Tinnitus is one of the most common symptoms before and/or after the surgical removal of a vestibular schwannoma (VS) affecting almost half of the patients. Although there is increasing evidence for the association of hearing impairment and VS-associated tinnitus, the effect of hearing deterioration due to surgery and its relation to the postoperative tinnitus (postTN) is poorly investigated. This knowledge, however, might (i) enlighten the pathophysiology of VS-associated tinnitus  and (ii) improve preoperative patient counseling. The aim of this study was to understand the predisposition factors for a postTN in relation to hearing outcome after surgery.This retrospective study analyzed the presence of tinnitus in 208 patients with unilateral VS before and after surgical removal. A binomial logistic regression was performed to ascertain the effect of pre- and postoperative hearing as well as age, gender, tumor side, and size, and intraoperative cochlear nerve resection (CNR) on the likelihood of postoperative VS-associated tinnitus.Preoperative tinnitus was the strongest predictor of postTN. In addition, deterioration of functional hearing was increasing, while functional deafferentation  of non-functional hearing was reducing the risk of postTN. At the same time, patients with no preoperative tinnitus but complete hearing loss had the lowest risk to suffer from postTN. Patient age, gender, tumor side, and size as well as CNR played a subordinate role.per se.While the presence of preoperative tinnitus was the strongest predictor of postTN, there is a distinct relationship between hearing outcome and postTN depending on the preoperative situation. Functional or anatomical deafferentation due to surgical tumor removal does not prevent postTN  Tinnitus occurs in 63–75% of patients with unilateral vestibular schwannoma (VS) , 2 and iHowever, the association between postoperative hearing outcome and tinnitus remains elusive , 17–19. evolution of hearing and its relation to the presence of preoperative tinnitus. The aim of the study is to describe the relationship between pre- and postoperative hearing impairment and the occurrence of postTN in VS surgery under the prism of maladaptive neuroplasticity.We hypothesize that the best predictor of postTN is neither CNR nor hearing outcome itself but the surgery-associated via a retrosigmoidal-transmeatal approach. Continuous neuromonitoring of the brainstem auditory-evoked potentials (BAEP) was applied in all patients with preserved preoperative BAEP  by a semi-structured interview by experienced neurosurgeons. Finally, the presence of ipsilateral tinnitus symptoms was dichotomized for statistical analysis . The presence of tinnitus was evaluated preoperatively (preTN) and 3 months postoperatively (postTN).m) and hearing impairment was reclassified in (i) GRm1: functional hearing (GR1 and GR2), (ii) GRm2: non-functional hearing (GR3 and GR4), and (iii) GRm3: no hearing (GR5) (m) and postoperatively (postGRm). In addition, patients were classified according to their hearing outcome (ΔGRm), (i) unchanged hearing preGRm1 → postGRm1 or preGRm2 → postGRm2 (ΔGRm0), (ii) deterioration of preoperative functional hearing preGRm1 → postGRm2/3 (ΔGRm1), (iii) deterioration of preoperative non-functional hearing preGRm2 → postGRm3 (ΔGRm2), and (iv) preoperative complete hearing loss (ΔGRm3).Hearing impairment was classified according to the Gardner and Robertson (GR) scale  based onng (GR5) , 18. HeaIn all patients, a preoperative magnetic resonance image of brain with gadolinium contrast was available and the tumor extent was graded according to Hannover classification . VS weret-test or Chi-square test. Binary logistic regression analysis was used to determine the predictive value of gender, age, tumor side, tumor size, the presence of preoperative tinnitus (preTN) and preoperative (preGRm) and postoperative (postGRm) hearing impairment as well as the surgery associated change of hearing (ΔGRm) for the occurrence of postTN using a backward step-wise method. The backwards method removes explanatory variables from the first model, which includes all the specified variables based on the likelihood ratio criterion, which is considered the criterion least prone to error . Group differences in distribution of clinical attributes such as gender, age, tumor side, tumor size, and preoperative and postoperative hearing impairment were evaluated by Student’s to error . Predictm1), while 19.7% (41/208) presented non-functional hearing (preGRm2) and 17.3% (36/208) were deaf (preGRm3). 58.7% (122/208) complained about a preoperative tinnitus (preTN+). All patients underwent a surgical removal of the tumor via the retrosigmoidal-transmeatal approach supported by continuous neuromonitoring of the BAEP aiming anatomical preservation of the cochlear nerve. However, in 7.2% (15/208), CNR was noted. Postoperatively, functional hearing (postGRm1) was preserved in 48.1% (63/131) of patients.This retrospective study enrolled 208 patients with unilateral VS . 32.2% (67/208) presented a T1/2, 40.4% (84/208) a T3 and 27.4% (57/208) a T4 tumor according to the Hannover classification. Preoperatively, 63% (131/208) of the patients had functional hearing  of postTN+ patients were suffering from tinnitus preoperatively (preTN+) but only 40.6% (43/106) of postTN− patients were preTN+. In detail, in 67.5% (85/126) of preTN+ patients, the tinnitus sustained after surgery, while tinnitus disappeared in only 32.5% (41/126) of preTN+ patients. In contrast, 79.3% (65/82) of preTN− patients had no postTN, while 20.7% (17/82) developed a new-onset tinnitus after surgery.m3) were less likely than patients with residual preoperative hearing  to suffer from postTN. 26.4% (28/106) of postTN− patients had preGRm3; however, only 7.8% (8/102) of postTN+ patients had a preoperative hearing loss (preGRm3). While there were no significant differences in postoperative hearing level (postGRm), there was a significant difference in change of hearing level (ΔGRm) due to the surgery. Most postTN+ patients showed unchanged (ΔGRm0) or a deterioration of preoperative functional hearing (ΔGRm1), while postTN− showed a high rate of postoperative hearing loss , and postoperative hearing (postGRm) as well as the hearing change (ΔGRm) on the likelihood of postoperative VS-associated tinnitus, a binomial logistic regression was performed using a backward step-wise method. After seven iterations, the logistic regression model was statistically significant . Of the predictor variables, only preTN and ΔGRm were included in the model as significant predictors of postTN  and postoperative hearing loss in patients with preoperative non-functional hearing (ΔGRm2) are reducing the risk of postTN . In turn, this means that patients with unchanged hearing or deterioration of initial functional hearing have an increased risk of postTN.In order to ascertain the effects of age, gender, tumor side, and size, the presence of preoperative tinnitus (preTN), CNR, pre-  of preoperative non-functional hearing is reducing the risk of postTN, significantly. At the same time, patients with no preoperative tinnitus but complete hearing loss have the lowest risk to suffer from postTN. Patient age, gender, tumor side, and size as well as CNR play a subordinate role in predicting postTN.In the present study, 67.5% of patients with preoperative tinnitus were still suffering after the surgery, while in 32.5%, tinnitus disappeared postoperatively. In contrast, 79.3% of patients with no preoperative tinnitus, while 20.7% of patients developed a new-onset tinnitus. There are several studies evaluating the tinnitus occurrence after VS surgery showing a high variability in tinnitus outcome. This could probably be attributed to difference in surgical procedures and approaches . Howeverevolution of hearing after surgery than by the pre- or postoperative hearing level. Patients with hearing preservation or deterioration of preoperative functional hearing have the highest risk, while functional deafferentation of preoperative non-functional hearing and preoperative hearing loss is reducing the risk of postTN. Among the studies that performed a similar surgical strategy, none have shown a significant association between the postoperative hearing outcome and tinnitus .LT contributed to the analysis and interpretation of data, statistical analysis, and writing of the first draft. JS, KM, FE, and MT contributed to the data acquisition, interpretation of data, and the review and critique of the final manuscript. GN was responsible for the conception and design, data acquisition, analysis and interpretation, statistical analysis, as well as the review and critique of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A–ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A’s involvement in mutation of endogenous or exogenous DNA.The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)T APOBEC3 (A3) proteins restrict replication of retroviruses by inducing hypermutations in the viral genome7. A3s deaminate deoxycytidines in ssDNA into uridines during reverse transcription. This results in G to A hypermutations, as adenosines are transcribed across from uridines during second strand DNA synthesis. While all A3 enzymes deaminate deoxycytidines in ssDNA, they have differential substrate specificities that are context dependent, resulting in altered frequencies of mutation for the deoxycytidines. Some A3s deaminate the second deoxycytidine in a sequence containing CC while others deaminate deoxycytidine in a TC context10. However, not every cognate dinucleotide motif (CC or TC) in the ssDNA of the HIV genome is deaminated11. Nevertheless, hypermutation in a viral genome results in defective proteins and proviruses, thus decreasing the probability of further viral replication12.The APOBEC3  family of human cytidine deaminases provides a first line of defense against many exogenous and endogenous retroviruses such as HIV-1 and the retro-element LINE-113. Soon after, APOBEC3B was found to be correlated with a variety of other cancers such as ovarian, cervical, bladder lung, head and neck; signature sequence analysis was also a contributing factor that led to these conclusions15. Most recently APOBEC3H, which has a different sequence preference than APOBEC3B, has been identified to also play a role in breast and lung cancer16. Thus, defining A3 sequence specificity can be helpful in identifying A3s’ role in viral restriction and in cancer.Beyond restricting viral replication, the ability of A3s to deaminate deoxycytidines in ssDNA have made A3s a double-edged sword. When overexpressed, A3s can mutate the host genome resulting in a variety of cancers. The identities and patterns of the mutations observed in cancer genomes can define the source of these mutations. Recently, the search for the deaminase(s) responsible for kataegic mutations found in breast cancer was narrowed down to APOBEC3B, through the comparison of all known APOBEC mutational signatures and eliminating APOBEC3G and other deaminases