The cases of ear malformations, conductive, mixed, and single-sided deafness hearing loss are candidates for surgery and use of Bone-Anchored Hearing Aids (BAHA). Commonly, the literature highlights two procedures to assess the benefits and characteristics of amplification in users: functional gain (FG) and effective gain (EG).  Estimate and compare the EG and the FG to evaluate the benefits obtained by users of BAHA and, later, to compare tests of speech perception in silence and in noise. The sample (n=79) was divided into four groups, implanted from February 2014 to February 2021. The following tests were analyzed: pure-tone audiometry by air and bone; research of audiometric thresholds in free field; speech perception tests in silence and in noise. EG presented lower values than FG in all frequencies. The positive results of the speech perception tests were correlated with worse FG values. EG is the best method for evaluation, as it allows a proper comparison between devices, as well as a comparison with the prescription of validated rules. A better evaluation of results was observed on the EG values, indicating that it is a relevant method to assess auditory performance. In addition, the FG results were incompatible with the benefits obtained in the speech perception tests, showing that it is not a reliable tool for monitoring the results with the use of BAHA. However, cases in which the benefit of using hearing aids is limited or even non-existent. The advancement of technology allowed the development of surgically implantable hearing aids. Cases of ear malformations, conductive hearing loss, mixed hearing loss, and individuals with profound unilateral hearing loss, Single-Sided Deafness (SSD) are candidates for surgery and the use of BAHA.4In 1960, Von Békesy described the principle of sound transmission by bone and found no difference between air and bone sound transmission for the basilar membrane. This process was named air-bone cancellation.5 These can be objectively described in direct bone conduction (direct drive), in which the implantation of a pin called abutment or the implantation of a transducer, either electromagnetic or piezoelectric, occurs. Other types of BAHA have a system in which sound conduction occurs by skin (skin drive), by non-invasive transcutaneous vibration. When it comes to direct drive system, the technology of dental implants has allowed the creation of the technique in which a titanium abutment is implanted in the temporal bone, penetrating the skin and bone and conducting the sound by bone directly to the cochlea.The BAHA follow this principle and were introduced in the 1980s, allowing a greater notoriety on the subject.in-situ evaluation equipment of these devices are still needed, like with hearing aids. Commonly, the literature indicates two procedures to assess the benefits and characteristics of amplification in patients using BAHA: functional gain (FG) and effective gain (EG).6To verify if the adjustments and electroacoustic characteristics of the BAHA are in line with the amplification goal, measurements in couplers, skull simulators, are available, but many studies and development of 5 EG is estimated by the difference between the cochlear thresholds obtained during bone conduction research in pure-tone audiometry comparing them to the thresholds obtained in free field audiometry with these implantable devices.7FG is defined and estimated by the difference between the threshold obtained in free field with and without the use of BAHA (obtained during audiometry in free field). The frequencies included in this estimation are 500, 1000, 2000, and 4000Hz.8The hypothesis that guided this research was that EG is the best method to evaluate the benefits obtained by BAHA users, considering the results of speech perception tests in silence and in noise . EG can allow an adequate comparison between devices, as well as a comparison with the prescription of validated rules, it is also applied to sensorineural hearing losses, so when applied to conductive and mixed hearing losses, it considers poor middle ear functioning, expressed by the air-bone gap, which affects the FG values. The greater the gap, the greater the loss in these values. However, this does not mean that the gain obtained with the device is insufficient for the patient, but rather indicates a failure in the procedure for evaluating the benefit obtained with the BAHA.This research aimed to estimate and compare EG and FG to evaluate the benefits obtained by BAHA users and, later, compare them with the values obtained with speech perception tests in silence and in noise .This is a cross-sectional, retrospective study approved by the Research Ethics Committee under number 4,630,115. Data were analyzed prior to the BAHA implantation surgery and later in the activation of the processors.A total of 79 BAHA users from nine to 62 years of age were assisted from February 2014 to February 2021 and divided into four groups:Group 1: Active transcutaneous (n=21)Group 2: Passive transcutaneous (n=17)Group 3: Percutaneous 1 (n=20)Group 4: Percutaneous 2 (n=21)The Inclusion criteria are: unilateral user of BAHA, having updated audiometry for at least one year, having moderate to severe bilateral conductive or mixed hearing loss.The exclusion criteria are: bilateral BAHA user, not having updated audiometry and having sensorineural hearing loss, mild or profound degree of hearing loss, patients with unilateral hearing loss or single-sided deafness.Data were collected using a two-channel audiometer, with TDH-39 supra-aural headphones, B71W vibrating arc, and speaker. The analysis of the results and benefits obtained from the use of BAHA was based on the following criteria:Pure-tone threshold audiometry relative to the thresholds in the frequencies of 250 to 8000 Hz bilaterally by air for diagnostic purposes and by bone from 500 to 4000 Hz obtained via pure-tone modulated by frequency (warble);Research of logoaudiometric thresholds, to verify compatibility with pure-tone audiometry thresholds.Research of audiometric thresholds in free field, at frequencies from 500 to 4000 Hz, which were performed in a soundproof booth, with the individual positioned 1m from the sound source, in the 0º azimuth condition.9 This material consists of a list of 25 sentences (List 1A), seven lists of 10 sentences (1B to 7B), and a speech spectrum noise. It is recorded on a compact disc CD in which the sentences and the noise were recorded in independent channels, allowing its presentation both in silence and in noise. Continuous noise was presented at the level of 65 dB SPL. The presentation of sentences started at +5 dB S/N ratio (noise at 60 dB SPL and speech at 65 dB SPL). After the patient’s first correct answer or not, the sentence presentation level was respectively decreased or increased by 4dB and after the change in the response pattern, the additions or subtractions were always 2 dB. From the change to 2 dB, the presentation levels of each sentence were recorded and the mean of the presentation level was estimated. To obtain the S/N ratio, this value was subtracted from the competitive noise presentation level. In this study, the signal/noise ratios (S/N) were obtained, and 50% of the sentences presented in free field with and without BAHA were recognized.Speech perception test in silence and in noise with and without device. Lists of sentences in Portuguese (LSP).7The EG and FG values were estimated according to the studied literature:FG (by frequency) = Free field thresholds (without BAHA) – Free field thresholds (with BAHA)EG (by frequency) = Free field thresholds (with BAHA) – Bone conduction audiometric thresholds.Statistical analysis of the study data was performed using a sample of 79 individuals organized into four groups. This analysis aimed to:Investigate the correlation between FG and speech-in-noise tests and between EG and speech-in-noise tests for the total sample;Compare the groups in terms of age and gender;Compare the EG and FG for the total sample.10The statistical significance value adopted was equal to 5% (p≤0.05). The SPSS Statistics software, version 26.0 , was used.To estimate the 95% confidence intervals, the corrected and accelerated bias method was used based on 2000 bootstrap samples. The values in square brackets in the tables indicate the upper and lower limits of the 95% confidence intervals.11The comparison of the groups in relation to age and gender presents the measures of central tendency and dispersion of age according to the group, as well as the comparison of the four groups by means of ANOVA with an independent factor (parametric), since all groups obeyed the normality assumption . Once a violation of the homoscedasticity assumption was observed , the Welch correction for heteroscedasticity was performed in the estimation of the p-value. The effect size of the difference among the groups was measured using the r coefficient.12 For this analysis, statistically significant values were considered at the 5% level (p≤0.05).12, showed a difference between the Passive Transcutaneous group and the Active Transcutaneous groups , Percutaneous 1  and Percutaneous 2 , and, for all cases, the Transcutaneous Passive group was younger.The results in An analysis of the distribution of the study sample according to group and gender was performed. Fisher’s exact test was used to compare groups in terms of gender. The results showed no statistically significant difference among the groups regarding the proportion of male and female individuals in each group.The results in