Laparoscopic colorectal surgeries offer numerous advantages over their open counterparts. To compare these measurable short-time outcomes of open and laparoscopic resections in Hungary, data of colorectal surgeries were collected and analysed. The study focused on identifying patients’ characteristics that can influence the decision on laparoscopic colorectal resections and on comparing efficiency of Hungarian colorectal operations with international data.Using patients’ data of laparoscopic and open colorectal surgery performed in 2015 and 2016 from the National Health Insurance Fund of Hungary, a countrywide retrospective comparative analysis was done. Logistic regression was used to explore main influencing factors for laparoscopic colorectal surgery.A total of 17,876 colorectal surgical cases, including 14,876 open and 3,000 laparoscopic resections were selected and analysed. Laparoscopy was used only in 16.78% of all cases. Comparison of age groups showed that odds ratio (OR) of laparoscopic colorectal resections was significantly lower in over 40 years than in younger patients (18–39 years). In university institutes patients had higher odds (OR: 2.23 p<0.0001) for laparoscopic colorectal resections. Presence of comorbidity codes and preoperative treatment in internal medicine department decreased odds for laparoscopic colorectal operations.Patients’ age, comorbidities and hospital type influenced the likelihood of decision on laparoscopic colorectal resection. Selection of patients contributed to improved laparoscopic outcomes. Laparoscopic colorectal resection (LCR) techniques offer numerous advantages over their open counterparts, including shorter hospitalization, faster recovery and reduced morbidity. Short-term physical benefits include decreased pulmonary and gastrointestinal morbidity, improved pain control, less dehiscence and surgical site infections, less postoperative hernia and blood loss –7. ThereUsing the National Health Insurance Fund Administration database, we have collected and analysed data of elective colorectal surgeries between January 1, 2015 and December 31, 2016. This study involves only anonymized administrative medical data. According to the Article 18 of Act XLVII of 1997 on the processing and protection of health care data and associated personal data, there is no need for the approval of the Research Ethics Committee for the analysis of administrative data requested from the National Healthcare Service Center (Állami Egészségügyi Ellátó Központ- AEEK). According to the above statement, the request and analysis of administrative data under the contract between Semmelweis University and the National Healthcare Service Center (registration number: ÁEEK/000810-001/2017) are not subject to the need for an ethical permission request. Elements of database were collected from 70 hospitals in Hungary, where colorectal surgeries were performed. Our research units were colorectal surgeries. In order to identify the type of colon or rectal procedures performed , we usedProcedure codes were associated with patient characteristics  codes), 30-day mortality, and hospital identification code. ICD diagnoses were divided into two groups: main diagnosis (indicating the surgery) and secondary diagnoses (comorbidities and complications). The 70 inpatient hospitals involved, where colorectal surgeries were performed during the study time period were categorized by their type, and divided into three groups: university clinics and national centres, county centres and rural hospitals. Data of hospitalization (immediate before the operation) in department of internal medicine were also collected. Inclusion criteria were: age over 18 years, included open and laparoscopic colorectal surgical procedures , electivth version).To determine factors influencing surgical decision-making for open versus laparoscopic colorectal procedures, multivariable logistic regression analysis was performed. Odds ratio (OR) with a 95% confidence interval was calculated to determine the combined effect of various preoperative factors for laparoscopic colon and rectal operations. These variables included patient age and sex, comorbidities , main di2 test (χ2(1) = 2,363; p = 0,124). Patient sex and age data for the laparoscopic and open groups are given in Of the 17,876 colorectal resections that met inclusion criteria, 14,876 were open and 3,000 were laparoscopic. Overall, laparoscopy was used only in 16.78% of all colorectal cases. Mean age was 66.35 (SD: 12.68) years in the OCR group and 63.78 (SD: 12.62) years in the LCR group (p<0.0001). Distribution of sexes was statistically not significant between the two groups (p = 0.124) with Pearson ChiIn the study population, it seemed that the rate of LCR cases decreased in both sexes over the age of 70 (LCR% was between 14.9% and 9.4%). Although there was a low incidence of colorectal resection in women between the age of 18–39 and 40–49, this group had the highest proportion of LCR . Distribution of main diagnosis groups was analysed  in conteThe most common indication for surgery was malignant neoplasm of the colon (58.3%), of which 16.3% were performed laparoscopically. The condition with the highest proportion of patients receiving LCR was diverticulosis, with a rate of 23.9%. Colon and rectal malignant tumours were the most frequent reasons in men for open and laparoscopic procedures as well . We found some distributional differences of main diagnoses regarding open and laparoscopic surgeries in the two sexes. The rate of LCR in cases of female patients was higher than in male patients in all (main) diagnoses indicating the surgery, except for diverticulosis: male with 25.4% vs. female with 22.9%. . 40.2%  evaluated the use of laparoscopy for elective colorectal resections in 48 hospitals in the United States in 2010, and a rate of 41.6% was found . Askari The most important findings of our study are the relationship between age, comorbidities, main diagnoses, type of operating hospitals and laparoscopic colorectal procedures . HungariWe found that the most frequent main diagnosis codes were malignant colorectal tumours . These rWe noticed some limitation of our research at the beginning. We found a shockingly high rate of postoperative 1-30-day mortality (8.2%) in the OCR group, especially, if compared with 1.28% 30-day–mortality rate reported by Kellers nationwide study  or 2% inThe authors are confident that the present research will support the improvement of insurance data transparency and the development of patient care.


