While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating results between chemical, thermal, and mechanical unfolding. The limited knowledge of the relationship between these processes makes it challenging to compare results between studies if different refolding methods were applied. Our current work compares the energetic barriers and folding rates derived from chemical, thermal, and mechanical experiments using an immunoglobulin-like domain from the muscle protein titin as a model system. This domain, I83, has high solubility and low stability relative to other Ig domains in titin, though its stability can be modulated by calcium. Our experiments demonstrated that the free energy of refolding was equivalent with all three techniques, but the refolding rates exhibited differences, with mechanical refolding having slightly faster rates. This suggests that results from equilibrium-based measurements can be compared directly but care should be given comparing refolding kinetics derived from refolding experiments that used different unfolding methods. The seminal work of Christian Anfinsen and Cyrus Levinthal ,2 initiaA critical step in protein refolding studies is the denaturation of the purified protein, which is most commonly accomplished either by chemical denaturants or by thermal denaturation. These two approaches disrupt existing protein structure differently and so it has often been hypothesized that different unfolding methods result in different unfolding states due to differences in the types and number of intramolecular contacts existing in the unfolded state ,10,11. FIn recent years, a growing number of studies have applied force as a mechanism to unfold the protein of interest ,18,19,20One of the most studied proteins by force spectroscopy has been the titin protein, which has over 90 unique immunoglobulin (Ig) domains. One of the first domains from this protein to be characterized was the I91 Ig domain  ,21, thourefolding, as determined by chemical denaturation, is equivalent to the ΔGunfolding [We have been using the I83 domain, the terminal C-domain in the N2A region of titin, to further explore the question of how closely single molecule folding experiments correlate with chemical and thermal denaturation studies. Previous work by our lab has demonstrated that while the I83 domain is structurally similar to the I91 domain, it has a free energy of unfolding that is 2–3 times lower than I91 ,27. We hnfolding ,27. MostWe chose to use magnetic tweezers to explore the mechanical unfolding and refolding pathway of I83 for two primary reasons. First, it provides the ability to monitor discrete unfolding and refolding events at physiologically relevant forces (<20 pN) with high resolution  without unfolding and the ΔGrefolding are statistically equivalent regardless of the approach used for unfolding. In contrast, mechanical unfolding exhibited faster unfolding kinetics and faster refolding kinetics than chemical denaturation kinetics. We also demonstrate that including calcium at pCa ~4.3 stabilizes the I83 domain in force spectroscopy experiments, consistent with previously observed stabilization in chemical and thermal denaturation experiments. Most interestingly, this stabilization is observed under weak forces but not at higher forces, denaturant concentrations, or temperatures. This suggests that the calcium binding site disappears as more of the overall structure of the domain is lost. Overall, our results demonstrate that all three unfolding approaches tested are thermodynamically equivalent, but they are not kinetically equivalent.The focus of this work was to correlate the thermodynamic and kinetic properties of the I83 domain as measured following chemical, thermal, and mechanical unfolding. Our results demonstrate that the ΔGReagents: Chemicals were obtained through standard chemical suppliers such as Fisher Scientific, Waltham, MA, USA and Sigma-Aldrich, St. Louis, MO, USA. The coding sequences for the three Ig domains were codon optimized for Escherichia coli and synthesized by GeneArt. The synthesized genes were subcloned into the pET151-D-TOPO vector for expression. The NCBI sequence for the N2-A isoform of titin (NP_035782) was used as the reference for the sequences of the individual Ig domains.6 containing an N-terminal Halo-tag and C-terminal Avi-tag were transformed into chemically competent Escherichia coli BL21 (DE3) cells. Autoinduction media cultures were inoculated by 0.5% (v/v) overnight cultures and grew at 30 °C and 230 rpm for 16–18 h. Purification of Halo-(183)6 followed the same method as for the monomeric I83 [6. Size exclusion chromatography fractions containing purified Halo-(183)6 were pooled and dialyzed into storage buffer  with 20% glycerol, flash frozen, and stored at −80 °C.Expression and purification of the monomeric I83 domain was as published in our previous work . Expresseric I83 , sans cl2+) with varying urea concentrations (0–7.6 M urea). Samples were placed in a quartz 96-well plate and incubated at room temperature (22 ± 2 °C) for one hour prior to data collection to ensure equilibrium was reached. Samples were excited at 280 nm and the emission spectra were collected in 2 nm steps from 300 to 450 nm at 25 °C using a SpectraMax M3 plate reader. The Center of Mass (CoM) of each spectrum was calculated using Equation (1), where I is intensity and Purified protein was diluted to a final concentration of 100 µg/mL in storage buffer (+/− 50 µM CaGunfolding using Equation (2)x is the concentration of urea. A two-way t-test analysis of variance was used to determine statistical significance using a value of p < 0.01 as the cutoff for significance.CoM was plotted as a function of urea concentration and fit using linear extrapolation  to deterθ) using Equation (3):M is the molecular weight, L is the path length of the cell in centimeters, and C is the concentration of the protein in g/L. Values reported are the average of four scans and were background subtracted before deconvolution.Circular Dichroism (CD) spectra of 100 µg/mL samples of the purified and I83 domains were measured using a JASCO J-1500 CD Spectrophotometer . Variable temperature measurements were made at 222 nm and 208 nm every 0.1 °C nm from 15–95 °C while samples were placed in a sealed quartz cuvette with a 0.1 cm path length and heated at a rate of 1 °C/min using a Peltier temperature controller. The signal (mdeg) was converted to molar ellipticity (mH°) and unfolding midpoint (Tm) using Equation (4):T is the melting temperature of the protein, such that the midpoint temperature (Tm) yields a free energy of zero [The free energy for the two-state refolding processes were determined by using a derivation of the Gibbs-Helmholtz, in which the specific heat capacity (Cp) of the folded and unfolded state are assumed to be the same . CD data of zero .y in the signal intensity, c is the y-intercept, A is the amplitude of the curve, k is the rate constant, and t is time in seconds.Unfolding kinetics were measured by the addition of various concentrations of urea to achieve a final protein concentration of 100 µg/mL. Samples were excited at 280 nm and the emission spectra were collected at 20 °C. The unfolding emission spectra were monitored at 354 nm every 3 s using a SpectraMax M3 plate reader and rate constants were determined by fitting data to a single exponential using Equation (5):v/v, Sigma-Aldrich, St. Louis, MO, USA), and glutaraldehyde  as described by Popa et al. [6 protein was diluted in 20 mM HEPES buffer and added to a functionalized flow cell. Chambers were washed with TRIS buffer and then streptavidin coated paramagnetic beads  were added to attach the biotinylated C-terminus of the protein.Magnetic tweezers measurements were performed in fluid chambers that were functionalized by (3-aminopropyl)-trimethoxysilane in ethanol , as induced by applying pulling forces in the piconewton (pN) range, was measured vs. time using an imaging rate of 1800–2000 frames per second (fps). The load rate of the magnetic force was negligible (<0.01%) as compared to the overall unfolding rates observed. Noise-reduction was applied using the AutoStepFinder fitting algorithm in MatLab (R2021a), developed by Kerssemaker [A Z-stack library of both a protein-tethered paramagnetic bead and a fixed reference bead were initially acquired prior to each measurement and this was used to calculate the real-time Z-position displacements using the image processing method developed by the Fernandez Lab . The extssemaker . Five inssemaker .6 in absence and presence of calcium were conducted by exchanging the buffer components in the flow cell while taking measurements on a single molecule.The rate of homopolymer unfolding and refolding was determined using the same single exponential equation sed for the fluorescent kinetic experiments to fit the stepwise unfolding and refolding events over time when initiated by a change in force (Equation (5)). Force-induced folding comparisons of Halo-(I83)ku) and refolding rates (kf) in for the equilibrium constant (K) in the free energy equation, as shown in Equation (7) using 293 K as T.Mechanical and chemical stabilities were compared by substituting unfolding . This