Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in  E. coli. This study provides insights into molecular events that occur in carrier protein-dependent pathways.Using structural and computational analysis, Bartholow et al. perform a comprehensive residue-by-residue comparison of the protein-protein interactions in de novo fatty acid biosynthesis that occurs in  These are generally organized as type I or type II enzymes, with the type I “megasynthases” containing multiple enzymatic domains and carrier proteins housed as large multi-domain proteins3. In contrast, type II synthases exist as discrete proteins that must recognize and associate with one another in solution through an organized choreography of metabolic steps  more than 25 partner proteins (PPs) are known to functionally bind to the acyl carrier protein (AcpP)  with multiple partners, efficiently chaperoning intermediates through 30–35 discrete enzymatic steps to produce the membrane lipids that maintain homeostasis and facilitate cellular reproduction9. Simultaneously FAB generates fatty acid intermediates for cofactor biosynthesis and secondary metabolism10. This study further illustrates AcpP•PP recognition through unique PPIs with each of the FAB enzyme players  Fig. 4–6, a smers Fig.  while pr12, but they are sequestered within the hydrophobic pocket of the AcpP helices, protecting intermediates from non-specific reactivity15. Reactions are controlled through this sequestration and presentation of the substrates appended to 4′-phosphopantetheine, a process termed chain flipping16. The rapid doubling times and relatively narrow distribution of fatty acid products require an efficient, high fidelity FAB17, suggesting that stochastic binding events of AcpP with its binding partners are unlikely. Activity studies and mechanism-based crosslinking experiments have demonstrated that acyl-AcpP binding and enzyme turnover are highly specific  thioester linkagefic Fig. 17–20. A 1H-15N HSQC-NMR titration studies to collect residue-by-residue information for six de novo FAB partner enzymes to characterize each intrinsic PPI with the E. coli AcpP. Experiments were performed to study the interfaces of AcpP with elongating ketosynthases FabB and FabF, reductases FabG and FabI, dehydratase FabA, and thioesterase TesA. These spectroscopic data combined with a combinatorial docking protocol benchmarked with crosslinked structures of AcpP in complex with FabA, FabZ, FabB, and FabF provide atomic-resolution information on which residues of AcpP mediate each step in iterative de novo FAB. This combinatorial method was able to overcome the unique challenges of modular synthases, with substrate identity effecting carrier protein structure and each enzyme forming unique interactions with the carrier. Due to the high sequence homology of AcpP with carrier proteins from other species25 and polyketide synthases28, this protocol is expected to extend for characterization of ACP•PP interactions for engineering and drug design across multiple systems.Here we used 1H-15N HSQC-NMR titrations in the study of rapid and intricate PPIs31. In this study, uniformly labeled and perduterated 15N-C8-AcpP (octanoyl-AcpP)  Figs.  and S11 P) Figs.  and S1–3P) Figs.  S1–3b32.ng Figs. . Furtherrs Table . Previou1H-15N HSQC-NMR titrations shown here can provide specific information of the AcpP residues involved in PP binding; however, the residues on the PP that mediate binding cannot be determined by this process. While titrations provide ample evidence of the carrier protein’s interacting residues, no information is gained about the residues of the PP with which they are interacting. We have recently elucidated the X-ray crystal structures of several FAB enzymes crosslinked to AcpP, including FabA, FabZ, FabB, and FabF. These structures can indicate residues involved in PPIs on both proteins; however, each structure requires prior development of enzyme-specific crosslinking probes, which are not available in all cases. We sought to develop protein–protein docking protocols with Molsoft’s ICM software to predict structures of the AcpP•FabI, AcpP•FabG, and AcpP•TesA complexes that have eluded experimental structural characterization38. Crystal structures of previously crosslinked AcpP-PPs were used to optimize this protocol, described more fully in the “Methods” section. Briefly, it was identified that to accurately re-create complexes it was necessary to produce a water box in which the PPs were minimized. Docking simulations were carried out between the X-ray structural model of heptanoyl-AcpP-C7 (PDB 2FAD), to which a methylene was added to the acyl chain simulate C8-AcpP, and crystal structural models of binding PPs from which cofactors had been removed. Using expanded calculations to assist the general docking protocol  when comparing docked models with FabB (962 Å2), perhaps consistent with its broader activity and the wider impact on CSPs compared to FabB53. This further agrees with data demonstrating a tighter binding for FabF than the previous calculated FabB Kd of 37.6 ± 6.6 µM. Though FabB and FabF share particularly similar structures and activity39, their interactions with AcpP are unique.FabB performs the same ketosynthase reaction as FabF, but performs the first unsaturated elongation step, making a case study in specificity54. For NMR titrations, NAD+ was added along with FabG, as previous studies showed a difference in AcpP binding efficiency in the presence and absence of NAD+  FAB is catalyzed by the enoylreductase, FabI, which produces a saturated acyl-AcpP through NADH–dependent reduction of enoyl-AcpP Fig. 58. FabI Ps Table , with pePs Table . UniquelE. coli, mature acyl-AcpPs are instead steered directly into other biosynthetic pathways via acyl transfer from AcpP, primarily for phospholipid biosynthesis. However, E. coli does possess the thioesterase TesA, which localizes in the bacterial periplasm59. Though TesA is not believed to be involved in the terminal step of E. coli FAB, it can hydrolyze acyl-AcpP in vitro and has been used as a tool for FAB engineering to increase free fatty acid titer when overexpressed within E. coli60. Upon titration with TesA, the pattern of C8-AcpP CSPs occurred predominantly in residues different from those perturbed by FAB enzymes  used in preparation of perdeuterated growth was purchased from Sigma Aldritch. All unlabeled proteins were grown on Luria broth from Teknova.The E. coli BL21 (DE3). Cells and grown in LB media. Cells were grown in the presence of 50 mg/L kanamycin before induction with 1 mM IPTG at OD600 = 0.8 and incubated at 18 °C for 12–18 h. Cells were pelleted in a JLA-8.1 rotor at 800 RCF. Cells were re-suspended and lysed by sonication in 50 mM Tris-HCl (pH 7.4), 250 mM NaCl, and 10% glycerol. The lysate was then spun at 10,000 RCF in a JA-20 rotor for 1 h to pellet the membrane and insoluble materials. Proteins were purified using Ni-IMAC (Bio-Rad) after a 30-min batch-binding time rotating at 4 °C. The general protocol used two solutions, a wash of 40 mL of lysis buffer followed by a wash of 40 mL lysis buffer with 15 mM imidazole added. This was followed by three 5 mL elutions with lysis buffer containing 250 mM imidazole. Unless stated in the purification specifics below this was the method used in all purifications. After purification proteins were concentrated to ~2 mL and purified using size exclusion chromatography on a Superdex 75 column. The PP was purified into NMR buffer and concentrated to the concentrations listed below before addition to the tube for NMR.All PPs were generated through overexpression in E. coli BL21 (DE3) cells. Cells were grown at 37 °C in M9 minimal media containing 1 g of 15N NH4Cl and 8 g of glucose. Perdeuteration was achieved by growing E. coli in increasing ratios of D2O. Starting by growing 5 mL overnight in 25% D2O/75% H2O, this was used to inoculate 5 mL cultures which were 50% D2O/50% H2O. This same technique was used to inoculate 75% D2O, 90% D2O, and finally 100% D2O starters. This starter was used to inoculate the liter of deuterated media. Once the growth reached an OD600 of 0.8 they were induced with 1 mM IPTG and allowed to grow for an additional 4 h at 37 °C.AcpP was grown on a His-tagged pET-22b vector in Pseudomonas aeruginosa ACPH in a solution with 5 mM MgCl2, and 0.5 mM MnCl2, and 1 mM DTT. This reaction was performed overnight at 37 °C. Apofication was confirmed by conformationally sensitive UREA-PAGE. Following this, loading was performed using three E. coli biosynthetic enzymes CoaA, CoaD, and CoaE and the Bacillus subtilis SFP. The reaction is performed with 12.5 mM MgCl2, 10 mM ATP, 0.1 µM CoaA, 0.1 µM CoaD, 0.1 µM CoaE, 0.2 µM Sfp, 0.02% Triton X, 0.01% Azide, and 0.1% TCEP. The reaction was performed overnight at 37 °C, with loading confirmed by conformationally sensitive UREA-PAGE. Stable C8 acyl loaded ACP analogs were achieved through loading of an octanoyl pantethenamide probe.Following purification AcpP was dialized overnight into 50 mM Tris, 250 mM NaCl, and 1 mM DTT buffer, in order to remove the imidazole before subsequent reactions. AcpP was first prepared as uniformly apo by reaction with −1 cm−1. FabF was concentrated to 10.1 mg/mL using Amicon Ultra-15 10 kDa spin filters, using the extinction coefficient 25,900 M−1 cm−1. These were used to create a 0.042 µM AcpP zero-point sample and a 0.042 µM AcpP 0.0837 µM FabF saturated sample. FabF was purified for titration the day before the experiment, ensuring a “fresh” sample for maximum stability. Perdeuterated AcpP was used to boost signal from sensitivity lost due to the titrated PP. This also lowers the concentration of PP necessary to accommodate stability concerns. The proteins were purified into a 10 mM potassium phosphate pH 7.4 buffer with 0.5 mM TCEP and 0.1% NaN3 and any buffer added to the tubes was taken from the same FPLC buffer solution. The total volume of both the zero point and saturated samples was 450 µL, and 50 µL of D2O was added to both tubes for locking.ACP was concentrated to 3.87 mg/mL using Amicon Ultra-15 3 kDa centrifugal filters, and a Nanodrop was used to measure concentrations with the extinction coefficient 1490 M−1 cm−1. FabI was concentrated to 22.1 mg/mL using Amicon Ultra-15 10 kDa spin filters, using the extinction coefficient 15930 M−1 cm−1. These were used to create a 0.0669 µM AcpP zero-point sample and a 0.0669 µM AcpP 0.2689 µM FabI saturated sample. This high equivalent concentration was used to ensure that there would be at least a 1:1 ratio of ACP: FabI tetramer. The FabI was purified the day before the experiment and concentrated to the high molarity necessary the morning of the experiment in order to ensure the sample was as stable as possible. Perdeuterated AcpP was used to boost signal form quenching. The proteins were purified into a 10 mM potassium phosphate pH 7.4 buffer with 0.5 mM TCEP, 0.5 mM NAD+ and 0.1% NaN3 and any buffer added to the tubes was taken from the same FPLC buffer solution. The total volume of both the zero point and saturated samples was 450 µL, 50 µL of D2O was added to both tubes for locking.ACP was concentrated to 2.38 mg/mL using Amicon Ultra-15 3 kDa centrifugal filters, a Nanodrop was used to measure concentrations with the extinction coefficient 1490 M−1 cm−1. FabG was concentrated to 11.21 mg/mL using Amicon Ultra-15 10 kDa spin filters, using the extinction coefficient 11460 M−1 cm−1. These were used to create a 0.0538 µM AcpP zero-point sample and a 0.0538 µM AcpP 0.2199 µM FabG saturated sample. This high equivalent concentration was used to ensure that there would be at least a 1:1 ratio of ACP:FabG