Among the following pairs of variables, a statistically significant positive correlation was observed, that is, the variables presented a directly proportional linear correlation, and the increase in one variable was associated with the increase in the other variable:FG 500, 750, 1000, 3000, and 4000 Hz and S/N ratio without device;FG 750, 1000, 1500, 2000, and 3000 Hz and SRT without device;Among the following pairs of variables, a statistically significant negative correlation was observed, that is, the variables presented an inversely proportional linear correlation, with an increase in one variable being associated with a decrease in the other variable:FG 1000 and 2000 Hz and S/N ratio with device;No statistically significant correlation was found among the other pairs of variables. Therefore, these variables lacked a linear correlation with each other.It shows that the mean values of the effective gain are lower than the values of the functional gain, except at the frequency of 3 kHz in the group of passive transcutaneous device. The positive results for the effective gain show that the lower the numerical value of the gain, the better the benefit that the user has with the use of BAHA.The first finding  showed a13The surgical indication for the placement of BAHA follows criteria previously defined by guidelines that include the age at implantation as a criterion. The main indications for BAHA are a minimum age of five years at the time of implantation and ≥ 3 mm of skull bone thickness. Failure to fix the prosthesis is observed in 40% of children under five years against 8% for children from five to 10 years and 1% for children over 10 years of age, which confirms the rate observed in adults.13For children under five years of age who do not meet the skull bone thickness criterion, we indicate testing with audio processor(s) positioned by a preoperative fixation system with softband. Patients fitted with these elastic bands were excluded from this study. These findings in the literature, compared with the younger age of patients in the Passive Transcutaneous group, show that, despite the difference among the groups, the indication criteria and age of implantation are the same for all groups, that is, for different forms of anchorage. of prostheses (direct drive or skin drive).14The comparison between the groups regarding gender showed no statistically significant difference. Thus, the groups were similar considering gender. These data show that the sample was homogeneous. In a retrospective study to characterize the etiologies and audiological status of hearing impairments in Brazil, the populations studied showed that hearing loss is more frequent in males. However, we should mention that neither this study nor our research was population-based, therefore, the results suggest that the findings regarding gender vary according to the origin of the population studied.15 Our study found a predominance of mixed hearing losses, since patients who undergo BAHA surgery have craniofacial malformations as etiologies, due to the specificity of the service.Regarding the predominance of the most common types of hearing loss, and reporting the context found in the literature, epidemiological data from neonatal hearing screenings, without gender differentiation, show a predominance of sensorineural losses, accounting for 87.3% of hearing loss diagnoses, conductive hearing losses for 6.7%, and mixed losses for 6.0%.When comparing the gains, we found a statistically significant difference between the functional and effective gains for all frequencies , and, in16Regarding FG estimation, the status of middle and external ear malfunctions in mixed or conductive hearing loss, as expressed by the air-bone gap, and which affects FG value, is disregarded. Thus, the greater the air-bone gap, the greater the FG of any device that transmits sound by bone. Therefore, to assess the effectiveness in conductive and mixed hearing loss, the FG must be reconsidered. As a viable alternative for these cases, EG is the priority measure of gain, defined as the thresholds obtained by bone conduction minus the thresholds in free field with the use of the implants.17The thresholds obtained in free field with the prosthesis reflect the audibility benefits, after bypassing the middle ear pathology, representing the cochlear response to bone stimulation transmitted by the BAHA. Therefore, the amount of gain provided by the transducer results from the difference between the free field thresholds with the prosthesis and the bone conduction thresholds at each frequency. The literature showed descriptions of lower free field thresholds than those obtained by bone, due to the transducer used, such as active transcutaneous prostheses, which eliminate the participation of the skin, adipose tissue, and bone density of the skull.6The FG results can also be erroneous when comparing different BAHA. A device could be better at threshold levels, but this certainly does not guarantee that the same device will be better at levels of speech understanding and benefits perceived by users. This theoretical basis states that the improvement of the hearing threshold made possible by the prosthesis when measured in a free field considers the cochlear function.6If the patient has sensorineural hearing loss, the hearing threshold with and without a free-field hearing aid deteriorates at the same rate. Thus, the difference between the threshold remains constant and, consequently, FG remains unchanged.6In patients with conductive hearing loss, the “unaided” free field hearing threshold is impaired, whereas the “with aid” threshold remains unchanged, this means that the FG improves when the conductive loss is lower, thus, it is dependent on the remaining air-bone gap.6Authors show that the EG is used as the main measure of the BAHA effectiveness, as the bone conduction thresholds obtained in pure-tone audiometry are properly masked to eliminate the participation of the best cochlea. When the individual is evaluated in free field, the response is nonspecific, with participation of the best ear, due to the low attenuation during bone conduction and vibrations in the skull bone.18On the other hand, the EG has the proven advantage that it can be compared to validated prescriptive rules, for example, the NAL  rule or the Desired Sensation Level (DSL) Rule.19Studies with bilateral BAHA users suggest that interaural attenuation is sufficient to evoke different responses of the two cochleae. Therefore, the reasoning allows us to conclude that we need to consider the VO obtained in ATL, as these are properly masked and named for compatible ears, also with the differentiation between right and left cochlea.Our study also found that a higher FG correlates with a lower signal-to-noise ratio and a speech recognition threshold in silence , that is20The literature found positive results in speech perception tests in silence and in noise in patients using BAHA, data that confirmed the user satisfaction questionnaire of these prostheses, showing that patients benefit from and improve speech recognition with the use of the devices.20In a comparative study between two BAHA, the results showed that the speech perception scores for both devices improved in silence and noise. All users reported that their devices improved their quality of life. Overall satisfaction scores between device fitting and audiological testing were also statistically significant. This study shows that speech perception tests are linked to user satisfaction, that is, the better the speech recognition, the better the satisfaction.21The literature also highlights that speech tests are established parameters to compare the performance with the current and previous hearing aid. Speech recognition tests in noise and in silence were carried out to compare the performance of patients with the individually adapted BAHA and with the previous hearing aid. All patients in this study answered a questionnaire on speech recognition in quiet and noisy environments. Audiological and questionnaire results were comparable.23A recent systematic review of 29 studies showed that free-field hearing thresholds with BAHA are linked to better speech understanding and higher satisfaction rates with the Active Transcutaneous device.Speech perception tests are excellent parameters for the speech therapist, audiologist, and otolaryngologist to visualize the benefits and the performance of the user in situations that try to simulate everyday life, as in tests in presence of noise. Thus, the results show that worse values of FG mean better performance in speech tests. The objective for estimating FG is to verify if the patient benefits from the use of the BAHA; however, the data of this gain do not confirm the benefits obtained in the speech test and with the reasoning found in the literature.The limitations of the study are related to the bias of speech perception ability being associated with auditory processing ability and duration of hearing loss without amplification and age of use of auditory amplification. These biases were disregarded for the separation of groups.Thus, we found a better evaluation of results on the EG values, showed by the lower value of this gain in all frequencies, indicating that it is a relevant method to evaluate and compare auditory performance. Furthermore, the FG results were incompatible with the benefits obtained in the speech perception tests in silence and in noise, showing that it is an unreliable tool for monitoring and verifying the results with the use of the BAHA.


