathway where Sgs1-dependent COs require Mlh1-Mlh3 [mms4, slx4, yen1 strains deficient in Top3 or Rmi1 [Additional support for the above model was obtained from analysis of the STR complex , 46, 49.lh1-Mlh3 . Similar or Rmi1 , 31. TheA role for Exo1 in crossing over is supported by genetic studies that show Exo1 and Mlh3 acting in the same CO pathway . Interesmlh3 alleles in S. cerevisiae. Five alleles predicted to disrupt the Mlh1-Mlh3 endonuclease motif conferred defects in both MMR and crossing over, providing further support that endonuclease activity is required for both functions. Importantly, we identified five mlh3 mutations that specifically disrupted MMR, and one mutation that specifically disrupted crossing over. By performing biochemical and genetic analyses of the separation of function Mlh1-mlh3 complexes we suggest that the defects seen in our mutants can be explained by a weakening of protein-protein interactions, which can be tolerated in meiosis, but not MMR. Importantly, our high-resolution recombination mapping of these mutants revealed ways in which defective meiotic components can alter the fate of meiotic recombination intermediates.In this study, we created a structure-function map of Mlh3 by analyzing 60 new S. cerevisiae MLH3 gene to create 60 mlh3 variants  to formfficient ; Fig 3A)ild-type ; Table 1ntervals –8. It isntervals .mlh3 alleles and a structure-function analysis of MLH1, we found that MLH3 MMR functions were more sensitive to mutagenesis than CO functions  conferred a nearly wild-type phenotype for MMR and a null phenotype for crossing over on chromosome VIII . The remaining five mutations  conferred null MMR phenotypes and nearly wild-type levels of crossing over . As indicated below, mlh3-23 displayed different CO phenotypes in different strain backgrounds, and so its designation as a separation of function allele is less clear.Comparison of the MMR and CO assay results for each individual allele led to the identification of six separation of function mutations, defined as showing strong defects in one function (e.g. MMR) relative to another (e.g. CO), in the Mlh3 ATP-binding motifs, N-terminal domain beyond the ATP-binding motifs, linker arm, and the interaction domain (E. coli MutL (LN40) and E. coli MutS was recently solved. This work showed that conformational changes license MutS-MutL interaction and are essential for MMR [The phenotypes observed in the separation of function mutants may result from a defect in DNA binding/substrate specificity, endonuclease activity, interactions with specific MMR and meiotic CO factors, or changes in protein conformation. It is important to note that a co-crystal structure of the N-terminal domain of  for MMR , 58.S. cerevisiae Mlh1-Pms1 and Mlh1-Mlh3 and human MLH1-PMS2 display latent endonuclease activities dependent on the integrity of a highly conserved metal binding motif DQHA(X)2E(X)4E [B. subtilis endonuclease MutL 2E(X)4E –17. This)2E(X)4E . Two add)2E(X)4E . In the )2E(X)4E ). This onserved; ).B. subtilis MutL and found all three possess conserved metal binding motifs, with the following five residues predicted to form the endonuclease active site in Mlh3: H525, E529, C670, C701, and H703. In addition, we constructed a homology model of S. cerevisiae Mlh3, and found the C-terminal domain can potentially fold in a similar manner to Pms1 such that these five conserved residues form a single putative metal binding site , -40, -57(C670A), -58(C701A), and -59(H703A)) conferred null phenotypes for MMR and crossing over, indicating they are essential for Mlh3 function. Thus, these genetic data, combined with the high sequence homology, suggest that H525, E529, C670, C701, and H703 in the C-terminal domain of Mlh3 form the catalytic active site 4E endonuclease motif  were analyzed by yeast two-hybrid for interaction with Mlh1. We also tested these alleles because a previously characterized mutation in the DQHA(X)2E(X)4E endonuclease motif (mlh3-E529K) disrupts Mlh1-Mlh3 interactions [mlh3-39, -40, and -41 were caused by specifically mutating residues that comprise the Mlh1-Mlh3 dimerization interface without causing a gross disruption in protein folding.The endonuclease motifs in Mlh3 overlap with the C-terminal Mlh1 interaction domain. To determine if mutations in the Mlh3 endonuclease motifs disrupted interaction with Mlh1, three alleles spanning the DQHA(X)if mlh3-E29K disruractions . We cannmlh3-60, mlh3-60 mutation on MMR or on crossing over  contained mutations that mapped to the Mlh1 interaction interface. We performed a two-hybrid assay to test whether these mutations affected Mlh1-Mlh3 dimerization. The mlh3-45 mutation did not alter Mlh1-Mlh3 interactions; however, both the mlh3-42 and mlh3-54 mutations disrupted this interaction  using traditional genetic map distance analyses  for accmlh3 alleles  in a genome-wide meiotic recombination assay [mlh3-23 and mlh3-32 were chosen as they confer stronger defects in one function relative to the other. The mlh3-D523N endonuclease mutation contains an aspartic acid to asparagine substitution in the DQHA(X)2E(X)4E metal binding motif of Mlh3. This mutation does not disrupt formation of the Mlh1-Mlh3 complex; however, it conferred a null phenotype for MLH3 functions in MMR and meiotic CO assays, and the Mlh1-mlh3-D523N complex is defective for endonuclease activity [We characterized three on assay . mlh3-23activity .MLH3, mlh3Δ, mlh3-23, mlh3-32, and mlh3-D523N alleles were analyzed in the S288c/YJM789 hybrid background , mlh3-32 (82%), and mlh3-D523N (82%) were similar to mlh3Δ (80%) and the wild-type hybrid containing SK1-MLH1/3  that appear to depend on strain background [The SK1 SK1 MLH1 . The spo1/3 82%; . Why do ckground , 67. Thickground .mlh3-23 and mlh3-32, and ten four-viable spore tetrads each of mlh3-D523N, mlh3Δ, and MLH3 were sequenced  events  events ; such anates see .We used groupEvents ; 5 kb th and 5B, mlh3-23 displayed the same average number of CO events (E2+E3) (p = 0.99), and mlh3Δ, mlh3-D523N, and mlh3-32 displayed CO levels lower than wild-type (p <0.01). mlh3-D523N and mlh3Δ displayed similar average CO levels (p = 0.23), but mlh3-32 displayed levels higher than mlh3Δ (p = 0.05) , mlh3-D523N  and mlh3-32  . An analto mlh3Δ . Signifiund Figs  and 5C.mlh3Δ (2.19 kb) compared to MLH3 (1.79 kb) and mlh3-32 (1.84 kb), but were not significantly longer (p ≥ 0.18) for mlh3-23 (1.96 kb), and mlh3-D523N (1.96 kb). We found this interesting because MLH3 and mlh3-32 mutants appeared similarly functional for MMR, whereas mlh3Δ, mlh3-23, and mlh3-D523N all displayed MMR defects , mlh3-23 (51), mlh3-32 (56), and mlh3-D523N (50) were significantly higher than the wild-type value . Significant increases in NCOs on chromosomes were observed for mlh3-32 (X and VII), mlh3-DN , mlh3Δ . No increase in NCOs on specific chromosomes was observed for mlh3-23 (The average numbers of NCOs (E1 