The variants of chronic inflammatory demyelinating polyneuropathy (CIDP) differ not just in their clinical, pathological and electrophysiological characteristics, but often in their indifferent response to conventional immunosuppressive agents which are effective in typical CIDP. High quality evidence is lacking as far as the management of these atypical variants is concerned. In this review, we summarize the treatment approaches to each of these CIDP variants based on existing data. Distal acquired demyelinating symmetric polyneuropathy (DADS) has the phenotype of a symmetric, demyelinating sensory, length-dependent polyneuropathy and is frequently associated with paraproteinemia and anti myelin associated glycoprotein (MAG) antibodies. While the management of idiopathic DADS (DADS-I) is the same as CIDP, DADS-M responds suboptimally and has a favorable response to rituximab. Multifocal acquired demyelinating sensory and motor neuropathy (MADSAM) manifests as a chronic progressive demyelinating mononeuropathy multiplex which can evolve to a confluent pattern indistinguishable from CIDP. Evidence favors treating MADSAM with conventional immunomodulatory therapy (IMT), but this disorder responds less favorably than CIDP. Some patients present with purely sensory symptoms, known as pure sensory CIDP or chronic inflammatory sensory polyradiculoneuropathy (CISP), the latter localizing to a pre-ganglionic pathology. Both respond well to first line IMT, particularly to intravenous immunoglobulin (IVIG), but patients relapse without maintenance therapy. Pure motor CIDP resembles multifocal motor neuropathy with conduction block (MMNCB), but the previously reported worsening status after steroid treatment was not reproduced in recent studies, and IVIG remains the first-line therapy. Some focal forms of CIDP defy exact classification, but respond well to first-line IMT including IVIG. Overall, atypical CIDP responds to treatment with first-line IMT, but has a suboptimal response compared to CIDP. There is evidence for effectiveness with agents such as rituximab, especially in DADS-M, and this medication can also be used in cases refractory to conventional IMTs. Rituximab is also effective in CIDP with IgG4 antibodies which has distinct clinical features and is mostly refractory to first-line IMT. The term “atypical CIDP” generally denotes those variants that deviate from the classical symmetrical, proximo-distal, motor, and sensory presentation of CIDP. The EFNS/PNS criteria list the following types of “atypical CIDP”: predominantly distal  or asymmetric , focal , pure motor and pure sensory   1). Stu. Stu1). Distal acquired demyelinating symmetric neuropathy (DADS) is defined by the symmetrical presentation of sensory or sensorimotor symptoms starting distally in the lower limbs without proximal limb or cranial nerve involvement and having abnormally increased distal motor latencies on nerve conduction studies (NCS) . DADS coDADS without monoclonal paraproteinemia or anti-MAG antibody, also referred to as DADS-I, is considered as a variant of CIDP. The initial report by Katz et al.  demonstrA causal association of monoclonal gammopathy with DADS is significantly stronger for IgM than for IgG or IgA monoclonal gammopathies . From thThe pathology of anti-MAG positive DADS, involves deposits of IgM and complement, with splitting of myelin lamellae resulting in demyelination and conduction block. The response to treatment in DADS with paraproteinemia (DADS-M) to the first line agents effective in CIDP is suboptimal , 8. InitMADSAM is a painless, demyelinating, mononeuropathy multiplex, and is the most frequently encountered variant of CIDP in most series. The distinct nature of MADSAM pathology is that the brunt of the macrophage-mediated demyelination is multifocal and distributed mainly in mid-limb or proximal nerve segments , 25. In The pure sensory variant of CIDP was initially recognized by Oh et al.  who firsCISP is often considered a pure sensory CIDP due to its similarities in clinical presentation, but with the distinctive feature of sensory pre-ganglionic root involvement as evidenced by normal sensory NCS, abnormal sensory evoked potentials (SEP), and thickened spinal roots on MRI. In the initial case series of 15 patients with CISP, six patients were significantly disabled and required treatment, four of whom received IVIG and two received steroids . All of Pure motor CIDP resembles multifocal motor neuropathy (MMN) in its clinical presentation but is more symmetrical and is classified as an atypical form of CIDP. Motor conduction blocks are the most common electrophysiological finding in this entity, and in many cases there is an absence of sensory nerve conduction abnormalities, again similar to MMN , 35. Fron = 84) and Japan (n = 40)  , 5. The (n = 40) . It was (n = 40) , all but(n = 40) –48. In a(n = 40) . Focal CInvestigations for possible biomarkers of CIDP have led to the identification of pathogenic autoantibodies directed against several nodal and paranodal antigens amongst the subset of patients with CIDP . IgG4 anThe data on the treatment of atypical variants of CIDP are of low-quality and limited in patient numbers. Large prospective series or clinical trials are non-existent other than possibly for the DADS phenotype. Furthermore, the diagnostic separation of these variants is not clearly demarcated and some entities such as DADS or pure motor CIDP and focal CIDP may represent overlapping phenotypes. These variants can be impossible to distinguish from other entities such as MMN or MADSAM. Other than in anti-MAG positive DADS, the