suggests that, while the energy requirements to unfold the protein are similar from either chemical or thermal denaturation, there is a subtle difference in the overall nature of the unfolded state that decreases the free energy requirement of refolding following thermal denaturation [Protein refolding experiments generally start by unfolding the protein using either chemical or thermal denaturation or by the application of mechanical force ,37. Eachkcal/mol A and theturation ,42.2+ due to a non-canonical calcium binding site involving the F–G loop [unfolding of 1.0 kcal/mol at pCa 4.3 . A similar shift was observed for refolding as well, with refolding occuring faster following mechanical unfolding (2.72 ± 0.66 s−1 to 0.99 ± 0.13 s−1). Interestingly, since there is a comparable shift to both the folding and unfolding rates, there is not a statistically significant difference in the ΔGunfolding  to 3.5 ± 0.62 , p-value = 0.56). These experiments were conducted at pCa ~4.3 and a similar effect was observed. The impact of calcium appears to be greatest at mid-range denaturant concentrations (3–5 M urea) or forces (10–30 pN), where calcium is likely shifting the equilibrium toward the folded state. This is illustrated by the minima of the chevron plot for experiments conducted in the presence of calcium being shifted toward a higher concentration of either denaturant or force. Calcium reduces the extrapolated natural log of the rate of unfolding in the presence of denaturant and had no significant impact on refolding in the presence of denaturant. It is interesting to note that, while calcium impacts both the folding and unfolding in the presence of force, there is a larger change in the extrapolated natural log for the refolding rate, suggesting that either calcium is impacting the folding process more strongly than the unfolding process or that calcium has more of an impact in the presence of the low forces used to measure refolding.A statistically significant difference is observed between the rate of chemically induced unfolding and mechanically induced unfolding , with chSingle-molecule force-based refolding studies offers unique insights into the folding pathway of individual molecules, which differs from the insights that can be gained in bulk refolding studies initiated from a chemically or thermally denatured state. Bulk refolding experiments have the advantage of being relatively easy to conduct and can generally be done with equipment available in most laboratories. It is also relatively easy to alter variables such as temperature and buffer conditions in these types of experiments. These types of studies have provided valuable insights into protein folding over the years and have shown relatively good correlation with more recent single-molecule experiments ,9,44,45.Immunoglobulin domains make an ideal model system to explore the question of correlation between single-molecule and bulk solution measurements. There is a growing body of work exploring the physiological role of force-induced unfolding and refolding events in mechanical proteins such as titin that have repeated Ig-domains ,43,49,50unfolding and ΔGrefolding regardless of the method of denaturation. This suggests that it would be possible to draw correlations between free energies reported in different studies, even if the studies relied on different methods of denaturation. This is significant since techniques such as intrinsic fluorescence are faster and easier than single-molecule techniques and can be used in some cases in lieu of single-molecule experiments.The focus of this study was to compare both the ΔGs and rates of both folding and unfolding derived using chemical, mechanical, and thermal denaturation. Our results demonstrated that there is not a statistically significant difference between the ΔGOur results indicate that kinetic studies do not exhibit the same degree of correlation. Mechanical denaturation resulted in statistically significant increases in the rates of both unfolding and refolding compared to chemical denaturation. This could be simply due to a stochastic difference since chemical denaturation is generally used for bulk refolding experiments using tools like intrinsic fluorescence. In these types of experiments, the measured rate is the average of all the molecules in the reaction compared to the rate determined from a single molecule. Alternatively, this difference may reflect a difference in the unfolded state, changing either the path or distance required to refold. Our current data cannot distinguish between these two possibilities.Our model system, the I83 immunoglobulin (Ig) domain from titin, has been shown to be relatively unstable compared to most other Ig domains in titin but it is stabilized by the presence of calcium. Mechanical unfolding and refolding experiments conducted in the presence of calcium as part of this study correlated with our previous results under lower forces. Mechanically induced unfolding rates measured in the presence of calcium exhibited a statistically significant decrease in unfolding rate for forces ≤40 pN , as wellmdm mice, a model system that exhibits altered titin behavior [The N2A region of titin has become a region of particular interest in recent years as it has become apparent that this region is a signaling hub for a number of regulatory proteins, including the Muscle Ankryin Repeat proteins (MARPs), the calpain-3 protease, the SET and MYND-containing lysine methyl transferase 2 (Smyd2) and actin ,55,56,57behavior ,59. An imdm mice also points to the importance of this region, especially since recent studies have shown the deletion of the regions of the PEVK that are lost in mdm mice does not reproduce the mdm phenotype [It is interesting that the I83 domain has the lowest stability given that binding of calpain-3 occurs in this region. It is possible that the domain is stabilized by binding of calpain-3 and that this interaction helps to regulate titin function, similar to how CARP binding to I81 stabilizes the interaction between N2A and actin ,60. The henotype . Taken tThe canonical calcium binding site is an EF-hand motif , which dImmunoglobulin domains are highly conserved structural elements used as building blocks by many multi-domain proteins, from cellular receptors to immune system proteins to mechanical/elastic proteins like titin ,65,66,67The role of Ig-domain unfolding in passive tension has been debated for a number of years. The force required to unfold the Ig domains studied in early AFM experiments were larger than could be obtained physiologically and therefore it was questioned whether Ig domains would become unfolded during eccentric contractions . Recent unfolding values, while slightly different rate constants were observed between chemical and force refolding experiments. Interestingly, the rate of both unfolding and refolding was faster for force-induced unfolding and refolding. This implies that the act of tethering may increase the rate of transition between the folded and unfolded state. This is important to other biological processes where the protein might be tethered, like folding on the ribosome during protein synthesis or unfolding/refolding events in elastic proteins like titin. Recent findings suggest that the folding mechanism of a newly synthesized protein while tethered to a ribosome can influence the final folded structure in vivo [Protein folding is important in a variety of physiological processes, including the impact on the extension of muscle proteins. We found that thermal, chemical, and force unfolding yielded similar ΔG in vivo . Most pr


Workers in iron and steel factories in Tanzania are exposed to noise levels above recommended limit values, without using hearing protection devices. Exposure to noise levels above 85 dB(A) is associated with temporary threshold shifts (TTS) of human hearing. Nevertheless, there are few studies of noise and hearing from African countries.To determine whether the normal hearing workers in Tanzania experiences TTS after full-shift occupational noise exposure of 85 dB(A) and above.A total of 55 workers were included. Full-shift personal noise measurements were conducted. Pre- and post-shifts pure-tone audiometry were conducted for each worker. TTS was defined as a 10 dB or greater change at 1000, 2000, 3000 or 4000 Hz in either ear.Aeq,8h) of 90.4 dB(A) (SD = 2.7). The difference in mean hearing thresholds was higher at 4000 Hz [Arithmetic Mean (AM) = 10 dB SD = 4 dB] compared to that of 1000 Hz (AM = 4 dB SD = 3 dB), 2000 Hz (AM = 4 dB SD = 4 dB), and 3000 Hz (AM = 9 dB SD = 6 dB), respectively.We found that 85.5% of the workers developed TTS across the work shift. There was significant increase in mean hearing thresholds across shift at 1000, 2000, 3000 and 4000 Hz among the workers exposed to an average personal noise exposure (LInterventions to reduce occupational personal noise exposure are warranted to reduce the high risk of developing a permanent threshold shift with persistent high noise exposure. An intervention study is planned for this group of workers. Noise-induced hearing loss (NIHL) is a slowly progressive, sensorineural hearing decrement, which typically occurs at