tetramer. The FabG protein was purified the day before titration and concentrated the morning of the experiment to ensure a stable sample for the experiment. Perdeuterated AcpP was used to boost signal form quenching. The proteins were purified into a 10 mM potassium phosphate pH 7.4 buffer with 0.5 mM TCEP, 0.5 mM NAD+ and 0.1% NaN3 and any buffer added to the tubes was taken from the same FPLC buffer solution. The total volume of both the zero point and saturated samples was 450 µL, and 50 µL of D2O was added to both tubes for locking.ACP was concentrated to 2.25 mg/mL using Amicon Ultra-15 3 kDa centrifugal filters, a Nanodrop was used to measure concentrations with the extinction coefficient 1490 M−1cm−1. TesA was concentrated to 3.2 mg/mL using Amicon Ultra-15 10 kDa spin filters, using the extinction coefficient 40450 M−1 cm−1. These were used to create a 0.0538 µM AcpP zero-point sample and a 0.0699 µM AcpP 0.104 µM TesA saturated sample. TesA was prepared the day before the experiment in order to have a fresh and stable sample for the experiment. Due to the small size of TesA, perduteration was likely not necessary but was maintained for consistency. The proteins were purified into a 10 mM potassium phosphate pH 7.4 buffer with 0.5 mM TCEP, and 0.1% NaN3 and any buffer added to the tubes was taken from the same FPLC buffer solution. The total volume of both the zero point and saturated samples was 450 µL, and 50 µL of D2O was added to both tubes for locking.ACP was concentrated to 6.5 mg/mL using Amicon Ultra-15 3 kDa centrifugal filters; a Nanodrop was used to measure concentrations with the extinction coefficient 1490 M35 of the C8-AcpP were used to assign the backbone peaks on the HSQC for all experiments. Each experiment was performed at 37 °C, with each titration including at least five titration points in order to observe the full movement of peaks. CSPs were quantified using the formula32Experiments were performed on a Bruker 800 MHz spectrometer equipped with a cryo-probe at the UCSD Biomolecular NMR facility. Previous assignmentsα value of 0.2 was used in all CSP calculations. Titrations were performed by the preparation of an initial saturated and zero-point sample. To create intermediary samples for the titration these two samples were mixed. AcpP is an extremely stable protein, with no denaturation observed. However, in some cases a degree of PP crash was seen in the later titrations. To mitigate this when not in the spectrometer samples were kept refrigerated at 4 °C and the spectra were collected one after another over the course of ~12 h. Through this cautious approach and fresh PP preparation we were able to collect all saturated samples with no observed crashed partner, though in the case of FabF and FabG a small amount of crashed protein was observed for the final “middle” spectra’s sample . All HSQC were acquired with a 1.5 s recycle delay and 2048 data points in the spectra. Spectra were processed using in NMR Pipe61 and NMRFAM-SPARKY62. Spectra were visualized and all NMR spectra figures were generated in SPARKY. CSP calculations and the CSP heat map were generated in the Matplotlib python utility63. NMR titration data were further analyzed using the TITAN 2D lineshape analysis program. For the analysis in all cases a flexible stoichiometry model was used. Both to allow for examining the stoichiometry of the interactions and in order to allow the concentration of the AcpP to vary in the calculation. This was done because AcpP has a very low extinction coefficient, making quantifying exact AcpP concentrations difficult. In the analysis five titrations steps were used in each analysis. The calculations were performed by selecting each region of interest on the spectra. Following this the initial fitting was performed, with the parameters first estimated at 10 µM with a koff rate of 5000 s−1. After the fitting each peak chosen was hand checked, in order to ensure that the peak had been properly fit. Though the TITAN program self fits peaks, in more crowded regions of the spectra the program can improperly fit the wrong peak. After the initial fitting jackknife error analysis was performed, this was done in order to verify the program ran without issue. Improperly fit peaks were identified in the more rapid bootstrap error analysis, peaks which had very small migrations or which migrated into other peaks displayed high error and were hand chosen to not be fitted. In this way Jackknife analysis was used to identify user error and occasional problematic peaks before the much longer bootstrap analysis was run. After these problematic peaks were hand checked a final bootstrap analysis was performed, in all cases 300 steps were performed in the analysis. The results of this bootstrap analysis were used as the error in reporting values, a set of simulated and “real” peaks are presented for each analysis below. A selection of two contour plots and two 3D contour plots for each analysis is supplied. It was observed that the FabG, FabF, and TesA resulted in greater error in the analysis. We suspect the error in FabG, FabF, and TesA is due to the instability of the PP seen in the final titration step. FabG in particular had all of the components at extremely high concentrations in order to ensure saturation.An In the case of FabF, FabB, FabI, FabG, FabA, and TesA structures for the PP were acquired from the protein data bank: 2GFW, 1G5X, 4CV3, 1Q7B, 1MKB, and 1IVN were used respectively. For FabZ the only structure available is 6N3P, a crosslinked crystal structure. As such this was used but AcpP was deleted. 2FAD was used as a starting crystal structure, with an additional carbon added in ICM to elongate the 7 carbon acyl chain and create a C8 acyl chain. All PPs were used in the subunit structure which it adopts in solution, especially given that in many cases AcpP binds multiple subunits of a dimer or tetramer. Specifically: FabI and FabG were docked as tetramers. FabA, FabZ, FabF, and FabB were docked as dimers. TesA was docked as a monomer.Before docking the proteins were prepared by solvation and minimization. The AcpP and all PPs were solvated with the ICM quickflood procedure in order to generate a water box. After this the proteins were minimized in ICM to optimize side chain orientations and hydrogen bonding with the water box and any ligands. This was performed by running the optimizeHbonds and optimize HisProAsnGlnCys protocols in order to form more solution relevant conformations of the residues. After this the cofactors, including the AcpP substrate, were deleted and the proteins were docked. However, the structural effects of the AcpP substrate remain reflected in the docking, with the acyl pocket remaining during the simulation.E. coli FAB we leveraged studies which mutated the AcpP interface and saw diminished activity. As an example in the docking of FabG the work of Price et. al., 2004 was used. In their paper they identified the region of NADP binding and proposed an interface. R15 was identified at the edge of the NADP pocket. The R129 and R172 were not chosen because they perform a more complicated docking with the AcpP from the adjacent chain. However, the final model generated did dock such that these two identified residues were interacting with AcpP. This same methodology is very broadly applicable in carrier protein-mediated biosynthesis, given the depth of the literature. For the calculations interface residues on the AcpP were chosen by selecting the largest perturbations whose position was such that they were likely hydrogen bonding at the interface. Docking poses were sorted by lowest energy, it was examined if scoring based on Van der Waals or electrostatic interactions specifically would yield more accurate structures. But we noted that the ICM energy scoring function performed best. Table Docking was performed both with and without focus residues using the ICM fast Fourier transform protein docking protocol. The “informed” docking procedure was performed by specifying experimentally known interacting residues on the AcpP and PP. Explicitly, the “uninformed” docking jobs are performed by docking the exact same input structures, without focus residues. In more detail, given the history of mutational study in 44, HADDOCK46, and Rosie48 with the PP FabF. The results are reported in Table It should be noted that the methodology used is not unique to ICM, as such we have performed three docking experiments with commonly available online docking servers. Docking was performed with three common servers: ClusPro1H-15N HSQC experiments. Chemical shift perturbations were calculated using the supplied equation and α value. TITAN analysis was performed according to the method outlined by the developers. Calculation of error was performed using only the recommended bootstrapping method, with 300 steps of calculation. RMSDs were calculated using the Pymol align command, in all cases of alignment the 10 lowest energy states are reported. Starting structures were taken from publicly available repositories.NMR analysis was performed on single sets of Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1–4Supplementary Data 5Reporting Summary


Mucormycosis is a rare, life-threatening, angioinvasive opportunistic fungal infection. Most common sites involved are rhino cerebral, pulmonary, cutaneous followed by gastrointestinal. We report a successful management of rare gastrointestinal ileal mucormycosis with perforation peritonitis managed surgically. Later histopathology revealed the ileal mucormycosis with transmural necrotizing inflammation. Histopathological demonstration of the fungus in surgical specimens remains cornerstone of the diagnosis of mucormycosis in view of its non-specific symptoms, low isolation rates of mycological culture and lack of other rapid tests. Mucormycosis is an uncommon angioinvasive fungal infection caused by the Mucorales predominantly in immunocompromised and intravenous drug addicts, and carries a poor prognosis . Rhino-cA 49-year-old male addicted to intravenous drug abuse, presented with severe pain abdomen, abdomen distension, constipation and bilious vomiting for four days. On examination, abdomen revealed a peritonitis sign and was having tachypnea and tachycardia and blood pressure was 90/60 mmHg. Blood parameters showed leucocytosis with TLC- 24,000 cubic millimeters, ABG showed mild metabolic acidosis pH being 7.23, and renal and liver profile was normal, blood culture was sterile. Anti-hepatitis C virus (HCV) enzyme-linked immunosorbent assay (ELISA) was positive. Erect X-ray of the abdomen showed air under the diaphragm. He was resuscitated and with the diagnosis of perforation peritonitis underwent exploratory laparotomy, intra-operative findings revealed around 1 litre biliopurulent free fluid in the peritoneum along with ileal perforation of size approx. 1 x 1 cm which was proximal to the stricture segment of the ileal loop Figure.  and B inMucormycosis is an angioinvasive, life-threatening opportunistic fungal infection caused by fungi of the subphylum Mucormycotina, order Mucorales . RhizopuRisk factors for mucormycosis are neutropenia, immunosuppression, diabetes, penetrating trauma, prematurity, malnourishment, hematologic malignancies, burns, iron overload and illicit intravenous drug use as in our case . HIV infGastrointestinal mucormycosis is a rare angioinvasive fungal infection associated with high mortality. High index of suspicious should be kept in cases of immunosuppressed and intravenous drug addiction patients with acute abdomen, early diagnosis and surgical therapy is of utmost importance. LAmB is the drug of choice.