events) in ants see . Compare mlh3-23 .mlh3 mutants was due to the presence of discontinuous gene tracts that arose from the same initiating DSB. Such discontinuous tracts could have resulted from defective MMR but are not included in the E1 NCO class above. The E4 class (mlh3 mutants were significantly different from wild-type (p = 0.33 to 0.80). This suggests that discontinuous tracts are unlikely to significantly contribute to the increased level of NCO events seen in mlh3 mutants.It is possible that some of the increase in NCOs seen in E4 class  is a minmlh3 mutants analyzed . However, both Kolmogorov–Smirnov and Wilcoxon Rank-Sum tests indicated that an apparent enrichment of short NCO tract lengths in mlh3 mutants, compared to wild-type, was not significant. A similarly weak pattern (an insignificant increase in mlh3 mutants compared to wild-type) was seen for the E5A class (The average NCO (E1) tract lengths were similar to wild-type (1.46 kb) in all of the ild-type . We foun5A class .mlh3Δ, mlh3-23, mlh3-32 and mlh3-D523N mutants is that these mutants experienced meiotic progression delays that resulted in the continued accumulation of NCOs, possibly through increased DSB formation. Increases in NCO events and a meiotic delay were observed in ndt80 and the ZMM zip1, zip3 and msh5 mutants as a result of impeding feedback circuits that inhibit DSB formation [mlh3 mutants displayed a significantly higher value than wild-type, but mlh3-D523N  and mlh3Δ  displayed lower values.One explanation for the increase in NCO events seen in ormation , 71, 72.ormation . As showMLH3, mlh3Δ, mlh3-32, mlh3-23 and mlh3-D523N SK1 strains by measuring the completion of the first meiotic division. This would be difficult to do in S288c/YJM789 strains because they do not show the highly synchronous and efficient meiotic progression profile seen in SK1. As shown in MLH3, mlh3Δ, mlh3-32, mlh3-23, and mlh3-D523N mutants showed similar kinetics for completion of at least the first meiotic division (MI+MII). These data suggest that the increase in NCO events in mlh3Δ, mlh3-32, mlh3-23, and mlh3-D523N cannot simply be explained by an increase in DSB levels, MMR defects, or a meiotic progression delay.We also examined meiotic progression in mlh3 mutants is consistent with a possible role for SSNs and/or the STR complex , Mlh1-mlh3-6, and Mlh1-mlh3-45 . Mlh1-mlh3-45, located in the C-terminal Mlh1 interaction domain, was chosen because it is the only separation of function mutant in that domain that displayed wild-type Mlh1-Mlh3 interactions as measured in the two-hybrid assay  to EAY3339  and conferred a more apparent MI nondisjunction pattern . Suppression or enhancement of phenotypes was not observed upon overexpression of the sgs1-K706A helicase defective mutant protein (sgs1-hd), indicating that these effects were dependent on Sgs1 helicase activity (mlh3Δ+sgs1-hd-2μ and 78% in mlh3-32+sgs1-hd-2μ). In addition, no effect was seen upon overexpression of Sgs1 or sgs1-hd in MLH3 (MLH3+2μ vs. 95% in MLH3+SGS1-2μ and 96% in MLH3+sgs1-hd-2μ), mlh3-23 (86% in mlh3-23+2μ vs. 90% in mlh3-23+SGS1-2μ and 87% in mlh3-23+sgs1-hd-2μ) and mlh3-D523N  strains. Although mlh3-32 and mlh3-D523N both confer null-like meiotic defects, they did not confer phenotypes similar to mlh3Δ in response to Sgs1 overexpression. This observation suggests potential roles for Mlh1-Mlh3 that are independent of its enzymatic activity.The increase in NCO events seen in in vitro  in terms of homology and intrinsic behavior iewed in ). Obtainiewed in , 50, 74.mlh3-32  and mlh3-45 , support the above hypothesis that protein-protein interactions are critical for directing Mlh1-Mlh3 endonuclease activity (- CO+mlh3 mutants (mlh3-42 and -54) in which the mutant mlh3 protein fails to interact with Mlh1. For the Mlh1-mlh3-32 complex, the MSH interaction and enhancement is retained, but interaction with other critical meiotic factors is likely lost, possibly resulting in an unstable complex that cannot resolve dHJs.Our analysis of activity ; Fig 7. S. cerevisiae, MLH1-PMS2 in humans) to act in strand-specific repair [in vitro studies performed with yeast proteins showed that RFC-loaded PCNA can activate Mlh1-Pms1 but not Mlh1-Mlh3 endonuclease on circular plasmids  betw mutants .mlh3Δ compared to wild-type . Mlh1-Mlh3 does not share conservation with the known endonuclease superfamilies , and does not appear capable of resolving model HJ substrates , 74. As MLH3 genes have been associated with male and female infertility [mlh3-23 mutation, which only weakly affected crossing over, conferred an alteration in meiotic recombination outcomes that was similar to that seen in mlh3 mutants that conferred more severe defects  or minimal complete media at 30°C [ at 30°C . For sel at 30°C , 88. Celmlh3 alleles were constructed, resulting in the mutagenesis of 139 amino acids in the 715 amino acid Mlh3 polypeptide . mlh3-60, in which the last 11 residues of Pms1 (DWSSFSKDYEI) were inserted before the MLH3 stop codon, was also made by QuickChange. Mutations were confirmed by sequencing the entire open reading frame (Sanger method), as well as 70 bp upstream and 150 bp downstream. Primer sequences used to make and sequence these variants are available upon request.60 ypeptide . The sinS. cerevisiae Mlh3 (YJM789) was used to construct a homology model from HHpred (http://toolkit.tuebingen.mpg.de/hhpred) and Modeller software. PyMOL was used for imaging.The amino acid sequence of mlh3 MMR and meiotic crossing over phenotypes. It carries a spore autonomous fluorescent protein marker (RFP) on chromosome VIII to monitor chromosome behavior  as wellz et al. . At leasis study . These hing over . Diploid+ as described in Tran et al. [mlh3Δ controls. Analyses were performed for two independent transformants per allele. Reversion rates were measured as described [The haploid strains described above were analyzed for reversion to Lysn et al. . At leasescribed , 91, andescribed .mlh3 allele were counted to determine the % tetratype. Two independent transformants were measured per allele. A statistically significant difference (p<0.01) from wild-type and mlh3Δ controls based on χ2 analysis was used to classify each allele as exhibiting a wild-type, intermediate, or null phenotype.Diploids in the EAY3255/EAY3486 background described above  were spoMLH3), EAY3255/EAY3486 (mlh3Δ), EAY3534-35/EAY3486 (mlh3-23), EAY3552-53/EAY3486 (mlh3-32), and EAY3819-20/EAY3486  and mutant derivatives, and then sub-cloned into the target vector pEAM98 (S288C MLH3) to make pEAM234 (SK1 MLH3) and mutant derivatives  assay as described in [The L40 strain  was co-tivatives . pEAM98 ribed in .8 cells; aliquots from the final heparin purification were frozen in liquid N2 and stored at -80°C. Protein concentrations were determined by Bradford assay [mlh3-6, mlh3-32 and mlh3-45 mutations were introduced into pEAE358 , pEAE383 (Mlh1-mlh3-32) and