higher frequencies 3–6 kHz) as a result of prolonged exposure to high intensity sound levels . It is o2–6 kHz as4Aeq,8hr of 98 SD = 9.7 with 53.8% subjective hearing loss) [Aeq of 70–104 dB[A]) where the subjective hearing loss was 54% ) ) , and a h2 dB[A]) 13.Temporary threshold shift (TTS), which is the reversible hearing loss that occurs immediately after exposure to intense sound levels, and which recovers over a period of several hours or days after exposure, is considered an important indicator for the early development of irreversible hearing loss . Thus, mFew field studies have been conducted in humans on TTS and none among the iron and steel workers in Tanzania. These studies show increased hearing thresholds, and a high prevalence of TTS among noise-exposed workers. For example, a study in the United States involving male, hockey officials found a TTS of 86.2% at an equivalent noise exposure of 93 dB(A) ranging from 88–97 dB(A) . A TTS pAlthough the Tanzanian Occupational Safety and Health Act (OSHA) requires the employer to provide and maintain effective personal protective equipment (including Hearing Protection Devices [HPDs]) for use by employees who are exposed to hazardous levels of noise , a recenIn this study, we assumed that the iron and steel workers exposed to 92 dB(A)would develop TTS , and tha14This study involved male production workers in one iron and steel factory in Dar es Salaam, Tanzania. No women were working on the production line. The factory has a total of 200 workers on the production line and its production capacity is about 20,000 tons of rebars per year. The factory is among four iron and steel factories in Tanzania that have been previously studied for workers’ exposure to noise in the production area and their respective NIHL 13. The wThe factory administration allowed us to perform the study at their site. From a recent list of workers provided by the factory administration in 2020 together with the information available from the previous study, i.e. job titles, work sections, shift pattern, description of tasks and work environment . We invi19pL,A]), and were set up at a 3-dB exchange rate. The dosimeters logged noise data each minute during the measurement period. We calibrated the dosimeters before and after each sampling period. During the measurement period, we attached the dosimeters to the workers’ shoulders approximately 10–15 cm from the ear and instructed the participants to handle the dosimeters carefully while working, by not touching, tampering, or shouting into the dosimeter’s microphone. In addition, we conducted several follow-ups during the measurement period to check for any dosimeter mishaps. After each measurement day, we recorded the start- and end-time of the sampling period, the A-weighted equivalent noise level for the duration of the measurement  and the C-weighted peak noise level . The data were normalized to daily noise exposure levels  by noise exposure groups (job groups) and working sections, using the following formula:We conducted full-shift personal noise measurements using personal noise dosimeters  following the ISO standard 9612:2009 . The dospL,A,eqTe is the A-weighted equivalent continuous sound pressure level from dosimeter, Te is the measurement period and T0, is the reference duration, equal to eight hours. This was done to see if there was any variability in personal noise exposure with the previous personal noise exposure profile  compared to that of 1000 Hz (AM = 4 dB SD = 3 dB), 2000 Hz (AM = 4 dB SD = 4 dB), and 3000 Hz (AM = 9 dB SD = 6 dB), respectively . Also, the largest proportion of hearing threshold shifts >10 dB was recorded at 4000 Hz  compared to about 54%, 13% and 18% at 3000, 2000 and 1000 Hz respectively (not shown in the Table).In this study, there was a statistically significant increase in the mean hearing threshold shifts from pre- to post-shifts  at audiometric frequencies of 1000, 2000, 3000, and 4000 (Table 1). There was no statistically significant association between TTS and personal equivalent noise exposure, number of noise peaks >130 dB (C), age, work duration, current smoking and use of earphones/headphones (Table 1). The participant’s age and duration of work were strongly correlated . Likewise, the number of noise peaks was significantly correlated to personal noise exposure .Overall, 85.5% of workers had a TTS defined as a 10 dB or greater change at 1000, 2000, 3000 or 4000 Hz . The largest recorded difference in hearing threshold in individual audiometry were 30 dB at 3000 Hz and 4000 Hz followed by several others of 20 – 25 dBs, many of which were recorded at 4000 Hz .Furthermore, the individual PTA measurements showed that the hearing threshold shifts varied between workers, and generally increased from 1000 Hz to 4000 Hz  was 85.5%. There was a significant increase in mean hearing thresholds across the work shift at all measured noise frequencies. The largest increase in hearing threshold was found at 4000 Hz, suggesting that this audiometric frequency was more sensitive than the lower frequencies to noise exposure. To the best of our understanding, this study is the first to be conducted among iron and steel workers in Tanzania and indicates that registration of TTS might be a useful tool to validate the effect of the planned intervention to reduce occupational noise exposure among workers. If successful, the intervention might reduce the prevalence of TTS, and thereby indicate that the risk of permanent threshold shift (PTS) due to repeated high noise exposures among the workers will be reduced.Although we could not find any analogous studies from industrial work environments, our study shows a comparable prevalence of TTS as reported among male hockey officials in the U.S i.e., 86.2% . This su313216In the present study the prevalence of TTS did not increase with an increase in noise exposure. This is in agreement with a study that found no significant correlation between occupational noise exposure of 85 to 90 dB(A) and TTS among iron workers in Norway . These fWe found a significant increase in hearing threshold across work shift, which was more pronounced at 4000 Hz than at the lower audiometric frequencies. This is analogous with several studies involving human subjects in different sectors, although the magnitude of threshold change varies in different studies. However, what seems to be common to the different studies is the significant role of the noise exposure to observed results and that the higher audiometric frequencies, especially at 4000 Hz, indicates a noise effect on hearing 40. For iThere was a large individual variability in cross-shift change in hearing threshold among the iron and steel factory workers which conforms to the already available body of knowledge on the individual variations in susceptibility to noise in various studies 1941. Dif191627The strengths of the present study include the use of standard methods and devices in data collection, i.e., the use of calibrated noise dosimeter and audiometer for collecting information in the field while adhering to the ISO standard 9612:2009 and ISO 8253–1:2010 as well. All the audiometric tests were all done in a background noise controlled mobile booth located within the factory during measurement, using similar standard protocol for validity of the results. Potential determinants for TTS were identified and their relationship with TTS was analyzed. In addition, only workers with normal hearing were included. It might have been a strength if we had been able to conduct a follow-up audiometry after 48 hours or more to confirm the presence or absence of TTS and establish a complete recovery to baseline threshold level. However, this was not possible in our setting as workers were obliged to continue working on daily basis afterwards.The TTS studies are, in some cases, quite complex. One challenge is the availability of standard definitions across studies. Some researchers use the higher noise sensitive frequencies of 3000 – 6000 Hz and others focus on lower audiometry frequencies both as average and or single frequency metrics 1943. How19To our knowledge, this is the first study in Tanzania exploring the relationship between occupational noise exposure and TTS among iron and steel workers. However, our results are most likely to be representative for the workers in the same kind of working environments with the same kinds of noise characteristics. Our results seem to agree with findings from similar studies in other parts of the world and seem to be related to the noise levels and not to geography or type of industry. However, it is important to underline that the results are only representative for workers without any protective hearing equipment.The findings of our current study were from screened iron and steel workers with normal hearing (with PTA better than 25 dB Hearing Level) who were exposed at an average personal noise exposure of 90.4 dB(A) and above. We found 85.5% of workers had TTS after completing a work shift. Interventions to reduce the occupational personal noise exposure among workers are warranted to reduce the high risk of developing permanent threshold shift with persistent high noise exposure.