Occupational noise exposure accounts for approximately 16% of all disabling hearing losses, but the true value and societal costs may be grossly underestimated because current regulations only identify hearing impairments in the workplace if exposures result in audiometric threshold shifts within a limited frequency region. Research over the past several decades indicates that occupational noise exposures can cause other serious auditory deficits such as tinnitus, hyperacusis, extended high-frequency hearing loss, and poor speech perception in noise. Beyond the audiogram, there is growing awareness that hearing loss is a significant risk factor for other debilitating and potentially life-threatening disorders such as cardiovascular disease and dementia. This review discusses some of the shortcomings and limitations of current noise regulations in the United States and Europe. The World Health Organization (WHO) estimates that ~16% of all disabling hearing loss stems from occupational noise exposure ,3,4,5. Decades of research has led to the development of guidelines designed to protect individuals from hazardous occupational noise exposure ,7,8. CurSome of the prominent governmental bodies that have established regulations/recommendations to avoid hazardous noise exposure include the Occupational Safety and Health Administration , the Nateq), which represents the energy mean of the noise level averaged over a defined period. When Leq acoustic measurements are made for 8 h with the A-weighting filter, the term LAeq,8h is used to indicate the type of frequency weighting and the duration of the exposures. Finally, the fourth factor, which is mainly considered in environmental noise ordinances, is the time of day. Several studies have suggested that nighttime noise exposure increases the risk of hearing loss, annoyance, risk of cardiovascular disease, sleep disturbance, and cognitive impairment [DEN (day/evening/night level), which indicates the average A-weighted sound pressure level over a 24-h period with an additional 10-dB penalty during nighttime hours (11:00 p.m.–7:00 a.m.) or a 5-dB penalty during evening hours (7:00 p.m.–11:00 p.m.) [eq measure to reflect night or evening exposure, respectively). The combination of these factors is the basis for understanding current occupational and environmental noise regulations. Contemporary regulations consider four major interacting variables when determining if a noise dose is hazardous. First, and most obvious, is the noise sound pressure level (SPL), represented in the logarithmic unit of decibels (dB). Second is the spectral content; noise levels are most commonly measured using the dB(A) scale, which attenuates sounds below 1 and above 8 kHz. This filtering is done because high-frequency noise (namely those centered around 4 kHz) pose a significant risk for auditory threshold shifts , althougpairment ,24. For 00 p.m.)   as 90 dB(A) for 8 h. The PEL is adjusted for exposure durations shorter or longer than 8 h using a 5-dB exchange rate. Accordingly, the PEL would be 95 dB(A) for an exposure duration of 4 h, whereas the PEL would be 85 dB(A) for a 16-h exposure . FurtherWhile OSHA is responsible for establishing regulations, the Occupational Safety and Health Act of 1970 (Public Law 91-596) tasked NIOSH with providing recommendations to avoid hazardous exposure to occupational noise. Importantly, these recommendations were meant to include occupational noise dosages that would reduce the risk of employees having “diminished health, functional capacity, or life expectancy as a result of his or her work experience” , health Aeq,8h  reaches 80 dB(A) or a peak pressure value of 135 dB(C). If noise reaches a LAeq, 8h of 85 dB(A) or has a peak sound pressure value of 137 dB(C), it is classified as an upper action limit level. Finally, the ceiling threshold for acceptable noise exposure is an LAeq, 8h of 87 dB(A) or a peak sound pressure limit of 140 dB(C), and there is no permissible exposure above this ceiling noise limit. The lower and upper action levels are distinct from each other in that they require increasing conservation practices, respectively. When noise reaches the lower action level, employers must make hearing protection devices available to employees and make annual hearing monitoring available; whereas if noise exceeds the upper action level, these practices stay in place and the employer must also take actions to actively reduce the noise level, either through engineering controls  or organizational practices .In the European Union, EU-OSHA regulates occupational noise based on the directive 2003/10/EC promulgated by the European Parliament . The dirDEN and 45 dB LNIGHT, (2) railway noise be limited to 54 LDEN and 44 dB LNIGHT, (3) aircraft noise be limited to 45 LDEN and 40 dB LNIGHT, (4) wind turbine noise be limited to 45 dB LDEN, and that (5) other leisure activities be limited to 70 dB LAeq,24 h [Aeq, 24 h, that community indoor levels be maintained below 45 dBLeq, and that certain community outdoor areas be maintained below 55 dBLeq [Recently, the WHO recommended guidelines for environmental noise exposures in the European Region . It is rAeq,24 h . In the 55 dBLeq . It is iThe regulations outlined above were not designed to protect the entire working population from noise-induced hearing loss. Rather, they are meant to protect against a substantial portion of occupational NIHL while maintaining a feasible cost–benefit ratio.For instance, a 40-year lifetime exposure to NIOSH’s REL still holds a risk of developing NIHL of ~8% and a 40-year lifetime exposure to OSHA’s PEL criteria still has a risk of NIHL of 25% ,8. DeploThe human cochlea can process a wide range of frequencies from ~20–20,000 Hz; however, standard clinical audiometric evaluations only sample hearing acuity from 250 to 8000 Hz, since they are putatively the most important for speech comprehension. The cochlea appears especially sensitive to noise-induced damage in the frequency regions between 3–6 kHz . ConsequThe cochlea possesses a finely-tuned, biological electro-mechanical system that amplifies sound-induced pressure fluctuations. This inherent non-linear amplification of the cochlea lowers hearing thresholds and enhances frequency resolution, thereby improving hearing abilities in both quiet and noise . CochleaIndeed, several studies have indicated that DPOAEs could serve as a more sensitive tool for identifying noise-induced damage in occupational settings ,55,56,57In addition, test re-test reliability in OAEs also demonstrates less variance than pure-tone thresholds ,58. In aHistorically, OHCs were considered the cochlear structure most sensitive to noise-induced damage. However, recent animal studies now suggest that synaptic connections between inner hair cells (IHCs) and type I afferent nerve fibers could be more sensitive to noise damage than OHCs ,60. ImmuTinnitus is a phantom auditory perception, described as a ringing, buzzing, or hissing sensation that can occur following excessive noise exposure . ApproxiHyperacusis, an abnormally low tolerance to moderate-intensity everyday sounds , is anotThe exact pathophysiological mechanism(s) responsible for tinnitus and hyperacusis remains enigmatic. However, many researchers believe tinnitus emerges from excessive spontaneous neural activity ,88,89,90Hearing in the presence of background noise is the most common hearing-related complaint and can occur in individuals with and without normal audiometric thresholds ,94. DiffThe pathophysiological mechanisms that make it difficult to discriminate in background noise are not fully understood; however, mounting evidence suggests it could be due to selective damage to IHCs and/or auditory nerve fibers. When chinchillas are treated with carboplatin, a commonly used anti-cancer drug, they exhibit a unique pattern of damage that selective destroys IHCs, while leaving OHCs intact. Despite this damage, animals display normal threshold acuity in quiet . HoweverDifficulty hearing in noise is commonly linked to the discussion of hidden hearing loss (a term some use synonymously with cochlear synaptopathy). If findings in animal models hold true in the translation to human studies, cochlear synaptopathy may provide an explanation for why noise exposure can impair hearing in noise. For example, evidence suggests that selective damage to IHCs and auditory nerve fibers does not affect thresholds, but does impair supra-threshold processing in noise ,106,107.Taken together, several auditory deficits occurring from noise exposure can occur beyond the standard pure tone audiogram and are not considered in today’s hearing conservation programs. Outside of the auditory realm, recent studies also indicate several non-auditory consequences of excessive noise exposure, some of which are discussed in the following section.Unregulated stress responses can have large implications for numerous biological functions. The body’s stress response system attempts to maintain homeostasis through hypothalamic–pituitary–adrenal (HPA) axis negative feedback. In general, the paraventricular nucleus of the hypothalamus activates the HPA axis through the secretion of corticotropic releasing hormone (CRH), which provokes the release of adrenal corticotropic hormone (ACTH) from the anterior pituitary. ACTH then travels via the blood stream to the adrenal glands, which sit atop the kidneys, and activate the release of stress hormones such as cortisol, adrenaline, and noradrenaline When levels of stress hormones are sufficiently high they interact with the hypothalamus and hippocampus to shut off the HPA axis, resulting in negative feedback . ChronicLAeq if the noise was disrupting to active processes . During sleep, traffic noise levels as low as 30 dBLAeq have been shown to increase cortisol release [Both occupational noise and chronic environmental noise can result in increased stress hormones such as cortisol, adrenaline, and noradrenaline ,117. Int release . The rel release ,122Cardiovascular disease (CVD) is an umbrella term used to refer to a host of disorders of the heart and blood vessels. CVDs are the number one cause of death globally, responsible for ~31% of all deaths , and posBoth occupational and environmental noise exposure can increase the risk of CVDs. For example, in a large sample of industrially noise-exposed workers, the relative risk for developing coronary heart diseases was as high as 1.48, significantly higher than in age-matched, unexposed workers . SimilarThere is growing evidence to suggest that age-related hearing loss increases the risk of cognitive decline in older adults. This once provocative finding is supported by several meta-analytic reviews ,137,138.LAeq, 8 h) for extended periods, they can display poorer performance on learning and memory tasks [Since it is extremely difficult to disentangle the contribution of past noise exposures on age-related hearing loss, it is plausible that noise could play a role in developing dementia. Indeed, animal studies support the notion that exposure to noise can disrupt cognitive functions such as learning and memory. Rats’ exposure to traumatic noise shows reduced neurogenesis in the hippocampus, a brain region critically important for spatial orientation and memory. Moreover, reduced neurogenesis is associated with deficits on learning and memory tasks ,140,141.ry tasks . Similarry tasks . Exposure to occupational noise poses a significant public health concern. It is well documented that exposure to noise above a critical level results in unrepairable sensorineural hearing loss. Noise regulations established for occupational settings do not protect 100% of workers from NIHL, but rather strike a balance between hearing conservation and economic development. The current occupational and environmental noise regulations may significantly underestimate the adverse health and societal cost of noise-induced hearing loss. There is growing awareness that noise exposures, even at moderate levels, may contribute to a host of other auditory and non-auditory consequences that can go undetected in annual hearing conservation monitoring programs. Taken together, these results suggest that the true fiscal consequences to our healthcare systems may be far greater than anticipated. Further research is needed to fully characterize the auditory and non-auditory consequences associated with occupational noise. Future research could uncloak (1) methods for earlier detection of auditory damage, (2) risks associated with combined noise exposure sources  and (3) establish dose–response relationships between various noise conditions and non-auditory consequences.


Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Weekly serum and daily oral fluid samples were collected through 56 days postinoculation (dpi). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period. Analysis of ELISA performance at various cutoffs found that the manufacturers’ recommended cutoffs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives. In study 2, the 3 best-performing ELISAs from study 1 were compared using serum samples generated under field conditions. Ten 8-week-old pigs were intratracheally inoculated with M. hyopneumoniae. Matched serum and tracheal samples (to establish the true pig M. hyopneumoniae status) were collected at 7- to 14-day intervals through 98 dpi. Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current M. hyopneumoniae ELISAs and an understanding of their use in surveillance. Mycoplasma hyopneumoniae is an agent in the porcine respiratory disease complex and the cause of enzootic pneumonia in pigs. M. hyopneumoniae colonization of the respiratory cilia can result in suppurative bronchiolitis and lymphoplasmacytic peribronchiolitis and suppression of the immune defenses afforded by the pulmonary mucociliary apparatus, thereby creating an environment in which other pathogens can proliferate and induce more-severe respiratory disease  estimated the economic burden of M. hyopneumoniae to the pork industry to be ∼$400 million per year. While disease caused by M. hyopneumoniae is manageable using medication and vaccination, M. hyopneumoniae elimination programs have become more frequent due to their high chances of success  becomes increasingly important. Given that various commercial M. hyopneumoniae enzyme-linked immunosorbent assays (ELISAs) are available on the market, the objective of this study was to compare their performance under experimental and field conditions.eillance , but its(i) Study 1. Six commercial Mycoplasma hyopneumoniae ELISAs were evaluated using serum samples (n = 680) derived from an experiment previously conducted at Iowa State University (ISU) under the supervision of the Institutional Office for Responsible Research (n = 10), (ii) Mycoplasma flocculare (n = 10), (iii) Mycoplasma hyorhinis (n = 10), (iv) Mycoplasma hyosynoviae (n = 10), and (v) Mycoplasma hyopneumoniae (n = 10). Longitudinal sampling of group M. hyopneumoniae allowed for the temporal evaluation of antibody detection, and samples from non-M. hyopneumoniae groups permitted the evaluation of M. hyopneumoniae ELISAs for nonspecific reactions. At the termination of the experiment, all pigs were humanely euthanized by penetrating captive bolt followed by exsanguination. Lung tissues collected at necropsy from group M. hyopneumoniae were tested by PCR to verify M. hyopneumoniae infection. The performance of six commercial ELISAs was evaluated at various cutoffs and scored in terms of misclassification error rate, i.e., indices of false positives and false negatives.Research . In brie(ii) Study 2. Three commercial M. hyopneumoniae ELISAs from study 1 were further evaluated using serum samples (n = 362) collected over the course of an M. hyopneumoniae field study. Briefly, 10 8-week-old animals in one pen (inoculated pen) in a room housing 1,250 pigs in 46 pens (∼28 pigs per pen) were intratracheally administered an M. hyopneumoniae inoculum. Thereafter, paired individual serum and tracheal samples were collected from one noninoculated pig in each of the 46 pens at 7- to 14-day intervals for 98 days. At the termination of the study, samples were tested for DNA  and antibody (serum). The performance of the three commercial ELISAs was evaluated using a logistic mixed regression.(i) Study 1. Animals were allocated to 5 rooms (treatments) with 5 pens per room and 2 pigs per pen. Prior to inoculation, they were determined to be free of mycoplasmal infections on the basis of mycoplasma-specific ELISA and quantitative real-time PCR (qPCR) testing of serum, oral fluid, or tonsil scraping (M. hyosynoviae group only) samples , xylazine , and ketamine  at a dose of 1 ml per 4.4 kg of body weight.Pigs were inoculated as described below in M. hyopneumoniae (strain 232) inoculum consisted of lung tissue homogenate  from M. hyopneumoniae-inoculated CDCD pigs diluted in 1:100 of a modified Friis broth media (6 color-changing units (CCU)/ml by titration in Friis broth media (10M. hyopneumoniae group as previously documented (th media  to a con media 1012. The dcumented .M. flocculare (strain 27399) inoculum consisted of a live culture propagated in Friis broth media in a shaking water bath (37°C for 48 h) diluted to a concentration at 1.0 × 105 CCU/ml, as determined by titration in Friis media. The inoculum was administered intranasally (1 ml in each nostril) and intratracheally (1 ml in the trachea). In addition, on 0 and 4 days postinoculation (dpi), the inoculum was “scrubbed” on the tonsils of the soft palate using a 16-in. (41-cm) large-tip cotton swab  saturated with 2 ml of the inoculum.M. hyorhinis (strain 38983) inoculum consisted of a live culture propagated in Friis broth media (37°C for 48 h) and diluted to a concentration of 3.2 × 108 CCU/ml, as determined by titration in Friis agar media. The inoculum was administered into the peritoneal cavity (2 ml) and “scrubbed” on the tonsil of the soft palate using a 16-in. (41-cm) large-tip cotton swab (Scopettes 16-in. absorbent) saturated with 2 ml of the inoculum.M. hyosynoviae (strain 34428) field isolate was cultivated in Difco medium containing turkey serum (d-TS) broth (37°C for 48 h) and diluted to a concentration of 2.1 × 109 CCU/ml, as determined by titration in Friis agar media. Pigs were inoculated intranasally (1 ml per naris) and intravenously (1 ml via the auricular vein) and “scrubbed” on the tonsils of the soft palate using a 16-in. (41 cm) large-tip cotton swab (Scopettes16-in. absorbent) saturated with 2 ml of the inoculum.(ii) Study 2. The M. hyopneumoniae (strain 232) inoculum used in study 2 was identical to that in study 1 except that the concentration was estimated to be 1 × 105 CCU/ml, and each of the 10 M. hyopneumoniae-inoculated pigs was intratracheally administered 10 ml.g for 15 min), and the serum was aliquoted into 2 ml cryogenic tubes  for storage at −80°C.Blood samples were collected using single-use collection tubes. In the laboratory, samples were centrifuged  100% cotton rope  suspended from a bracket fixed to the side of the enclosure . The oraTracheal mucosal samples were collected using a sheathed, single-use porcine cervical artificial insemination catheter . After sampling, the tip containing the collected material was severed from the catheter and placed in a tube containing 1 ml sterile phosphate-buffered saline (PBS). In the laboratory, tracheal samples were transferred to 2 ml cryogenic tubes (Cryo.s) for storage at −80°C.In study 1, Blood samples were collected from all pigs on −3, 0, 3, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, and 56 dpi, and pen-based oral fluid samples were collected daily. Lung tissues were collected at necropsy (56 dpi) and stored at −80°C until processed for PCR testing.M. hyopneumoniae-inoculated pigs and 1 noninoculated pig. In the remaining pens, tracheal and blood samples were collected from one pig on 15, 24, 42, 56, 71, 85, and 98 dpi.In study 2, In the inoculated pen, tracheal samples  and blood samples  were collected from the 10 M. hyopneumoniae serum antibody ELISAs were included in the comparison . Two assays were blocking ELISAs based on the detection of antibody against a conserved epitope of the M. hyopneumoniae 74-kDa protein . Distinct from the other ELISAs, one assay  was based on a two-well indirect ELISA format. As described by the manufacturer, one well contained an M. hyopneumoniae-specific antigen and the other contained generic mycoplasma antigen. The response was then determined as the proportion of M. hyopneumoniae-specific antibodies in the sample.Six commercial mparison . Three aM. hyopneumoniae ELISAs: (i) M. hyorhinis (n = 129), (ii) M. hyosynoviae (n = 131), (iii) M. flocculare (n = 140), (iv) M. hyopneumoniae (n = 140), and (v) negative control (n = 140). To avoid bias, each sample was randomly assigned to a specific ELISA plate and well .In study 1, we tested 680 serum samples from the 5 inoculation groups on each of the six commercial M. hyopneumoniae-inoculated (n = 44) and noninoculated pigs (n = 318) on three commercial M. hyopneumoniae ELISAs .In study 2, we tested 362 serum samples from M. hyopneumoniae ELISA was run per day following the instructions provided by the manufacturer. Assay validity criteria were checked and verified for each plate according to the instructions provided by each manufacturer. Serum samples were randomized to avoid systematic errors, and no samples were retested.The same ELISA equipment, i.e., plate washer , ELISA reader , and reader software  were used throughout. One commercial Mycoplasma species-specific DNA and antibodies initially to confirm pigs’ negative status and later to corroborate M. flocculare, M. hyorhinis, and M. hyosynoviae infections in inoculated animals (0 to 56 dpi). Mycoplasma DNA extraction was performed using a commercial kit  on the Kingfisher Flex system  and amplified on Applied Biosystems 7500 real-time PCR (Thermo Fisher Scientific). Antibody responses against M. flocculare and M. hyosynoviae were assessed using Tween 20-extracted surface protein-based ELISAs. M. hyorhinis-specific antibody testing used a chimeric VlpA to VlpG recombinant protein-based ELISA  from M. flocculare, M. hyorhinis, M. hyosynoviae, and negative control groups were tested for M. hyopneumoniae DNA to establish that they remained free of the pathogen. In total, 332 oral fluid samples from the following 5 inoculation groups were tested for M. hyopneumoniae DNA: (i) M. hyorhinis (n = 33), (ii) M. hyosynoviae (n = 39), (iii) M. flocculare (n = 44), (iv) M. hyopneumoniae (n = 276), and (v) negative control (n = 36).In study 1, daily oral fluid samples were tested for M. hyopneumoniae DNA detection was based on commercial kits  performed as directed by the manufacturer. DNA was extracted on the Kingfisher Flex system and amplified on Applied Biosystems 7500 real-time PCR. Each plate included a known M. hyopneumoniae-positive sample and a negative control (RNA-free water). A test result was considered valid when the internal positive cycle threshold (TC) values were ≤36. A sample was considered M. hyopneumoniae positive when TC values were ≤40.g).Lung tissues were collected at necropsy (56 dpi) and stored at −80°C until they were processed for PCR testing. As described elsewhere , lung  performed as directed by the manufacturer. DNA was extracted on the Kingfisher Flex system and amplified on Applied Biosystems 7500 real-time PCR. Each plate included a known M. hyopneumoniae-positive sample  and a negative control sample (RNA-free water). A test result was considered valid when the internal positive TC value was ≤36. A sample was considered M. hyopneumoniae positive when TC values were ≤37.