pEAE384 (Mlh1-mlh3-45), in which the MLH1-FLAG gene is downstream of the p10 promoter and the His10-mlh3-HA gene is downstream of the pPH promoter. The sequence of the restriction fragments inserted into pEAE348 were confirmed by DNA sequencing (Cornell Biotechnology Resource Center). Msh2-Msh3 was purified as described previously [Mlh1-Mlh3 and Mlh1-mlh3 mutant derivatives were purified from Sf9 cells infected with Bac-to-Bac baculovirus expression system using pFastBacDual constructs . Mutant rd assay  using BSa et al. ) by Quiceviously .2 for 1 h at 37°C. Reactions were quenched by incubation for 20 min at 37°C with 0.1% SDS, 14 mM EDTA, and 0.1 mg/ml proteinase K (New England Biolabs) . Samples were resolved by 1% agarose gel with 0.1 μg/ml ethidium bromide run in 1X TAE buffer for 50 min at 95 V. All quantifications were performed using GelEval . The amount of nicked product was quantified as a fraction of the total starting substrate in independent experiments. bkg indicates that amount of nicked product was not above background levels established by negative controls. ATPase assays were performed as described [Mlh1-Mlh3 nicking activity was assayed on supercoiled pBR322 or pUC18 (Thermo Scientific). DNA (2.2 nM) was incubated in 20 μl reactions containing indicated amounts of Mlh1-Mlh3 and Msh2-Msh3  in 20 mMescribed .MLH3/mlh3Δ::NATMX) and EAY1848/2413 (mlh3Δ::KANMX/mlh3Δ::NATMX) were used as wild-type and null controls respectively  using standard techniques [mlh3Δ::NATMX) giving rise to diploids carrying markers suitable for genetic distance measurements (http://molbio.uoregon.edu/~fstahl/).Diploids from the SK1 congenic strain background EAY1112/1108 –7 were uectively . mlh3 alchniques . The resurements . Two indmlh3 mutations, including the wild-type and mlh3Δ controls in the presence of SK1 MLH1 in the S288c/YJM789 hybrid. Strain genotypes are shown in MLH1 and MLH3 alleles were introduced into wild-type YJM789 and S288c mlh3Δ::natMX4, respectively, using plasmids pEAA214 and pEAI254. The SK1 MLH1 specific SNPs were confirmed by Sanger sequencing. The mlh3-23::kanMX4, mlh3-32::kanMX4, and mlh3-D523N::kanMX4 mutations were introduced using plasmids pEAI347, pEAI356, and pEAI252, respectively into the S288c mlh3Δ::natMX4 background. The S288c mlh1Δ::hphMX4 and YJM789 mlh3Δ::kanMX4 strains were made using an mlh1Δ::hphMX4 construct amplified by PCR and the mlh3Δ::kanMX4 deletion construct pEAI168, respectively.Whole genome sequencing was performed using S288c/YJM789 hybrid diploids. The Mlh1 protein sequence has four amino acid differences between SK1 and YJM789 strains and five amino acid differences between SK1 and S288c strains. The SK1 Mlh3 protein has 11 amino acid differences with respect to S288c Mlh3 and seven with respect to YJM789 Mlh3. To avoid genetic incompatibilities between Mlh1 and Mlh3, we analyzed the SK1 mlh3 point mutants, isogenic wild type, and isogenic mlh3Δ mutants as described previously [Genomic DNA was extracted from spore colonies of four viable spore tetrads of the eviously . Whole geviously  and Chakeviously , with 4:eviously  . Sequence data are available from the National Centre for Biotechnology Information Sequence Read Archive (Accession numbers SRP096621 and SRP110341).The segregation files, CrossOver program output files, groupEvents output files and the custom R scripts are available online at the Dryad digital repository  was PCR amplified from SK1 genomic DNA obtained from NKY730  and cloned into the high copy vector pRS426  to make pEAM266 (sgs1-K706A the helicase defective variant of SGS1 (sgs1-hd) was made by site-directed mutagenesis of pEAM266 (New England BioLabs Q5 kit). The correct sgs1-hd, 2μ, and URA3 sequences were confirmed by Sanger sequencing. Primer sequences used to make these constructs are available upon request. The high copy vector (with or without the SGS1 or sgs1-hd insert) was transformed into stable diploids of EAY3534-35/EAY3468 (mlh3-23/mlh3Δ), EAY3552-53/EAY3486 (mlh3-32/mlh3Δ), EAY3819-20/EAY3486 (mlh3-D523N/mlh3Δ), EAY3252/EAY3486 (MLH3/mlh3Δ), and EAY3255/EAY3486 (mlh3Δ/mlh3Δ) (2 test. pEAM266 . The corΔ/mlh3Δ) . MeiosisS1 FileS288c and YJM789 SNPs are shown in red and blue, respectively.(PDF)Click here for additional data file.S2 FileThis file is cited in the corresponding table and figure legends.(XLSX)Click here for additional data file.S1 FigMLH3 (blue) vs mlh3Δ (red) (lys2-A14 reversion assay [MLH3 (1X). The vertical bars indicate the approximate position of the mlh3 mutations analyzed in this study with the height of each bar corresponding to the phenotype relative to MLH3 (1X). Red indicates a null phenotype, blue indicates wild-type (WT), and green indicates intermediate. For mismatch repair (top), bars represent reversion rates of at least 10 independently tested cultures from two independently constructed strains presented here normalized to MLH3 median rate 1X = 1.43x10-6 (n = 140). For crossing over (bottom), bars represent percent tetratype of at least 250 tetrads from two independently constructed strains presented here normalized to MLH3 percent tetratype 1X = 36.7% (n = 226). Blue and red dotted lines represent MLH3 and mlh3Δ respectively. Black star indicates separation of function mutants (Mismatch repair (top) and crossing over (bottom) phenotype of 3Δ (red) . Mismatcon assay  and croson assay  depicted mutants .(TIFF)Click here for additional data file.S2 FigA. and C. Scatter plot of average crossover  and noncrossover  counts per chromosome against chromosome size . The equ−6 x chr. size + 0.65; NCO = 2.99 x 10−6 x chr. size + 0.28);wild-type ;mlh3-32 ;mlh3-D523N ;mlh3Δ .B. and D. Bar plot of average crossover and noncrossover counts per chromosome. The asterisk symbol (*) marks chromosomes that have significant difference  in crossover / noncrossover counts compared to wild-type. Chromosomes are arranged by size from left to right. Error bars are mean ± standard error .(TIFF)Click here for additional data file.S3 Figmlh3Δ to mlh3-32. In A and B the minimum, first quantile, median, third quantile, and maximum count are indicated in the box plot. Outlier points are not shown (A. Distribution of gene conversion tract lengths associated with simple NCO (E1) and CO (E2) events . B. Distot shown .(TIFF)Click here for additional data file.S4 Figmlh3-23, mlh3-32, mlh3-D523N and mlh3Δ strains. Cells with two, three, or four nuclei were counted as having completed MI (MI+MII). All strains for a single time course were grown in the same batch of media under identical conditions. Two independent transformants were measured per allele (Representative time courses showing the completion of the MI division (MI+MII) in wild-type, r allele .