from potential mutational contributors21. A recent high-throughput assay suggested the preferred quad-nucleotide motif for A3A to be CTCG20. Although A3s are known to have varied sequence preference, quantitative and systematic studies of sequence specificity are incomplete. Recently, crystal structures of APOBEC3A (A3A) and APOBEC3B-CTD (an active site A3A chimera) with ssDNA have been solved22. However, despite these breakthrough structures, the molecular mechanism underlying substrate sequence specificity flanking the TC dinucleotide sequence remains unclear.A3 signature sequences proposed for deaminating deoxycytidines range between di-nucleotide to quad-nucleotide motifs23 and a known restriction factor for the retroelement LINE-1 and HPV25. A3A can also contribute to carcinogenesis with increased expression or defective regulation26. A3A is the only A3 where both the intact apo and substrate-bound structures have been determined28. Initial substrate specificity studies have shown selectivity for DNA over RNA, suggested by NMR chemical shift perturbation19. Since A3A is the best biochemically characterized A3 human cytidine deaminase and thus a critical benchmark within the family, we chose A3A to elucidate the extended characteristics of ssDNA specificity.A3A is a single-domain enzyme with the highest catalytic activity among human APOBEC3 proteinsC(A/G) and found binding correlated with enzymatic activity. Also, we determined that A3A can bind RNA in a sequence specific manner. Surprisingly, A3A’s signature sequence was necessary but not sufficient to account for A3A’s high affinity for ssDNA. Significantly, A3A bound more tightly to the motif in longer oligonucleotides, and in the context of a hairpin loop. Using recently published structures of A3As complexed with ssDNA from our lab and others, we propose a structural model for the molecular mechanism for this enhanced affinity where inter-DNA interactions contribute to A3A recognition of the cognate sequence. This model provides insights into how the nucleotides flanking the canonical TC sequence may contribute to substrate sequence preference of A3A.To determine the substrate specificity of A3A, we systematically quantified the affinity of A3A for nucleic acid substrates as a function of substrate sequence, length, secondary structure, and solution pH. We identified the A3A preferred ssDNA binding motif, (T/C)T28. First, to ensure that the affinity for substrate was due entirely to the sequence of interest and not due to nonspecific binding or undesired secondary structure effects, an appropriate control background sequence was identified. The dissociation constants (Kd’s) for homo-12-mer ssDNA sequences, Poly A, Poly T, Poly C, were determined . Poly G was not tested due its propensity to form secondary structure elements. Poly T (750 ± 44 nM), which had previously been used in background sequences28, bound to A3A with 2-fold higher affinity than Poly C . Thus without a greater context for A3A to target, Poly C was only weakly bound. A3A had the lowest affinity for Poly A with a Kd of >11,000 nM   is similar to the affinity for Poly A-U (5A-1U-6A)  and even the background Poly A. This is in contrast to A3A’s specificity for binding a single C over U in a Poly T background, which is more than ten-fold (35 ± 2 nM and 500 ± 23 nM respectively)  Fig. , as we p30 and set a reference pH for subsequent experiments. The Kd of A3A for TTC in a Poly A background was determined at pH ranging from 4.0 to 9.0 in 0.5 pH increments  on A3A affinity for substrate -TC-(6 A), AC, CC, GC in a Poly A background were determined. A preference for TC (143 ± 4 nM), followed by CC (250 ± 14 nM) was identified. Interestingly, AC and GC had similarly very weak binding affinities for A3A , validating a preference for pyrimidines (T or C) over purines (A or G) at −1 position with T as the strongest binder.To study the effect of the nucleotide identity at position −1 relative to target deoxycytidine  on affinity of A3A for ssDNA was determined by first testing the change in affinity for all nucleotide substitutions at −2 position (3A)-NTC-(6 A). A3A has a preference for pyrimidine over purine at −2 position  compared to that of purines ATC and GTC (145 ± 2 nM and 150 ± 3 nM respectively). While not as strong as for −1 position, there is a preference for the smaller pyrimidines at position −2. Next, the effect of +1 position on affinity of A3A to TC was determined  was determined  were those that contained purines at −2 with pyrimidines at +1 position. Thus, the substrates can be broadly classified as high (80–130 nM), medium (150–165 nM), and weak (210–220 nM) affinity binders, with (T/C)TC(A/G) identified as the preferred sequence for ssDNA recognition by A3A.Finally, to identify if there was any interdependency between nucleotide identity at −2 and +1 positions, the affinity of A3A for (3A)-NTC(A/G). The affinity of three substrate sequences, TTCA, ATCG and ATCA, were compared to the corresponding product sequences . For all three sequences, a substantial loss of binding affinity was observed for the corresponding TTUA, ATUG and ATUA, with the most substantial loss with ATUA. Thus, the decrease in affinity for product over substrate was context dependent.A3A’s affinity for substrate C was compared to product U in the context of variations of the signature A3A substrate sequence (T/C)Tces Fig. . For all27. Thus A3A’s deamination activity was determined in the context of variations of the signature sequence (T/C)TC(A/G) using a 1H NMR based A3 deaminase activity assay. High (TTCA and TTCG), medium  and low (ATCT and GTCT) affinity sequences were tested (Table ). This indicates that in general those substrates sequences with varying binding affinity  are also processed in a similar order.Although enzymatic activity and binding affinity are not expected to be directly correlated, the trends for specificity would likely be similar. The NMR assay is a highly quantitative method of observing product concentration and/or substrate reduction directly by NMR signal volumes throughout the reactioned Table . This inC(A/G), crystal structures of A3A bound to ssDNA recently determined by our group and others (PDB ID: 5KEG and 5SWW) were analyzed22. The target deoxycytidine is well coordinated and buried within the active site of A3A TC(A/G), neither of the co-crystal structures has the optimal nucleotide identity at the −2 and +1 positions22. Specificity for purine at the −2 position was not evident in the available A3A–ssDNA structures, presumably as neither structure contains an optimal ssDNA sequence. For instance, even though the 5KEG structure contains a preferred pyrimidine in the −2 position, the thymidine is disordered in this complex. However, in both structures22, the base at +1 position (pyrimidine T in 5KEG and a purine G in 5SWW) stacks with the critical His29 Ts29 Fig. 20,22. Th22. This U shape of the bound polynucleotide may be conserved among deaminases, including adenosine deaminases34. In both A3A–ssDNA structures, the U shape of the ssDNA orients the −2 and +1 bases in close proximity to each other. Thus, we hypothesized that the observed sequence preference  signature sequence, molecular models were developed based on the crystal structures of A3A bound to ssDNA (PDB ID: 5KEG and 5SWW)22. These models orient the bases of the −2 and +1 nucleotides so that they form hydrogen bonds at an angle of approximately 120 degrees and distance of less than 3.5 Å, with the larger purine at +1 position stacking on His29 and the smaller −2 pyrimidine coordinating the +1 base TC(A/G) and suggests stabilizing inter-DNA interactions may further increase the affinity.To determine the potential for intra-DNA interactions when A3A is bound to a (T/C)Tase Fig. . The revCA (TTCAA and ATTCA) to 3 nucleotides flanking each end, increasing by one nucleotide addition on either end. Surprisingly, a single nucleotide flanking TTCA signature sequence was not enough to permit binding  . As expected, A3A had a higher affinity for the DNA hairpin with loop region containing TTC compared to one with AAA (26 nM vs ~676 nM respectively)  is comparable to that for a single C in a polyT background (35 nM)28. This may imply that the Poly T DNA adopts a hairpin structure in solution, as has been reported36.Another implication of this model would be that pre-bent DNA could be a better substrate for A3A binding, as A3A would not have to pay the entropic cost of bending the DNA. This bending of DNA could be achieved either by the inter-DNA interactions modeled in Fig. ly) Fig. . InteresC in the loop of this hairpin versus a modified SDHB hairpin RNA replacing the AUC with AAA was compared. A3A had specific affinity for the hairpin RNA containing UC compared to AA (37 nM vs 202 nM respectively)  Fig. . In cont23, a known restriction factor25, and also likely contributes to carcinogenesis26. In this study we quantified the ssDNA specificity of A3A, and identified the consensus signature sequence as (T/C)TC(A/G). The dinucleotide sequence preference for A3A, TC, which was previously found through activity assays21 was confirmed and expanded to a preference for pyrimidine-TC-purine. Surprisingly context matters, in that the background nucleotide sequence impacts binding affinity, with essentially no binding observed for Poly A 1 C TC(A/G) is imbedded within also modulates affinity TC(A/G), not only account for the discrepancies in the A3A target sequences reported in the literature such as TT38.Designing inhibitors or activators for A3s has been extremely challenging. Our results implicate a need to incorporate the structural context of the target deoxycytidine in the therapeutic design. Larger macrocycles may serve as more appropriate starting scaffolds in designing cancer therapies targeting A3s, which would mimic the “U” shape of the bound ssDNA. Macrocycles have recently been shown to have good drug-like properties and may be a strategy to target these critical enzymesThe pColdII His-6-SUMO-A3A(E72A) was constructed by first cloning the SUMO gene from pOPINS His-6-SUMO into pColdII His-6 vector (Takara Biosciences) using NdeI and KpnI restriction sites. Human APOBEC3A coding sequence from pColdIII GST-A3A was then cloned into the pColdII His-6-SUMO vector with KpnI and HindIII. The C171A mutation in the A3A construct was reverted to wild type residue by site directed mutagenesis resulting in the pColdII His-6-SUMO-APOBEC3A(E72A) catalytically inactive over-expression construct used in this study.Escherichia coli BL21 DE3 Star (Stratagene) cells were transformed with the pColdII His-6-SUMO-APOBEC3A(E72A) vector described above. The E72A mutation was chosen to render the protein inactive. Expression occurred at 16 °C for 22 hours in lysogeny broth medium containing 0.5 mM IPTG and 100 µg/mL ampicillin. Cells were pelleted, re-suspended in purification buffer  and lysed with a cell disruptor. Cellular debris was separated by centrifugation . The fusion protein was separated using HisPur Ni-NTA resin (Thermo