existing literature indicates a favorable treatment response with conventional first-line IMT used in typical CIDP. Given reports of some patients with pure motor and pure sensory CIDP worsening with steroids, IVIG may be preferred as the first-line agent in these variants. Agents such as rituximab are currently being re-explored in anti-MAG neuropathy and in IgG4 antibody associated CIDP earlier in the treatment algorithm. A revision of the guidelines to clearly define the diagnostic criteria for these entities is required in order to undertake prospective clinical trials and improve our understanding of atypical CIDP.DM was involved in the review of literature, drafting the work, and revising it critically for important intellectual content. HK was involved in drafting the work and revising it critically for important intellectual content. VB was involved in designing, review of literature, revising it critically for important intellectual content, and final approval of the version. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Disability is an important and often overlooked component of diversity. Individuals with disabilities bring a rare perspective to science, technology, engineering, mathematics, and medicine (STEMM) because of their unique experiences approaching complex issues related to health and disability, navigating the healthcare system, creatively solving problems unfamiliar to many individuals without disabilities, managing time and resources that are limited by physical or mental constraints, and advocating for themselves and others in the disabled community. Yet, individuals with disabilities are underrepresented in STEMM. Professional organizations can address this underrepresentation by recruiting individuals with disabilities for leadership opportunities, easing financial burdens, providing equal access, fostering peer-mentor groups, and establishing a culture of equity and inclusion spanning all facets of diversity. We are a group of deaf and hard-of-hearing (D/HH) engineers, scientists, and clinicians, most of whom are active in clinical practice and/or auditory research. We have worked within our professional societies to improve access and inclusion for D/HH individuals and others with disabilities. We describe how different models of disability inform our understanding of disability as a form of diversity. We address heterogeneity within disabled communities, including intersectionality between disability and other forms of diversity. We highlight how the Association for Research in Otolaryngology has supported our efforts to reduce ableism and promote access and inclusion for D/HH individuals. We also discuss future directions and challenges. The tools and approaches discussed here can be applied by other professional organizations to include individuals with all forms of diversity in STEMM. A diverse scientific workforce leads to increased creativity and productivity by introducing a wider range of perspectives, experiences, and skill sets . DiversiIndividuals with disabilities offer a unique perspective drawn from their experiences of living with a disability that can guide novel and innovative scientific progress or improve medical care for people with that particular disability. They frequently have extensive experience navigating healthcare settings as patients, utilizing creativity to overcome accessibility challenges, managing limited time and resources, and advocating for themselves and others with disabilities. This experience translates to valuable attributes such as better communication skills, persistence, empathy, planning, creativity, and adaptability . IndividAs defined by the We are a group of more than 110 deaf and hard-of-hearing (D/HH) engineers, scientists, and clinicians, most of whom are active in clinical practice and/or auditory research . Our netOur peer mentorship network for D/HH individuals endorses a biopsychosocial view of hearing loss (defined below) that incorporates medical and social models of disability. While these disability models are not mutually exclusive, each model has distinct approaches to disability.Medical models of disability address the physical differences associated with specific diagnoses and focus on preventing, curing, remediating, or accommodating the physical underpinnings of a given disability  who view hearing loss as a key part of their personal identity and often use sign language as a primary form of communication . More broadly, disability culture includes individuals with any and all disabilities and encompasses values, arts, and political stances in addition to experiences of shared discrimination and prejudice see .Diversity models of disability contributed to the rise of the field of critical disability studies, which focuses on how different diverse identities can intersect with disabilities and draws parallels between the disability rights movements and other civil rights movements . This fiProponents of diversity models and the field of disability studies often advocate for the use “identity-first language”  which enables disabled individuals to express belonging and pride as members of a disabled community and to define what it means to be disabled instead of letting it be defined by negative stereotypes . The AmeBiopsychosocial models integrate aspects of individual, social, and diversity models of disability. The biopsychosocial model has the potential to be transformative because it acknowledges that there are limitations to our ability to address hearing loss and other disabilities through medicine. By combining medical, societal , and diversity