(TIFF)Click here for additional data file.S5 Fig32P-γ-ATP. Reactions were performed in duplicate for two separate purifications of each, and the average values, +/-SD, are presented . B, C. Mlh1-Mlh3 and Mlh1-mlh3-6  were incubated with 2.2 nM supercoiled pBR322 DNA, analyzed in agarose gel electrophoresis, and endonuclease activity was quantified (average of 6 independent experiments presented +/-SD) as described in the Methods . C. ATParesented .(TIFF)Click here for additional data file.S1 TableEAY3252, EAY3255 and derivatives, and EAY3486 are SK1 strains that contain spore autonomous fluorescent markers described in Thacker et al. . EAY1112(PDF)Click here for additional data file.S2 TableThe indicated haploid strains were mated to form the diploids with the relevant genotype shown.(PDF)Click here for additional data file.S3 TableMLH3 mutagenesis plasmids are derived from pEAI254, a 7.8 kb MLH3SK1::KANMX integrating vector. pEAI254 was mutagenized by QuickChange to create the alleles listed. The DNA sequence of the entire ORF, and 70 bp upstream and 150 bp downstream, were confirmed by DNA sequencing using primers EAO318, EAO319, EAO1778 and EAO321. B. For the two-hybrid analysis, pEAM105 contains the entire MLH1 gene derived from the SK1 strain inserted immediately after the lexA binding domain in pBTM116. All GAL4 activating domain-mlh3 plasmids are derived from pEAM234, which contains DNA sequence encoding SK1 MLH3 amino acids 481 to 715 inserted immediately after the GAL4 activating domain in pGAD424. C. SGS1 was cloned into pRS426 to make pEAM266 as described in the Methods. Site directed mutagenesis of pEAM266 was performed to make pEAM270 carrying the helicase defective sgs1 allele as described in the Methods.A. All (PDF)Click here for additional data file.S4 Tablemlh3 mutants are isogenic derivatives of EAY1112/EAY2413 (http://molbio.uoregon.edu/~fstahl/) was used to calculate standard error (SE) around the genetic distance for tetrads. n; number of single spores, N; four spore viable tetrads analyzed; Par, parental single spores; Rec, recombinant single spores.All /EAY2413 ; Methods(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 TableCrossovers are E2+E3 events and noncrossovers are E1 events  S2 FileS2 File.(PDF)Click here for additional data file.S7 TableCrossovers are E2+E3 events and noncrossovers are E1 events  S2 FileS2 File.(PDF)Click here for additional data file.S8 Tablemlh3 alleles compared to mlh3Δ using the Wilcoxon rank sum test Click here for additional data file.S9 Table(PDF)Click here for additional data file.


Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE. Lawsonia intracellularis is a Gram negative, obligate intracellular bacterial pathogen which infects porcine intestinal crypt cells and causes proliferative enteropathy (PE), an economically significant disease of the pig industry worldwide . Re. ReLGR5 t damage , 55–57. t damage , 59. Oned damage , 47, 56.d damage –45. Mored damage . Taken td damage . We haver damage  61]. Fu. FuLGR5 L. intracellularis infection. This may occur in a similar way to that observed in S. typhimurium induced murine colonic crypt hyperplasia [et al.,) interpretation was based on the enhanced SOX9 and β-CATENIN transcript levels and PCNA (proliferating TA cells marker) staining intensity, as well as the lack of changes to LGR5 transcript levels, which is similar to our observation in L. intracellularis infected ileums [H. pylori, suggesting the importance of SOX9 expression in H. pylori-induced gastric carcinogenesis [Low SOX9 expression in the context of active Notch signalling confers a gene expression profile typical of actively dividing TA progenitor cells , 56. In erplasia . The autd ileums . Moreoveogenesis .ATOH1 and MUC2 at the peak of L. intracellularis infection. This observation is consistent with active Notch signalling suppression of ATOH1 and the inhibition of secretory lineage commitment during infection [L. intracellularis infection [L. intracellularis infection also impedes the maturation of absorptive enterocytes. As described previously in mice, Notch signalling appears to be dispensable for absorptive enterocyte maturation after lineage specification [L. intracellularis infection. We have also observed that ileum infected with the greatest bacterial load manifested the greatest changes in activity in the β-catenin/Wnt and Notch signalling pathways, including expression of downstream target genes, indicating a potential link between infection burden and the kinetics of changes in downstream signalling  School of Veterinary Studies.S1 Table(DOCX)Click here for additional data file.S1 FigL. intracellularis bacterial load (L. intracellularis isolate LR187/5/83 16S rRNA per ng of genomic DNA) in pigs 325 (7 dpc), 352 (14 dpc) and 353 (14 dpc) to the other pigs euthanized at 7 (324 and 327) and 14 dpc (354 and 355). Data was derived from previous study by Smith et al. (2014) and provided by Dr. Tahar Ait-Ali with the authors’ permission. B) Comparisons of HES1, OLFM4, SOX9 and AXIN2 mRNA transcript levels in pigs 325 (7 dpc), 352, 353 (14 dpc) with uninfected pigs and other pigs euthanized at 7 (324 and 327) and 14 dpc (354 and 355). Mean values ± standard deviation are shown. C) Comparison of MUC2 and ATOH1 mRNA transcript levels in pigs 325 (7 dpc), 352, 353 (14dpc) with uninfected pigs and other pigs euthanized at 7 and 14 dpc. Mean values ± standard deviation are presented. Note that while MUC2 and ATOH1 expression in 325 is similar to that of other pigs euthanized at same time point (7dpc), greater discrepancies in MUC2 and ATOH1 expression can be observed between 352 and other pigs euthanized at 14dpc. D) Table showing AXIN2, HES1, SOX9 and OLFM4 mRNA transcript levels in pigs 325 (7dpc), 352 (14dpc) and 353 (14dpc). All RTqPCR results were normalised to GAPDH housekeeping transcript and to uninfected samples thus the mRNAs transcripts levels in uninfected pigs are 1.00 [p-values shown were based on comparison of the mRNA transcript level between uninfected pigs and pigs 325, 352 and 353. P-value shown for Hes-1 derived from comparison between uninfected pigs and pig 352 and 353.A) Comparison of (DOCX)Click here for additional data file.S2 FigA) IF detection of L. intracelluaris antigen, LI  and MUC2 in two contiguous sections from L. intracellularis infected crypts at 7 dpc. LI staining and anti-MUC2 were detected using FITC (green) and Alexa-647 (red)-conjugated secondary antibodies, respectively. Nuclei counterstained with DAPI. Insets (blue rectangles) represent the region with with MUC2 signal and mucin-containing vacuoles (black circles). The white dash line represents the outline for the apical sides of the crypt. Scale bar: 10μm. B) IF using VPM53 antibody on L. intracellularis infected crypt at 7 dpc. Green: LI; Blue: DAPI. White dashed lines represent the outline of a crypt. Scale bar: 10μm.(DOCX)Click here for additional data file.S3 FigMean values ± standard errors are shown. All RTqPCR results were normalised to GAPDH housekeeping transcript and to uninfected samples .(DOCX)Click here for additional data file.