Scientific). The His-6-SUMO tag was removed by means of a Ulp1 protease digest overnight at 4 °C. Untagged A3A(E72A) was separated from tag and Ulp1 protease using HisPur Ni-NTA resin. Size-exclusion chromatography using a HiLoad 16/60 Superdex 75 column  was used as a final purification step. Purified recombinant A3A was determined to be free of nucleic acid prior to binding experiments by checking OD 260/280 ratios, which was at 0.54.Labeled and unlabeled oligonucleotides used in this assay were obtained through Integrated DNA Technologies (IDT). Labeled oligonucleotides used in the fluorescence anisotropy based binding assay contain a 50-TAMRA flourophore at their 5′ end and were resuspended in ultra-pure water at a concentration of 20 µM. Unlabeled oligonucleotides used for the competition assays were resuspended in ultra-pure water to a concentration of 4 mM.28 with minor alterations. A fixed concentration of 10 nM 50-TAMRA-labeled oligonucleotides was added to A3A(E72A) in 50 mM MES buffer (pH 6.0), 100 mM NaCl, 0.5 mM TCEP in a total reaction volume of 150 µL per well in nonbinding 96-well plates (Greiner). The concentration of A3A was varied in triplicate wells. Plates were incubated for overnight at room temperature.Fluorescence anisotropy based DNA binding assay was performed as describedFor the pH dependence experiments the buffer reagent used for testing was pH 4.0–5.0 sodium acetate, pH 5.5–6.5 MES, pH 7.0–8.0 HEPES, pH 8.5–9.0 TRIS. Assay was performed as described above. For the competition assays, a fixed concentration of 300 nM A3A(E72A) was used and unlabeled oligonucleotide of varied concentration was added from 0–6.1 μM. A3A(E72A) was pre-incubated with unlabeled oligonucleotide for an hour in assay buffer, then labeled DNA was added and incubated overnight at room temperature.d values, Prism (GraphPad) was used for least-square fitting of the measured fluorescence anisotropy values (Y) at different protein concentrations (X) with a single-site binding curve with Hill slope, a nonspecific linear term, and a constant background using the equation d is the equilibrium dissociation constant, h is the Hill coefficient, and Bmax is the extrapolated maximum anisotropy at complete binding.For all experiments, fluorescence anisotropy was measured using an EnVision plate reader (PerkinElmer), exciting at 531 nm and detecting polarized emission at 579 nm wavelength. For analyzing data and determining K1H NMR. Active A3A protein (50 nM) was assayed against linear DNA substrates (200 µM) in buffer with 50 mM MES pH 6.0, 100 mM NaCl, 0.5 mM TCEP, and 5% D2O. Experiments were performed on 9-mer substrates containing the target sequences AA(A/G/T)TC(A/G/T)AAA and at 40 °C to prevent the DNA from oligomerizing due to high concentration. Experiments were performed using a Bruker Avance III NMR spectrometer operating at a 1H Larmor frequency of 600 MHz and equipped with a cryogenic probe. Product concentration was estimated from peak integrals with Topspin 3.5 software  using an external standard. Activity was determined from the initial rate of product formation via first-order exponential fitting of the progress curve. Rate errors were estimated by Monte Carlo simulation using 100 synthetic data sets and taking the residuals of the initial fit to the experimental data as the concentration error.Deaminase activity was determined for A3A protein by assaying active enzyme against linear DNA substrates and measuring the product formation using 22. The DNA sequence was first mutated using Coot39. The complex structure was then prepared, energy minimized with ProteinPrep Wizard in Maestro (Schrödinger) using the OPLS3 force field, at pH 6.0 with all other settings kept as default.The crystal structures of A3A bound to ssDNA (PDB ID: 5KEG and 5SWW) were used for molecular modelingSupplementary Information


Cognitive hearing science is a relatively new field, which developed in response to an increasing awareness of the critical role of cognition in communication  was to encourage submissions that could push the field forward by suggesting behavioral and neural mechanisms that are important for online language processing, and for long-term cognitive change. Each of the 34 papers that are included in this research topic have contributed toward meeting this goal, and to furthering our understanding of the complex interplay between cognition and language. In addition to papers reporting original research, the research topic also includes both review and opinion and theory articles, giving us not only new empirical evidence, but novel approaches and theories drawn from existing knowledge and data.Carroll et al. showed that age-related differences in lexical access efficiency modulates successful speech recognition in noise in listeners with normal hearing, even when vocabulary size is matched between younger and older listeners. Karawani et al. investigated auditory perceptual learning in older adults with and without hearing loss. While both groups of listeners showed significant improvements on the trained conditions (compared to untrained listeners), generalization to non-trained tasks was limited. Heinrich et al. compared behavioral and self-report measures of aided speech perception in older adults with mild-to-moderate hearing loss. The results suggest that behavioral and self-report measures correlate more highly when they relate to similar speech situations; and that only behavioral speech perception measures correlate with cognition. Two articles focussed on sentence comprehension in older adults; DeCaro et al. report that once hearing acuity and working memory capacity have been accounted for, age does not significantly predict comprehension accuracy. Amichetti et al. were interested in whether aging and/or hearing loss affected the extent to which listeners relied on formal syntax vs. plausibility to successfully comprehend sentences. Like DeCaro et al., Amichetti et al. also found that  age did not significantly predict comprehension once hearing acuity and working memory capacity had been accounted for, but that age and hearing acuity may affect which comprehension strategy is used. Meister et al. investigated the effects of cognitive load on speech recognition with one or two competing talkers in older listeners with and without hearing loss. The results showed that listeners with hearing loss performed particularly poorly, and demonstrated a different pattern of errors, in conditions with two (compared to one) competing target talkers. These differences are attributed to impaired object formation and an increased demand on working memory.Many of the papers included in this research topic focus on the impact of aging on cognition and language. Souza et al. investigate the relation between hearing aid processing and working memory, concluding that evidence for a link between memory and wide-dynamic range compression is strong, yet further research is needed to investigate the links between working memory and other hearing aid processing strategies. Pisoni et al. also highlight the need for more research, in this case into the cognitive factors predicting speech and language outcomes in cochlear implant users, suggesting that research into basic domain-general learning abilities is particularly lacking.In their review article, Moulin and Richard investigated the role of context and lexical factors on a standard clinical spondaic word recognition in quiet task in 160 adult listeners with hearing loss. Their results indicate that the use of context decreased with hearing loss once the pure tone average exceeded 55 dB HL, and that there is a significant age effect on the relation between word recognition and word frequency. Koch et al. were also interested in the clinical and real-world utility of the speech materials used in their study of acceptable noise level test outcomes. They found that acceptable noise levels were correlated with self-reported hearing problems, and that the repeatability of the acceptable noise level test was not affected by the use of more natural speech materials. Frtusova and Phillips also focussed on the impact of methodological choices on test outcomes, showing that older adults obtained benefit to performance in a working memory task when stimuli were audiovisual as opposed to audio-only. Both behavioral and electrophysiological measures indicated that this benefit was more pronounced for listeners with poorer hearing than for those with better hearing.Schneider et al. compared the short-term memory performance of older and younger native English speakers to a group of younger people for whom English was a second language. The results showed that older adults had poorer memory scores than younger adults, but that there was no difference in performance between the younger adults for whom English was a native language, and those for whom English was a second language. Schmidtke investigated the speech understanding in noise scores of monolingual English and Spanish-English bilingual young adults. The results indicated that speech understanding in noise improves with greater language exposure and that working memory does not provide additional predictive power, likely due to the large amount of variance shared between working memory and language proficiency. In their review, Calvo et al. argue that while many previous studies have observed that bilingualism does not affect working memory, certain aspects of working memory may be enhanced by bilingualism, yet methodological choices often mean that such improvements are difficult to observe.A number of papers in the research topic focussed on native and non-native language use, or bilingualism and its relation to cognition. Holmer et al. also investigated the effects of native and non-native language, but in signed rather than spoken language. The results are used as support for a developmental version of the Ease of Language understanding model, the D-ELU, which is outlined in the article. Rudner et al. also focussed on sign language by investigating the effects of load and distinctness on performance on a sign-based memory task by young adult listeners with no previous experience of sign language. The results showed that working memory load increased when sign distinctness decreased, providing support for an amodal mechanism active even when a pre-existing semantic representation is missing.Füllgrabe and Rosen caution against assuming that working memory has the same importance to speech-in-noise processing in young adults with normal hearing as it does in older adults with hearing loss. The results of their meta-analysis suggest that variations in working memory capacity account for only approximately 2% of speech-in-noise recognition scores in younger adults with normal hearing. In contrast, Hadar et al. report that working memory is of importance for speech processing in younger adults with normal hearing, using processing time as opposed to accuracy as an outcome measure in their eye-tracking study.Marsh et al.  report results from a