(i) Study 1. Serum antibody data from 680 samples were identified by inoculation group , pig number (1 to 50), and the standardized response for each of the six ELISAs, i.e., sample-to-positive ratio (S/P), percent blocking (%B), or relative index (RI). Evaluation of the six ELISA results revealed that the data were not normally distributed . Transformation using logarithms or the Box-Cox method was not successful but was achieved using “ordered quantile normalization” (P > 0.05).ization” . Orderedization”  in R  and the delayed antibody response in these pigs. Initially, test results from non-M. hyopneumoniae groups (n = 40 pigs) were used to evaluate the impact of alternative cutoffs on the false-positive rate for each manufacturer’s assay. As described elsewhere , i.e., the values localized at the 97.5% and 99.7% quantiles of the normal distribution , respectively. Because Oxoid ELISA defined positives as results with <50% blocking, the alternative cutoffs were established as (x̄ − 2 and 3 SDs), i.e., the values located at the 2.5% and 0.3% quantiles of the normal distribution, respectively . Thereafter, these alternative cutoffs were back transformed to the original ELISA units , using the predict function in R.The evaluation of test performance was constrained by the small number of lsewhere , alternaM. hyopneumoniae ELISAs were determined by Poisson regression using generalized estimating equations . For the misclassification analysis, false-positive results were defined as any positive result from non-M. hyopneumoniae-inoculated pigs or from M. hyopneumoniae-inoculated pigs at <21 dpi . False-negative results were defined as negative results from M. hyopneumoniae-inoculated pigs ≥21 dpi. Two pigs in the M. hyopneumoniae group did not become M. hyopneumoniae infected via inoculation or contact, i.e., were free of M. hyopneumoniae infection on the basis of M. hyopneumoniae PCR testing of lung homogenate collected at the termination of the experiment, and were excluded from the misclassification error rate analysis.Overall misclassification error rates  for the six commercial P ≤ 0.05.The Poisson regression model used the count of misclassification responses as the dependent variable, ELISA as the independent variable, and dpi as a repeated measure, and it assumed a compounded symmetry correlation structure across dpi . The mod(ii) Study 2. Serum samples  were tested on BioChek, IDEXX, and Hipra ELISAs. For noninoculated pigs, individual animal M. hyopneumoniae status was established by PCR testing of tracheal samples (positive or negative).M. hyopneumoniae ELISAs were estimated by logistic regression using GEE  with the qualitative ELISA result as the dependent variable, sample status  as the independent variable, and pen as a repeated measure (1 to 46), and assumed a heterogeneous first-order autoregressive covariance-variance structure  were observed in seven pigs, and increased joint fluid volume was observed at necropsy. One M. hyosynoviae-inoculated pig died during blood collection on 10 dpi.Clinical signs and pathological lesions at necropsy were reported in a previous study . In brieM. hyopneumoniae DNA testing of pen-based oral fluid samples collected daily from M. hyopneumoniae-inoculated animals (n = 276) confirmed productive M. hyopneumoniae infection in the inoculated group. Overall, 80 of 276 (29.0%) oral fluid samples were positive for M. hyopneumoniae DNA, with the first PCR-positive at 9 dpi. At the individual pig level, necropsy and DNA testing of individual pig lung homogenates revealed that 8 of 10 pigs had been infected, i.e., 2 pigs showed no gross lesions compatible with M. hyopneumoniae, were M. hyopneumoniae DNA negative, and showed no evidence of an antibody response against M. hyopneumoniae. Therefore, these 2 pigs were removed from the study, and, for this reason, the statistical analyses were based on 8 M. hyopneumoniae-infected pigs. M. hyopneumoniae DNA testing of weekly pen-based oral fluid samples from M. flocculare (n = 44), M. hyorhinis (n = 33), M. hyosynoviae (n = 39), and negative control groups (n = 36) were all negative, i.e., no inadvertent contamination of these pigs or samples from these groups with M. hyopneumoniae occurred during the experiment.M. hyorhinis DNA was detected in one serum sample (10 dpi) from the M. hyorhinis group at 10 dpi and consistently in pen-based oral fluid samples from 2 dpi through 56 dpi. M. hyosynoviae DNA was detected in two serum samples (3 and 7 dpi) from two M. hyosynoviae-inoculated pigs and intermittently in pen-based oral fluid samples from 4 dpi through 15 dpi. M. flocculare DNA was not detected in serum or oral fluid samples collected during the study. M. hyorhinis and M. hyosynoviae antibody responses were detected at earlier 10 dpi in serum samples. No M. flocculare antibody response was detected in serum during the study. Thus, M. hyorhinis and M. hyosynoviae groups were infected using the inoculation procedures described in M. flocculare-exposed pigs.M. hyopneumoniae inoculated pigs were used to evaluate the false-positive rates in the 6 ELISAs. Four cutoffs were considered: (i) manufacturer's cutoff with suspect considered negative, (ii) manufacturer's cutoff with suspect considered positive, (iii) cutoff based on the mean of the results from non-M. hyopneumoniae groups plus two standard deviations (x̄ + 2 SD), and (iv) cutoff based on the mean of the results from non-M. hyopneumoniae groups plus three standard deviations (x̄ + 3 SD).A total of 680 serum samples were collected over the course of the study (−3 to 56 dpi) from the pigs in the 5 treatment groups. For the analysis of the ELISAs, the 540 serum testing results from the non-x̄ ± 2 or 3 SD were generally less stringent than the manufacturer's cutoffs and, with exception of IDvet ELISA, resulted in more false-positive results. No patterns in false-positive responses were observed between any of the ELISAs and non-M. hyopneumoniae-inoculated groups, i.e., negative control, M. flocculare, M. hyorhinis, and M. hyosynoviae.As shown in M. hyopneumoniae antibody over time in the 8 individual pigs colonized by M. hyopneumoniae is shown in M. hyopneumoniae antibody-positive pigs, the overall ELISA comparison was based on misclassification errors under two scenarios, i.e., with suspect results interpreted as negative or positive (P < 0.05) in the misclassification rate was detected among BioChek, IDEXX, Hipra, and Oxoid ELISAs, whereas significantly higher misclassification rates were observed in Eurofins Ingenasa and IDvet ELISAs. With suspect considered negative, the misclassification error rates for Eurofins Ingenasa and IDvet ELISAs were 2.07 and 3.28, respectively, i.e., 107% and 228% higher than the other ELISA used in the comparison. With suspect considered positive, the misclassification rates for Eurofins Ingenasa and IDvet ELISAs were 2.36 and 3.00, respectively, i.e., 126% and 200% higher than the comparison ELISAs.The detection of an positive . For botM. hyopneumoniae DNA testing of tracheal samples from M. hyopneumoniae-inoculated pigs (n = 10) confirmed productive infection, i.e., all M. hyopneumoniae-inoculated pigs were positive for M. hyopneumoniae DNA testing at 7 dpi and a noninoculated pen mate at 15 dpi. In noninoculated pens, M. hyopneumoniae DNA testing of tracheal samples verified transmission to other animals throughout the room over time, with the first positive at 28 dpi (t 28 dpi .n = 8) from inoculated pigs were antibody negative at 0 dpi, with the exception of one sample positive on BioChek ELISA. At 15 dpi, 5 of 10 serum samples were antibody positive on IDEXX and Hipra ELISAs, and 7 of 10 were antibody positive on BioChek. All M. hyopneumoniae-inoculated pigs were antibody positive on all the three ELISAs at 42 and 56 dpi. In the noninoculated pigs, all ELISAs were negative until 42 dpi, including pigs sharing the pen with the 10 M. hyopneumoniae-inoculated pigs, with the exception of one BioChek-positive sample at 15 dpi  and then compare the best performing assays using field samples (study 2).The objectives of this study were to compare the performance of commercial M. flocculare, M. hyorhinis, M. hyosynoviae, or M. hyopneumoniae under experimental conditions, which were used to achieve these objectives. The use of CDCD pigs, i.e., animals raised and maintained in a highly controlled environment free of common swine pathogens , swine mycoplasmas, etc.), provided assurance that all pigs were mycoplasma free at the beginning of the study. Moreover, using colostrum-deprived (CD) pigs is a good alternative strategy to circumvent passively acquired immunity against mycoplasmas or exposure to any mycoplasma that may occur shortly after birth. Collecting and testing serum and oral fluid specimens over the course of the observation period and lung tissue recovered from pigs at euthanasia allowed for establishing the timeline and infection status of individual animals. Serum samples of a precisely known status were then used to evaluate and compare ELISA performance.Study 1 was performed with samples from cesarean-derived, colostrum-deprived (CDCD) pigs inoculated with M. hyopneumoniae group were intratracheally inoculated with using lung homogenate strain 232 (1.0 × 106 CCU/ml), a well-characterized and mildly virulent isolate , with the first PCR-positive sample collected at 9 dpi. At euthanasia, no lung lesions were observed, but M. hyopneumoniae was confirmed in lung homogenate by PCRs in 8 of 10 pigs. Two M. hyopneumoniae-inoculated pigs were PCR negative for lung homogenate and likewise showed no detectable antibody response on any of the ELISAs. These data supported the conclusion that these animals did not become infected either through inoculation or through exposure to M. hyopneumoniae-infected animals housed in the same room. Therefore, the analyses were based on the 8 pigs demonstrated to have become infected with M. hyopneumoniae.Animals in the ntrolled , 16 and ntrolled , 25. Infe models , 27. TheM. hyorhinis and M. hyosynoviae, with pen-based oral fluids first positive for M. hyorhinis and M. hyosynoviae DNA by 2 and 4 dpi, respectively  and the absence of antibody in serum samples (n = 140) evaluated  from 50 CDCD pigs in 5 defined mycoplasma exposure groups. Two sources of variation in M. hyopneumoniae antibody detection were apparent: pig-to-pig variability in response to exposure to M. hyopneumoniae and assay variability performance. As described by Pieters and Maes  for 5 ELISAs  and 2.5% and 0.3% quantiles  for Oxoid ELISA, as described in reference The and Maes , M. hyop results . Most ty results . In thisM. hyopneumoniae ELISAs were analyzed in terms of misclassification error rate based on the manufacturers' cutoffs. The misclassification error rate was calculated as the total number of false negatives and false positives among samples tested for both suspect conditions, i.e., as positive or as negative  were observed in all assays evaluated. No significant difference in misclassification error rate was observed among BioChek, IDEXX, Hipra, and Oxoid ELISAs  free of M. hyopneumoniae, porcine reproductive and respiratory syndrome virus, and influenza A virus infection by intratracheal inoculation of 10 pigs with M. hyopneumoniae. Thereafter, the collection and DNA testing of tracheal samples from all pens over time was used to establish the M. hyopneumoniae infection status of individual animals and compare serum ELISAs.In study 2, the three ELISAs  that provided the highest performance in study 1 were evaluated under field conditions. Specifically, M. hyopneumoniae from commercial production systems requires ongoing testing to establish the true status of populations and detect the introduction of the pathogen. In study 1 (CDCD pigs), the three ELISAs  with the best performance were equivalent when comparing diagnostic specificity and false-positive rates. Notably, IDEXX and Hipra performed best by interpreting suspect results as positive. Likewise, in study 2, although the BioChek ELISA produced 2 false-positive results based on DNA testing, no statistically significant differences were detected in the diagnostic sensitivity or specificity of BioChek, IDEXX, and Hipra ELISAs. The point of conflict in the monitoring process has been (and continues to be) maximizing early detection while minimizing false-positive reactions. Future research should focus on improving diagnostic methods in order to be able to improve time to detection and overall diagnostic sensitivity. For the present, the data reported in this study will help users understand ELISA performance and select the assay (or combination of assays) most suited to their testing objective(s).The control and/or elimination of