Knowing how to classify retinopathy of prematurity is essential as it provides information on the prognosis and guides decision making about screening and treatment.The severity of the ROPThe zone in the retina where ROP is foundThe extent of the ROPWhether the retinal blood vessels are dilated and/or tortuous (pre-plus or plus disease)Whether aggressive posterior ROP is presentIt is important to classify retinopathy of prematurity (ROP) in each eye, at each screening session. Doing so makes it possible to screen babies consistently and to make decisions about whether further screening is required and when, or whether laser treatment or surgical management is needed. The International Committee for the Classification of ROPROP can develop when the immature retinal blood vessels have not reached the edge of the retina, known as the ora serrata.Stage 1 ROP: Demarcation line. A whitish line is visible between the normally vascularised retina and the peripheral retina in which there are no blood vessels  and are visible in the ridge  is nearly always in zone I. The proliferating blood vessels are flat and difficult to see, and plus disease is always present .NOTE: It is very important to recognise AP-ROP as it can progress extremely quickly to retinal detachment. Treatment should be given within 48 hours.If immature retinal vessels are present, screening should be repeatedIf ROP is in zones II or III (further away from the optic disc) and is at stage 1 or 2, without any plus disease, the prognosis is good and the ROP is likely to resolve without treatment. Repeat screening is required in 1–2 weeks.If ROP is in zone I, or if it is Stage 3 with plus disease, or aggressive posterior ROP is present, urgent treatment is needed as the disease is very likely to progress to retinal detachment.Classification of ROP guides decision making about screening and treatment. For example:Untreated ROP can sometimes heal with scarring in the peripheral retina and vitreous. This distorts the retina, leading to macular dragging or retinal folds. These signs are not included in the International Classification of ROP, but can be associated with loss of vision .


Colorectal cancer (CRC) is third most commonly diagnosed cancer worldwide. The aim of the prospective study was to evaluate mitosis and apoptosis of epithelial cells at each stage of colorectal neoplasia.A total of 61 persons were enrolled into the study: 18 patients with non-advanced colorectal adenoma (non-a-A), 13 patients with advanced colorectal adenoma (a-A), 13 patients with CRC and 17 controls: individuals with normal findings on colonoscopy. Biopsy samples were taken from pathology (patients) and healthy mucosa . Samples were formalin-fixed paraffin-embedded and stained with haematoxylin-eosin. Mitotic and apoptotic activity were evaluated in lower and upper part of the crypts and in the superficial compartment. Apoptotic activity was also assessed using detection of activated caspase-3.In controls, mitotic activity was present in lower part of crypts, accompanied with low apoptotic activity. Mitotic and apoptotic activity decreased  in upper part of crypts. In superficial compartment, increase in apoptotic activity was observed. Transformation of healthy mucosa into non-a-A was associated with significant increase of mitotic activity in lower and upper part of the crypts and with significant increase of apoptotic activity in all three compartments; p < 0.05. Transformation of non-a-A into a-A did not lead to any further significant increase in apoptotic activity, but was related to significant increase in mitotic activity in upper part of crypts and superficial compartment. A significant decrease in apoptotic activity was detected in all three comparments of CRC samples compared to a-A; p < 0.05. No differences in mitotic and apoptotic activity between biopsies in healthy controls and biopsy samples from healthy mucosa in patients with colorectal neoplasia were observed. Detection of activated caspase-3 confirmed the above findings in apoptotic activity.Significant dysregulation of mitosis and apoptosis during the progression of colorectal neoplasia, corresponding with histology, was confirmed. In patients with sporadic colorectal neoplasia, healthy mucosa does not display different mitotic and apoptotic activity compared to mucosa in healthy controls and therefore adequate endoscopic/surgical removal of colorectal neoplasia is sufficient. Colorectal cancer (CRC) is the third most commonly diagnosed cancer with incidence of 1.36 million and mortality of 694,000 cases worldwide , 3.The colorectal mucosa is formed into the crypts of Lieberkühn, which increase in number between the birth and adulthood . In eachColorectal carcinomas arise in 70–80% sporadically , usuallyThe aim of our prospective study was to evaluate the degree of mitotic and apoptotic activity at each stage of colorectal neoplasia.A total of 18 patients with non-advanced colorectal adenoma , 13 patients with advanced colorectal adenoma  and 13 patients with colorectal carcinoma  were included into the prospective study. Inclusion criteria for those with colorectal neoplasia were: sporadic neoplasia, absence of previous colorectal surgery, negative history of inflammatory bowel disease, absence of any type of colitis , absence of serrated lesion, absence of hyperplastic polyps proximal to the rectosigmoid colon, absence of diverticulitis and postradiation proctopathy. Two patients with well differentiated carcinoma and 11 patients with moderately differentiated carcinoma were included. A total of 7 patients with dedifferentiated carcinoma were excluded from the original group of 20 patients with CRC as mitotic and apoptotic activity could not be evaluated due to presence of excessive necrotic tissue. A total of 17 controls, individuals with normal findings on colonoscopy, with no previous history of colorectal neoplasia or inflammatory bowel disease , were enrolled into the study.Biopsy samples were taken during diagnostic and/or therapeutic colonoscopy. Paired biopsies were obtained in patients with colorectal neoplasia: one sample was taken from the pathology and the second sample was taken from a healthy mucosa, 10 cm distally from the side of neoplasia. In healthy individuals, one biopsy sample was obtained, usually in the rectum, after the exclusion of a colorectal neoplasia in the colon and the rectum.All biopsy samples were formalin-fixed , embedded into paraffin , and tissue sections 5 μm thick were cut . Sections were stained with haematoxylin-eosin  and evaluated using a BX-51 microscope . The grade of mitosis, conveyed as a mitotic index [mitotic index (%) = number of mitotic figures × 100 / number of all evaluated cells] and grade of apoptosis, conveyed as an apoptotic index [apoptotic index (%) = number of apoptotic cells × 100 / number of all evaluated cells] were assessed in three different compartments: in the superficial compartment (which is in the direct contact with the lumen of the large intestine), in the upper part of intestinal crypts and in the lower part of intestinal crypts. Single apoptotic fragments of similar size, when compared to the adjacent non-apoptotic cells, or a cluster of at least three small apoptotic bodies were considered to be apoptotic cells. A minimal amount of 750 cells were evaluated in each compartment in each biopsy sample.Apoptotic activity was also assessed immunohistochemically by detection of activated (cleft) caspase-3, which is a key element regulating the apoptotic process. For that, a rabbit monoclonal antibody  and standard peroxidase technique in 5 μm thick tissue sections were used . After tThe original effort was to evaluate apoptosis similarly to the samples stained with haematoxylin-eosin. Nevertheless, high false positivity in marginal areas was observed Fig. , probablPreliminary data without controls in each group of patients with colorectal neoplasia and without any data on mitosis have been published previously .Obtained data were tested statistically by means of descriptive statistics and Mann-Whitney rank sum test using Statistica software, version 13, 2013, Tulsa, OK, USA.All patients included in the study were given the necessary information and provided informed consent via a signed form. The project was approved by the Joint Ethical Committee ; approval number: 201107 S54. For all obtained data, all personal identification information was removed in compliance with the Czech laws for protection of confidentiality.In healthy controls, mitotic activity was observed in the lower part of crypts, where it was accompanied by low apoptotic activity. Both activities decreased  in the upper part of crypts Fig. , whereasTransformation of healthy mucosa into a non-advanced adenoma (non-a-A) was