study showing that background noise interferes with gist processing of spoken messages. Finally, Beaman and Jones discuss mechanisms involved in forgetting in short-term memory, focussing on different forms of overwriting.In relation to the interplay between communication, working memory and cognition, Murphy et al. looked at phonological and non-linguistic auditory training on the phonological skills of children with speech sound disorder. While neither training condition led to improvements in phonological skills, the non-linguistic auditory programme did lead to improvements in both auditory and cognitive measures. Söderlund and Jobs investigated differences in speech recognition thresholds in children with and without attention deficit hyperactivity disorder when exposed to noise. The findings indicated that children with attention deficit hyperactivity disorder had higher speech recognition thresholds than a control group, and that this difference disappeared when the children were exposed to white noise. Frölander et al. investigated theory of mind and executive function in adults with Alström syndrome (a genetic disorder associated with a variety of symptoms including vision and hearing loss), finding that the group with Alström syndrome performed significantly poorer than a control group in both types of task. Usher syndrome is another genetic disorder associated with hearing and vision loss, and was the focus of research reported by Henricson et al. The findings showed that adults with Usher syndrome performed more poorly than a control group on tests of phonological processing, and on tests involving fast visual or phonological processing. Henricson et al. suggest that these difficulties may contribute to explaining the high levels of fatigue often reported by individuals with Usher syndrome.A number of articles in this research topic report results pertaining to individuals with communication-related disorders other than hearing loss. Wagner et al. used pupillometry and response times to show that lexical competition is associated with effort, and that degraded speech affects the timing of information processing, leading to increased effort. McMahon et al. also used pupillometry, along with alpha power, to investigate listening effort. The findings showed that these two measures show similar trends when participants processed highly intelligible speech, but seemed to diverge with degraded speech. Wendt et al. were also interested in comparing different measures of listening effort, finding that pupillometry and subjective rating scales index different aspects of effort, and that the syntactic complexity of speech can affect effort even when intelligibility is high. Francis et al. also report differences in subjective and physiological measures of listening effort, finding that masking affects physiological measures to a greater extent than subjective measures of listening effort.Listening effort was the subject of a number of papers in the research topic. Cardin's review of the effects of aging and hearing loss on cortical auditory regions, parallels are drawn between the cortical mechanisms that are engaged when young listeners with normal hearing engage in effortful listening, and those that are engaged in all listening for older listeners and those with hearing loss. Marsh and Campbell were also interested in the neurophysiological mechanisms underlying auditory processing. Their article introduces the new early filter model which suggests that complex sounds are processed early on by a subcortical filter under cholinergic top-down control.In Kishida et al. reported work on the synthesis of intelligible noise-vocoded Japanese speech. Ruigendijk and Friedmann investigated the effects of hearing impairment on the comprehension and repetition of movement-derived sentences in German-speaking children. Heald et al. introduce a new framework for perceptual plasticity relating to auditory object recognition, focussing on its context-dependent nature. Skoog Waller et al. investigated estimates of a speaker's age, finding that speakers were estimated as having a younger age when the speech rate was fast, and an older age when the speech rate was slow.In addition to the themes outlined above, a number of other topics were the focus of articles included in this research topic. Taken together, the manuscripts included in this research topic represent important advances in the field of cognitive hearing science. Specifically, the papers demonstrate evidence of the involvement of cognition at all levels of speech perception, from basic word recognition through to comprehension. The variety of outcome measures included has enabled us to gain further insight into the impact of methodological choices on the likelihood of observing an effect of cognition, and indeed how the effect of these choices may vary depending on the participant group being tested. Further evidence is provided of the negative impact of hearing loss and aging on communication, and the role of cognition in ameliorating these negative effects. An important contribution of this topic is finding evidence for the importance of cognition on communication not just for older listeners or those with hearing loss, but also for listeners with a variety of communicative impairments, and indeed young listeners without hearing loss . We also see a lot of evidence for the moderating role of linguistic factors  on the relation between cognition and communication.Together these results provide support for five of the six predictions based on the ease of language understanding (ELU, see for example Rönnberg et al., We hope that this research topic will help to inspire many more studies in the field of cognitive hearing science. This area will continue to advance and contribute to both our understanding of the mechanisms underlying language understanding, and to improving assessment and treatment options for people with a communicative impairment.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hearing loss is a global public health problem with a high prevalence, significantly impairing communication and leading to a decrease in the quality of life. The association between moderate alcohol consumption (MAC) and hearing impairment has been addressed in several studies with inconsistent results. The intent of our study is to clarify the correlation between MAC and the hearing threshold and further investigate the interplay between MAC and the hearing threshold categorized by gender. The study included 4,075 participants aged 20–69 years from the 1999–2004 data of National Health and Nutrition Examination Survey (NHANES). The associations among MAC, gender differences, and high-frequency and low-frequency hearing thresholds were analyzed. We found that current female drinkers with MAC tended to have lower hearing thresholds. There is a significant protective effect of MAC on hearing threshold shifts in the US adult population, especially in females. Our research was the first study to further indicate that there is a gender difference in the association between MAC and hearing impairment. In accordance with our results, if people drink, they should consume moderate rather than higher amounts, especially in women, which may result in a reduced risk of hearing loss. The widely known causes of hearing loss include aging, exposure to noise, ototoxic agents, inherent genetic determination, diabetes, hypertension, and cardiovascular disease2. By analyzing the possible contributing factors to hearing loss, many studies attempted to identify the possible modifiable factors that can potentially help to prevent or slow the development of hearing loss7. Notably, alcohol consumption seemed to have a protective effect on hearing impairment.Hearing loss is a highly prevalent sensory disorder and global public health problem. It significantly impairs communication and leads to a decrease in the quality of life because it negatively affects social interaction, leading to progressive isolation and withdrawal8. The plausible mechanisms about the protective effect of alcohol consumption on cardiovascular disease include increasing levels of plasma high-density lipoprotein (HDL) cholesterol10, improving insulin sensitivity12, the potential salutary effects of endothelial function14, fibrinolysis/coagulation16, and anti-inflammatory action18.The most impressive effect of alcohol consumption is its protective association with cardiovascular disease. The accumulating evidence of consistent observational and mechanistic studies reveals that moderate alcohol consumption  has beneficial effects on cardiovascular disease20, some found a beneficial effect or negative association26, and others observed no remarkable association29. Different study methods or purposes or different definitions of alcohol consumption might have been responsible for these inconsistent results. In general, the protective effect of alcohol consumption on hearing impairment was addressed, and the decreased protective effect or increased risk of hearing loss was noted regarding alcohol consumption at higher levels than moderate levels.The association between alcohol consumption and hearing impairment has also been reported in several research works. These studies appear to contradict one another; some of them noted a detrimental effect or positive association32. Compared with men, women develop cirrhosis of the liver and more cognitive and motor impairment at lower levels of exposure to alcohol33. Gender differences in alcoholic cardiomyopathy, alcohol related behavioral or medical problems, and alcohol use and misuse in adults have also been reported34. Within the extensive research on alcohol consumption, comparatively little has focused on the gender differences in the association between MAC and hearing impairment. Among the studies indicating that MAC had a protective effect on hearing impairment, some did not find different effects between women and men23, while others did not analyze gender effects24.Gender differences in the consequences of alcohol use have been extensively studied. Women are more susceptible to the effects of alcohol consumption than men because women have higher blood alcohol concentrations after drinking equivalent doses of alcohol per kilogram of body weightBy studying the possible beneficial or risk factors for hearing impairment, we would be able to understand the etiologic factors of hearing loss, develop ideas for basic research, and make public health efforts for hearing preservation. Knowledge regarding the MAC, hearing impairment, and gender differences is rare. Given the theoretical positions taken for the study and the status of the field as briefly reviewed above, the aim of our study was to further clarify the interactions between MAC, hearing thresholds, and gender distribution using the National Health and Nutrition Examination Survey(NHANES) dataset.The present study included 4,075 participants obtained from the 1999–2004 NHANES dataset. The clinical and demographic characteristics of the study group were analyzed according to the patterns of alcohol consumption 2. Some studies proposed that the beneficial cardiovascular effects of alcohol consumption lead to the decreased risks of hearing loss25. Second, alcohol consumption may increase plasma HDL cholesterol concentrations10, contribute to better endothelial function14, and reduce coagulation16, which contribute to an optimal cochlear circulation and an intensifying anti-atherosclerotic condition and result in decreased hearing impairment. Third, alcohol consumption had anti-inflammatory action and neuroprotection35 that may help to enhance cellular survival of the cochlea. Finally, a U-shape association between alcohol consumption and hearing impairment was reported and the lack of enhanced benefits of higher levels of alcohol consumption or AMAC on hearing impairment is likely due to the adverse effects on other risk factors for hearing loss24.The protective effects of alcohol consumption on hearing impairment may occur through several possible pathways or mechanisms. First, as far as we know, cardiovascular disease is associated with hearing loss36, which is characterized by histopathologic findings with atrophy of the stria vascularis in the whole cochlea and by clinical results of decreased hearing sensitivity across the whole frequency spectrum. In other words, the protective effect of MAC may be related to the involvement of the stria vascularis, affecting the microvasculature of the cochlea. Notably, this is consistent with the possible mechanism about the protective effects of alcohol consumption that we described in the second point above; further study is required to investigate whether the stria vascularis of the whole cochlea is affected by alcohol consumption.The tonotopic distribution of the cochlear hair cells leads to the base part of the cochlea responding to high frequencies and the apical part to low frequencies. We found a protective effect of MAC on the hearing threshold shift in both low and high frequency. It indicated this effect was influencing the whole cochlea. It was consistent with the strial hearing loss proposed by Schuknecht37. Current drinkers with MAC were observed to pay more attention to their health using preventive care or improving their health behaviors than never and heavy drinkers38. Other confounding factors from MAC, such as socioeconomic status, have also been suggested39.Lifestyle variables may also be confounding factors of the hearing protective effects between current drinkers with MAC and never drinkers. Emerging studies have reported that never drinkers had a higher body mass index, less regular exercise, lower vegetable consumption, and higher fat intake comparing with all drinkers32. There are several differences between men and women in the pharmacokinetics of alcohol. Women have lower gastric alcohol dehydrogenase; thus, the first stage of alcohol metabolism is decreased and more alcohol is absorbed into the blood stream31. Women have a higher percentage of body fat32 and a lower percentage of body water than men40; thus, each drink is more concentrated in women’s blood circulation. Therefore, gender differences should always be considered when studying the effects of alcohol consumption.The gender differences in the association of MAC and hearing threshold shifts is likely due to innate biological differences. Women are more susceptible to the effects of alcohol consumption than men because women have higher blood alcohol concentrations after drinking equivalent doses of alcohol per kilogram of body weightThere are some limitations in our study. First, causality cannot be claimed because of the cross-sectional design of our study. Second, we could not exclude potential recall bias for information about the frequency and quantity of past and current alcohol consumption and other personal and medical history data. Third, the questions in the Alcohol Use Questionnaire are about drinking alcoholic beverages, including liquor, wine, beer, and any other type of alcoholic beverage. The types of alcoholic beverages cannot be differentiated in our dataset. Fourth, there was no information about genetic or congenital hearing impairment in the NHANES data.Our study highlighted that there is a significant protective effect of MAC on hearing threshold shifts in the US adult population, especially in female individuals. This study may lead to a better understanding of the interplay between gender differences, MAC, and hearing threshold shifts. If people drink, they should consume moderate rather than higher amounts, especially in women, which may result in a reduced risk of hearing loss. Further research is warranted to clarify the benefit of MAC on hearing threshold shifts and gender differences.NHANES is a special cross-sectional survey that combines personal interviews, standardized physical examinations, and numerous laboratory studies. It was directed by the Centers for Disease Control and Prevention’s National Center for Health Statistics and designed to gather information about the health, nutritional status, and health behaviors of the non-institutionalized civilian resident population of the United States. Starting in 1999, it became an ongoing annual survey; the data sets have been released every two years on the NHANES website.We limited our study subjects to 1999–2004 NHANES individuals who had completed both the personal questionnaires about alcohol consumption and audiometric examinations. Individuals were excluded if they had current or past occupational noise exposure and if their relevant data were incomplete. The final analytic data sets included 4,075 subjects aged 20–69 years, including demographic data, the results of physical investigations and laboratory tests, questionnaire information, and audiometric examinations.41.According to the 1999–2004 NHANES protocol, half of the included participants were randomly arranged for audiometric examinations after household personal interview and medical examinations. The participants were not included if they could not remove their hearing aids or bear with the headphones. Audiometric measurement was performed in a mobile examination center’s sound-isolated room by technicians who had been trained by a qualified audiologist from the National Institute for Occupational Safety and Health. Audiometric equipment included an interacoustic model AD226 audiometer with EARTone 3 A insert earphones and standard TDH-39P headphone. Each ear was tested from 500 to 8000 Hz with an intensity-range of −10 to 120 dB to determine the air-conduction pure tone hearing thresholds. The average of pure tone hearing thresholds at 3,000, 4,000, 6,000, and 8,000 Hz was defined as the high-frequency pure-tone average (High-PTA). The average of pure tone hearing thresholds at 500, 1,000, and 2,000 Hz was defined as the low-frequency pure-tone average (Low-PTA). The worse ear with the lower pure-tone average was chosen for the regression analysis42. We classified individuals into the following four groups44: (I) lifetime abstainers- < 12 drinks in entire life; (II) former drinkers− ≧ 12 drinks in the past but none during the past 12 months; (III) current drinkers with MAC- < 0 to ≦2 dkspd in men and <0 to ≦ 1 dkspd in women during the past 12 months; and (IV) current drinkers with AMAC- > 2 dkspd in men and >1 dkspd in women during the past 12 months.The Alcohol Use Questionnaire (ALQ) in NHANES was designed to collect information about the frequency and quantity of past and current alcohol consumption. The definition of a drink was an ounce of liquor, a 4-ounce glass of wine, or a 12-ounce beer. The variable of drinks per day (dkspd) was calculated by the ALQ dataDemographic data, including age, gender, race, and medical history were collected. Race was classified as non-Hispanic white, non-Hispanic black, or other. Individuals were considered to have diabetes if there was a physician’s diagnosis by self-report medical history, random and fasting glucose level greater than or equal to 200 and 126, respectively, and/or the use of diabetic medications. Heart disease was considered positive if an individual had a diagnosis of or had experienced congestive heart failure, coronary artery disease, myocardial infarction or angina. Stroke was ascertained by self-reports. The present use of ototoxic medication, including non-steroidal anti-inflammatory drugs, anticancer drugs, aminoglycoside, or loop diuretics was defined by self-reports. Waist circumference was determined at the top of the hip bone after breathing out normally to the nearest 0.1 cm. Abnormal otoscopy was recorded if there were abnormalities in screening otoscopic examination of the eardrums and ear canals before the audiometric measurement. Tympanometry was determined as abnormal if the compliance was less than or equal to 0.3 ml or middle ear peak pressure was less than or equal to −150 daPa using an audiometric tympanometer. Measurement of serum uric acid was determined using a Hitachi 737 analyzer. The level of total cholesterol was measured using a Hitachi 704 analyzer. The level of serum fasting glucose was measured using the Cobas Mira assay. All associated information about biospecimen collection and processing protocols is available on the NHANES website.41. First, age, gender, and race were adjusted in model 1. Model 1 subsequently adjusted for waist circumference, uric acid, fasting glucose, and total cholesterol was model 2. Model 2 further adjusted for abnormal otoscopy and tympanometry, current use of ototoxic medication, history of diabetes, heart disease and stroke was model 3.SPSS  was used to perform the statistical analyses. The significant differences were defined as two-sided p-values < 0.05. Covariates, including age, waist circumference, uric acid, fasting glucose, and total cholesterol were managed as continuous variables and presented as the mean value with standard deviation. Other covariates, such as gender and race were treated as categorical variables and expressed as numbers with percentages. Logarithmic transformation was performed for the values of the PTA hearing thresholds to approximate a normal distribution. Our study used patterns of alcohol consumption-base analysis by dividing participants into four groups with the lifetime abstainers as the referent group. The linear regression model was used to evaluate the effect of patterns of alcohol consumption on the hearing threshold. A multiple-model analysis was performed for adjustments of covariatesThe Research Ethics Review Board of National Center for Health Statistics approved the NHANES study and underwent annual review. All eligible subjects provided their written informed consent before participating the study. Because de-identified data were used in our study, it was exempt from IRB review.


Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ≥1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC.HIGHLIGHTSM. hyopneumoniae.Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of The adhesive effect of EF-Tu is partly meditated by fibronectin. Mycoplasma hyopneumoniae is the primary pathogen responsible for swine enzootic pneumonia. This infection is highly prevalent (ranging between 38 and 100%) in almost all areas of pig production worldwide, and causes significant economic losses , which is one of the target tissues of M. hyopneumoniae. Importantly, the results of this study could facilitate uncovering new biological activities or unknown functions of known proteins that can lead to a more complete understanding of the virulence-associated proteins and the complex biological and infectious processes taking place during infection. For example, the canonical function of the cytoplasmic protein, EF-Tu is acting as a GTPase on the binding of aminoacyl-tRNA to the ribosome-mRNA complex during translation elongation in protein synthesis . All experimental procedures conformed to the guidelines of Jiangsu Province Animal Regulations (Government Decree No. 45) in accordance with international law.M. hyopneumoniae strain 168 (GenBank accession CP002274) was originally isolated in 1974 from an Er-hua-lian pig  exhibiting typical clinical and pathogenic characteristics of swine mycoplasmal pneumonia in Gansu Province, China  containing 20% (v/v) swine serum at 37°C  and inoculated with M. hyopneumoniae strain 168. A series of 1:10 dilutions of cultures in broth with a metabolic indicator was used to estimate titers of M. hyopneumoniae strain 168. The dilution of the last tube to show growth was taken as the number of CCU (color change unit)  = 20] were washed with sterile PBS and resuspended in RPMI-1640 medium with 2% (v/v) FBS and incubated with STEC for 48 h. Supernatants were collected from each well for M. hyopneumoniae separation. M. hyopneumoniae strain 168 was cultured in RPMI-1640 medium with 2% (v/v) FBS in 24-well plates for use as the control. The assay was performed three times.STEC were seeded in 24-well plates and cultured in RPMI-1640 medium  supplemented with 10% (v/v) fetal bovine serum . The cells were grown at 37°C in 5% COTM Protein Assay Kit .The obtained culture was centrifuged at 12,000 rpm for 20 min at 4°C. The precipitates were washed three times with sterile 10 mM Tris-HCl (pH 7.4) and resuspended in 30 μL protein extract . After vortexing for 15 s, the mixture was placed in an ice bath for 30 s, and the procedure was repeated for 15 min. The whole protein in the supernatant was obtained by centrifugation at 12,000 rpm for 30 min at 4°C. A ReadyPrep 2-D cleanup kit  was used to clean up the protein samples. The protein concentration was determined using a BCAThe purified proteins were quantified and redissolved with 350 μL rehydration solution , and then centrifuged  to remove insoluble components. The samples were loaded into a 17-cm strip , and positively rehydrated at 50 V for 12 h at 20°C. Isoelectric focusing (IEF) was then carried out at 20°C as follows: slow to 250 V for 1 h; rapid to 1,000 V for 1 h; linear to 10,000 V for 3 h; rapid to 10,000 V to total 90,000 Vh; rapid to 500 V for the preservation of samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to a previously described protocol  analysis . Peptide mass fingerprinting data were analyzed using the MASCOT server (http://www.matrixscience.com). Peptides with a rank of 1 in the MASCOT search were considered significant and used for the combined peptide score.After Coomassie blue staining, the protein spots from each gel were detected and matched automatically by the PDQuest V8.0 software followed by additional visual analysis. The intensity of individual spot for each gel was normalized relative to total valid spot intensity to eliminate gel-to-gel variation. Only spots whose abundance changed by ≥1.5-fold and a value of M. hyopneumoniae and to filter the most important virulence associated factors, we summarized the putative virulence factors reported in the previously published papers , defining a “confidence score” that can be used to evaluate the confidence in the interaction. The interactions derived from textmining and experiments with a confidence score of at least 0.4 were considered for analysis. The protein-protein interaction network was visualized using Cytoscape (3.5.1).To determine how the differential proteins contribute to virulence in ef-tu gene encoding one of the most important novle virulence associated factors was cloned into pET-32a using homologous recombination technology . The sequence that overlapped with the end of the cloning site was added onto the insert through a PCR step. Table Escherichia coli BL21 (DE3) for isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression, and the induced proteins were purified by Ni-chelating chromatography . Polyclonal antibody against the recombinant EF-Tu (rEF-Tu) was prepared by subcutaneously immunizing 1-month-old New Zealand white rabbits. Each rabbit was immunized a total of three times with 1 mg of purified recombinant protein emulsified in Freund's adjuvant  at 2-week intervals. Sera were collected 1 week after the third immunization. The animals used in this study met the legal and ethical requirements and were treated humanely.The M. hyopneumoniae incubated with STEC is expressed as the percentage of that in M. hyopneumoniae cultured in KM2 medium and the three replicates were subjected to statistical analysis by SPSS 20.0.Equal amounts (40 μg for each lane) of the protein samples were separated on a 12% SDS-PAGE gel. The proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes  and developed with Ponceau-S as the loading control. After washing with TBST buffer  and blocking with TBST buffer containing 5% skimmed milk, the membranes were incubated with primary antibody against rEF-Tu , followed by horseradish peroxidase (HRP)-conjugated secondary antibody . Signals were detected with the ECL substrate  using the ChemiDoc XRS+ system . Image J software was used to calculate the optical density (OD) of the corresponding bands. The OD of EF-Tu from different samples was normalized to that of the Ponceau-S stained membrane. The level of abundance of EF-Tu in M. hyopneumoniae strains. In brief, three pathogenic M. hyopneumoniae strains, 168, NJ and WX (108 CCU) were incubated with anti-rEF-Tu primary serum at a 1:100 dilution (preimmune rabbit serum was used as the negative control). The blank control was incubated with PBS instead of antibody. Then, M. hyopneumoniae cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-IgG. Finally, the fluorescence intensity was detected using a flow cytometer (BD Accuri® C6)  were washed three times with PBS and pre-incubated with the polyclonal antibody (1:20 dilution) against rEF-Tu at 37°C for 30 min. M. hyopneumoniae that had been pre-incubated with preimmune sera (1:20 dilution) were used as the control. The bacterial suspension in RPMI-1640 medium was added to each well of 24-well cell plates with confluent STEC, and the plates were centrifuged at 800 × g for 10 min and incubated at 4°C for 2 h. Following incubation, the wells were washed three times with PBS to remove unbound M. hyopneumoniae cells. Then the cells in the wells were treated with lysis buffer containing 0.1% trypsin and 0.025% (v/v) Triton X-100, followed by bacterial genome extraction and real-time PCR for bacteria counting. Real-time PCR was performed using a QuantStudio® 5 Real-Time PCR System according to a previous method, with a slight modification  was performed. Total 20 μg recombinant proteins, including rEF-Tu and a negative control MRP-D2, which was previously verified not to bind to fibronectin  experiment on a Biacore™ X100 Plus instrument . Fibronectin was diluted to 10 μg/mL in 10 mM sodium acetate (pH 4.0) and covalently linked to the carboxylmethylated dextran matrix of a sensor chip CM5 as ligands using an amine coupling kit (Biacore AB). Immobilization of soluble fibronectin generated resonance units (RU) of 2868. The binding kinetics was measured with the increasing concentrations (0–200 μg/mL) of the analyte (rEF-Tu) in running buffer  surfactant P20]; Biacore AB) at a flow rate of 30 μL/min for 180 s over immobilized fibronectin at 20°C. The dissociation phase was monitored for 1,000 s by allowing buffer to flow over the chip. Association kinetics were analyzed manually using Biacore™ X100 Control Software  were washed three times with PBS. The bacteria resuspended in RPMI-1640 medium was added to the 24-well cell plates and incubated at 4°C for 2 h. After incubation, the wells were washed three times with PBS to remove unbound M. hyopneumoniae cells. Then the cells in the wells were treated with lysis buffer containing 0.1% trypsin and 0.025% (v/v) Triton X-100. The following bacterial genome extraction and real-time PCR for bacteria counting were conducted according to the method in section Adherence Inhibition Assay of Antibody Against rEF-Tu. The assay was performed in triplicate, and the data were analyzed using Student's t-test using SPSS 20.0. A value of p ≤ 0.05 was considered statistically significant.The confluent STEC in 24-well cell plates was washed three times with PBS and pre-incubated with the antibody against fibronectin (1:20 dilution) at 37°C for 30 min. The RPMI-1640 medium was used instead of fibronectin antibody as the negative control. M. hyopneumoniae-infected STEC were subjected to MALDI-TOF-MS/MALDI-TOF-TOF-MS analysis. Six novel differential abundance proteins were successfully identified. They were YX2, pyruvate dehydrogenase E1-alpha subunit, EF-Tu, hypothesis protein MHJ-0662, enolase, and adenine phosphoribosyltransferase. Among them, EF-Tu showed a relatively higher expression in M. hyopneumoniae-infected STEC in 2D gels  in the M. hyopneumoniae virulence and to filter the most important virulence associated factors. The results revealed a total of 32 direct physical interactions among the 14 nodes  by which the EF-Tu surface protein affected virulence, we used indirect immunofluorescence to determine whether or not rEF-Tu could adhere to STEC. Significant fluorescence was detected on the cell surface of STEC incubated with rEF-Tu Figure ; meanwhiM. hyopneumoniae. Polyclonal antibody against rEF-Tu and preimmune sera were used to incubate with M. hyopneumoniae cells respectively before adherence to STEC. The level of adherence of M. hyopneumoniae incubated with anti-rEF-Tu sera is expressed as the percentage of M. hyopneumoniae adherence with preimmune sera. The results revealed that after incubation with anti-rEF-Tu antibody, the adherence efficiency of M. hyopneumoniae to STEC showed a 46% (P < 0.05) reduction compared to that incubated with preimmune sera  and to fibronectin, while no specific reaction was observed in the negative control MRP-D2, a non-related recombinant protein which was verified not to bind to fibronectin in a previous study  reduction compared to that incubated without antibody . These results demonstrate that M. hyopneumoniae may increase abundance of EF-Tu to help better attach to the host cells to some extent. Thus, M. hyopneumoniae EF-Tu, in addition to its