considerations we can maximize opportunities for the success of disabled individuals. For example, combining cochlear implants  with accommodations such as closed captioning and training of colleagues/teachers on how to facilitate communication  as well as introducing the disabled individual to other disabled peers (diversity) has a higher likelihood of success than any of these strategies in isolation.Hearing loss ranges from mild to profound. For example, an individual with mild hearing loss may experience difficulty understanding speech in noisy environments, whereas an individual with profound hearing loss may have little to no functional hearing. Communication modalities found within the hearing loss community can include audio-visual, where speechreading is combined with residual hearing, and visualonly, such as sign language and Cued Speech. The diversity of hearing loss etiologies and communication modes means the hearing loss community is heterogeneous and accessibility strategies for a particular individual  may not work for another individual.Differences in the wording of questions across surveys makes it difficult to estimate the true prevalence of hearing loss . NeverthThere are strong parallels in the history and consequences of discrimination between individuals with hearing loss and other marginalized communities. Strategies developed to recruit and support members of the D/HH community in STEMM fields can serve as a template for efforts to recruit and support members of other historically disadvantaged or otherwise marginalized communities. The approach presented below is targeted towards D/HH individuals but can easily be adapted for the specific needs of other marginalized groups.The Association for Research in Otolaryngology (ARO) is a scientific organization focused on hearing and balance research that was founded in 1973. In 1992, D/HH attendees at the ARO Midwinter Meeting formed an informal networking group dedicated to increasing the representation of D/HH individuals in hearing research. This group has grown to more than 110 members globally and has encouraged ARO to develop five pillars that support diversity and inclusion among their members see . These pwww.deafearscientists.org). Most of the mentorship and interactions occur via email, which is ideal for a global group of individuals who often find written communication to be preferable to speaking and listening in a large group.Informal interactions between marginalized peers have been shown to promote academic and social growth in higher education settings . DiversiHI-ARO includes researchers at all career stages and STEMM disciplines, from trainees to senior scientists, medical students to clinicians, as well as audiologists and other leaders in healthcare and industry. HI-ARO members are highly diverse, spanning race, gender, nationality, socioeconomic status, religion, and disability . The registration form ARO uses has a specific field where registrants can describe the accommodations they need. ARO’s management company will then reach out to the individual to set up the necessary arrangements.Of particular relevance to many professional societies that host meetings within the United States is the Americans with Disabilities Act . The ADAWhile the ADA requires equal access for these meetings, there is a difference between the professional society complying with the letter of the law, which places tremendous burden on the disabled individual to advocate for themselves and seek services and being proactive about reaching out to disabled attendees and securing accommodations for them. By being proactive about accommodations, professional societies can nurture and support diversity within their communities while avoiding potential conflicts between disabled individuals and conference management.The optimal approach may vary from organization to organization. For example, ARO is aware that they have approximately 20 D/HH members from our group attending the annual meeting, along with an unknown number of attendees who may not wish to disclose their hearing difficulties. Thus, the proactive approach for ARO is to automatically arrange for captioning of podium talks, ensure assistive listening devices are available and set up the conference facilities with the goal of improving acoustics. During the registration process, ARO captures information about additional access services that may be required. For other organizations who may feel they do not have a critical mass of D/HH attendees, they can still be proactive by including questions in the registration form to identify anyone who might need special support services. Inclusive practices such as captions and designing the conference with acoustics in mind benefit some members with age-related hearing loss, auditory processing difficulties, or other listening challenges  but who do not self-identify as D/HH. Finally, being proactive can greatly increase the likelihood of increasing diversity in the organization by encouraging future participation of marginalized individuals.Increasing access to opportunities for diverse trainees with additional avenues for financial support is essential for cultivating personal and professional career success . Many scThe organizational committees of scientific and professional organizations provide an opportunity for trainees, faculty, and other members to take leadership roles within the organization. At ARO, D/HH trainees and faculty have been recruited to multiple committees, including Council, student-postdoc leadership committees, and those that target diversity and accessibility