In the literature, the positive effect of ICT on labour productivity and, in general, economic growth is vast and well consolidated. This paper wants to go beyond the general term of ICT and look inside the "black box." In particular, broadband adoption among Italian firms is critical for productivity. Hence, we focus on broadband adoption and internet facilities and how they affect the firms' total factor productivity in the Italian business sector firms at NUTS2 over the period 2003–2018. Italy is indeed still characterized by a robust North–South divide. Our question is: can we exploit the digital advantage for filling the productivity gap? To answer, we are going to use a classical two-stage approach. In the first one, the TFP is filtered out using both a semi-parametric approach and a parametric one . The second step investigates its determinants, using broadband firms' adoption as a covariate in an ECM augmented by spatial spillovers, controlling for competitivity, internationalization, and human capital. Our results show a positive relationship between TFP and broadband adoption (cointegration), including regional spillovers; this positive effect spreads to GVA. Moreover, our results show that digitalization makes Southern regions more resilient to external shocks. The Next Generation EU plan is the ambitious social and economic strategy that should drive the EU out of the Covid crisis. In Italy, the Government has launched the National Plan for Recovery and Resilience, representing the implementation of the EU plan. The Italian plan is composed of six pillars. In particular, the pillar related to digitalization, innovation, and competitivity (Mission 1) plays a prominent role in the recovery phase, including the rebalance of the North–South divide in Italy. The financial resources for such mission amount to 21% of the entire national plan budget . In particular, the investments for the business sector's digitalization and innovation amount to 38 billion euros. In mission 1, a specific point is devoted to developing broadband and 5G in the SMEs (4.2 billion). Such investments should speed up the Italian Strategy for the ultra-broadband network launched in 2015. It addressed to cover, within 2020, 85% of the population with a speed of at least 100 Mbps (NGA-VHCN -Very High-Capacity Networks) and the full coverage of the population with at least 30 Mbps.As said, the broadband investments lie under the mission of increasing the competitiveness of the business sector; nonetheless, the National Plan does not underline how broadband coverage and adoption will be transferred in a higher competitivity. Presumably, the policymaker refers to the economic literature related to the positive effect of ICT investments on firms' productivity, notably labour. This literature is vast and well consolidated. As Italy is concerned, Iammarino and Jona-Lasinio  analyse From a theoretical point of view, human capital and technology have been crucial drivers of economic growth since the pioneering contributions by Lucas , Romer , and AghConnectivity—Fixed broadband take-up, fixed broadband coverage, mobile broadband, and broadband prices.Human Capital—Internet user skills and advanced skills.Use of Internet—Citizens' use of internet services and online transactions.Integration of digital technology—Business digitization and e-commerceDigital public services—e-Government.From the policymaker viewpoint, fostering the digital economy has been on the European agenda since the Industry 4.0 action plan, until the "Shaping Europe's Digital Feature" in February 2020. In the last few years, many efforts have been devoted to providing data and measures of digitalization. The Digital Economic and Society Index (DESI) is an example. Following the vast literature on ICT and human capital, the index covers five dimensions of the digital revolution, summarized in Table The 2020 DESI for Italy raises several questions about points 1–5. First, looking at the general index, Italy performs poorly. It is 25 out of the 28 EU members. However, this poor performance is mainly due to the low human capital: "Compared to the EU average, Italy records shallow levels of basic and advanced digital skills. ICT specialists and ICT graduates are also well below the EU average. These gaps in digital skills reflect the poor use of online services, including digital public services. Similarly, Italian enterprises lag in the use of technologies such as cloud and big data, as well as in the uptake of e-commerce" page (3). About the connectivity sub-index, Italy ranks 17th among EU countries (we will come back on the point in the following section).This paper will focus on points 1 and 2 and their relationship with Italian firms' Total Factor Productivity (TFP). We want to investigate how broadband adoption among Italian firms affects their productivity. Answering this question is crucial to assess whether the Recovery Plan will effectively support firms' competitiveness, hence growth. Theoretically, the widespread of broadband fosters the sharing and availability of information and data across multiple locations; moreover, it opens to a new opportunity for firms in e-commerce, information availability, market perspectives, etc. Beyond the reduction of costs of existing business processes, high-speed internet enables new business and firm-cooperation models that rely on the spatial exchange of local information, which fosters competition and innovation processes. On the demand side, broadband internet may increase market transparency and thus additionally intensify competition. However, an empirical answer for the Italian case has to be provided.When combined with information technologies, the broadband infrastructure can also affect firm productivity and economic growth in additional ways. The development of information technologies fundamentally changed and improved information processing, resulting in significant productivity growth of IT-using firms Stiroh, . The recMore recently, Gal et al.  providedNonetheless, there are no specific empirical contributions on the effects of broadband adoption on the TFP in Italy; this is the novelty of our contribution. We address the question from a regional perspective for two reasons. Firstly, our primary goal is to assess the effect of broadband adoption in Italian firms in fostering regional development. Broadband coverage and adoption lie in the EU strategic framework for regional development and social cohesion and is an instrument for such a policy, even before the Next Generation EU. Italy is part of this framework; both the Italian Digital Agenda plan and the Italian Strategy for Ultra-Broadband are examples. The Minister for Infrastructures and Transports (MIT), the one for Economic Development (MISE) and the one for the Technological Innovation and Digital Transition (MITD) manage the targets that Italy has established with the EU. Secondly, reliable time series on firms' network adoption does not exist at the firm level. The official data comes from the Italian National Institute for Statistics (Istat—which provides Eurostat data to the EU commission for monitoring the implementation of the network) but only at a regional level . Moreover, the regional dimension allows us to analyse the possibility of spatial spillovers generated by widespread internet firms' adoption and higher human capital; more connected territories represent another channel through which new ideas, processes, and organizations spill over into the firms.Our results confirm a positive relationship between ICT facilities and TFP. Under this point of view, it seems evident that policies fostering broadband, pc, and internet adoption in the Italian firms increase their productivity. Such a result holds both for Northern and Southern firms, although differences remain. Especially in the South, the increase in broadband usage has contrasted with the fall of TFP after the 2008 financial crisis. Since 2014, regions have shown a gross value-added recovery trend (GVA). For the Northern areas, this recovery was mainly linked to improved employment levels, and a less extent, in the TFP (except Far North–East). By contrast, in the Southern regions, the expansion of TFP has supported the recovery of the GVA. This is because the Southern companies have gained more from digital infrastructures.In general, broadband adoption sustains TFP, and it positively impacts GVA, tempering the negative impact of a recession. Moreover, the spatial estimate shows that positive spatial spillovers among neighbouring regions are at work. Under this point of view, sustaining the digital economy could be promising for the post-Covid age.The paper is organized as follows. In the next section, we present some stylized facts about firms' broadband adoption in Italy. Sections 3 and 4 estimate the TFP, while Sect. 5 investigates the relationship between TFP and ICT component. Discussion of main findings and policy implications are in Sect. 6. Section 7 concludes.Istat, the National Institute for Statistics, collects data on broadband adoption in private firms with more than ten employed since 2003; although this firm size covers only 10% of the total, they represent more than 50% of GDP and employment. However, the definition of broadband itself is not fully clear; in Italy,The European Digital Agenda defines three levels of broadband speeds: 2, 30, and 100 MBPS. On the third of March 2015, the Italian Government signed the "Strategia Italiana per la banda ultralarga"  addressed to cover, within 2020, 85% of the population with a speed of at least 100 Mbps (NGA-VHCN -Very High-Capacity Networks) and the full coverage of the population with at least 30 Mbps. The European Cohesion Fund finances the Strategy with 3,5 billion and a further 1,8 billion from national and regional development funds. Additional funds will come from the National Plan for Recovery and Resilience. However, the target has not been achieved.While the 30 MBPS coverage appears satisfactory, Italy is still behind in the Ultra-broadband (at least 100 MBPS), essential to fostering enterprises' digitalization. According to Istat, in 2019, 41% of firms with at least ten employed using a broadband connection (30 MBPS), and only 13.8% accessed 100 MBPS. The picture is made even more complicated by the geographical distribution of the Ultra-broadband, with "white area" (no coverage), "grey area" (only one provider), and "black area" (at least two providers); these areas are currently part of the Italian Strategy for Ultra-broadband that points to reducing the gap in the next three years. However, two out of three Italian SMEs are in the grey area where the ultra-broadband is practically missing. In 2019, Italy completed Phase I of the ultra-broadband Italian plan for white areas and awarded the last three tenders to the wholesale-only operator Open Fiber. Nevertheless, severe delays remain, mainly due to existing infrastructure and obtaining regional permits.Broadb), rather than coverage. Istat provides regional data on the firms' network adoption since 2003, but only since 2019 by type of connection. Figure This paper focuses on net firms' broadband adoption , and coarse entrepreneurship. Despite this, it ranks at the top of the regions with firms connected in ultra-broadband (more than 100 MBPS) in 2019. So, for some regions, broadband adoption is not enough for boosting their economy. Is this a characteristic of Southern regions that cannot exploit this digital advantage for filling the productivity gap? Is this the lack of other dimensions, such as entrepreneurship, human capital, firms’ strategy? We try to answer by an empirical investigation. Unfortunately, before 2019, Istat did not provide detailed data by type of connection. The only regional data available concerns firms' share connected to a fixed network, from 2 MBPS, although the primary connection type is 30 MBPS.At first glance, the Southern regions show an adoption share not so distant from the North levels (except