major cytoplasmic, biosynthetic, and metabolic roles, can translocate to the surface and moonlight as an important adhesion factor.EF-Tu is the most abundant bacterial protein . Interaction with the ECM components, with the most common being fibronectin, may influence the initiation, facilitate colonization, and establishment of infection, and express important virulence factors (Patti and Höök, M. hyopneumoniae adherence to cell surface significantly. Immunohistochemical staining of bronchioles showed that fibronectin is more abundant in the ciliary borders of the bronchioles, which is a prefered place for M. hyopneumoniae colonization. Thus, fibronectin was verified one of the host cell receptors for M. hyopneumoniae adhesion.Using far-WB analysis, rEF-Tu was found to be able to bind to fibronectin. The dissociation constant of rEF-Tu determined by SPR displayed values with a specific and moderately strong interaction. Furthermore, block of fibronectin in STEC decreased M. hyopneumoniae. Further analysis revealed that M. hyopneumoniae displays EF-Tu on the surface to bind to host fibronectin with a moderately high affinity, thereby promoting adherence to STEC. This binding was markedly reduced by pretreatment of bacterial cells with anti-rEF-Tu sera or pretreatment of STEC with anti-fibronectin antibody. The binding between EF-Tu with fibronectin was proven to contribute to the adhesion of M. hyopneumoniae to STEC.In summary, using system-wide methodologies, EF-Tu was found to act as an important hub in the virulence-associated network of M. hyopneumoniae, it can be speculated that when EF-Tu is blocked, the adhesion of M. hyopneumoniae still partly exists.However, in the competitive adhesion inhibition assay, only partial inhibition of EF-Tu was observed. Since several other proteins, such as P97 (Zhang et al., M. hyopneumoniae virulence. A lot remains to be understood about how proteins, especially those lacking signal motifs, localize on bacterial cell surfaces. M. hyopneumoniae moonlighting proteins and their roles in infection and immunity should be further studied in the future.In conclusion, moonlighting proteins located in various parts of the cell, not only in the cytoplasm but also on the cell membrane, have been suggested to be associated with YY completes the study of the pathogenic mechanism of EF-Tu and prepares the manuscript. The comparative proteomics analysis is done by HW. JW, and BL perform the cell adhesion experiments. MW prepares the polyclonal antibody. ZF, JX, and GS modify the manuscript. QX and ML supervise and guide this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intracellular signaling through cyclic nucleotides, both cyclic AMP and cyclic GMP, is altered in colorectal cancer. Accordingly, it is hypothesized that an underlying mechanism for colorectal neoplasia involves altered function of phosphodiesterases (PDEs), which affects cyclic nucleotide degradation. Here we present an approach to evaluate the function of selected cyclic nucleotide-PDEs in colonic endoscopic biopsies from non-neoplastic appearing mucosa.Biopsies were obtained from patients with and without colorectal neoplasia. Activities of PDEs were characterized functionally by measurements of transepithelial ion transport and their expression and localization by employing real-time qPCR and immunohistochemistry.p = 0.006). Furthermore, real-time qPCR analysis showed overexpression of subtype PDE4B (p = 0.002) and subtype PDE5A (p = 0.02) in colorectal neoplasia patients. Finally, immunohistochemistry for 7 PDE isozymes demonstrated the presence of all 7 isozymes, albeit with weak reactions, and with no differences in localization between colorectal neoplasia and control patients. Of note, quantification of PDE subtype immunostaining revealed a lower amount of PDE3A (p = 0.04) and a higher amount of PDE4B (p = 0.02) in samples from colorectal neoplasia patients.In functional studies PDE subtype-4 displayed lower activity in colorectal neoplasia patients  contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is a major global health problem and has one of the lowest survival rates for all types of cancers . Colorecde novo disease development. The exact mechanism by which NSAIDs exert their anticarcinogenic effect is not completely understood [The pathogenesis and etiology of CRC and CRN are still not fully understood. With regards to CRC, non-steroidal anti-inflammatory drugs (NSAIDs) decrease mortality rate and inhibit derstood , 4. Invederstood , 6.Recently some COX-independent targets have been identified as being involved in the anticancer effects of NSAIDs , 7–10. MStudies on human colon cancer cell lines show decreased levels of cGMP and elevated levels of PDE5 mRNA , 14, indIn order to identify a potential predisposition in colonic mucosa for development of CRN in non-neoplastic colon, we decided to study the function, expression and localization of several PDE subtypes in specimens of endoscopically non-neoplastic appearing colonic mucosa.The aim of this study was to investigate function, expression and localization/abundance of selected PDEs in biopsies from non-neoplastic appearing colonic mucosa obtained from patients with and without CRN. Thus, questioning if a possible predisposition to the cancer disease exists in non-neoplastic appearing colonic mucosa.Patients referred for a colonoscopy on suspicion of CRC, were included and divided into two groups. The first group consisted of patients with present or history of CRN, while the second group was control patients (i.e. CTRL) with no present or history of CRN. Patients with incomplete colonoscopy, hemorrhagic diathesis, and inflammatory bowel disease or with previous sigmoid resection were excluded from the study. A total of 27 subjects were enrolled, hereof 12 CRN-subjects (4 women) and 15 CTRLs (7 women). For each patient, we noted age, body mass index (BMI), previous illnesses, medication, all signs of earlier colorectal disease and the findings during the colonoscopy. Age and BMI were well balanced between groups. Mean age (±SEM) for CTRL patients was 58 ± 3.7 and for CRN patients 66 ± 3.7 years. Mean BMI was 24.9 ± 1.5 in CTRL patients and 26.2 ± 0.9 in CRN patients. Eight patients from the CTRL and 6 patients from the CRN group had comorbidities such as ischemic heart disease, heart failure, hypertension, atrial fibrillation, diabetes, kidney failure, chronic obstructive lung disease, dyslipidemia, intermittent claudication, peptic ulcer disease, osteoporosis, rheumatic arthritis, polycystic ovary syndrome and pacemaker implantation. There were no apparent differences between the two groups in comorbidity. Eight patients in the CTRL group and 8 patients in the CRN group were using regular medications e.g. anti-thrombotic, ACE inhibitors, statins, levothyroxine, proton pump inhibitors, angiotensin II receptor antagonists, glucocorticoids, β-blockers, β-agonists, anti-histamines, xanthine oxidase inhibitors, diuretics, antifolate and anti-emetics. There were no apparent differences between the two groups in prescribed medications. None of the drugs has a direct effect on the PDE metabolism.The Scientific Ethical Committee of Copenhagen (H32013107) and The Danish Data Protection Agency approved the study protocol . The study was conducted by the Helsinki declaration. All patients participating gave written informed consent.+ (140), Cl− (117), K+ (3.8), PO43− (2.0), Mg2+ (0.5), Ca2+ (1.0), glucose (5.5) and HCO3− (25). Before obtaining biopsies, the media pH was adjusted to 7.4 by gassing with 95% O2/5% CO2.Six biopsies from each patient were obtained during endoscopy from non-neoplastic appearing colonic mucosa using standard biopsy forceps . Biopsies were obtained approximately 30 cm orally from the anal verge and at least 10 cm from endoscopically abnormal tissue (i.e. neoplasia). The biopsies were immediately transferred to an iced, oxygenized bicarbonate Ringer solution with the following composition (in mM): NaTheophylline, indomethacin, acetazolamide, zaprinast, cilostamide, rolipram, cAMP and cGMP were purchased from Sigma-Aldrich . Sildenafil, db-cAMP, cGMP, and antibodies for PDE3B (cat. no.: SC-376823), PDE4A (cat. no.: SC-74428), PDE4B (cat. no.: SC-25812), PDE4D (cat. no.: SC-25097) and PDE5A (cat. no.: SC-32884) were purchased from Santa Cruz Biotechnology . Antibodies for PDE3A (cat. no.: LS-A732) and PDE4C (cat. no.: LS-C185777) were purchased from LifeSpan BioSciences, Inc. . Primer sequences were synthesized by TAGCopenhagen . The human PDE10 real-time PCR primers were purchased from Qiagen. Bumetanide was obtained from Leo Pharmaceuticals . All other chemicals were of analytical grade.Up to 4 biopsies from each patient were mounted in Mini-Ussing-Air-Suction-Chambers (MUAS chambers) for measuring transepithelial electrolyte transport captured as short circuit currents (SCC). The method used has previously been described in detail , 22. CorIn one part of our functional study, db-cAMP (16 μM) was applied on the basolateral side of biopsies. After 10 min, biopsies were then exposed to cilostamide  or rolipram . PDE3 and PDE4 primarily degrade cAMP , 24, andIn a second part of the functional study, cGMP (16 μM) was applied to biopsies. Previous reports have provided data showing more consistent responses when cGMP is applied vs db-cGMP . After 1260/A280 and A260/A230 absorbance ratios.One biopsy from each patient was obtained for RNA-analysis. The mean wet weight of each biopsy was 5.8 mg. Thus we were only able to perform qPCR on a selected number of PDE isoforms. Biopsies were immediately transferred to RNA Later . Biopsies were homogenized using a TissueLyser II , and RNA was extracted using NucleoSpin RNA® . RNA concentration and purity were determined using a NanoDrop® ND-1000 , and the latter as the ARNA was converted to cDNA using the iScript™ cDNA Synthesis Kit  according to the manufacturers protocol.http://frodo.wi.mit.edu/primer3/input.htm) based on sequences obtained from Ensembl (http://www.ensembl.org/). The primer sequences were synthesized by TAGCopenhagen : PDE3A forward (5′-CAGAGTGAATCCCGTCACTTC-3′) reverse (5′-TGGTCCAAGTGGAAGAAACTC-3′); PDE3B forward (5′-TATGATCACCCAGGGAGGAC-3′) reverse (5′-GTTGTATTCTGGGCGAGAAAG-3′); PDE4A forward (5′-TGAAGACCGATCAAGAAGAGC-3′) reverse (5′-GTAATCCGACACGCAAAAGAT-3′); PDE4B forward (5′-ATCTCACGCTTTGGAGTCAAC-3′) reverse (5′-TTAAGACCCCATTTGTTCAGG-3′); PDE4C forward (5′-GCGATATCTTCCAGAACCTCA-3′) reverse (5′-CTTGTCACCTTCTTGGTCTCC-3′); PDE4D forward (5′-TTTCACGGTGGCACATACAT-3′) reverse (5′-GTGGACAAAATTTGCTTGGAG-3′); PDE5A forward (5′-TTGTGCAGAACTTCCAGATGA-3′) reverse (5′-TTTAGAGCAGCAAACATGCAC-3′); β-Actin forward (5′-ACCCAGCACAATGAAGATCA-3′) reverse (5′-CGTCATACTCCTGCTTGCTG-3′).Primers against genes of interest and against β-actin  were desDilution series of cDNA from HEK293 cells were run to verify acceptable amplification efficiencies and specificities by standard and dissociation curves for all primer sets.cDNA was amplified on a 7900HT Fast Real-Time PCR System  using Fast SYBR® Green