associated with a significant increase of apoptotic activity in all three compartments Figs. , 6 and 7Transformation of a non-a-A into an advanced adenoma (a-A) did not affect apoptotic activity Figs. , 6 and 7Finally, there was a statistically significant decrease in apoptotic activity in all three compartments of carcinoma samples (CRC) compared to a-A group Figs. , 6 and 7Further statistical analysis was conducted in order to compare mitosis and apoptosis of neoplastic epitheĺial cells between the right  and the left colon : no differences were found in each pathology; p > 0.05, Figs. Mitotic and apoptotic activity in biopsies from the normal tissue taken in patients with colorectal neoplasia in the right and left colon was assesed: there were no differences, p > 0.05.No differences in mitotic and apoptotic activity between controls from healthy individuals and control samples in each group of patients with colorectal neoplasia in each compartment were found; p > 0.05, Figs. A total of eight CRC patients had the neoplasia localised in the rectosigmoid junction or in the rectum and the biopsy from the healthy mucosa was taken in the rectum. Mitotic and apoptotic activity from these samples were compared with mitotic and apoptotic activity of eight randomly chosen controls, whose biopsies were taken in the rectum: no differences were found out; p > 0.05.No differences in mitotic and apoptotic activity between females and males were observed; p > 0.05.Immunohistochemical detection of activated caspase-3 showed significant increase in apoptotic activity in the upper part of the crypts during the transformation of healthy mucosa into a non-a-A and an a-A; p < 0.05. This was followed by a significant decrease of activated caspase-3 positivity in the same compartment in CRC samples when compared to a non-a-A and an a-A; p < 0.05 Table .Table 1IDysregulation of epithelial proliferation and apoptosis is typical for a neoplastic process. Recently, therapeutic strategies involving induction of apoptosis have become an effective part of therapy of an established carcinoma –18, but The “programmed cell death” was first proposed by Lockshin and Williams in 1965 .In healthy mucosa, mitotic activity was observed in the lower part of crypts only, where it was accompanied by a certain level of apoptotic activity. Apoptotic activity in this part of crypts might be a mechanism contributing to the maintenance of genetic information providing stability of mutiplying stem cells and progenitors , 22. TheIn this study, mitosis and apoptosis increased significantly almost in all evaluated compartmens of non-advanced and advanced adenomas. Resembling trends in both neoplasias suggest that both these processes are triggered by genetic alterations. On the contrary, mitotic activity remained unaffected at the luminal surface of non-advanced adenomas indicating maintenance of terminal differentiation in this compartment despite altered morphology, which is in agreement with the literature . This waApoptosis is regulated by two major mechanisms: Bcl-2 family, which consists of pro- and antiapoptotic members and Inhibitor of apoptosis (IAP) family . SurviviMutations in the WNT/beta-catenin pathway are typical for sporadic colorectal cancer and apoptosis in CRC cells with these mutations is induced by histone deacetylase inhibitors . ButyratDue to Warburg effect, the cells of colorectal neoplasia prefer glucose as the energy source. Butyrate therefore accumulates in the nuclei of cancerous colonocytes and induces apoptosis via the inhibition of histone deacetylase . Our stuNo differences in mitotic and apoptotic activity were found between right sided and left sided neoplasias, between normal tissue obtained in patients with right sided and left sided neoplasias, and between males and females. Still, we are very carefull to draw any firm conclusions - despite a very high number of cells (> 750) were evaluated in each compartment - as the numbers of participants in each subgroup  and women in the group with advanced adenoma (3/13)) were small.Another limitation worth noting is that most of the samples taken from healthy patients were from the rectum and we are aware that rectal samples do not have to be fully representative of the entire colorectum. Yet, based on our study, the observation of no difference in mitotic and apoptotic activity between controls from healthy individuals and control samples in each group of patients with colorectal neoplasia is of a very high clinical significance: adequate endoscopic/surgical removal of colorectal neoplasia is warranted, but should be sufficient in case that we deal with a sporadic colorectal neoplasia in a patient without inflammatory bowel disease.Significant dysregulation of mitosis and apoptosis during the progression of colorectal neoplasia, corresponding with histology, was confirmed. In patients with sporadic colorectal neoplasia, healthy mucosa does not display different mitotic and apoptotic activity compared to mucosa in healthy controls and therefore adequate endoscopic/surgical removal of colorectal neoplasia is sufficient.


Impaired hearing and cognition are disabling conditions among older adults. Research has presented inconsistent conclusions regarding hearing impairment posing a risk for cognitive impairment. We aimed to assess this from published evidence via searching PubMed and Embase, from the inception of the databases indexed to December 2, 2016. For those high-quality studies retrieved, relative risk (RR) and 95% confidence intervals (CIs) were combined to estimate the risk of cognitive impairment. Eleven cohort studies were included in the present study. Pooled results found that elderly people with disabled peripheral and central hearing function had a higher risk of cognitive impairment  compared to those with normal hearing function. We also recorded a dose-response trend for cognitive impairment as hearing thresholds rose. No evident bias from potential confounding factors was found with one exception: the length for clinical follow-up. Although results are preliminary because qualifying studies were few, statistical findings were consistent with older people identified as having greater levels of hearing loss, having a corresponding higher risk of cognitive impairment. Depending upon the auditory pathways involved, ARHL can be categorized as peripheral ARHL or central ARHL, and the clinical manifestations are often mixed2. ARHL has become a major sensory condition, within the world’s rapidly growing aging population1. American prevalence rates showed that hearing impairment affected 29.3% of the population at age 60 to 69 years3 and the percentage was increased to 63.1% in people aged 70 years or over4. Surveys from other countries also revealed the leading role of ARHL among disabling conditions associated with older demographics6. Apart from reduced hearing sensitivity and speech understanding, ARHL has a series of consequences including reduced ability to detect and localize safety and/or warning alarms1, and compromised communication efficiency linked to comorbid psychosocial issues such as social isolation and depression7. Although there are numerous rehabilitative alternatives for ARHL, patients and their families may not always seek such options; therefore, the condition is largely underestimated, especially during an early stage of ARHL2. Patients generally fail to obtain sufficient screening and intervention probably because they regard ARHL as simply a part of entering their senior years, unaware of its potentially far-reaching consequences8.Age-related hearing loss (ARHL), a hearing impairment caused by aging and neurodegeneration, is characterized in older adults as difficulty in understanding speech and detecting sound9. For instance, ARHL serves as one of the substantial markers of frailty  in older age with adverse outcomes like cognitive impairment7. Cognitive impairment affects many domains such as: memory, attention, executive function, perception, and semantic knowledge, which also constitute some of the primary targets of dementia and Alzheimer’s disease (AD)11. Epidemiological evidence supports the high possibility of cognitive impairment progressing into dementia and AD12, both of which have rapidly-increasing prevalence with age and the absence of disease-modifying treatment13. Prevention of dementia and AD has become a public health priority due to its irreversibility and its burden upon individuals, families, and society14.However, ARHL may be widely associated with neurodegenerative, functional, physical, and psychosocial impairment7. Thus, collecting evidence from observational studies could be an important initiative to assess whether ARHL serves as a modifiable factor among the strategies of