Over the treatment period, average change in BCVA was −0.053 logMAR, and average change in CMT was −51.1 µm, neither of which had significant differences between retina specialists. BCVA and CMT at initial visit were found to be significantly associated with improved BCVA and CMT over the treatment period (p < 0.001). Number of injections administered and interval between injections were not found to be significant factors affecting change in BCVA or CMT. Despite significant differences in injection dosing regimen, retina specialists achieved similar outcomes in change in BCVA and CMT over the treatment period.Intravitreal anti-VEGF injections are the current gold standard for treating diabetic macular edema (DME). However, injection practice patterns of retina specialists have varied markedly based on physician discretion. This retrospective study analyzes the impact of injection protocol selection on change in best-corrected visual acuity (BCVA) and central macular thickness (CMT) in 170 eyes treated by 4 retina specialists practicing a pro re nata (PRN) strategy between 2010 and 2020. DME patients received an average of 7.25 injections every 6.24 weeks over 56.6 weeks. There were significant differences between retina specialists in mean number of injections ( Diabetes mellitus (DM) currently affects 463 million adults worldwide, with an expected rise to 700 million by the year 2045 . DM may Because patients with DME represent a heterogeneous group that have varied responses to anti-VEGF therapy, there are no established guidelines on standardized injection protocols for retina specialists to follow. The common clinical injection protocols practiced are fixed monthly injections for the first three months followed by either continuation pro re nata (PRN), in which patients are treated as-needed, or with a treat-and-extend (T&E) strategy, in which treatment interval is incrementally increased until maximal length of quiescence of disease is achieved . In receFrequent intravitreal injections not only carry ophthalmological risks but may also negatively affect the quality of life of the patient. Patients have to worry about practical inconveniences, such as cost of injections, absences from work, and reliance on family members and caregivers to accompany them to the frequent visits, as well as emotional stressors of frustration, treatment anxiety, and needle phobia . IncreasInjection protocols of retina specialists have varied markedly based on physician discretion, patient preference, and treatment outcomes. Because of this, there are no established clinical practice guidelines in place for treatment of DME patients. Such heterogeneity in the DME patient population along with large variance among treatment regimens between physicians has led to ambiguity in how to best treat patients. Although multiple studies have analyzed the effects of differences in anti-VEGF drugs on clinical outcomes, the impact of specific injection protocol selection of individual retina specialists has not been studied ,21,22,23Ethical approval was obtained the Institutional Review Board of University of Texas Medical Branch in accordance with the tenets of the Declaration of Helsinki.A retrospective chart review using Epic Systems Electronic Medical Records were used to identify patients with ICD codes E11.321, E11.331, E11.341, and E11.351 for clinically diagnosed DME with visits between January 2010 and July 2020 by retina specialists practicing at University of Texas Medical Branch, Galveston. All practicing retina specialists were trained in Retina Fellowships with Accreditation Council for Graduate Medical Education (ACGME) accreditation in the United States.Patient inclusion criteria included diagnosis of DME, age 18–100 years, and administration of aflibercept, ranibizumab, and bevacizumab in a PRN pattern. Exclusion criteria included patients with coexisting conditions that complicated the evaluation of DME, including wet AMD, retinal artery occlusion, retinal vein occlusion, high myopia (>6 diopters), trauma, and history of anti-VEGF injections or laser photocoagulation. Patients with no CMT data available during the treatment period, patients who switched between retina specialists, and patients whose physicians treated fewer than five patients over the treatment period were also excluded. Each qualified eye was counted separately for analysis.All patients initially received loading doses of monthly anti-VEGF injections for three consecutive months after which retina specialists proceeded with an individualized PRN regimen. Patient demographics, comorbid conditions, total number of injections, time interval between injections, and BCVA and CMT at each injection visit were collected. The mean CMT obtained from Heidelberg Spectralis ocular coherence tomography (OCT) for each eye was retrieved up to 30 days before and after starting treatment for each eye. CMT is defined as the mean thickness measured at the point of intersection of the 6 radial scans on optical coherence tomography. Comorbid conditions including smoking history, chronic kidney disease, hyperlipidemia, hypertensive retinopathy, and pseudophakia were also collected for each patient. BCVA was recorded as lines from Snellen chart and converted to logMAR. Changes in BCVA and CMT over the treatment period were calculated as the difference between these values from the first and last injection (last injection–first injection).Statistical analyses were performed to determine the relationship between physician injection protocols and changes in BCVA and CMT over the treatment period using Chi-square tests for categorical data and Kruskal–Wallis tests for continuous data. Multivariate generalized linear modeling was utilized to determine factors associated with achieved difference in visual acuity and macular thickness over course of injection treatment.Epic Systems EMR identified 176 clinically diagnosed DME patients that met the inclusion criteria seen between January 2010 and July 2020 by 8 identified retina specialists. Out of 176 patients, 52 patients were excluded due to being treated by multiple retina specialists. Four retina specialists were excluded because they had fewer than five DME patients over the time frame, which subsequently resulted in the exclusion of 2 additional patients from the study. A total of 170 eyes from 122 DME patients between 4 retina specialists labeled as A, B, C, and D were included in the data analysis.The mean age of the 122 included DME patients was 62.2 ± 11.3 years, who were equally divided in gender with 61 males (50%) and 61 females (50%). As far as racial distribution, 83 patients (68.03%) were Caucasian, 38 (31.15%) were African American, and 1 (0.82%) was Asian. Assessment of comorbidities and medical history revealed 35 (28.69%) were smokers, 62 (50.82%) had chronic kidney disease, 92 (75.41%) had hyperlipidemia, and 38 (31.15%) had hypertensive retinopathy .p = 0.07). The remaining patients received a combination of 2 or 3 drugs in no defined pattern. With respect to choice of specific anti-VEGF drug, for retina specialists A, B, C, and D, respectively, bevacizumab was used most frequently in 100, 95.5, 100 and 100% of patients (p = 0.08), followed by aflibercept for 16.7, 4.6, 20.7 and 3.2% of patients (p = 0.046), and ranibizumab least frequently for 0, 9.1, 2.7 and 0% of patients (p = 0.26)  .p = 0.036). The mean length of injection treatment in weeks was 17.57 ± 7.67, 34.00 ± 9.33, 71.29 ± 6.94, and 27.57 ± 5.31 for retina specialists A, B, C, and D, respectively, with an overall average of 56.6 weeks (p = 0.0007). The mean interval period between injections in weeks was 3.7 ± 0.89, 5.29 ± 1.21, 6.67 ± 0.47, and 5.87 ± 1.03 for retina specialists A, B, C, and D, respectively with an overall average of 6.24 weeks; however, these differences were not statistically significant (p = 0.381)  .p = 0.036). However, the mean difference in BCVA from first to last injection measured in logMAR units was −0.08 ± 0.07, 0.005 ± 0.09, −0.05 ± 0.04, and −0.1 ± 0.11 for retina specialists A, B, C, and D, respectively, with an overall average of −0.053 logMAR, and differences were found not to be significant (p = 0.226)  .p < 0.001)  .p = 0.131). Mean difference in CMT from first to last injection visit was 9.3 ± 35.1 μm, −165.8 ± 63.2 μm, −26.0 ± 22.5 μm, and −71.4 ± 51.9 µm for retina specialists A, B, C, and D, respectively, with an overall average of 51.1 µm and was also not significantly different amongst the four retina specialists (p = 0.06)  .p of 0.03, 0.03, and 0.01, respectively. Caucasian race is also a factor shown to be significant in this model with an effect estimate of 58.38 (p = 0.05) when compared to other races as the reference (Multivariate generalized linear modeling was additionally used to determine which factors played a significant role in change in CMT from first to last visit. Factors included in analysis were CMT at first visit, retina specialist, age, sex, race, smoking status, injection number, interval between injections, hyperlipidemia, and hypertensive retinopathy. Retina specialist B was made to be the reference to highlight significant differences compared to the other retina specialists. The analysis showed that compared to retina specialist B, retina specialists A, C, and D showed significant differences in terms of their effect on change in CMT with effect estimates of 151.34, 81.65, and 114.20 and eference .We provide a comparison of anti-VEGF PRN treatment regimens between four retina specialists with respect to change in BCVA and CMT in DME patients. While there are multiple published studies comparing the efficacy of the different anti-VEGF drugs on the treatment of DME, a “head-to-head” comparison of injection protocols between retina specialists has never been done ,21,22,23This study first categorically compared treatment regimens