Puglia and Basilicata). In some cases, Southern areas show a high degree of ultra-broadband adoption (more than 100 MBPS)Our paper's first step involves estimating a traditional Cobb Douglas production function augmented by a Hicks neutral technical progress (TFP). The estimation of a production function is debated in the literature. Several approaches are used, from simple growth account decomposition of GDP to parametric estimates , semi-parametric approach  is the technical change (or TFP). Let us assume that Let us start by assuming that the market output of a representative firm The "endogeneity of input choice" or "simultaneity bias" arises because productivity shocks are potentially known to the firms when they make input decisions, such as the managerial ability or an expected shortage in intermediate inputs and so on. Hence, the inputs choice—Usually, researchers assume nd Pakes —OP and lnd Pakes —LP, by gIn OP s of Eq. . NeverthAs said, whenever we can assume Concluding, both approaches (semi-parametric vs. parametric) have lights and shadows for our aim. On one hand, the former is well-founded in the firms' behaviour while the latter is more suitable for regional data. For such a reason, in this paper, we present both estimation strategies. The semi-parametric approach uses the GMM approach by ACF, described briefly in Appendix In this section, we are going to estimate Eq.  both by Nevertheless, the Cobb Douglas estimate at the regional level involves the knowledge of labour and capital at NUTS2 level. Unfortunately, an official time series of the stock of capital at NUTS 2 does not exist, while the gross fixed investment measure at NACE level is available. So, following the Bank of Italy . Moreover, the J test on the instruments’ validity of the GMM is successful. The sum of the estimated coefficients is slightly higher than one in both estimations  and broadband adoption cover the connection and the adoption of digital solutions. As previously discussed, we are interested in the first two DESI components and, partly, in point 4—connection, human capital, and digital economy integration in the Italian firms. Stem and the population with tertiary education account for the human capital. Moreover, productivity is also affected by physical investment (FI), which can be considered a proxy for firms' competitiveness. Finally, the degree of internationalization and number of newborn firms in knowledge-intensive sectors are further controls that could impact the TFP of private firms. Data are provided by Istat that collects, since 2003, information at NUTS2 level on a series of variables related to statistics for the local development and social cohesion. In particular, the section Information Society records data on the digital economy for firms with more than ten employed.The first variables , as they are complementary and related to each other, especially Broadb, Pc and Int.To clarify the question, let us start from a general equation for the TFP model  approach, with fixed effect. We regress Broadb on Pc and Stem, using Int as an instrumental variable for Pc. Stem is treated as exogenous variable, but it could be helpful in gaining efficiency of the Broadb estimate. Results are in Table  PC and Stem positively impact the firms' broadband adoption through the Internet facilities, as expected. In addition, the growing share of graduates in Stem disciplines in most regions has effectively encouraged companies to use ICT instruments and adopt broadband. The F-test on the instrument validity is higher than 10, confirming that Int can be treated as an instrumental variable.Once having estimated Broadb in a consistent way, the fitted values will be used as a covariate in the TFP equation for the reasons previously stressed. This is performed in the following section.According to Engle and Granger , if two To investigate the question, we must firstly check the unit root both in TFP and in the fitted value of the IV estimation, where The cointegration equation acts as a long run constraint between the TFP and the broadband adoption in an empirical model. Engle and Granger  show thawhere We estimate the ECM by a ML panel with fixed effect , is justified by the different hypothesis about spatial externalities. In the SEM case, spatiality in the error component captures possible common factors and possible cross-dependence in omitted variables. In a SAR model, spatial correlation arises through externalities related to the endogenous variable. Such an assumption fits our basic assumption: the TFP embodies ideas and knowledge that are spread in the economy; it generates positive spillovers that are beneficial also for others.The results are in Table The spatial autoregressive coefficient is relatively high . The impact of the covariate is hence divided in indirect and direct impacts; the first one accounts for the effect on region j of a change in region i, while the second one of the region i on the region itself. The sum of direct and indirect impacts produces the total impact, providing the right estimate coefficient measure. Each impact’s statistical significance must be calculated using a bootstrap algorithm, as discussed in LeSage and Pace , firms' entrepreneurship , and human capital (in terms of education and training both in the youngest and the workers). The policymakers should carry out employment and entrepreneurship policies to make regions more resilient to external shocks and reduce the North–South divide. The ICT facilities are not enough for the Southern economies that need a boost in employment by creating new businesses. Therefore, within the Recovery Plan framework, the pillar of Social Inclusion is also crucial for sustainable development, especially in Southern regions.


Ascaris, PDE occurs during the 4 to 16 cell stages of embryogenesis, when germline chromosomes are fragmented and specific DNA sequences are reproducibly lost in all somatic cells. PDE was identified in Ascaris over 120 years ago, but little was known about its molecular details until recently. Genome sequencing revealed that approximately 1,000 germline-expressed genes are eliminated in Ascaris, suggesting PDE is a gene silencing mechanism. All germline chromosome ends are removed and remodeled during PDE. In addition, PDE increases the number of chromosomes in the somatic genome by splitting many germline chromosomes. Comparative genomics indicates that these germline chromosomes arose from fusion events. PDE separates these chromosomes at the fusion sites. These observations indicate that PDE plays a role in chromosome karyotype and evolution. Furthermore, comparative analysis of PDE in other parasitic and free-living nematodes illustrates conserved features of PDE, suggesting it has important biological significance. We summarize what is known about PDE in Ascaris and its relatives. We also discuss other potential functions, mechanisms, and the evolution of PDE in these parasites of humans and animals of veterinary importance.In most organisms, the whole genome is maintained throughout the life span. However, exceptions occur in some species where the genome is reduced during development through a process known as programmed DNA elimination (PDE). In the human and pig parasite  Ascaris is a parasitic nematode that lives in the small intestine of humans and pigs . In. InP. univision) , the copAscaris elimination mitoses. At metaphase, retained and eliminated DNA are both aligned at the metaphase plate. In early anaphase, the retained DNA segregates to the daughter cells while the eliminated DNA remains at the metaphase plate  and pig (A. suum) parasites [Several significant findings emerged from these some end  41]. In. InAscarsome end . No genoaris PDE ,41. Theiechanism ,40. ThirP. univalens and approximately 2,000 in the dog parasite T. canis . The internally eliminated regions in both Ascaris and Parascaris have sequence features that are common to the ends of nematode chromosomes [Parascaris and the 24 Ascaris germline chromosomes can be viewed as fusion products of the 36 ancient germline chromosomes. These ancestral germline chromosomes also likely underwent PDE with end remodeling  from Clade III [Strongyloides [Ascaris, P. univalens, and O. tipulae PDE is the removal of all chromosome ends, followed by new telomere addition. This leads to the question whether PDE is an ancient trait, or if it has convergently evolved in multiple nematode clades. It is known that the model organism C. elegans (Clade V) does not undergo PDE [In nematodes, PDE has been discovered in 3 distinct clades, including many  species ,56, Caenhabditis  from Clalade III , and Strgyloides ,101,102 ergo PDE ,104. Butergo PDE . PDE mayergo PDE ,106. Howergo PDE ,45,49. NAscaris, the ability to dissect specific germline and somatic tissues, and its slow, synchronous embryo development have enabled robust biochemistry, molecular biology, and genomics to be carried out. Other parasitic ascarids, including Parascaris and Toxocara, have also been used as comparative models to study PDE. Many features of PDE, including DNA breaks, telomere addition, and chromosome end removal and remodeling, are conserved in these nematodes [O. tipulae now adds genetic and functional approaches to study PDE [Ascaris.PDE violates the general view of genome constancy and integrity. However, PDE is a natural process found in many branches of animal phyla, suggesting its common presence and biological significance. The number of species described with PDE has increased with the use of genome sequencing and modern cytology. However, mechanistic insights into PDE are mostly limited to single-cell ciliates. For metazoans, the nematode group has been a leading model to study PDE since its discovery over 130 years ago. In particular, the large size of ematodes ,40. The tudy PDE . Overall


Horizontal gene transfer (HGT) in the microbiome has profound consequences for human health and disease. The spread of antibiotic resistance genes, virulence, and pathogenicity determinants predominantly occurs by way of HGT. Evidence exists of extensive horizontal transfer in the human gut microbiome. Phage transduction is a type of HGT event in which a bacteriophage transfers non-viral DNA from one bacterial host cell to another. The abundance of tailed bacteriophages in the human gut suggests that transduction could act as a significant mode of HGT in the gut microbiome. Here we review in detail the known mechanisms of phage-mediated HGT, namely specialized and generalized transduction, lateral transduction, gene-transfer agents, and molecular piracy, as well as methods used to detect phage-mediated HGT, and discuss its potential implications for the human gut microbiome. The human gut microbiome is a complex community with a vast network of microbe–host interactions. The ability to change and adapt to acute events is essential to maintain long-term stability. The microbial community, with its short generation times and intense selective pressure, evolves at a speed unimaginable for multicellular organisms, and can therefore respond more quickly than its host to changing environmental conditions . This isGenetic information is typically transferred vertically from parent to offspring and is subject to change through mutation. This is how a phylogenetic tree depicts evolution. Any other movement of genetic information is referred to as horizontal gene transfer (HGT). In recent years it has become increasingly clear that without taking HGT into account, it is impossible to describe the evolution of microbial communities , especiaHGT within the microbiome has potential consequences for human health and disease. The spread of antibiotic resistance genes happens mainly by way of HGT . VirulenBacillus subtilis  and Wellcome Trust under a Wellcome Trust Research Career Development Fellowship [220646/Z/20/Z] (A.N.S.). This research was funded in whole, or in part, by the Wellcome Trust [220646/Z/20/Z]. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.