preventing dementia. An initial review appeared to provide some support for the relationship between ARHL and cognitive impairment; however, it also identified some studies which argued against this correlation19. More recent studies22 have emerged. They applied more recognized measures to evaluate the auditory and mental status of various populations. However, research with negative results24 has prevented us from definitively concluding that hearing function is connected to the risk of cognitive impairment during the later life of adults.An increasing number of findings have suggested an association between ARHL and cognitive impairment, indicating that ARHL may be a potential early marker of AD26 have offered some perspectives demonstrating that hearing impairment and cognitive problems are associated. To our knowledge, the first meta-analysis to explore hearing loss and cognitive function25, without putting restrictions on age or methodology when retrieving studies, concluded that individuals with hearing loss had worse cognitive performance. A recent study26 gathered some prospective cohort studies to support the conclusion that hearing impairment increases the risk of both cognitive disorders and AD, though it analyzed only four heterogeneous studies. Categories of hearing function and possible confounding factors were not explored in either study.Previous systematic reviews and meta-analyses28. Follow-up durations were considered because both ARHL and cognitive impairment are conditions that deteriorate with age2. Apart from the hearing function-cognition relationship, we also analyzed the initial findings from these retrieved studies about the effect of hearing aid use on the incidents of cognitive impairment.The present meta-analysis of cohort studies was undertaken to explore research inconsistencies regarding ARHL and cognitive impairment, statistically. By including more recent data, we appraised the hearing function-cognition relationship in older adults and its dose-response trend with more participants. We also incorporated both peripheral and central hearing function as independent variables to carry out an analysis for the risk of cognitive impairment by category. Additionally, the present study considered potential confounding factors such as race and sex in meta-regression because, for example, melanin and estrogen may work in the pathogenesis of ARHL34 were included, seven of which31 were combined in the following meta-analyses. Eight studies34 were analyzed in meta-regression.Figure 32, North America33, Oceania24, and Asia34. All cohorts, except for one21, included both genders. Some studies evaluated hearing function with insurance records32 and self-reporting34, and the rest used pure-tone audiometry. Five cohorts were followed-up ≤6 years32. Cognitive status was qualified or quantified by the Mini-Mental State Examination (MMSE) and its revised version34, or the Diagnostic and Statistical Manual (DSM) and the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA)33. All studies were multi-adjusted of covariates with three exceptions34. Quality assessment is presented in Supplementary Table Table 29), the risk of cognitive impairment in older subjects increased 29–57% compared to those with normal hearing : 1.04–1.59; follow-up >6 years, RR = 1.57, 95% CI: 1.13–2.20, Fig. 35; RR = 1.29, 95% CI: 1.12–1.50, Fig. I2 = 0%, P = 0.45, in subgroup of follow-up >6 years, I2 = 0%, P = 0.71, Fig. I2 = 0%, P = 0.38, Fig. I2 = 45%, P = 0.16, Fig. Figure 31. The RR for incidence of cognitive impairment in older people was 2.42 in the moderate CAD group compared with the normal function group .Figure 29. Analyses were performed with groups stratified by the severity of hearing loss, evaluated with PTA results of thresholds in the better ear . Relative risk from the studies was combined with the risk ranging from 1.19 (95% CI: 0.96–1.48) to 4.94 (95% CI: 1.09–22.40) across three hearing loss categories.Table P = 0.004). The data for meta-regression were gathered in Supplementary Table Table Pheterogeneity and I2 was observed when evaluating the risk of cognitive impairment for every 10 dB increase in hearing loss.Table Publication bias analysis was not applied here due to a limited number of qualifying studies.Our major findings showed that, after statistical analyses, both peripheral and central hearing dysfunction appear to contribute to the risk of cognitive impairment in the aging population. The overall risk of cognitive impairment increased 29% (follow-up ≤6 years) or 57% (follow-up >6 years) in senior participants who had disabled peripheral hearing function compared with people of normal hearing function. The risk remained significant when adopting the World Health Organization (WHO) standard of hearing impairment or when PTA was modeled continuously. The analysis showed that the association between central hearing dysfunction and cognitive impairment was stronger . A dose-response trend was found between peripheral hearing function and cognition. There was no obvious heterogeneity among most studies, excluding the analysis combining RR for every 10 dB increase in hearing loss Fig. . After a26 had collected encouraging findings of the hearing function-cognition relationship, as it applied to cognition domains or the onset of AD. The first meta-analysis25 found that individuals with hearing loss had poorer cognitive manifestations whether hearing was treated or untreated. However, this conclusion was based on adults from a wide range of age groups, applying varied measures on hearing and cognitive status, and varied statistical methods. Another meta-analysis26 – which gathered limited but robust-appearing data from prospective studies – also chose a generalizing approach to analyze the relationship between hearing loss and AD. These two studies explored neither categories of hearing function nor possible confounding factors.Previous meta-analyses7, confirming  the hearing function-cognition relationship in older people. In addition, we included more recent high-quality cohort studies and explicitly-classified hearing function as an independent variable. The present study has sought to elucidate research inconsistencies associated with previous studies investigating potential links between ARHL and cognition in the following ways: firstly, there have been unresolved discrepancies regarding whether hearing loss clearly correlates to cognitive decline. This has been preliminarily reconciled after our statistical analysis. We also observed a trend of increased risk for cognitive impairment when extending follow-up. Secondly, CAD symptoms have not been well-considered in the hearing function-cognition relationship, as ARHL has appeared to be regarded in terms of mostly being peripheral hearing dysfunction36. However, from the evidence accumulated here, CAD has been shown to potentially increase the risk of cognitive impairment. Thirdly, we found that there was a dose-response association that more damaged peripheral hearing function contributed to a greater risk of cognitive impairment. Lastly, we statistically analyzed the influence of several potential confounding factors that previous studies did not cover. From the summarized data, we hope that our findings can help others understand the hearing function-cognition connection in older adults, in a clearer way. The possibility that hearing intervention may be a way to delay cognitive impairment could be further explored based on this relationship. This is especially essential because hearing rehabilitative interventions are largely underutilized37.Our combined estimates were consistent with previous reviews38. The association has been seen in additional observational studies since then. A preliminary summarizing study19 reviewed existing literature to explore this relationship, despite the varied methods and limited sample sizes those investigations had applied. However, considering at least three opposing views18 exist within the preliminary study, it is difficult to conclude the presence of a solid correlation. Although the risk from hearing impairment on cognition cannot be addressed by randomized controlled trials (RCT) due to ethical concerns regarding possible harm to participants’ wellbeing (studies could not be designed to deliberately assign “hearing impairment” to the participants), recent well-designed cohort studies31 have emerged to serve as an alternative to explore the problem as thoroughly as possible. Still, some studies drew negative conclusions about the hearing function-cognition relationship34. Furthermore, few studies included the tests of central auditory function as part of the hearing evaluations, although CAD is prevalent ; additionally, CAP and cognitive function are able to