with respect to the choice of anti- VEGF drug, the number of anti-VEGF injections, and the intervals between injections used by retina specialists. Of note, there was a significant difference in aflibercept use between the retina specialists; however, bevacizumab and ranibizumab were administered similarly . This diWe further explored whether the injection protocols used by the retina specialists impacted changes in BCVA and CMT in clinical outcome of DME management. At baseline before anti-VEGF treatment was initiated, visual acuities of the DME patients were significantly different among the four physicians. However, baseline CMTs of patients were statistically similar amongst the four retina specialists suggesting an anatomical consensus in when to initiate treatment. Despite differences in protocols of treatment regimens and initial BCVA, both mean changes in BCVA and CMT from first to last injection were statistically similar amongst the retina specialists . Such fiBased on the multivariate analysis, BCVA at first visit was the only variable that played a significant role in affecting change in BCVA over the treatment period. CMT at first visit, retina specialist, and race demonstrated significance in determining change in CMT over the treatment period. In agreement with previously published work, this study demonstrates that worse vision and thicker central macula at baseline is associated with more improvement in BCVA and CMT over time ,32. ThisAlthough patients treated by retina specialist B demonstrated the greatest improvement in CMT, they saw no improvement in BCVA on average. Other investigations have found that reduction of CMT has an inconsistent and weak association with outcome of BCVA; specifically, studies have shown a lag in potential visual improvement behind resolution of macular edema ,29,34. IAdministration of intravitreal injections poses significant clinical, humanistic, and economic burden on patients. Each recommended monthly visit requires a comprehensive ophthalmic exam, fundoscopy, OCT imaging, and intravitreal injections . PatientLike any other retrospective study, there are some limitations in study design. Small sample size, unequal subject distribution, retrospective nature, and missing data points may have influenced our observation in treatment outcomes. Furthermore, the sample size of retina specialists was also small with unequal patient distribution amongst retina specialists, likely leading to variances in data. Confounding variables that may have affected outcomes such as use of intravitreal steroids or poor systemic control and duration of diabetes were not considered and should be included in future studies. However, the strength of our study is the availability of four retina specialists in a single hospital-based setting where intravitreal injection clinics are controlled by the university’s strict guidelines. Therefore, every retina specialist abided by the same compliance policy with minimum influence from payors.In summary, this study revealed that despite significant differences in number of anti-VEGF injections administered and overall length of treatment of DME patients, all four retina specialists had similar outcomes with respect to changes in BCVA and CMT. Additionally, both baseline BCVA and CMT were found to be significant in determining change in BCVA and CMT, respectively, while number of injections and interval between injections were not. An extended large scale prospective study with incorporation of other co-factors that could affect clinical decision making of intravitreal injections may help to explore further possibility of having a standardized intravitreal injection regimen for DME treatment.


Previous research has shown deficits in vocal emotion recognition in sub-populations of individuals with hearing loss, making this a high priority research topic. However, previous research has only examined vocal emotion recognition using verbal material, in which emotions are expressed through emotional prosody. There is evidence that older individuals with hearing loss suffer from deficits in general prosody recognition, not specific to emotional prosody. No study has examined the recognition of non-verbal vocalization, which constitutes another important source for the vocal communication of emotions. It might be the case that individuals with hearing loss have specific difficulties in recognizing emotions expressed through prosody in speech, but not non-verbal vocalizations. We aim to examine whether vocal emotion recognition difficulties in middle- aged-to older individuals with sensorineural mild-moderate hearing loss are better explained by deficits in vocal emotion recognition specifically, or deficits in prosody recognition generally by including both sentences and non-verbal expressions. Furthermore a, some of the studies which have concluded that individuals with mild-moderate hearing loss have deficits in vocal emotion recognition ability have also found that the use of hearing aids does not improve recognition accuracy in this group. We aim to examine the effects of linear amplification and audibility on the recognition of different emotions expressed both verbally and non-verbally. Besides examining accuracy for different emotions we will also look at patterns of confusion (which specific emotions are mistaken for other specific emotion and at which rates) during both amplified and non-amplified listening, and we will analyze all material acoustically and relate the acoustic content to performance. Together these analyses will provide clues to effects of amplification on the perception of different emotions. For these purposes, a total of 70 middle-aged-older individuals, half with mild-moderate hearing loss and half with normal hearing will perform a computerized forced-choice vocal emotion recognition task with and without amplification. Hearing loss is among the leading causes of disability globally with an increasing prevalence with age. It leads to difficulties in communication which can contribute to social isolation and diminished well-being . In SwedEmotions can be defined as “episodic, relatively short-term, biologically-based patterns of perception, experience, physiology, action and communication that occur in response to specific physical and social challenges and opportunities” , p. 468)86]. The Emotions are vocally expressed through emotional prosody, as well as through non-verbal vocalizations . Chin, BDamage to any part of the auditory system can result in hearing loss . The mosConsequences of sensorineural hearing loss include reduced sensitivity, dynamic range, and frequency selectivity . ReducedThe most common intervention for people with hearing loss is the use of hearing aids . HoweverSeveral studies have found mild-to-moderate hearing loss to be related to deficits in vocal emotion recognition ability , 27, 28)The overall aim is to deepen our understanding of the effects of hearing loss and signal amplification on vocal emotion recognition. It is unclear how mild-moderate hearing loss and linear amplification through a hearing aid effects the perception of auditory cues important for (a) being able to perceive and (b) being able to discriminate between emotions.More specifically, by examining accuracy for different emotions (what emotions are easiest to accurately identify) and patterns of confusion (which emotions are mixed up when inaccurately identified), for individuals with normal hearing and for individuals with hearing loss (listening with and without linear amplification), and by comparing performance with acoustic analysis of sentences and non-verbal expressions with different emotional emphasis, the aims are to examine:Which acoustic parameters are important for detection of and discrimination between different emotions How the effect of hearing loss affects emotion recognition (by comparing performance of participants with and without hearing loss)How emotion recognition is affected by linear amplification (by examination of performance of participants with hearing loss using linear amplification)Based on the literature, we predict that:individuals with hearing loss will have poorer recognition compared to normal hearing individuals for emotions expressed verbally, regardless of acoustic features and regardless of the use of linear amplification, and for emotions expressed non-verbally when linear amplifications is not usedindividuals with and without hearing loss will not differ in accuracy for non-verbal expressions when linear amplification is usedpatterns of confusion will differ between individuals with and without hearing loss for both verbally and non-verbally expressed emotionsvocal emotions which are more distinct in terms of acoustic parameter measures, will be recognized with higher accuracy for both groups, but emotions that are distinguished mainly based on frequency parameters will be less correctly identified by the hearing loss groupthe more salient the amplitude-related acoustic parameters are for an emotion, the better that emotion will be identified when linear amplification is used compared to not.2 = .02 (f = 0.14); a next to small effect size. Power analysis was performed using G*Power version 3.1.9.7 Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.Reviewer #1: PartlyReviewer #2: Partly**********2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions  to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.Reviewer #1: PartlyReviewer #2: Partly**********3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?Reviewer #1: NoReviewer #2: Yes**********4. Have the authors described where all data underlying the findings will be made available when the study is complete?