Its cause and pathogenesis are still unknown. Among the theories proposed to explain its pathogenesis, we list cochlear viral infection, vascular occlusion and membrane ruptureThis patient had left side idiopathic sudden hearing loss at 40 years of age. He reports that when he woke up in the morning, he noticed a high frequency tinnitus on his left ear, he felt irritable and unable to concentrate enough to read the newspaper, as he had done every morning. Some hours later he felt ear fullness, diplacusis and sudden profound hearing loss, and he also had trouble keeping his balance. He did not report upper airway infection symptoms that day, nor the week before. Tonal and vocal audiometry showed profound hearing loss on the left side. His electrocochleogram (EcoG) showed no cochlear potentials on the left side and the electronystagmography (ENG) showed a vestibular function deficit to the right side. He was treated as having sudden sensorineural hearing loss of viral origin, and treatment details are unknown. Tonal and vocal audiometry carried out six months afterwards revealed 20 dB thresholds for frequencies 125, 250 Hz and of 80–90 dB in the high frequencies, with speech recognition of 40%. This patient died at 62 years of age, of cardiac cause. His temporal bones were removed 6 hours after his death and immediately fixed in 10% formalin solution.Histopathology analysis showed a very atrophic stria vascularis in all of its turns and mass loss of cochlear neurons in the basal turns, with greater dendritic loss when compared to central axons , consequent to a loss in the Organ of Corti and hair cells . CochleaThis patient developed profound left side sensorineural hearing loss, with a downsloping audiometric pattern which can be explained based on the loss of the Organ of Corti and hair cells in the basal turns. There was no microscopic evidence of viral involvement, vascular occlusion, perilymphatic fistula or any other known cause.


Identifying novel mediators of lethal myocardial reperfusion injury that can be targeted during primary percutaneous coronary intervention (PPCI) is key to limiting the progression of patients with ST-elevation myocardial infarction (STEMI) to heart failure. Here, we show through parallel clinical and integrative preclinical studies the significance of the protease cathepsin-L on cardiac function during reperfusion injury.n = 76) immediately post-PPCI leads to elevated serum cathepsin-L levels and that serum levels of cathepsin-L in the first 24 h post-reperfusion are associated with reduced cardiac contractile function and increased infarct size. Preclinical studies demonstrate that inhibition of cathepsin-L release following reperfusion injury with CAA0225 reduces infarct size and improves cardiac contractile function by limiting abnormal cardiomyocyte calcium handling and apoptosis.We found that direct cardiac release of cathepsin-L in STEMI patients (Our findings suggest that cathepsin-L is a novel therapeutic target that could be exploited clinically to counteract the deleterious effects of acute reperfusion injury after an acute STEMI. Primary percutaneous coronary intervention (PPCI) is the most effective treatment for restoring blood flow to ischaemic myocardium and limiting cardiomyocyte death following an acute STEMI.Cathepsins are proteases classified by structure and catalytic type into serine, aspartic and cysteine cathepsins. These enzymes were traditionally thought to be located exclusively within intracellular lysosomes and only play a role in homeostatic protein breakdown.Our integrative clinical and experimental study reveals the vital role and clinical relevance for cathepsin-L during myocardial reperfusion injury in rodents and humans.A detailed description of the methods and statistical analysis is reported in Figure S1). The study protocol and consent processes were approved by the local Research Ethics Committee (REC 14/WS/0085).A prospective single-centre cohort study was performed at a regional cardiac centre . Research staff screened patients with acute STEMI undergoing emergency invasive management. In all, 83 patients agreed to participate and provided written informed consent. The human studies conformed to the principles outlined in the Declaration of Helsinki. Systemic blood samples were taken at pre-reperfusion of the occluded culprit coronary artery; 20 min post-PPCI; 24 h post-PPCI; and at 6 months post-PPCI. Cardiac magnetic resonance imaging (MRI) was performed at 24 h and 6 months post-PPCI  study. Assent for participation was obtained at the time of PPCI. Blood samples were obtained at the end of the PPCI procedure from the coronary artery guide catheter, from a catheter placed in the coronary sinus, and from a peripheral vein.Serum levels of cathepsin-L were measured by enzyme-linked immunosorbent assay using the manufacturer’s recommended protocol (USCN Life Science Inc.) and normalized to the total serum protein content.The care and use of animals were in accordance with the UK Government Animals (Scientific Procedures) Act 1986. All animal procedures  were approved by the University of Glasgow Animal Welfare and Ethical Review Body and licensed by the Home Office, UK (project license numbers 600/4503 and P06FE1F82).Left intraventricular pressure was measured in Langendorff perfused hearts from adult male Wistar rats. Infarct size was measured using triphenyltetrazolium chloride staining. Cathepsin-L activity was determined using established protocols. The cathepsin-L inhibitor CAA0225 Adult rat cardiomyocytes were isolated as previously describedin vivo model of reperfusion injury involved male C57Bl/6 mice aged 9–12 weeks. These mice were anaesthetised in an induction chamber with 4% isoflurane and 100% oxygen. The anaesthetised mice were intubated and ventilated using a small animal respirator  and maintained with 1.5% isoflurane and underwent temporary coronary artery ligation via thoracotomy.The Figure S1). The demographic characteristics are shown in Table S1.In all, 83 STEMI patients agreed to participate in the current study . Infarct size and left ventricular (LV) function were assessed by MRI at 24 h and 6 months post-PPCI . Serum cathepsin-L levels increased 20 min post-PPCI to 135% of pre-PPCI levels (P < 0.05). Area under the curve analyses indicated that the 24-h log10 cathepsin-L levels were negatively correlated with contractile parameters measured by MRI at 24 h post-PPCI : LV ejection fraction ; stroke volume indexed to body surface area ; and cardiac index . The LVEF at 6 months, negatively correlated with cathepsin-L levels at baseline , which suggested prognostic significance of baseline serum cathepsin-L concentrations for long-term LV function. Elevated cathepsin-L levels were also associated with increased infarct sizes at 24 h  and 6 months .Angiography and electrocardiogram conducted pre-PPCI and post-PPCI confirmed the presence of STEMI (Table S2) and mean cardiac performance improved over time post-PPCI . The post-PPCI levels were 113% of the pre-PPCI levels , thus demonstrating the robustness of these findings across two geographically distinct locations. Placement of catheters in the second cohort enabled parallel collection of aortic root, coronary sinus and venous blood samples post-PPCI. Cathepsin-L levels in aortic root and coronary sinus post-PPCI were higher than those detected in venous blood samples taken before PPCI . Furthermore, post-PPCI serum cathepsin-L levels in coronary sinus samples were higher than those found in post-PPCI aortic root samples, resulting in a mean cardiac release ratio of 9.8 ± 3.3 . In contrast, serum cathepsin-L levels in post-PPCI venous blood and aortic root samples were similar .Serum cathepsin-L levels were also assessed pre-PPCI and immediately post-PPCI in a cohort of STEMI patients treated at a different cardiac care centre +CAA0225] vs. the control group , demonstrating that the coronary artery ligation placement was equivalent. However, the infarct size among mice treated with CAA0025 was 73% of that observed among control mice . M-mode echocardiography was performed before and after IR to assess cardiac contractile function . Fractional shortening was decreased in the control group over the 2-week period, whereas CAA0225-treated mice exhibited preserved fractional shortening . No statistically significant between-group differences were found for LV internal dimension at systole  or diastole . Similarly, LV posterior wall thickness at systole  and diastole  did not differ between the two groups.Given the findings that elevated levels of cathepsin-L were associated with larger infarct size and reduced cardiac function among STEMI patients, we next sought to determine whether blocking cathepsin-L activity using a specific cathepsin-L inhibitor (CAA0225)Figure ). Compared with the control mice, CAA0225-treated mice demonstrated increased intra-LV pressure parameters, including developed pressure (P < 0.05), maximum rate of intra-LV pressure rise , and minimum rate of intra-LV pressure fall (P < 0.05). No statistically significant between-group difference was found for end-systolic or diastolic pressure  or for end-systolic or diastolic volume .Pressure–volume (PV) loop measurements were also performed at 2 weeks to assess hemodynamic function . Fractional shortening was preserved in CAA0225-treated vs. control mice (P < 0.05); however, no statistically significant differences were found for LV internal diameter at systole or diastole, and wall thickness at diastole or systole . CAA0225 decreased infarct size to 69% of that in the IR+DSMO group . CAA0225 also improved overall LV developed pressure post-reperfusion . The Pdev in CAA0225-treated hearts at 120 min post-reperfusion was 178% of that in the IR+DMSO group . In the IR+CAA0225 group, the dP/dtmax and dP/dtmin  at 120 min were 169% and 151% of those in the IR+DMSO group, respectively . No statistically significant change was found between the IR+CAA0225 and IR+DMSO groups for Pmin (a measure of the ability of the heart to relax) during the ischaemic period. However, this measure was reduced during reperfusion with CAA0225 (53 ± 2 mmHg), which was 68% of that in the IR+DMSO group at 120 min . CAA0225 therefore reduced infarct size and improved the systolic and diastolic contractile function of ex vivo rat hearts post-reperfusion injury.To determine the direct effects of CAA0225 on cardiac contractile function, Langendorff-perfused Figure ) in a randomized and blinded study. CAA0225 applied during reperfusion decreased infarct size to 80% of that in the IR+DMSO ( 0.05). Furthermore, CAA0225 improved overall LV developed pressure, dP/dtmax and dP/dtmin at 120 min post-reperfusion to 276%, 198%, and 198%, respectively, of that in the IR+DMSO group .To assess therapeutic potential, CAA0225 was administered to Langendorff-perfused rat hearts early in reperfusion  improved overall LV developed pressure, dP/dtmax and dP/dtmin post-drug application , of that in the IR+DMSO group .In separate experiments, we confirmed our findings using a different highly selective cathepsin-L inhibitor called Cathepsin Inhibitor IV. Unlike CA00225, Cathespin Inhibitor IV is a potent reversible inhibitor of cathepsin-L. Cathepsin Inhibitor IV applied for 20 min during reperfusion . Cathepsin-L levels peaked at 20 min after the start of reperfusion, which was equivalent to the time when cathepsin-L was measured among patients post-PPCI. Recombinant cathepsin-L protein (RP) provided a positive control, with 0.68 nmol.L−1 giving an equivalent cathepsin-L activity level as that observed upon reperfusion injury .To corroborate the findings observed among STEMI patients regarding direct cardiac release of cathepsin-L during reperfusion injury, rat hearts were Langendorff-perfused and cathepsin-L activity measured in the coronary effluent. Control hearts had minimal detectable cathepsin-L activity whereas the reperfusion injury group exhibited increased cathepsin-L activity . As shown in Figures 7D, the calcium transient peak (systolic [Ca2+]i) with increasing concentrations of cathepsin-L RP was 81%, 74%, and 54% of the respective control values. The calcium transient minimum (diastolic [Ca2+]i) with increasing concentrations of cathepsin-L RP was 86%, 100%, and 127% of the respective control values . The changes in peak and minimum [Ca2+]i with increasing concentrations of cathepsin-L RP resulted in a calcium transient amplitude that was 80%, 70%, and 43% of the respective controls . Furthermore, the calcium transient rate constant with increasing concentrations of cathepsin-L RP was 90%, 82%, and 33% of the respective controls .We next determined whether similar extracellular levels of cathepsin-L might alter sarcoplasmic reticulum (SR)-mediated calcium release and contractile function .We next determined whether the reduced calcium transient observed with cathepsin-L was related to a reduced SR calcium content. The caffeine-induced calcium transient amplitude  in response to increasing concentrations of cathepsin-L RP was 79%, 90%, and 60% of the respective control values .Sarco-endoplasmic reticulum calcium ATPase (SERCA)-mediated calcium uptake is bypassed during application of 10 mM caffeine and cytosolic calcium removal occurs predominantly via the sodium calcium exchanger (NCX). To assess whether the reduction in SR calcium content by cathepsin-L was related to calcium extrusion from the cell via altered NCX, the rate constant for decline of the caffeine-induced calcium transient was measured. The caffeine-induced calcium transient rate constant with increasing concentrations of cathepsin-L RP was 102%, 95%, and 79% of the respective control values . Therefore, extracellular cathepsin-L can negatively affect both systolic and diastolic SR-mediated calcium release in cardiomyocytes.Spontaneous SR-mediated calcium release during diastole, which can result in calcium waves, is known to contribute to abnormal cardiomyocyte function including cell death. Using confocal microscopy, we established that cathepsin-L RP increased calcium wave frequency to 136% of the control levels .To establish if inhibition of cathepsin-L using CAA0225 altered calcium handling post-reperfusion, rat hearts were treated as outlined in ,Figure ). Following continuation of electrical stimulation, the steady state mean calcium transient peak was not appreciably changed . In contrast, the minimum diastolic calcium concentration was increased by reperfusion to 163% of the control but normalized to control levels by CAA0225 . These changes resulted in a reduced Ca2+ amplitude after reperfusion to 70% of the control value, which was restored to control levels by CAA0225 . The Ca2+ transient rate constant of decay decreased with reperfusion to 55% of the control but was reversed by CAA0225 . The SR Ca2+ content—determined by the amplitude of the caffeine-induced calcium transient at the end of the protocol—was not altered . The NCX activity—determined by the caffeine-induced calcium transient rate constant—was decreased to 66% of the control value, an effect that was reversed by CAA0225 . SERCA activity—assessed by the subtraction of the caffeine-induced calcium transient rate constant of decline . CAA0225 therefore had the ability to rectify abnormal systolic and diastolic calcium handling within cardiomyocytes and improve LV function post-reperfusion.Before electrical stimulation of cardiomyocytes, 10 mM caffeine was applied to empty the SR of calcium. The amplitude of the first electrically stimulated calcium transient post-caffeine is an index of calcium entry via the L-type calcium channel.Figure  were used. The percentage of cardiomyocytes producing spontaneous contractile events during reperfusion increased to 397% of the control value, an effect that was prevented by CAA0025 . The number of viable rod-shaped cardiomyocytes was decreased by reperfusion injury to 43% of the control value but restored by CAA0225 . Apoptosis and activation of caspases can lead to cell death post-reperfusion. CAA0225 normalized the reperfusion-induced increase of caspase 3 and 7 activity . PARP-1 [(ADP-ribose) polymerase-1] cleavage by caspase 3/7 is an established marker of apoptotic cell death.,Figure ). In separate experiments, the ability of CAA0225 to prevent apoptosis was assessed by TUNEL staining and hydrogen peroxide to induce apoptosis. CAA0225 prevented the hydrogen peroxide-induced increased in apoptosis .Increased diastolic calcium concentration and spontaneous release of calcium from the SR  has been linked to mechanisms that contribute to cell death post-reperfusion. We assessed whether inhibition of cathepsin-L using CAA0225 could alter the production of spontaneous contractile events that originate from calcium waves and cardiomyocyte cell death. Unstimulated (quiescent) populations of cardiomyocytes from hearts undergoing equivalent protocols to Here, using both clinical and integrative preclinical studies, we demonstrate that serum cathepsin-L is more than simply a consequence of a