initiatives. For scientific organizations to practice true inclusion and equity, diverse members need to be in organizational and leadership positions. Diverse perspectives at the senior level can yield innovative approaches to drive the organization forward and encourage constructive changes that advance equitable principles. These experiences allow for D/HH individuals to gain the confidence to seek similar leadership roles at their home institutions.The five pillars described above have focused on how scientific organizations can support their diverse members. However, support should also come from other members. Here, organizations have an opportunity to provide tools for all members to learn from diverse perspectives so that they can reflect, grow, and start to advance and advocate for inclusion and equity in their professional and personal lives. ARO has hosted several events aimed at increasing awareness of diverse perspectives, addressing personal biases, and providing tools that help mitigate negative situations for marginalized attendees. These include events where notable individuals with hearing loss share their experiences, roundtable discussions for Women and Allies, networking events, and hiring outside facilitators to provide bystander training for all ARO members. From the point of view of scientific organizations, it can be difficult to assess whether progress has been made in establishing a culture of inclusion and equity. One way organizations can track progress is through anonymous surveys, where members can report whether they feel that the organization has or has not been successful in fostering inclusivity, identify areas of improvement, or voice concern if exclusionary conduct has occurred. This gives the organization an opportunity to improve in areas that are lacking, intervene if there is a problematic situation/person, and track progress of efforts over time. In addition, organizations can track the number of diverse individuals on society committees and leadership roles to ensure that representation is maintained and/or exceeded over time. It should be noted that in other fields, such as deaf education, certain conferences place great emphasis on accessibility. For example, D/HH individuals are included in the meeting planning and keynote presenters are chosen who are themselves D/HH. ARO is working to emulate lessons learned at these conferences by setting up an accessibility committee consisting of D/HH and other disabled individuals. In the past year, greater emphasis has been placed on inviting individuals with hearing loss, such as a deaf comedienne, to present at AROorganized events.Over the last several years, an increasing number of D/HH clinicians and scientists have been presenting at auditory research conferences via posters and podium talks highlighting the need for expanded accessibility. Even within the field of hearing science and related clinical fields, some organizations still resist the need to accommodate a growing number of attendees with hearing loss. Nevertheless, the last decade has seen captioning at the biannual Conference for Implantable Auditory Prostheses (CIAP), the yearly American Cochlear Implant Alliance (ACIA) conference, the American Auditory Society annual conference, and the CI CRASH Midwest MiniConference on Cochlear Implants (hosted by University of Wisconsin-Madison). After three D/HH presenters had trouble understanding and answering questions at a CIAP meeting in the mid-2000s, one attendee declared that CIAP and related conferences should provide captioning. Since then, CIAP has received NIH funding for captioning which in 2019 benefited a record number of 13 D/HH attendees. By proactively seeking funding for captioning and providing access to all attendees, including those who are D/HH, these organizations stand out as truly being inclusive.Our experience illustrates the proclamation from the Disability Rights Movement, “Nothing about us without us.” D/HH scientists and clinicians should strive to be essential stakeholders in organizations that focus on hearing research and audiology so that the perspectives and experiences of D/HH individuals cannot be ignored. Moreover, equitable access in educational and workplace settings, and at conferences, will help establish productive research trajectories and increased diversity in these settings.For nearly 30 years, HI-ARO has worked with our professional organizations, especially ARO, to implement successful and practical strategies to increase inclusion of its D/HH members. With the guidance of our peer-network group, ARO leadership has continuously improved accommodations and provided essential support for academic, research, and career development success for D/HH members. ARO’s support for equity and diversity has served and may continue to serve as a model for other conferences and professional organizations.This article has described a highly successful model for promoting representation of D/HH individuals in hearing research. We have found that D/HH individuals have used their early career in hearing research, combined with mentorship from our group, as a stepping stone to other fields of study and medical specialties. This same support needs to be extended to inclusion of other disabilities, such as visual impairment, in their chosen field, and to the inclusion of D/HH individuals in STEMM fields beyond hearing research. Funding opportunities from the National Science Foundation  and the National Institutes of Health  gives professional societies financial support to implement the five pillars, such as with conference