interact centrally .The relationship between hearing function and cognition in older adults has been observed since at least the 1960s. At that time, the first research about this connection found that subjects with organic mental syndrome (deficits in memory and intellect) had a higher prevalence of deafness43. The peripheral-central impairment hypothesis suggests that poor encoding of sound from the impaired cochleae has severe consequences including: demanding more cognitive resources for auditory perceptual processing, influencing brain structure, and reducing social engagement49. Moreover, some manifestations of CAD itself could be a sign of cognitive dysfunction30 . Additionally, the common factors hypothesis shows that hearing and cognitive disorders are correlated because they share similar neurodegenerative processes resulting from aging, vascular diseases, and oxidative stress52.There are some hypothesized mechanisms that support the hearing function-cognition relationship. The frailty hypothesis states that ARHL is one of the markers of frailty – characterized by vulnerability to stressors – which could exert adverse health outcomes like cognitive frailty through inflammatory, vascular, hormonal, nutritional, and metabolic pathways28 although our analysis of some confounding variables did not result in significant heterogeneity.Our findings do have some limitations. Firstly, there were a limited number of the included studies, especially studies discussing CAD, which may make it difficult to show a conclusive connection. There were not enough studies for a publication bias analysis. Of the qualifying CAD-related studies, only two from the same research team were analyzed and only one CAP test was selected without further differential diagnoses to ensure CAD. Secondly, there was little data from cohorts with comprehensive controls including: ethnicity, gender, history of noise exposure, hereditary information, ototoxic drug use, lifestyle, socioeconomic status, and so on. This may impact the present findings because ARHL has been influenced by those factors54), nor do they predict mild cognitive impairment ultimately developing into dementia. Also, they cannot exclude cognitive difficulties from causes other than age-related cognitive decline58. Additionally, those cognition screening tools show a possible insufficient validity to find cognitive impairment across populations, compared with neuropsychological test batteries. For example, for ethnicity, MMSE had lower specificity within non-White groups59 despite previous evidence ascribing the ethnic differences to other causes61. Similarly, different norms of 3MS between White and Black populations may be worthy of consideration63. Therefore, though some studies29 ensured race was fully adjusted for in the cohorts, to eliminate possible bias, care should be applied when interpreting the cognitive function of the present included participants. Other limitations associated with evaluating cognitive status have included: a lack of studies which have extensively analyzed AD and other types of dementia as the outcomes, plus possible overestimation of cognitive impairment via verbal administration of some instruments and its dependence on preserved hearing function. The latter concern has been raised by previous studies65, although others66 have claimed that verbal administration is not an issue if questions are delivered by experienced examiners in quiet environments. Some studies have attempted to address this by cautiously applying multiple sources of data, and by adjusting total scores to account for any potential confounding aspects associated with sensory problems33. These aspects may make it difficult to reach a comprehensive conclusion until further studies emerge to clarify the field.Thirdly, the measures of cognition varied. Qualifying or quantifying instruments applied by the included studies incorporated the following classifications, diagnostic manuals, or screening tools: MMSE, Modified Mini-Mental State Examination (3MS), International Classification of Diseases (ICD), Cognitive Abilities Screening Instrument (CASI), DSM, and NINCDS-ADRDA. This could lead to potential heterogeneity for example, as screening tools such as MMSE, 3MS, and CASI neither play a role in diagnosing cognitive impairment 71, we searched the databases of PubMed and Embase from the inception of the databases indexed to December 2, 2016. Free text terms with the meaning of hearing loss without restriction were used: “auditory defect” or “auditory impairment” or “auditory dysfunction” or “deafness” or “deaf” or “hearing damage” or “hearing defect” or “hearing difficulty” or “hearing loss” or “hypacusia” or “hypacusis” or “impaired hearing” or “hearing impairment” or “hearing decline” or “hearing disability” or “poor hearing” or “presbycusis” or “hearing deficit” or “hearing trouble” or “hearing limitation” or “hearing handicap”. Mesh and Emtree words were also searched. The same strategies were applied to identify cognition disorder and dementia (the synonyms used for cognitive impairment and dementia were “cognition disorder” or “cognitive defect” or “cognitive deficit” or “cognitive disability” or “cognitive disorder” or “cognitive dysfunction” or “cognitive impairment” or “dementia” or “cognitive decline” or “cognitive difficulty” or “compromising cognition” or “cognitive compromise” or “cognitive trouble” or “troubled cognition” or “cognitive limitation” or “limited cognition” or “cognition limit” or “amentia” or “Alzheimer disease”). The age filters of the results were limited to middle-aged and aged individuals. For some major reviews19, we went through the contents and bibliographies, finding the related research by citation searching.According to the guidelines for the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA)74), missing data, mean age at baseline not reaching 60 years, being irrelevant to our aim, and being non-observational studies. Next, the full texts of the remaining studies were reviewed to find: (1) prospective cohort studies; (2) extracted estimates were OR, RR, HR, or with enough data to calculate them, and their 95% CIs ; (3) extracted results were possible to combine and adjusted with covariates. Divergences between the two investigators were discussed and resolved with the whole author group.Two investigators (J.Y. and Y.S.) separately screened the studies. Duplicates were excluded; studies were then removed for reasons such as: having a sample size of less than 100  participants of the study; (2) baseline information of the study; (3) the measures employed to evaluate hearing and cognitive function; (4) adjusted variables; (5) follow-up; (6) adjusted estimates of OR, RR, and HR with 95% CIs. If necessary, authors of some included studies were contacted for more detailed information.76. Selection (maximum 4 asterisks), comparability (maximum 2 asterisks), and outcome quality (maximum 3 asterisks) from the included studies were calculated. The scales were tabulated in values as the number of asterisks each study had gained. A maximum of 9 asterisks can be given for an individual study. The score for a high-quality study was defined as more than 5 asterisks77.Quality assessment was made with standards from the Newcastle-Ottawa Scale (NOS)Two investigators (J.Y. and S.S.) carried out data extraction and quality assessment independently.Q-statistic among studies: Pheterogeneity < 0.10 and I2 > 50% indicating significant heterogeneity80. In our analyses, we used the random-effect models because there was variation among these studies in characteristics like sample size, follow-up, population characteristics, and definitions for cognitive impairment. We estimated the dose-response trend based on risks calculated by categories of peripheral hearing function. Covariates as potential confounding factors were retrieved and analyzed in groups with meta-regression. A confounding factor was recognized with a meta-regression P < 0.0581. Sensitivity analysis was conducted by the one-study-out method: omitting one study at a time, sequentially, to test the combined RR from the remaining studies so as to see the stableness of each meta-analysis82.Studies which had used OR or HR to calculate the incidence of cognitive disorder were pooled as approximate RR because hearing loss resulting in cognitive impairment is a rare event. Meta-analysis was conducted with the inverse variance (IV) method. In the studies including covariates, we extracted the most comprehensively adjusted estimates. Heterogeneity was explored with the Cochrane P < 0.05 for all the analyses unless otherwise specified.Meta-analysis was performed by Cochrane Review Manager  and Stata . Statistical significances were set at Supplementary Information