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.Reviewer #1: AbstractThe authors’ rationale for the current study is that existing research cannot distinguish a specific emotion-perception deficit from a general prosody-perception deficit in listeners with hearing loss, because emotion identification of verbal materials depends on prosody perception. The authors plan to use both verbal and nonverbal emotional speech materials to examine if listeners with hearing loss have a true emotion-specific deficit, and not just a general prosody-related deficit.The abstract could be edited to make the rationale clearer and more coherent. To improve the flow of the abstract, I suggest focusing on the specific- vs general-deficit issue before moving on to the topics of audibility and hearing aids, instead of introducing information about hearing aid studies and then switching back to the first issue. It is also not clear in the abstract how changing the audibility would help to answer the question of whether listeners have a specific or general deficit, or how examining “mix-ups” would be helpful.Sentences that need editing include: “…deficits in individuals hearing loss also ha are not ameliorated…” and “…mix-ups between different vocal of individuals…”.IntroductionThe study aim of relating acoustic features to emotion recognition was mentioned in the “vocal emotion recognition” section and included in the Aims section, but was not described in the abstract.“Patterns of confusion” is a slightly different concept than “differences in accuracy among emotions”. The authors should consider which is most relevant for their purposes and be consistent in their terminology, instead of using these terms interchangeably.The authors could explain what they mean by “examining patterns of confusion [will lead to] deeper knowledge of emotion recognition” (end of Introduction); e.g., the relevant explanation at the end of the Aims section could be brought up earlier.- missing ’s’ in ‘material’ at the end of the first paragraph- pg 3 line 8: clarify what “in contrast” is contrasting- pg 3 line 10: “however” implies the pattern for prosody and non-verbal vocalizations should be different, but they seem more alike than different- proofread for extra punctuation, e.g., ‘;,’ at the end of page 3, or missing punctuation, e.g., missing period after ‘poorer pitch perception (18)’Aims- the phrase “for emotions expressed non-verbally when linear amplifications is not used” seems to be redundant, given that the first part of the sentence already both types of materials would be more poorly recognized regardless of amplification- extra ’s’ in ‘amplifications’Method - participantsRegarding the a priori power calculation, the meaning of the following statement is unclear: “we will be able to conduct separate analyses for different stimulus types and outcome measures”. I assume that it means that the same number of participants is appropriate for a 2 x 7 within-subjects design, for comparing amplified and non-amplified speech materials, and for comparing non-verbal vocalizations to sentences, but it would be clearer to say so explicitly.Method - task and study designThe sentence materials are described in detail, but there is no description of what the “non-verbal vocalizations” consist of. I see that the “neutral” option is also excluded; is there no possibility of a “neutral” non-verbal vocalization?What is the accuracy criterion for emotions to be recognized “well above chance” for pilot testing of the speech materials, and will it be in line with previous studies?The authors should clarify which parts of the text describe pilot testing and which parts describe the procedures for the actual study, e.g., by using a separate sub-header.For reader unfamiliar with this master hearing aid system, the authors could clarify if the “amplified” stimuli will be pre-processed and tailored to each participant’s audiogram before presentation in the session — is this the case?If participants are presented with 8 options instead of 7, including the extra option of “other emotion”, wouldn’t this technically create 8 levels in the condition ? This also seems to add an extra complication to the calculation of chance levels for recognition, given that actors were directed to create only 7 emotions.Method - analysesA very brief description of GeMAPS would help readers to understand why the authors chose to use this set of speech measures.How will the distance matrices of the acoustic measurements be integrated with the behavioral data (accuracy)?Reviewer #2: Introduction-page 2, first paragraph, they’ve been identified as an important question… why? Especially auditory rehabilitation .-maybe in this first paragraph you want to discuss the number of individuals (esp older adults) with hearing loss, the prevalence of this condition in that population makes addressing issues related to age-related hearing loss a pressing concern. You actually don’t really talk about hearing loss in middle-aged/older individuals (your target population for the proposed study) at all, or why it is so important to study these people.-page 3, first paragraph. There are a lot of data here about differences in recognition rates of emotions, and how they vary between emotional prosody and non-verbal vocalizations. Could be helpful to provide a table?End of first paragraph, page 3, reword “why we…”If less is known, is there any information that is known? Any previous studies on this?Add a line about how hearing aids are most common intervention but still such a low uptake. Suggests that many people (esp. older adults) with hearing loss are not accessing amplification.Top of page 4, Christensen and Goy papers, are these with older individuals experiencing hearing loss? Is there a confusion between age x emotion x hearing loss? Any expectations why aging might change ability to recognize emotion?Method2.2- can you break down into sub-heading first describing the pilot study, then another section describing the proposed full set of stimuli2.3 Analyses- can you break down into behavioural data (participant responses) and acoustical analysis of your stimuli, starting with “for each recording…”It would be helpful to describe where your data will be stored .**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  22 Oct 2021Reviewer #1AbstractReviewer comment 1:1The authors’ rationale for the current study is that existing research cannot distinguish a specific emotion-perception deficit from a general prosody-perception deficit in listeners with hearing loss, because emotion identification of verbal materials depends on prosody perception. The authors plan to use both verbal and nonverbal emotional speech materials to examine if listeners with hearing loss have a true emotion-specific deficit, and not just a general prosody-related deficit.The abstract could be edited to make the rationale clearer and more coherent. To improve the flow of the abstract, I suggest focusing on the specific- vs general-deficit issue before moving on to the topics of audibility and hearing aids, instead of introducing information about hearing aid studies and then switching back to the first issue. It is also not clear in the abstract how changing the audibility would help to answer the question of whether listeners have a specific or general deficit, or how examining “mix-ups” would be helpful.Sentences that need editing include: “…deficits in individuals hearing loss also ha are not ameliorated…” and “…mix-ups between different vocal of individuals…”.Response 1:1Thank you for the suggestions about the abstract, we have now rewritten the abstract to make the rationale clearer and more coherent. The new abstract now reads as follows: ” Previous research has shown deficits in vocal emotion recognition in sub-populations of individuals with hearing loss, making this a high priority research topic. However, previous research has only examined vocal emotion recognition using verbal material, in which emotions are expressed through emotional prosody. There is evidence that older individuals with hearing loss suffer from deficits in general prosody recognition, not specific to emotional prosody. No study has examined the recognition of non-verbal vocalization, which constitutes another important source for the vocal communication of emotions. It might be the case that individuals with hearing loss have specific difficulties in recognizing emotions expressed through prosody in speech, but not non-verbal vocalizations. We aim to examine whether vocal emotion recognition difficulties in middle- aged-to older individuals with sensorineural mild-moderate hearing loss are better explained by deficits in vocal emotion recognition specifically, or deficits in prosody recognition generally by including both sentences and non-verbal expressions. Furthermore a,, some of the studies which have concluded that individuals with mild-moderate hearing loss have deficits in vocal emotion recognition ability have also found that the use of hearing aids does not improve recognition accuracy in this group. We aim to examine the effects of linear amplification and audibility on the recognition of different emotions expressed both verbally and non-verbally. Besides examining accuracy for different emotions we will also look at patterns of confusion (which specific emotions are mistaken for other specific emotion and at which rates) during both amplified and non-amplified listening, and we will analyze all material acoustically and relate the acoustic content to performance. Together these analyses will provide clues to effects of amplification on the perception of different emotions. For these purposes, a total of 70 middle-aged-older individuals, half with mild-moderate hearing loss and half with normal hearing will perform a computerized forced-choice vocal emotion recognition task with and without amplification.”IntroductionReviewer comment 1:2The study aim of relating acoustic features to emotion recognition was mentioned in the “vocal