Master Mix (Applied Biosystems) by the manufacturer’s manual. All samples were run in triplicates with β-actin primers on all plates. Results were analysed using SDS 2.3 (Applied Biosystems), and expression was calculated by the 2-ΔCT method.One colon biopsy from each patient was put aside in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), embedded in paraffin, and cut into 4 μm sections. Sections were boiled in citric buffer (pH 6 or 9) in a microwave oven for 15 min, followed by preincubation in 2% BSA for 10 min, and overnight incubation at 4 °C with primary antibodies. Subsequently, the sections were incubated for 40 min with biotinylated secondary antibody immunoglobulins, followed by a preformed avidin and biotinylated horseradish peroxidase macromolecular complex  for 30 min. Three percent hydrogen peroxide was added after the second antibody layer. Finally, 3,3-diaminobenzidine  was added for 15 min, followed by a 2 min incubation in 0.5% copper sulfate  diluted in Tris buffer containing 0.05% Tween 20. Counterstaining was performed with Mayer’s hemalun.ctrl = 7, NCRN = 8). Images for quantification were recorded at 20x magnification and the area measured represented 176000 μm2 of tissue. The area of stained structures was measured by selecting a colored region of interest. Automatically, areas with same color were measured - one image from each biopsy was measured. Mean ± SEM for samples in each group were calculated. Images for localization were recorded using a Zeiss Axio10 Imager A1 microscope  fitted with a Zeiss AxioCam ICc 3 camera  and analysis was performed using Image-Pro 9.1 software. Only mucosal layers were analyzed.Images for quantification were recorded using a Zeiss Axioplan 2 plus microscope  fitted with a Photometrics CoolSNAP camera  and analysis was performed using Image-Pro Plus 7.0 software. A quantification of coloring was performed by to blinded investigators as a marker for protein abundance in biopsies .Values are expressed as the mean ± SEM. The mean value was calculated and used when identical experiments were performed on several biopsies from the same patient. The statistical significance of differences between two groups was tested using Student’s p = 0.006)  or sildenafil (1 μM), (p = 0.899). No changes in SCC were recorded at even the highest concentrations of sildenafil or zaprinast. All biopsies presented an increase in SCC when exposed to theophylline. A decrease was observed when bumetanide and oubain were applied at the end of experiments, verifying the viability of samples to elicit a response. Up to 4 biopsies from each patient were successfully mounted in the MUAS chambers. At baseline mean SCC (±SEM) for CTRL and CRN patients was 68.2 ± 12.6 μA/cm2 and 78.2 ± 24.9 μA/cm2. During the observation period, slope conductance ranged between 36 and 150 mS⋅cm−2. To better understand the functional connection between SCC, PDE activity and cNT activity and inhibition, we present a simplified model  with higher SCC increases compared to CRN patients ( μM with 6) Figs. . This in6) Figs. . No signp = 0.002), and PDE5A had a 49% higher expression level in the same group (p = 0.02). PDE3A had an expression level more than 10 fold greater than all other investigated PDEs (p < 0.0001), both in CRN and CTRL patients  , the generated dataset is available as Additional file All 8 investigated PDEs were detected in biopsies by qPCR. Normalized to ß-actin levels, a significantly higher expression was measured for PDE4B and PDE5A in biopsies from CRN patients compared with CTRLs , and for PDE4B, 4276 ± 640 vs. 6723 ± 724 (p = 0.019), respectively. No difference was observed for PDE3B, PDE4A, PDE4C, PDE4D and PDE5A (Table Blinded quantification of specific coloring in immunohistochemical biopsies revealed a difference between CTRL patients and CRN patients for PDE3A, 11 403 ± 1209 vs. 8313 ± 762 (Many studies conducted with cancer cell lines have evaluated the importance of PDEs, although most studies only examined one or a few PDE subtypes. The general conclusion from these studies is, yes PDEs are perturbed in transformed cells or tissues , 28.Of note, to our knowledge, there are no studies on the importance of PDE subtypes in non-neoplastic colonic mucosa obtained from patient with CRN.Our study of PDEs provides support for a CRN predisposition with both perturbed activities of PDE4 and of expression of PDE4B and PDE5A subtypes likely involved in regulatory functions of normal colonic mucosa.Based on a review of the literature, we selected 3 families of the PDE superfamily, PDE3, PDE4, and PDE5 to be evaluated. For members of each of these PDE families, 3 aspects were studied – their activity, although indirectly by SCC, expression by employing qPCR, and tissue localization by immunohistochemistry. Whilst collecting data for our study a publication by Li N et al., Oncogene, reported elevated expression levels of PDE10A in both colon tumor cell lines and clinical tumor specimens. Therefore we conducted an additional q-PCR run examining expression levels of PDE10A in our normal appearing colonic mucosa from patients with CRN and compared it to CTRL. Expression levels of all the remaining PDE subtypes must await later studies, but is relevant to examine to fully asses the role of the entire PDE-family.Two key intracellular signaling molecules in enterocytes are cAMP and cGMP  and theiAbnormal levels of either cAMP or cGMP are shown to play a potentially pivotal role in initiating cancer development and for its progression or abrogation , 30–32. Although it may be argued that use of enzymatic assays and Western blots would be more direct approaches to measurements of PDE activity and abundance than SCC and qPCR, the latter methods carry advantages. Both SCC and qPCR are by far more sensitive and yield much higher accuracy than enzymatic assays and Western blots. For instance, the SCC technique has a sensitivity of measuring changes in ion movement in the range of pmol per second, equal to changes of 0.1 μampere. No ELISA kit can match that. Moreover, the SCC is directly related to the PDE activity located in epithelial cells, elegantly circumventing activity in other locations of the studied tissue. Immunohistochemistry has an advantage over Western blotting by the visualizing location of specific proteins, sometimes even revealing subcellular positions, while often with a near equal sensitivity in the abundance of studied proteins compared with Western blots.Rolipram is a selective inhibitor of PDE4 family members. The response of SCC to rolipram was significantly larger in biopsies from CTRLs compared to CRNs. As PDE4 enzymes primarily hydrolyze cAMP, the result of rolipram-sensitive SCC indicates a reduced elimination of cAMP and reduced control with this signaling pathway in normal colonic mucosa from CRN patients.The SCC response to cilostamide showed no difference in activity of the PDE3 family of enzymes in non-neoplastic appearing colonic mucosa between patient groups. Meanwhile, the change in SCC due to cilostamide as an inhibitor was on average three times larger than that of rolipram, see results. The balancing control effect of cellular PDE on cNT signaling is thus dominated by PDE3 family members.Surprisingly, we did not find any SCC-sensitive activity of PDE5 in the biopsy material, despite applying high concentrations of potent inhibitors. An explanation for this lack of activity is low levels of basal intracellular cGMP concentration and failure to introduce significant amounts of cellular cGMP by our basolateral exogenous application of cGMP. Furthermore, applied PDE5 inhibitors sildenafil and zaprinast might not be inhibitors of PDE5 in whole human colonic epithelia tissue. In cell line studies, zaprinast was found not to be a specific PDE5 inhibitor, while specific effects of rolipram, a PDE4 inhibitor, were detected . In contOur expressional study detected PDE subtypes 3A, 3B, 4A, 4B, 4C, 4D, 5A and 10A, with PDE3A as the dominant PDE isoform in normal colonica mucosa in both CRN patients and CTRL patients. Nucleotide expression data from a cancer cell line published by Tsukahara et al. suggests PDE3B, and not PDE3A as the main PDE isoform . This isWe detected a significantly higher expression of PDE4B in the CRN patients, which has not been previously described in man. Meanwhile, our data on rolipram-sensitive SCC clearly show a lower activity of PDE isozymes in biopsies from CRN patients Fig. , while dThe literature describes PDE5A as overexpressed in breast and colon tumor cells while the expression of other cGMP PDE isozymes is decreased , 37. OurImmunohistochemical analysis confirmed that our qPCR was performed on epithelial cells specimens as no underlying nerve and muscle tissue were detected.Before the start of our study we conducted a search of the literature, which indicated that immunostaining for PDE3A-B, PDE4A-B-C-D and PDE5A often lacked images demonstrating the precision of the antibodies and with very few reports on the topic. We used several of the most recommended antibodies for each PDE. In our material/methods and result sections we present the best antibodies of those employed after consulting with several manufacturers of the PDE antibodies.We found that antibodies for PDE3A especially and most of the used antibodies were not specifically localized to colonic epithelial cells as underlying connective tissue cells showed high immunoreactivity as well. This result, therefore, demands further research for a better antibody for PDEs in human colonic epithelia.In regards to PDE5A our findings suggest that it can be seen in normal appearing epithelia while most studies so far have only observed PDE5 in smooth musculature in normal tissue . One stuDuring our immunohistochemical studies, we employed manual microscopic quantification to assess the abundance of PDEs as a surrogate marker for protein abundance in biopsies. The technique has some drawbacks as described in the literature . Most ofFurthermore, the abundance of PDE5A was lower in CRN compared to controls, but this result was not significant Table . In contIn light of recent reports in the neoplastic cell, we aimed to outline the role of the most prominent PDEs in normal appearing colonic mucosa from patients with CRN. We sought to investigate the function, expression and localization of PDE subtype 3, 4 and 5 in biopsies. PDE10 was investigated with qPCR only. Our data suggests that PDE4B activity is not only dysregulated in CRN per se but also dysregulated in normal appearing mucosa of patients with CRN. In specific PDE4 subtypes could potentially serve as promising pharmacological targets for diagnostic and therapeutic interventions for CRN, although a robust data-driven scientific rationale for selecting PDE4 or any other subtype remains to be challenged and explored accordingly. Furthermore, expression levels of PDE5A are elevated in normal appearing colonic mucosa from patients with CRN, however no differences were observed for this PDE with regards to activity or protein abundance.