The relationship between kidney function and hearing loss has long been recognized, but evidence of this association mostly comes from small observational studies or other populations. The aim of this study is to explore the association between reduced kidney function and hearing loss in a large population-based study among the middle-aged and elderly Chinese.2. Multivariable logistic regression was employed to adjust for potential confounders, including demographics, health-related behaviors, and cardiovascular risk factors.Data collected from the Chinese Health and Retirement Longitudinal Study (CHARLS) in 2015 were used for analysis. A cross-sectional study was conducted among 12,508 participants aged 45 years and older. Hearing loss, the outcome of this study, was defined according to interviewees’ responses to three survey questions related to hearing in the CHARLS. Estimated glomerular filtration rate (eGFR) was employed to assess kidney function, and participants were classified into three categories based on eGFR: ≥90, 60–89 and < 60 mL/min/1.73 m2, participants with eGFR of 60–89 mL/min/1.73 m2  and eGFR < 60 mL/min/1.73 m2  showed increased risk of hearing loss after adjusting for potential confounders.The overall prevalence of self-reported hearing loss in the study population was 23.6%. Compared with participants with eGFR ≥90 mL/min/1.73 mReduced kidney function is independently associated with hearing loss. Testing for hearing should be included in the integrated management of patients with chronic kidney disease. The World Health Organization (WHO) reported that approximately 432 million adults suffered from disabling hearing loss in 2018 and estimated that over 900 million people will have disabling hearing loss by 2050 [The effects of aging on the auditory system are considered the leading cause of adult-onset hearing loss . The assKidney disease has become a public health issue of global concern in recent years. Since the first report on Alport syndrome revealed that hereditary familial nephritis is related to sensorineural deafness in 1927 , severalThe kidney and cochlea have common antigenicity and similar physiologic mechanisms involving the transport of fluid and electrolytes, which might explain the hearing loss in patients with kidney disease . Some po2 were compared. However, Gupta et al. [Unfortunately, most of existing studies have small sample sizes or enrolled patients with kidney failure; moreover, few studies have focused on the association between hearing loss and mildly-to-moderately reduced kidney function in a large population , 25. Vila et al.  found thThe prevalence of hearing loss is consistently higher in middle-aged and older populations than in younger cases, but evidence of the association between reduced kidney function and hearing loss in the Chinese middle-aged and older population is limited. We conducted a cross-sectional population-based study to explore the relationship between reduced kidney function and hearing loss by taking advantage of a representative sample of the general population aged 45 years and older and the strict quality control process of the Chinese Health and Retirement Longitudinal Study (CHARLS) .cun) in rural areas and neighborhoods (shequ or juweihui) in urban areas. Eighty or more households with age-eligible members were selected within each PSU, and one age-eligible member was randomly selected from qualified households. If the chosen person was willing to participate, this person and his or her spouse were interviewed. All stages of sampling were conducted by a computer to avoid potential biases arising from human manipulation.The CHARLS, a nationally representative survey of China’s middle-aged and elderly population, provides a high-quality public micro-database with social, economic, and health information. Samples were selected by using multistage probability sampling taking into consideration regional and socioeconomic disparities. The probability-proportional-to-size (PPS) sampling technique was used to select 150 county-level units from all counties of China except Tibet. PPS sampling was also applied to select three primary sampling units (PSUs) from each county-level unit. PSUs represent the lowest level of government organization and consist of administrative villages  system, which could help substantially improve the quality of the surveyed dataset by detecting errors in data inputs. When the interviewer enters an input item with a logical error or abnormal value, the system will pop out a message to alert the interviewer to this dubious entry. In this way, the participants no longer need to read the questionnaire, and the interviewers would ask them face to face and help them understand the questions and corresponding options. Anthropometric and physical measurements were provided, and blood samples were collected by trained nurses from township hospitals or China’s Center for Disease Prevention and Control (CDC) according to the standard protocol.CHARLS was approved by the Biomedical Ethics Review Committee of Peking University (IRB00001052–11015), and all participants signed informed consent before participation.The exclusion criteria of the study were as follows: 1) demographic data were not recorded; (2) aged < 45 years old; and (3) creatinine or hearing status data were not recorded. A total of 12,508 participants were included in our final analysis. The participant selection process is shown in Fig.  demograpIn our study, hearing loss was identified through self-reporting. Objective measurement, such as audiometry, was not provided in CHARLS, but several studies have demonstrated the reliability of self-reported hearing loss , 29. We The estimated glomerular filtration rate (eGFR) is considered the best overall index of kidney function in health and disease . In this2, according to the different stages of CKD defined by the Kidney Disease: Improving Global Outcomes (KDIGO) guideline [2 and 8 (0.1%) participants with eGFR < 15 mL/min/1.73 m2, which may lead to limited statistical power to examine the association between eGFR and hearing loss. This is a study based on general population, and thus there are less people in lower eGFR groups. Therefore, we finally used only three eGFR groups of ≥90, 60–89, and < 60 mL/min/1.73 m2 for analysis, which represented normal kidney function, mildly reduced kidney function, and moderately to severely reduced kidney function, respectively.At the beginning of this study, we attempted to classify the eGFR into five groups: ≥90, 60–89, 30–59, 15–29, and < 15 mL/min/1.73 muideline . We calc2), normal weight (18.5 to 24.9 kg/m2), overweight (25.0 to 29.9 kg/m2), and obese (≥30 kg/m2) according to their BMI. In our study, central obesity was defined as a waist circumference of ≥80 cm for females and ≥ 102 cm for males. Hypertension was defined as mean systolic blood pressure (SBP) ≥140 mmHg, mean diastolic blood pressure (DBP) ≥90 mmHg, or a self-report of hypertension. Diabetes was defined as fasting plasma glucose ≥126 mg/dL, HbA1c concentration ≥ 6.5%, or a self-reported doctor diagnosis. Stroke was defined as a self-reported history of doctor diagnosis. HDL and LDL cholesterol levels were directly obtained from the results of laboratory tests.We chose predictor variables by referring to hearing loss risk factors reported in the existent literatures , 33 and t-test was used to compare the mean values of continuous variables between participants with and without hearing loss, and differences in hearing loss prevalence among different categorical variable groups were tested by using Pearson’s χ2 test. The association between reduced eGFR and hearing loss was modeled by using a logistic regression function, and odds ratios (ORs) with 95% confidence intervals (CIs) of hearing loss for different eGFR categories were calculated. Multivariable logistic regression models were constructed to adjust for potential confounding variables . To build the multivariable logistic regression models, we used a separate unknown category to represent missing data for BMI, central obesity, and stroke, which had missing rates greater than 1%. Other variables included in the models that had missing rates lower than 1% were not preprocessed as cases containing missing data were automatically deleted during logistic regression analysis.Descriptive statistics for continuous variables are presented by using means and standard deviations (SD), while frequencies and percentages are used to describe categorical variable characteristics. Student’s p values were based on two-sided tests with a significance level of 0.05.All analyses were performed by using STATA software (version 14.0). All p values < 0.001). The distribution of self-reported hearing loss prevalence is presented in Table 2 reported hearing loss. The prevalence of hearing loss in this group was nearly twice that in the group with eGFR ≥90 mL/min/1.73 m2 (19.4%).A total of 12,508 participants were included in the final dataset for analysis. The characteristics of participants are described in Table 2, the ORs of participants with eGFR of 60–89 mL/min/1.73 m2 and eGFR < 60 mL/min/1.73 m2 were 1.11  and 1.25 , respectively. The detailed multivariable logistic regression analysis results are shown in Table SThe results of logistic regression analysis of the association between eGFR and hearing loss after adjusting for potential confounders are listed in Table The results of our study indicated that 23.6% of middle-aged and older Chinese have hearing loss and that this prevalence was higher at older ages. In the process of exploring the association between eGFR and hearing loss, we observed that the odds of hearing loss are higher among participants with lower eGFR.Comparing the prevalence of hearing loss observed in this study with previous studies is difficult because different studies included participants belonging to different age groups and defined and measured hearing loss using different instruments. Pure-tone audiometry and self-reporting are two common ways to assess hearing status in hearing-related studies , 33–38. 2 has a significant independent influence on the hearing status of adults. The authors also reported that the OR of hearing impairment in participants with eGFR < 60 mL/min/1.73 m2 compared with those having eGFR ≥60 mL/min/1.73 m2 is 1.25 , after adjusting for age, sex, smoking, alcohol, BMI, diabetes mellitus, hypertension, dyslipidemia and microalbuminuria. Similarly, Vilayur et al. [2 and found an independent association between moderate CKD and hearing loss with an OR of 1.43 . In a prospective study of 1843 individuals, Gupta et al. [2 have higher odds of hearing loss, compared with participants with eGFR ≥90 mL/min/1.73 m2. These results suggest that, not only the CKD patients at stage 3–5, but also those at stage 2 have an increased risk of hearing loss. More attention should be paid to the hearing status of CKD patients from early stage to enable the implementation of suitable interventions at an appropriate time and reduce the burden of the disease.The association between reduced kidney function and hearing loss found in our study is consistent with the results of some previously published studies , 25. Seor et al.  defined a et al.  estimate+ cycling and endolymphatic K+, Na+, Ca2+, and pH homeostasis are expressed in both the inner ear and kidney [Possible mechanisms of the association between reduced kidney function and hearing loss have been reported. Some evidence indicates that many physiological, pathological and pharmacological similarities exist in the cochlea and kidney  and thatd kidney . Some std kidney –45 also d kidney  found thd kidney  reported2 or central obesity, and hearing loss. However, overweight (BMI in 25.0–29.9 kg/m2) appeared to be a protective factor of hearing loss, which may be due to the low rate of obesity in the middle-aged and elderly Chinese population. Based on the logic of deriving clinical risk scoring system in previous studies [Besides the reduced eGFR, our study also confirmed that health condition factors, including older age, hypertension, diabetes, and stroke, are significantly associated with hearing loss. Hearing loss in older persons may be caused by the death or damage of cochlear sensory hair cells and decreased functioning of the stria vascularis . Explana studies , the risThe main strengths of our study include the nationally representative large-scale dataset, high response rate, and strict quality control process. However, certain limitations must also be noted. First, causal inferences could not be drawn based on the current cross-sectional study. Second, information bias may exist as hearing loss was defined on the basis of self-reported results. However, the reliability of self-reported hearing loss has been previously confirmed , 29. ForThis study indicated that reduced kidney function is independently associated with hearing loss in the middle-aged and elderly Chinese and that patients with reduced kidney function have higher odds of hearing loss. We recommend that testing for hearing should be included in the integrated management among patients with CKD, and patients with CKD need to pay attention to their hearing status from early stage to enable the implementation of suitable interventions at an appropriate time and prevent the development of hearing loss.Additional file 1: Table S1. Multivariate Logistic regression analysis of hearing loss