emotion recognition” section and included in the Aims section, but was not described in the abstract.Response 1:2 Thank you for noticing and pointing this out. This aim is now included in the abstract. See Response 1:1. Reviewer comment 1:3“Patterns of confusion” is a slightly different concept than “differences in accuracy among emotions”. The authors should consider which is most relevant for their purposes and be consistent in their terminology, instead of using these terms interchangeably.Response 1:3We agree that there is a difference between the two terms. We distinguish between accuracy for different emotions and patterns of confusion throughout the manuscript. Reviewer comment 1:4The authors could explain what they mean by “examining patterns of confusion [will lead to] deeper knowledge of emotion recognition” (end of Introduction); e.g., the relevant explanation at the end of the Aims section could be brought up earlier.Response 1:4Thank you for pointing out the need for clarification here. In our revised aims section we now write (end of pg.4- pg.5): “More specifically, by examining accuracy for different emotions (what emotions are easiest to accurately identify) and patterns of confusion (which emotions are mixed up when inaccurately identified), for individuals with normal hearing and for individuals with hearing loss (listening with and without linear amplification), and by comparing performance with acoustic analysis of sentences and non-verbal expressions with different emotional emphasis, the aims are to examine:• Which acoustic parameters are important for detection of and discrimination between different emotions ?• How the effect of hearing loss affects that pattern (by comparing performance of participants with and without hearing loss)?• How that pattern is affected by linear amplification (by examination of performance of participants with hearing loss using linear amplification)? “We also introduce the topic of confusion between different emotions in individuals with hearing loss in the section “Vocal emotion recognition under mild-to-moderate hearing loss” in the introduction:“In individuals with hearing loss, confusions between different vocal emotions appear to be much more common in comparison to normal hearing individuals (31)” Reviewer comment 1:5- missing ’s’ in ‘material’ at the end of the first paragraphResponse 1:5Thanks for noticing this error. The ’s’ has been addedReviewer comment 1:6- pg 3 line 8: clarify what “in contrast” is contrastingResponse 1:6Thanks for pointing this inconsistency out. This part of the text has been removed from the current manuscript.Reviewer comment 1:7- pg 3 line 10: “however” implies the pattern for prosody and non-verbal vocalizations should be different, but they seem more alike than differentResponse 1:7Thank you for this accurate observation. The wording has been changed to:”However, with regard to emotional prosody specifically, Scherer (5) and Castiajo and Pinheiro (16) argue that anger and sadness are more accurately recognized compared to fear, surprise, and happiness.”Reviewer comment 1:8- proofread for extra punctuation, e.g., ‘;,’ at the end of page 3, or missing punctuation, e.g., missing period after ‘poorer pitch perception (18)’Response 1:8Thank you for noticing. Missing period after ’poorer pitch perception’ has been added. The extra semicolon on pg. 3 has been removed. AimsReviewer comment 1:9- the phrase “for emotions expressed non-verbally when linear amplifications is not used” seems to be redundant, given that the first part of the sentence already both types of materials would be more poorly recognized regardless of amplificationResponse 1:9Thank you for noticing this mistake. The preceding sentence refers to verbal expressions and this to non-verbal. We have rewritten the sentences and clarified it:“...we predict that: 1. individuals with hearing loss will have poorer recognition compared to the normal hearing group for emotions expressed verbally, regardless of acoustic features and regardless of the use of linear amplification, and for emotions expressed non-verbally when linear amplifications is not used”Reviewer comment 1:10- extra ’s’ in ‘amplifications’Response 1:10Thank you for noticing. An extra s has been added .Method - participantsReviewer comment 1:11Regarding the a priori power calculation, the meaning of the following statement is unclear: “we will be able to conduct separate analyses for different stimulus types and outcome measures”. I assume that it means that the same number of participants is appropriate for a 2 x 7 within-subjects design, for comparing amplified and non-amplified speech materials, and for comparing non-verbal vocalizations to sentences, but it would be clearer to say so explicitly.Response 1:11Thank you. We agree that this section was unclear. This text has been changed to: “Using a 2 x 6 mixed design (group x emotion), 80% power, 5% significance level, correlation between repeated measures of 0.5 and with N=56 participants (a reasonable number of participants from a recruitment point of view), we will be able to detect an effect as small as η2=.02 (f=0.14); a next to small effect size. Power analysis was performed using G*Power version 3.1.9.7 (32).” As is discussed in the analyses section, several of the planned analyses will be 2x6 mixed ANOVAs, but not all. However, we think this is the most demanding analysis in terms of number of participants, so we based our estimation on this, along with consideration regarding how many participants we will realistically will be able to recruit. Method - task and study designReviewer comment 1:12The sentence materials are described in detail, but there is no description of what the “non-verbal vocalizations” consist of. I see that the “neutral” option is also excluded; is there no possibility of a “neutral” non-verbal vocalization?Response 1:12Thank you for noticing this omission. We have added the following description in the Task and study design section ”For the non-verbal expressions the actors were instructed to imagine themselves experiencing the different emotions and to make expressions with sounds, without language which match those emotions. This entails some variation of the specific sounds expressed for specific emotions by different participants. The sounds which were perceived as most clearly expressing a given emotion were selected by the authors, and some were slightly edited (omitting pauses) such that all sounds are between 1-2 seconds long.”With regard to the exclusion of neutral, we believe that such expressions would be very difficult to achieve, if they are achievable. We have not found any previous study including neutral for non-verbal expressions. Reviewer comment 1:13What is the accuracy criterion for emotions to be recognized “well above chance” for pilot testing of the speech materials, and will it be in line with previous studies?Response 1:13There does not seem to be a generally accepted criterion for validation at the level of individual stimuli. The commonly used criterion of above-chance accuracy seems to be used at a category level such as overall accuracy for expressions of a particular category. We have simply chosen stimuli which are accurately classified by a majority of participants (>50%). The new description under the subheading Validation of stimulus material reads: “Only sentences and non-verbal expressions which a majority of participants (>50%) classify as expressing the emotion intended by the actor will be included in the experiment” Reviewer comment 1:14The authors should clarify which parts of the text describe pilot testing and which parts describe the procedures for the actual study, e.g., by using a separate sub-header.Response 1:14Thank you for this suggestion. The pilot study is now described under the new heading “Validation of stimulus material”. Reviewer comment 1:15For reader unfamiliar with this master hearing aid system, the authors could clarify if the “amplified” stimuli will be pre-processed and tailored to each participant’s audiogram before presentation in the session — is this the case?Response 1:14The master hearing aid system will process the auditory stimuli tailored to the participant’s audiogram during presentation, such that the amplified stimuli will be presented via head- phones to the participants, who at the time will not wear their own hearing aids. We have added the following description- pg. 6, §4 : ”.. with linear amplification using the Cambridge formula for linear hearing aids (37) in which each stimulus is tailored to each participants audiogram during presentation”Reviewer comment 1:15If participants are presented with 8 options instead of 7, including the extra option of “other emotion”, wouldn’t this technically create 8 levels in the condition ? This also seems to add an extra complication to the calculation of chance levels for recognition, given that actors were directed to create only 7 emotionsResponse 1:15The option of ’other emotion’ is only included in the pilot study/validation. We agree that adding the option of “other emotion” does add complication to the calculation of chance levels, although it seems to be standard to include such an option in validation studies to reduce bias. Our new criterion as described however does not take into account the number of options. Method - analyses Reviewer comment 1:16A very brief description of GeMAPS would help readers to understand why the authors chose to use this set of speech measures.Response 1:16The following description has been added- pg. 7, §2: ” The GeMAPS consist of a set of acoustic parameters which have been proposed as a standard for different areas of automatic voice analysis, including the analysis of vocal emotions. The parameters of GeMAPS have been shown to be of value for analyzing emotions in speech in previous research (40) and are