pathological process, but is directly released from the hearts of patients and is intrinsically involved in mediating cardiac dysfunction during reperfusion injury. Importantly, we also demonstrate that use of a highly specific, cell permeable, cathepsin-L inhibitor (CAA0225)Three major aspects regarding cathepsin-L were unknown among STEMI patients until now: whether cathepsin-L levels rise upon reperfusion; whether cardiac release of cathepsin-L occurs upon reperfusion; and whether cathepsin-L levels correlate with cardiac function post-reperfusion. We believe that the current study has addressed these three questions by taking the novel clinical approach of evaluating serum cathepsin-L, together with cardiac function using MRI, among patients undergoing PPCI. We demonstrated a remarkable similarity between our preclinical and clinical study in that cathepsin-L levels temporarily increased 20 min after reperfusion injury and were associated with a decreased cardiac contractile function . Cardiac release of cathepsin-L upon reperfusion is the main source of increased cathepsin-L after reperfusion injury. Furthermore, patients with elevated serum cathepsin-L levels had increased infarct sizes. Surprisingly, cathepsin-L correlated with both cardiac function at 24 h post-PPCI and LVEF at 6 months. Although further clinical work is required, our data suggest that serum cathepsin-L levels might hold promise as a future biomarker for assessing patient outcome and optimizing drug therapy post-PPCI for STEMI.,Figure 6). Importantly, release of cathepsin-L from human hearts was also substantially increased at 20 min following reperfusion induced by PPCI.Cathepsins are released from lysosomes into the cytosol and systemic circulation during pathological conditions.ex vivo hearts upon reperfusion, with the other two representing elevated levels (four-fold and eight-fold) observed in some hearts. These experiments demonstrated a dose-dependent reduction in the amplitude of SR calcium release (a surrogate for cardiomyocyte contraction), decreased SR calcium content and, at the two higher concentrations, a reduction of NCX and SERCA activity. Consequently, a raised diastolic calcium concentration in cardiomyocytes was observed, which is known to impair diastolic function of the heart.2+ signalling are unknown. However, the novel data reported in this current study demonstrating that cathepsin-L can adversely affect calcium handling are important given the substantial evidence linking calcium dysregulation to mitochondrial failure, cardiac contractile dysfunction, and cell death during reperfusion injury and post-MI (cardiac remodelling).We next examined the effect of extracellular cathepsin-L on isolated cardiomyocyte function. Three concentrations of cathepsin-L were chosen. The first was the mean level released by the To test our hypothesis, we used CAA0225 in Langendorff-perfused hearts subjected to reperfusion injury. Pre-treatment of whole hearts with CAA0225 before induction of reperfusion injury reduced infarct size and almost doubled cardiac contractile function post-reperfusion, strongly suggesting that cathepsin-L inhibition is beneficial to cardiac function during reperfusion injury. We can rule out an indirect positive inotropic effect as CAA0225 did not alter diastolic and systolic parameters during a 25-min pretreatment of the hearts (data not shown). The simplest explanation for the improved cardiac function was prevention of cell death and increased survival of viable contractile myocardium. Nonetheless, it is also possible that preventing cathepsin-L activity upon entry into the cytoplasm and extracellular space (with subsequent paracrine effects) prevents cathepsin-L-mediated modification of SR-mediated calcium release and contractility of surviving myocardium, resulting in improved cardiac function. Although the current study was the first to demonstrate that CAA0225 can reduce infarct size and improve cardiac function post-reperfusion injury, previous studies in different organs have shown that inhibition of other cathepsins can also reduce cell death. Cathepsin-B inhibitors attenuate reperfusion injury-induced hepatocyte death,,ex vivo hearts post-reperfusion injury. Given that CAA0225 can penetrate the cell membrane and that the cardiomyocytes were continuously superfused, these cells had little, if any, opportunity to be exposed to external cathepsin-L, therefore demonstrating the importance of intracellular cathepsin-L for cardiac dysfunction post-reperfusion injury.To investigate the potential of CAA0225 to improve calcium handling and limit cardiomyocyte death post-reperfusion injury, we treated hearts with this inhibitor and then isolated the cardiomyocytes post-reperfusion to examine calcium handling. We clearly showed that in the absence of CAA0225 reperfusion injury resulted in a reduction of calcium transient amplitude, reduced SERCA activity and reduced NCX activity . The decreased ability of the reperfused cardiomyocyte to extrude calcium from the cell or for calcium to return to the SR as efficiently as healthy cardiomyocytes led to an accumulation of calcium within the cytosol  and increased cardiomyocyte death (irreversible hypercontracture). Cardiac reperfusion injury is associated with an increase in cardiomyocyte activation of caspases and apoptosis, a form of programmed cell death, which contributes to adverse cardiac remodelling and reduced cardiac function.in vivo when introduced after induction of ischaemia but before reperfusion . Furthermore, other factors present in vivo could influence the efficacy of CAA0225, for example, inflammatory cells migrate to the heart post-reperfusion injury and contribute to tissue concentrations of cathepsin-L.in vivo has a sustained positive impact on cardiac function. Collectively, these results indicate that release of cathepsin-L is more than simply a bystander effect of cell death during IR injury, but rather intrinsically involved in the pathophysiology of cardiac dysfunction following IR injury.Despite the benefit afforded by the Langendorff preparation in demonstrating a cardiac-specific protective effect of CAA0225, it was important to establish whether CAA0225 was effective ,The substantial benefits afforded by cathepsin-L inhibition using CAA0225 during reperfusion injury in the current study might appear counterintuitive given previous studies using cathepsin-L whole-body knockout mice with persistent cathepsin-L deficiency from birth. These mice exhibit ventricular enlargement, chamber dilation and impaired cardiac contraction,Preserving cardiac contractility, limiting infarct size and preventing maladaptive remodelling are key factors to limit progression from STEMI to heart failure. The present study has identified cathepsin-L as a potential therapeutic target. Additional basic and translational studies are now required to determine whether blocking cathepsin-L release or activity could mitigate disease progression among patients with MI, thereby improving survival rates, quality of life and the health-economic burden.Cardiovascular Research online.Acquisition, analysis, or interpretation of data:W.H., C.S.M.C., K.N., K.F., K.M., A.R., D.O.T., A.Z., D.C., D.C., M.M.Y.L., M.M.E., A.D., R.G., M.M.L., H.E., P.R., S.W., S.H., A.S., L.M.A., E.B.E., J.M.C., M.C.P., K.G.O., C.H., Oxford Acute Myocardial Infarction (OxAMI) Study, K.M.C., C.B.Substantial contribution to the conception/design of the work/Drafted the work/Substantially revised it:T.M., G.L.S., S.A.N., K.G.O., K.M.C., C.B., C.M.L.Conflict of interest: none declared.Chief Scientist Office , ISSF and The Wellcome Trust (097821/Z/11/Z). W.H. was supported by the China Scholarship Council. K.M. (FS/15/54/31639) and D.C. (FS/12/28/29417) are supported by Fellowships from the British Heart Foundation (BHF). K.N. was supported by a PhD studentship from the BHF. The OxAMI study is supported by the Oxford BHF Centre of Research Excellence (RG/13/1/30181), BHF Chair Award (CH/16/1/32013), to K.M.C and by the National Institute for Health (NIHR) Oxford Biomedical Research Centre. D.T. was supported by a PhD studentship from the University of Glasgow School of Veterinary Medicine and AZ is funded by the Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Al-Baha, Saudi Arabia and Saudi Arabian Cultural Bureau, London, UK. C.M.L is supported by a BHF programme grant (RG/20/6/35095).The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.cvab204_Supplementary_DataClick here for additional data file.