Scientific Reports 10.1038/s41598-019-41143-y, published online 14 March 2019Correction to: The Legend for Figure 10 is incomplete, as it does not specify that the panels for Figure 10b and 10c overlap. As a result,A–C) Abundant TCs were identified in the interstitial stroma. TCs had cell bodies (arrowheads) Podomeres were ramified and formed a 3D network, note: epithelium (EP), macrophage.”“(should read:A–C) Abundant TCs were identified in the interstitial stroma. TCs had cell bodies (arrowheads) Podomeres were ramified and formed a 3D network, note: epithelium (EP), macrophage. Panel 10b shows one telocyte cell under the epithelium (Ep), associated with a macrophage (selected square); Panel 10c provides a more focused image of the same area under the epithelium, showing another telocyte (arrow head) in addition to the previous one.”“(


PD is a complex, multifactorial disorder and its pathogenesis involves multiple pathways and mechanisms such as α-synuclein proteostasis, mitochondrial function, oxidative stress, calcium homeostasis, axonal transport, and neuroinflammation. Motor symptoms manifest when there is already an extensive dopamine denervation. There is therefore an urgent need for early biomarkers to apply disease-modifying therapeutic strategies. Visual defects and retinal abnormalities, including decreased visual acuity, abnormal spatial contrast sensitivity, color vision defects, or deficits in more complex visual tasks are present in the majority of PD patients. They are being considered for early diagnosis together with retinal imaging techniques are being considered as non-invasive biomarkers for PD. Dopaminergic cells can be found in the retina in a subpopulation of amacrine cells; however, the molecular mechanisms leading to visual deficits observed in PD patients are still largely unknown. This review provides a comprehensive analysis of the retinal abnormalities observed in PD patients and animal models and of the molecular mechanisms underlying neurodegeneration in parkinsonian eyes. We will review the role of α-synuclein aggregates in the retina pathology and/or in the onset of visual symptoms in PD suggesting that α-synuclein aggregates are harmful for the retina as well as for the brain. Moreover, we will summarize experimental evidence suggesting that the optic nerve pathology observed in PD resembles that seen in mitochondrial optic neuropathies highlighting the possible involvement of mitochondrial abnormalities in the development of PD visual defects. We finally propose that the eye may be considered as a complementary experimental model to identify possible novel disease’ pathways or to test novel therapeutic approaches for PD.Parkinson’s disease (PD) is characterized by motor dysfunctions including bradykinesia, tremor at rest and motor instability. These symptoms are associated with the progressive degeneration of dopaminergic neurons originating in the  Parkinson’s disease (PD) represents the second most common neurodegenerative disorder after Alzheimer’s disease. The prevalence of this condition in industrialized countries is generally estimated at 1% in people over 60 years of age representing an important burden for health systems considering that to date treatment options are mostly symptomatic . PD is aSNCA gene that encodes α-Syn and genes associated with mitochondrial function  proteostasis, mitochondrial function, oxidative stress, calcium homeostasis, axonal transport, and neuroinflammation. Of note, many PD mutations affect the function .substantia nigra pars compacta (SNpc), from which neurons project to the corpus striatum and to other brain regions of the mesocorticolimbic system, and by the accumulation of cytoplasmic inclusions, called Lewy bodies, mainly consisting of aggregated α-Syn and ubiquitin (PD is characterized by the death of dopamine (DA) neurons localized in the biquitin .SNCA gene leads to the formation of α-Syn aggregates (oligomers and fibrils), which induce a time-dependent loss of DA neurons  that constitute three cellular layers in the vertebrate retina: rod and cone photoreceptors in the outer nuclear layer (ONL), horizontal, bipolar, and amacrine interneurons and Müller glial cells in the inner nuclear layer (INL), and ganglion and displaced amacrine cells in the ganglion cell layer (GCL) . α-Syn astimuli: their heterogeneous nature makes this process rather complex from a physiological perspective  and horizontal cells. The latter are a class of inhibitory neurons fulfilling feed-back and feed-forward inhibition on both photoreceptors and bipolar cells. Downstream of bipolar cells, the electric signal is sent to RGCs whose axons form the optic nerve responsible for transmitting information to the brain. Intermingled among bipolar and RGC, amacrine cells can provide feed-forward and feed-back inhibition to both cell types by releasing γ-aminobutyric acid (GABA) and glycine. Muller cells, seeded with their bodies in the INL, are the main glial cells of the retina and support neuronal functions.As in the brain, dopamine plays a key role also in the retina and is involved in a variety of processes such as modulation of light adaptation, color vision, retinal development, synaptic formation and transmission .DA in the retina is synthetized in a subpopulation of amacrine cells (A18) localized in the IPL. Amacrine cells represent ∼35% of all cell types and they are classified based on the synthetized neurotransmitters, which mainly include glycine and GABA. Most DA amacrine cells (DACs) also synthetize GABA. DACs constitute only a small part of amacrine cells and they often co-express GABA and glycine . DACs haBased on this set of experimental evidence highly specific DA-dependent vision deficits might be expected in PD patients. However, the visual impairment can be worsened by deficits related to α-Syn pathology and/or mitochondrial related dysfunction that, although might preferentially impair DA neurons function in the initial stage of the pathology, are also harmful for other cell types leading to widespread neurodegeneration and impaired visual function.As mentioned before, immunohistological studies on PD patients’ postmortem retinas revealed the presence of α-Syn aggregates and Lewy bodies in dispersed amacrine cells at the border of the INL and in the RGC layer, and diffuse α-Syn depositions and Lewy neurites in the IPL . DiffereAmong the visual disfunctions described in PD patients, visual acuity, contrast sensitivity, and color vision impairment are more directly linked to the retinal alteration observed in PD patients showing α-Syn aggregation, retinal neuron degeneration and reduced retinal DA levels.Visual acuity refers to the ability to discern the shapes and details. Defects in this task were reported in about 30% of PD patients and this effect is not corrected by DA .Reduction of contrast sensitivity has been found to be one of the earliest signs of PD suggesting that this defect could be used as a premotor biomarker . PatientLRRK2) gene mutations show a more severe impairment of color discrimination compared with idiopathic PD patients  resolution  allows measurements of retinal layers solution . In partsolution  that, nosolution . In contsolution . OCT anasolution . Howeversolution . These dElectrophysiology techniques such as electroretinography (ERG) and visual evoked potentials (VEP) allow the analysis of selective retinal circuits and the determination of dysfunction of specific retinal cell types.A close electrophysiological examination of the retina has shown indeed a genuine impairment at the level of the local circuitry in PD subjects. ERG measures the electrical response evoked by a brief visual stimulus recorded from the retina. The analysis of the resulting electrical potential gives information about the function of different retinal cell types including cones, rods, photoreceptors and retinal interneurons . ERG is However, it is necessary to remember that by recording at the level of the visual cortex, VEP does not provide information exclusively about the retina but rather about the whole visual pathway. To date, the precise mechanisms responsible for the alterations described above are not fully understood. It has been suggested that decreased levels of DA can account (at least in part) for these abnormalities. In agreement with this suggestion, lower levels of TH or DA in the retina of PD patients have been also described .Different studies have demonstrated that PD patients display ERG profiles in which the amplitude and the latency are altered as compared to control age-matched subjects . InteresThese limitations in terms of mechanistic issues are compensated by the high clinical relevance that the combination of these ophthalmic diagnostic tools  may represent for early and non-invasive diagnosis of PD.To date a limited number of studies has been performed on the retina of PD animal models; all these studies, however, consistently report visual and retinal abnormalities as summarized in Ocular abnormalities were studied in the classical neurotoxin-induced rodent models generated by systemic administration or intracranial injection of drugs, such as Rotenone, 6-hydroxydopamine (6-OHDA), or 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).in vivo OCT analysis and detection of apoptotic retinal cells (DARC) demonstrated retinal neurodegeneration at 20-days post-rotenone injection, while degeneration of DA neurons in the SN and striatum became evident at day 60. These data indicate that neurodegeneration occurs first in the retina and then in the brain supporting the idea that the retina can be used as a potential biomarker tissue for early diagnosis.Rotenone is a naturally occurring pesticide and a potent inhibitor of the Mitochondrial Respiratory Chain (MRC) complex I and is used to model PD in animals. Rotenone-treated rats showed a decreased number of RGC and DA amacrine cells as wells as decreased thickness of the INL and ONL . These a6-OHDA and MPTP are toxins that selectively destroy DA containing neurons and are widely used to induce PD in animal models. Unilateral injection of 6-OHDA into the substantia nigra induced a decrease in DA levels in the retina of a rat PD model . AbnormaInjection of MPTP in mice and monkeys causes a reduction in the number of TH-positive amacrine cells and in