described in table 1.”Reviewer comment 1:17How will the distance matrices of the acoustic measurements be integrated with the behavioral data (accuracy)?Response 1:17We will discuss differences in accuracy and patterns of confusion in relation to descriptive graphical plots of distance matrices for the central acoustic parameters. We have added the following under Behavioral analyses, pg. 8, §1 of the subsection: ” To relate linear amplification and acoustic parameters to changes in performance, differences in performance over hearing condition (and emotions) will be described in relation to the distance matrices and patterns of confusion from the acoustic analyses”Reviewer #2:Reviewer comment 2:1Introduction-page 2, first paragraph, they’ve been identified as an important question… why? Especially auditory rehabilitation .Response 2:1Picou et al (2018) identified that the subject of emotion recognition in hearing loss has been slightly neglected in audiology. They highlight the importance of emotion recognition due to it’s important for interpersonal interaction and social functioning and discuss a number of topics for which there is a need for research, including emotion recognition (more knowledge of how it is affected by hearing loss), the effects of amplification (long term and short) and different rehabilitation strategies (what works and how) with regard to vocal emotion recognition. With “auditory rehabilitation” we initially meant all types of interventions including amplification. We now realize however that auditory rehabilitation . Therefore, we now separate between the two in the text. We have added the following to pg. 2, §1: ”Because of the importance of emotion recognition for interpersonal interaction and, thus, indirectly to measurements of well-being such as perceived quality of life and the presence/absence of depression, the effects of hearing loss, amplification, and auditory rehabilitation on the recognition and experience of vocal emotions, have been identified as important research topics (3)”Reviewer comment 2:2-maybe in this first paragraph you want to discuss the number of individuals (esp older adults) with hearing loss, the prevalence of this condition in that population makes addressing issues related to age-related hearing loss a pressing concern. You actually don’t really talk about hearing loss in middle-aged/older individuals (your target population for the proposed study) at all, or why it is so important to study these people.Response 2:2Thank you for this suggestion. We agree that this will be a good intro and have therefore added such information to the first paragraph of the Introduction. We have here also clarified the importance of investigating hearing loss in middle-aged individuals. As a consequence, the part introducing the meaning of emotions has been moved down under the subheading ’vocal emotion recognition’. The first part of the introduction now reads: ”Hearing loss is among the leading causes of disability globally with an increasing prevalence with age. It leads to difficulties in communication which can contribute to social isolation and diminished well-being (1). In Sweden, it has been estimated that approximately 18% of the population have a hearing loss. While the prevalence is comparatively much higher in the oldest age groups (44.6 % in the age group of 75-84 and 56.5% in the age group of 85+ years) the prevalence in the age groups of 55-64 and 65-74 (middle-age-to-old) is also relatively high (24.2 and 32.6%) (2). This shows that hearing loss is not only a concern for the oldest in the population. Because of the importance of emotion recognition for interpersonal interaction and, thus, indirectly to measurements of well-being such as perceived quality of life and the presence/absence of depression, the effects of hearing loss, amplification, and auditory rehabilitation on the recognition and experience of vocal emotions, have been identified as important research topics (3).”Reviewer comment 2:3-page 3, first paragraph. There are a lot of data here about differences in recognition rates of emotions, and how they vary between emotional prosody and non-verbal vocalizations. Could be helpful to provide a table?Response 2:3We agree that this paragraph was dense and difficult to follow. We have considered providing a table but decided to keep the information as text. However, we have revised this section to make it clearer, as follows: ”Research on how well different emotions of both stimulus types are recognized compared to other emotions has so far not yielded consistent results (see for example 4 and 15). However, with regard to emotional prosody specifically, Scherer and Castiajo and Pinheiro argue that anger and sadness are more accurately recognized compared to fear, surprise, and happiness  Reviewer comment 2:4End of first paragraph, page 3, reword “why we…”Response 2:4This part has now been rewritten and reads as follows: “Therefore, in the present study, recognition of non-verbal expressions as well as emotional prosody will be investigated.” Reviewer comment 2:5If less is known, is there any information that is known? Any previous studies on this?Response 2:6We have not found any study using non-verbal expressions with older individuals with hearing loss. We have added the following, with the addition of a new reference (nr 18) on pg. 3, end of §1, lines 29-32: ” The ability to accurately recognize non-verbal emotion expressions declines with aging (18), as does the ability to recognize emotional prosody in speech (19). Amorim et al. suggest that this decline happens due to age-related changes in brain regions which are involved in the processing of emotional cues (18). Hearing loss and aging both independently and conjointly contribute to a diminished ability to accurately recognize speech embedded vocal emotion expressions generally (19). To our knowledge, however, no previous study has examined the effects of mild to -moderate sensorineural hearing loss on non-verbal emotion expressions..” Reviewer comment 2:7Add a line about how hearing aids are most common intervention but still such a low uptake. Suggests that many people (esp. older adults) with hearing loss are not accessing amplification.Response 2:7We have added the following at -pg. 4, §2, with an added new reference: ”However, many individuals with hearing loss do not have access to or do not use hearing aids (25)”.Reviewer comment 2:8Top of page 4, Christensen and Goy papers, are these with older individuals experiencing hearing loss? Is there a confusion between age x emotion x hearing loss? Any expectations why aging might change ability to recognize emotion?Response 2:8The Goy et al. Paper examines the correlations between age and emotion identification and between PTA and emotion identification independently. Both age and PTA correlate significantly with emotion identification. In Christensen et al. participants are divided into different age groups. They found that aging does impact vocal emotion recognition negatively and that aging and hearing loss independently have negative effects on vocal emotion recognition accuracy, however the effects of hearing loss and old age are also additive. The suggested reasons for the age-related decline include both biological and social factors, we have added a brief discussion of one such proposed explanation, along with a very brief discussion of the effects of hearing loss and aging (see response 2:6): ”The ability to accurately recognize non-verbal emotion expressions declines with aging (18), as does the ability to recognize emotional prosody in speech (19). Amorim et al. suggest that this decline happens due to age-related changes in brain regions which are involved in the processing of emotional cues (18). Hearing loss and aging both independently and conjointly contribute to a diminished ability to accurately recognize speech embedded vocal emotion expressions generally (19).”MethodReviewer comment 2:92.2- can you break down into sub-heading first describing the pilot study, then another section describing the proposed full set of stimuliResponse 2:9The pilot study, stimuli material and the experimental study design are now presented under separate sub-headers (see pg. 6).Reviewer comment 2:102.3 Analyses- can you break down into behavioural data (participant responses) and acoustical analysis of your stimuli, starting with “for each recording…”Response 2:10Analyses have been broken down into ’behavioral data’ and ’acoustic analyses’ under separate sub-headings Reviewer comment 2:11It would be helpful to describe where your data will be stored .Response 2:11We intend to store data on the Open Science Framework (OSF) website. However, we have not yet created a link for this as the study has not started.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 1 Dec 2021Effects of mild-to-moderate sensorineural hearing loss and signal amplification on vocal emotion recognition in middle-aged–older individuals.PONE-D-21-13058R1Dear Dr. Ekberg,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Qian-Jie Fu, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.Reviewer #1: Yes**********2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions  to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.Reviewer #1: Yes**********3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?Reviewer #1: Yes**********4. Have the authors described where all data underlying the findings will be made available when the study is complete?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.Reviewer #1: The authors' revisions have strengthened the rationale and improved the clarity of the manuscript overall; I have no further suggestions.There is a minor typo in the abstract, "Furthermore a,," and a missing period before "Preliminary results" on p6.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: No