To bring out the diagnostic attitude of hearing professionals in Cameroon towards congenital hearing impairment (CHI), assess availability of tests, neonatal screening, and create a national map of availability of treatment opportunities.We conducted a cross-sectional online-based survey from June to December 2021, concerning ear–nose–throat (ENT) specialists, hearing care professionals, speech therapists and ENT nurses. A Google Forms online questionnaire was used to collect data, filled by eligible professionals involved in hearing care in Cameroon.A total of 93 professionals working in 31 different health facilities participated. A cumulative percentage of 79.9% of ENTs were found in just two out of 10 regions. Specialists sought by ENTs for assessment of patients with CHI included neurologists/neuro-pediatricians (96.8%), pediatricians (47.6%), other ENTs (34.9%), and psychologists (3.2%). Investigations requested included auditory-evoked brainstem response , otoacoustic emissions recording , and tympanometry (66.7%). There were eight OAE and nine ABR machines in the country. Twenty-five (88.6%) out of 31 facilities with otolaryngologists did not carry out systematic neonatal screening. Reasons included unavailability of equipment , and administrative delays . Sixteen (51.6%) facilities had ENTs with additional training in otologic surgery and 11 (35.5%) were equipped to perform ear surgery. Three centers (9.7%) specialized in hearing aid provision and maintenance services. Three hospitals (9.7%) had performed cochlear implantation.Our results show scarcity and overt unevenness in distribution of specialists, equipment and solutions to CHI in Cameroon. A serious negative health care consequence of this shortage is the unavailability of universal newborn hearing screening and implementation programs. Congenital hearing impairment (CHI) remains a major public health problem in sub-Saharan Africa (SSA). According to the World Health Organization (WHO), an estimated 466 million people worldwide have disabling hearing impairment (HI), among whom 49 million live in SSA and 34 million are children . CHI affMuch of the impact of HI can be mitigated through early detection and interventions ; managemIn SSA, HI is usually given less priority despite its significant burden . There iThe objective of this study was to bring out the diagnostic attitude of hearing professionals in Cameroon towards CHI, assess availability and distribution of neonatal screening programs, related tests, and treatment possibilities. This would raise awareness among practitioners and policy-makers about hearing care in Cameroon, enabling possible improvements in policies and the general approach to CHI. This study would also provide data for the creation of a national map of availability of investigations and treatment opportunities, facilitating referrals among physicians.We conducted a cross-sectional online-based survey from June to December 2021, concerning ear–nose–throat (ENT) specialists (otolaryngologists), hearing care professionals , speech therapists and ENT nurses. The survey was carried out with the help of a pre-tested Google Forms®  questionnaire designed to be filled online by all consenting professionals involved in hearing care in Cameroon, irrespective of age or sex. We excluded non-permanent professionals, for instance those working in Cameroon in the context of a fellowship or a cooperation agreement because of the temporary and inconstant nature of their services. Any questionnaire with unclear or incomplete responses was equally excluded when clarification was impossible despite contacting the concerned participant. The questions were written in English and French, the two official languages, in clear and simple medical terms. Compulsory field settings were used for essential questions, while optional question types were used for less important questions or those requiring some explanations. Rapid and effective dissemination of the questionnaire was ensured with the help of various social media groups that comprised hearing professionals. Following this, members of these groups were individually reminded by phone or e-mail to maximize study completion. Contacts and response rates were obtained by consulting the latest version of the national directory of hearing specialists designed by the Cameroonian Society of Otolaryngology-Head and Neck Surgery. Disparities in information provided by participants working in the same health facility were resolved by calling the concerned workers. Answers recorded were exported in a Microsoft Excel®  format for analysis.Answers to the following items obtained from participants comprised variables; socio-demographic data , professional information , approach and attitude , availability of diagnostic and investigative equipment in health facilities according to administrative region , auditory-evoked brainstem response (ABR), behavioral audiometry, pure tone audiometry (PTA), computerized tomography (CT) scan, magnetic resonance imaging (MRI), genetic workup), availability of treatment opportunities . Data were analyzed using STATA® version 13 . Results were presented as mean for quantitative variables, and percentages for qualitative variables.The survey comprised details on the objectives of the study, benefits of the results and intended use of information collected. A checkbox was used for informed consent, to be ticked by participants prior to filling the questionnaire. Information about participants was kept confidential by replacing names with assigned codes. Ethical clearance to carry out this study was granted by the Institutional Review Board of the Faculty of Health Sciences, University of Buea, Cameroon (Ref: 2021/1398B-11/UB/SG/IRB/FHS).A total of 93 professionals of various hearing-related specialties working in 31 different health facilities across the country participated in this study. Ages varied from 28 to 69 years (mean = 42 ± 8.1 years), with male predominance (54.8%). All 10 regions of Cameroon had specialists. A cumulative percentage of 79.9% of ENTs were found in the Center and Littoral regions. We obtained responses from 63 ENTs (response rate 70.8%), two speech therapists (response rate 66.7%), seven hearing care professionals (response rate 70%) and 21 ENT nurses (response rate 65.6%). Distribution of specialists according to region and specialty based on data collected in this study is represented on Table Otolaryngologists received most of the children suspected of having hearing loss as direct consultations in 61.9% as opposed to referrals from other specialists in 38.1% of cases. Concerning referrals, a proportion of 69.8% of ENTs received patients mostly from pediatricians, 15.9% mostly from neuro-pediatricians and 14.3% from general practitioners. Other specialists sought by ENTs for an opinion and/or team management included neurologists/neuro-pediatricians (96.8%), pediatricians (47.6%), other ENTs (34.9%), psychologists 3.2%), and ophthalmologists (1.6%). Investigations requested by ENTs included ABR (87.3%), OAE (71.4%), tympanometry (66.7%), CT scan (30.1%), and MRI (3.2%). Table %, and opTwenty-five (88.6%) out of 31 facilities with otolaryngologists did not carry out systematic neonatal screening. Reasons for not implementing screening included unavailability of equipment , implementation pending and delayed due to administrative reasons , and not applicable due to nature of facility .Sixteen 51.6%) facilities had ENTs with some additional training in otology/otologic surgery and 11 (35.5%) were equipped to perform ear surgery. Six of these (54.5%) were located in the center region, four (36.4%) in the littoral and one in the east. Three centers (9.7%) specialized in hearing aid provision and maintenance services, two in the Center and one in the Littoral regions. Three hospitals performed cochlear implantation, with the possibility of providing maintenance for these implants. Figure .6% facil2, a population of approximately 27 million and an estimated overall physician–patient ratio of 1 in 50,000 [Cameroon is a tropical sub-Saharan African country with a surface area of 475,000 kmn 50,000 . It is an 50,000 . Cameroon 50,000 , with a n 50,000 , and 45%n 50,000 . The coun 50,000 , 14. Labn 50,000  and bettContact between the specialist and children with potential CHI was mostly through direct consultations, for various hearing- or speech-related complaints. This is indicative that many children are missed at birth, since these children are brought by their parents who are usually unsure about their children’s hearing status. Other personnel who predominantly intervene in the diagnosis chain included pediatricians, neuro-pediatricians and general practitioners. These categories of physicians generally refer to or seek an opinion from ENTs when faced with possible cases of CHI. In Cameroon, hearing investigations are done by or under the supervision of otolaryngologists. Subsequently, confirmation of suspicion of CHI or any form of hearing disorders invariably passes through them. Interestingly, ENTs generally requested the opinion of other specialists for diagnosis and/or team management, including neurologists/neuro-pediatricians, pediatricians, other ENTs, psychologists, and ophthalmologists. A multidisciplinary approach is indispensable for adequate assessment, including careful prenatal and neonatal history, complete head and neck examination, exhaustive general physical examination, to be complemented by paraclinical tests . This caThe uneven distribution of clinical and paraclinical diagnostic equipment was evident, with a concentration of both hearing test and diagnostic imaging equipment in the Center and Littoral regions (see Table Availability and accessibility to treatment and rehabilitation was yet again problematic, with clustering of the few facilities providing solutions around the economic and political capital cities of the country see Fig. . InteresThis study gives a picture of sites offering hearing-related services for the benefit of patients. An increase in recruitment of specialists, with specific emphasis on genetic consultants and speech therapists, enhanced provision of diagnostic and surgical equipment, and better distribution of the few resources currently available would improve accessibility to hearing care. Implementation of special measures such as incentives for specialists working in semi-urban settings could encourage professional mobility. Implementation of universal newborn screening, treatment and rehabilitation programs would be greatly beneficial.Limitations of this study include the nature of data collection, making it difficult to verify certain details. This was mitigated by individually contacting participants to clarify discordant information. In addition, though acceptable, survey response rates per professional group were all below 80%, making findings not exhaustive.Our results show scarcity and overt unevenness in distribution of specialists, equipment and solutions to CHI in Cameroon. The paucity of essential diagnostic equipment such as OAE and ABR instruments raises concerns about effective diagnosis. There is an imperative need for the implementation of comprehensive hearing management programs, including universal newborn hearing screening, treatment and rehabilitation, which can guarantee a leap in diagnosis and outcome amelioration of CHI. Specialist societies and policy-makers should advocate and institute such programs that have proven to be possible even in resource-limited settings.