retinal dopamine levels . In MPTPTakatsuna and collaborators observed a reduction in the amplitude of oscillatory potentials and b-wave after intraperitonel injection of MPTP and ERG analysis 10 and 30 days after MPTP injections in C57BL/6J mice . PrimateLRRK2 mutations, and knockout models of PINK-1 and DJ-1.Besides the toxin-induced models, ocular abnormalities were also studied in animal models of PD including transgenic models overexpressing both wild-type (wt) and mutated α-Syn, Similar to what has been observed in human PD retinas, the analysis of the (Thy-1)- hu-A30P- α-Syn transgenic mouse showed the expression of the α-SYN transgene in the inner retinal layers while phospho-α-Syn was found in INL cells, in the cell bodies of the GCL, and in neurites in the IPL . No expeRecently, intravitreal injection of adeno-associated viral (AAV) vectors over-expressing the wt human α-Syn (hu-α-Syn) has been used to evaluate the effects of α-Syn over-expression in the mouse retina . AAV-hu-Drosophila and retinal functions have been examined. Among the mutations tested, one of the most common is the LRRK2-G2019S mutation. Its impact on visual function has been assessed by ERG following a 500 millisecond (msec) stimulation with blue light. Electrophysiological experiments revealed that compared to currents recorded from controls flies, the decrease in the peak-to-peak amplitude of the ERG observed in mutated flies was age-dependent, since it was detected starting at 10 days, reaching a minimal value at 28 days  is that the animal retina recapitulates the retina of patients and the complexity of the neuronal populations present in vivo. Furthermore, in vivo studies on disease mechanisms or on drug screening on the retina allows to test vision. Differently from the standard in vivo mesencephalic models the retina in animals is accessible with non-invasive methods  that allow a comprehensive analysis of the histological and functional progression of neuronal loss. This makes it particularly useful to follow the time-course of novel therapeutic strategies for months in the same subjects, which is one of the main limitations in animal models of neurodegeneration. Another important aspect is that it allows the local application of drugs, which is important for generating proof-of-concept evidence with novel pharmacological or genetic therapeutic approaches without using invasive (like intra-brain injection) and/or systemic injections that might have peripheral side effects. Therefore, the retina-model might be the first step for in vivo testing of disease modifiers. This aspect is also important for translating new therapeutic approaches to patients. Gene or pharmacological local treatments of the eyes are already at an advanced stage. There is the possibility of testing the efficacy of a treatment for a single patient by treating with local applications one single eye and evaluating it by in vivo imaging and functional tests. This would also contribute, together with studies on the patient IPS cells, to developing personalized therapies. For all these reasons we propose the retina as a useful complementary experimental model for the identification and study of pathways involved in the disease pathogenesis or to test novel therapeutic approaches for PD.Moreover, due to its anatomical organization, the retina could be an ideal structure to study the molecular mechanisms underlying PD pathology. The retina-model of PD does not replace the nigrostriatal models of PD, as it cannot recapitulate the cognitive and motor deficits as well as the complexity of the synaptic changes occurring in the mesocorticolimbic system in PD. However, it could have some advantages that combine those of ED and AI conceived and wrote the first draft of the manuscript. RP wrote the electrophysiological part of the manuscript. BF edited the manuscript. All authors shaped the final version of the manuscript.The authors declare that this study received funding from Fondazione Roche. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.


Little is known about the role of frailty in the recovery process of disability among older adults. We examined the association between frailty and recovery from activities of daily living (ADL) and instrumental ADL (IADL) disability among community-dwelling Chinese older adults.Data were from the China Health and Retirement Longitudinal Study. Three waves were used. Participants ≥60 years, had frailty assessment at baseline, and had incident disability in ADL or IADL in 2013, and had disability assessment in 2015 were included. Recovery from ADL and IADL disability were used as outcome measure. Multivariable logistic regression was used to evaluate the potential association between frailty and recovery from ADL or IADL.We included 516 and 598 participants in the ADL and IADL analysis, respectively. In total, 237 participants recovered from ADL disability and 293 recovered from IADL disability. Nearly half of the non-frail persons recovered from ADL disability, while less than one-quarter of the frail persons had recovery. Over half of the non-frail persons had IADL disability recovery, while only 30% of the frail recovered. After adjustment, the odds of recovery from ADL disability were 59%  lower among frail participants than those who were non-frail; the odds of recovery from IADL disability were 52% lower among frail persons than those who were non-frail and the association did not reach statistical significance.Frailty is an independent predictor of poor recovery from disability among nondisabled community-dwelling older adults in China. The prevalence of disability in activities of daily living (ADLs) and instrumental ADL (IADLs) has declined during the past several decades in China –3. This A growing body of literature has suggested that disability is a dynamic rather than static process and transitions among different states of disability are common –15. PrioIn the present study, we examined the association between frailty and recovery from ADL and IADL disability among community-dwelling Chinese older adults without pre-existing disability from the China Health and Retirement Longitudinal Study (CHARLS). We hypothesized that frail older adults would have lower ability to recover from disability than non-frail ones.Data are from the CHARLS, an ongoing longitudinal cohort study of a nationally representative sample of community-dwelling adults from 28 provinces of China. A total of 17,708 residents aged 45 years or older were interviewed at baseline 2011–2012), with a response rate at 80.5%, and followed every two years afterwards (2013–2014 and 2015–2016 waves). All participants gave informed consent; the protocol was approved by the Ethical Review Committee at the Peking University. Further details about the recruitment strategies and study design have been published elsewhere [11–2012, Frailty was measured at baseline (2011–2012); data on disability were collected at baseline and two follow-ups. We included participants who (i) were ≥ 60 years old, (ii) had frailty assessment at baseline, (iii) had no disability in 2011, (iii) were disabled in 2013, and (iv) had disability assessment in 2015. Among 611 participants who had frailty assessment and were not disabled in ADL in 2011, and developed ADL disability in 2013, we excluded 95 individuals who were lost to follow-up, resulting to an analytic sample of 516 for ADL analysis. Among 701 participants who had frailty assessment and were not disabled in IADL in 2011, and developed IADL disability in 2013, 103 individuals were lost to follow-up, leading an analytic sample of 598 for IADL analysis.Frailty was assessed by a modified version of the Fried’s Physical Frailty Phenotype (PFP) , which w2 .Slowness was measured by usual gait speed over a 2.5-m course. Two trials were performed and the average walking time was used to calculate gait speed (m/s). Slowness was defined as sex- and height-specific gait speed ≤20th percentile of the weighted sample distribution . HandgriFrailty was categorized into non-frail, prefrail, and frail according to the number of criteria met. Non-frail was defined when none of the criteria was met; prefrail was defined when one or two criteria were met; frail was defined when three to five criteria were met.Disability was assessed using five ADL tasks  and five IADL) tasks , by asking “Do you have difficulty in” performing the task? Participants who answered “I have difficulty but can still do it”, “Yes, I have difficulty and need help”, or “I cannot do it” to one or more tasks were considered having ADL or IADL disability. Recovery from ADL (or IADL) disability was defined as regaining independence in all ADLs (or IADLs) in 2015 after being disabled in 2013.Demographic factors included age, sex, education , and marital status (married vs. not married). Smoking status was categorized as never, former or current smokers. Body mass index (BMI) was calculated as weight (kilograms) divided by height (meters) squared, and classified into underweight (BMI < 18.5), normal (BMI = 18.5–24.0), overweight (BMI = 24.0–28.0), and obese (BMI ≥ 28.0) . Historyn = 516) and IADL analysis (n = 598), separately. We used logistic regression models to examine the association between frailty  and recovery from disability. Age, sex, and disability severity (severe vs. mild) were included in the minimally adjusted models; education, marital status, BMI, smoking status, depression, cognitive function, and number of chronic conditions were additionally adjusted in the fully adjusted models. ADL and IADL were analyzed separately. In addition, we examined the association between frailty and recovery from IADL disability among 336 persons who had incident IADL disability in 2013 but did not have any difficulty in ADLs. All covariates were measured at the time of frailty assessment instead of at the most recent assessment of covariates prior to the onset of incident disability because covariates measured after frailty could be in the causal pathway between frailty and disability recovery. Adjustment for such mediators could lead to underestimation of the overall effect of frailty.We first described the baseline characteristics of participants included in the ADL analysis .We included 516 and 598 participants in the ADL and IADL analysis, respectively. Among participants included in the ADL analysis, 62.0% were 60–69 years of age, 57.4% were female, and 74.2% were married/living together; 37.8, 55.4, and 6.8% were non-frail, prefrail, and frail, respectively  recovered within 2 years (Table Recovery from IADL disability within 2 years was observed for 293 49.0%) of the 598 newly disabled participants  recovered within 2 years  and persistent frailty. In addition, we excluded elders who were initially disabled because of the fundamental differences between primary frailty and secondary frailty induced by disability. Moreover, although the modified version of the PFP approach has been validated among Chinese older adults, modifying the criteria could potentially lead to misclassification of frailty status . FurtherIn summary, we examined the association between frailty, assessed by the PFP scale, and recovery from ADL and IADL disability among community-dwelling older adults in China. Our results provided additional evidence that frailty is an independent predictor of regaining independence after being disabled and demonstrated the construct validity of the PFP scale for assessing frailty, a clinical syndrome characterized by reduced reserve and resilience to stressors. Most of the frailty instruments were validated for predictive validity only. We found that frailty, as assessed by the PFP approach, independently modified older adults’ ability to recovery from disability. This type of empirical investigation has been considered the best way to evaluate the validity of a frailty assessment . Taken t