costs  and mentoring activities.The fact that the strong D/HH network arose in the auditory field is not surprising. After all, auditory researchers and clinicians understand communication challenges and many of us have been fortunate to have been mentored by normal hearing individuals who advocated for access within ARO. However, many other scientific societies have unfortunately not yet followed the lead of ARO. To fully realize equal participation of D/HH individuals in STEMM will require administrative champions in the leadership of other societies given the cost of assistive technology. The hearing research field is relatively small, and STEMM will benefit when D/HH individuals have full access to career opportunities in the field of their choice-not limited by the accessibility of the field or others’ perceptions of what would be a “natural” choice based on their disability. Thus, the scientific community needs to come to this realization and do more for D/HH individuals across disciplines.Professional organizations, including those that are not STEMMfocused, can include individuals with disabilities by inviting them to provide input on policies such as disability accommodations at scientific meetings. The COVID-19 pandemic provided an opportunity to reshape accommodations for those with disability when in-person conferences were shifted to virtual. For a virtual poster, a presentation is often recorded, without background noise, and can easily be captioned. Digital platforms can facilitate asynchronous text-based research interactions. This mode can be easier for many D/HH individuals and other individuals with disabilities than in-person sessions and conversations in noisy conference halls. Hybrid conferences adopting universal design approaches combined with remote manual correction and formatting of automatic captioning might proactively reduce isolation while still affording these benefits.Another opportunity for professional organizations to foster diversity is to create mentoring programs for diverse middle school and high school students, including those with disabilities. Many students from historically excluded groups have the potential to succeed in STEMM fields but do not always receive the guidance and support needed to pursue those paths. Having a mentor who has faced similar challenges can bring confidence in learning and self-advocating and provide encouragement and inspiration. Existing models such as AG Bell’s Leadership Opportunities for Teens (LOFT), Oregon Health Science University’s On Track OHSU! and others  provide Our peer-mentorship network of D/HH engineers, scientists, and clinicians is foundational to our success in STEMM. Trainees have successfully transitioned to being leaders in academia, healthcare, and industry, often receiving significant research funding. Through this network we discuss the challenges we face and strategies to overcome difficulties. We also form a cohesive group to advocate for changes in our fields and at professional conferences to address the underrepresentation of individuals with hearing loss in STEMM. We believe that the adoption of this peer-mentorship network model by other underrepresented minority groups, and collaboration among and between peer-mentorship networks and professional societies, will drive changes that will promote diversity and equity in STEMM and other fields.


In contrast, in users with average bone-conduction (BC) threshold of 25 dB or more, speech understanding in noise was improved by +0.8 dB to +1.1 dB (p < 0.03) when using the higher MPO level. In this user group, there may be an additional, but very small benefit of +0.1 dB to +0.4 dB when using the lower rather than the higher CT value, but the difference was not statistically significant (p > 0.27). In users with better average BC thresholds than 25 dB, none of the improvement was statistically significant. Higher MPOs and possibly, to a lesser degree, lower CTs seem to be able to improve speech understanding in noise in users with higher BC thresholds, but even their combined effect seems to be limited.Bone-anchored hearing systems (BAHS) transmit sound via osseointegrated implants behind the ear. They are used to treat patients with conductive or mixed hearing loss, but speech understanding may be limited especially in users with substantial additional cochlear hearing losses. In recent years, BAHS with higher maximum power output (MPO) and more advanced digital processing including loudness compression have become available. These features may be useful to increase speech understanding in users with mixed hearing loss. We have tested the effect of 4 combinations of two different MPO levels (highest level available and level reduced by 12 dB) and two different compression thresholds (CT) levels (50 dB and 65 dB sound pressure level) in 12 adult BAHS users on speech understanding in quiet and in noise. We have found that speech understanding  For over 4 decades, bone-anchored hearing systems (BAHS) have been used successfully to treat conductive and mixed hearing loss, especially in cases where conventional hearing aids cannot be used or where they are not effective , 2. BAHSFor the first decades of their existence, the technology of BAHS did not allow for sophisticated fine tuning. Modern BAHS systems use digital signal processing and a considerable number of parameters, such as MPO and gain settings in different frequency bands have become accessible to the audiologist and have opened new possibilities for fine tuning. It was shown that meeting adequate prescriptive targets can improve speech understanding , 4. On tOne important limit to a wider application of BAHS is low speech understanding in users with a mixed hearing loss, especially if the sensorineural component is substantial, i.e., above approximately 25-40 dB HL. As the maximum power output (MPO) levels of BAHS are inherently and substantially lower than those of conventional hearing aids, the dynamic range, i.e., the difference between the bone conduction threshold of the user and the MPO of the BAHS can become very narrow and may limit speech understanding considerably. Fortunately, BAHS with higher MPOs  have started to provide better preconditions for these difficult fittings. Nevertheless, the increase in speech understanding reached in this way is still limited .Dynamic range compression has been shown repeatedly to improve speech understanding in users of air conduction hearing aids –9. HowevThe rationale behind our study was that optimizing loudness compression parameters of BAHS might improve speech understanding in everyday life. Specifically, we wanted to test the hypothesis that choosing lower compression thresholds  might improve speech understanding of BAHS users. We are not aware of any other studies looking into this effect, and we aimed to start to close this gap.Because compression sets in already at lower input levels with lower CT-levels, they help to expand the dynamic range of the incoming acoustic signal, which is ultimately available at the inner ears of the users. We hypothesized that this might lead to better speech understanding, as dynamic ranges are generally narrow in fittings with BAHS, when compared to air conduction hearing aids , 13 and The study was approved by the local ethical committee of Bern (KEK-BE 2018-01521) and carried out in accordance with the Declaration of Helsinki.4) were 29 to 110 (mean 78) dB HL for the side tested with the BAHS and 28 to 103 (mean 59) dB HL for the contralateral side. BC thresholds were 5 to 71 (mean 36) dB HL on the BAHS side and 5 to 50 (mean 29) dB HL for the contralateral side. For data analysis and visual representation in the figures, we use the BC thresholds of the ear with the better BC threshold, as this threshold correlates better with the aided outcome with BAHS than that of the ipsilateral ear [Twelve regular BAHS users participated in the study after giving their written informed consent. Four of the volunteers were female, and 8 were male. Their ages ranged from 36 to 79 years (mean 63 years). All were German speaking, and all had a bilateral conductive hearing loss with an additional sensorineural hearing loss. Their average AC pure tone thresholds in the frequency range 500-4000 Hz . The processor was fitted individually for each participant using BC-direct threshold measurements. Automatic sound classification, position compensation, microphone directionality, and noise reduction were deactivated.Four different combinations of settings of the parameters were programmed for each volunteer. MPO was either maintained at the highest level possible by the hardware of the processor used (“High MPO”) or reduced by 12 dB over the entire frequency range (“Low MPO”). These MPO settings are shown in After an initial pure tone audiometry (AC and BC thresholds), all measurements were performed with a single audio processor mounted on the abutment of each subject. In those 3 participants, who had bilateral implants, the side with the better BC thresholds was chosen for all tests. During the measurements, the ear contralateral to the BAHS was plugged  in all subjects with the exception of 3 participants, who had a complete atresia of the external auditory canal.3) with an average reverberation time of 0.14 s. Four JBL Professional ControlVR 1 PRO loudspeakers  were placed to the left, right, front, and rear of the listener at a distance of 1 m. This setting was identical to that used in a previous study [All measurements took place in a soundproof chamber  were defined as fixed effects. For the post hoc analyses in p = 0.05, power 80%).Sample size was calculated for speech understanding in noise. As the standard deviation is below 1 dB , a smally-axis is limited to 0-100%), fitted Sigmoid curves are shown. For speech reception thresholds in quiet and in noise, where there is no such theoretical upper limit for the values on the x-axis, second-order polynomial functions were used.In 4 BC threshold  between 0 and 25 dB (N = 6) and group B between 25 and 50 dB (N = 6).For a part of the analysis, the study population was divided into 2 subgroups. Subgroup A included all participants with a mean PTAp < 0.001). However, there are no significant differences between the SRTs when using the higher or lower MPO or CT level  and all fitted curves run close together.p < 0.001). Again, different MPO and CT levels have no significant  impact on speech understanding and the 4 fitted curves lie close together.p < 0.001). In contrast to the measurements in quiet, the fitted curves for the 4 different test conditions are now somewhat further apart and statistically significant differences emerge, as shown in An increase of the MPO level by 12 dB improves speech understanding in noise in group B significantly by +0.8 at CT = 65 dB to +1.1 dB at CT = 50 dB. No other change in the fitting parameters under investigation causes an improvement that reaches statistical significance. The impact of a higher MPO is smaller for group A than for group B. While there is no statistically significant effect of lowering the compression threshold from 65 dB to 50 dB, it does improve SRTs in noise in both groups and for both MPO levels by +0.1 to +0.7 dB on average. As a result, the combination of the higher MPO level and the lower CT level give the best average SRTs in both groups.Our results show that choosing a higher MPO in BAHS users with a mixed hearing loss and a sensorineural component of more than approximately 25 dB HL can improve speech understanding in noise significantly, but modestly by approximately 1 dB in signal-to-noise ratio (SNR). The benefit for users with a less pronounced sensorineural component is smaller and does not reach statistical significance in our study group. These results are comparable to those of an earlier study using BAHS and different MPOs . They arAs to compression thresholds (CT), our rationale was that a lower CT might lead to better speech understanding as a widened range of the acoustic input levels could be mapped on the dynamic range of the BAHS users. This range may be rather narrow, being limited by their BC thresholds at one end and the MPO of their BAHS at the other end. Indeed, as For speech understanding in quiet, no effect of the choice of different MPO or CT levels was found. One reason for this finding may be that speech in quiet is generally easier to understand and distortions caused, e.g., by too narrow dynamic ranges are less detrimental, when only a single target signal is present, and not a mix of speech and noise. Finding no significant effect on speech understanding in quiet is rather typical for several types of studies involving BAHS and individual fitting parameters, such as MPO or compression ratio , 6 or evIn contrast, considerable improvements in speech understanding with BAHS by improving their fitting have been found by Hodgetts et al. . In theiHigher MPO levels lead to increased speech understanding in noise in BAHS users with BC thresholds above approximately 25 dB. The improvement is in the order of magnitude of 1 dB in SNR for an increase of 12 dB in MPO. No significant effect on speech understanding in quiet or in users with better BC-thresholds was found. Lowering compression thresholds from 65 dB to 50 dB may increase speech understanding in noise additionally by small amounts of +0.1 to +0.7 dB in SNR, but statistical significance was not reached in any condition in our study. For BAHS fitting, it may be beneficial to choose devices with high MPOs when treating patients with poor BC thresholds (approximately 25 dB or more) and using lower rather than higher compression threshold may give a small additional advantage.


Bacillus licheniformis can cause foodborne intoxication due to the production of the surfactant lichenysin. The aim of this study was to measure the production of lichenysin by food isolates of B. licheniformis in LB medium and skimmed milk and its cytotoxicity for intestinal cells. Out of 11 B. licheniformis isolates tested, most showed robust growth in high salt (1M NaCl), 4% ethanol, at 37 or 55°C, and aerobic and anaerobic conditions. All strains produced lichenysin (in varying amounts), but not all strains were hemolytic. Production of this stable compound by selected strains  was subsequently determined using LB medium and milk, at 37 and 55°C. Lichenysin production in LB broth and milk was not detected at cell densities < 5 log10 CFU/ml. The highest concentrations were found in the stationary phase of growth. Total production of lichenysin was 4–20 times lower in milk than in LB broth (maximum 36 μg/ml), and ∼10 times lower in the biomass obtained from milk agar than LB agar. Under all conditions tested, strain B4094 consistently yielded the highest amounts. Besides strain variation and medium composition, temperature also had an effect on lichenysin production, with twofold lower amounts of lichenysin produced at 55°C than at 37°C. All three strains produced lichenysin A with varying acyl chain lengths (C11–C18). The relative abundance of the C14 variant was highest in milk and the C15 variant highest in LB. The concentration of lichenysin needed to reduce cell viability by 50% (IC50) was 16.6 μg/ml for Caco-2 human intestinal epithelial cells and 16.8 μg/ml for pig ileum organoids. Taken together, the presence of low levels (<5 log10 CFU/ml) of B. licheniformis in foods is unlikely to pose a foodborne hazard related to lichenysin production. However, depending on the strain present, the composition, and storage condition of the food, a risk of foodborne intoxication may arise if growth to high levels is supported and such product is ingested. Bacillus licheniformis is a facultative anaerobic endospore-producing bacterium that is ubiquitously found in the environment, plant material, and soil. The organism belongs to the Bacillus subtilis group and is known for its use in the production of enzymes or antibiotics , while spore preparations of selected strains are used as crop bioprotectants and feed additives , but sporadic cases of human infection related to B. licheniformis have been reported  followed by mild gastrointestinal symptoms  lasting for 6–24 h  of ∼0.9 and a broad temperature range (∼10°C – 58°C) for growth  activity level  medium and skimmed milk at different temperatures. Additionally, we assessed the cytotoxicity of lichenysin with surfactin as a reference.In this study, we determined the types and amounts of lichenysin produced by selected B. licheniformis from the NIZO culture collection were used: B4089, B4090, B4091, B4092, B4094, B4121, B4123, B4124, B4125, and B4164. Details related to their isolation sources and whole-genome accession numbers were reported  using the Benchling biology software, available at https://benchling.com. B. licheniformis DSM13T type strain was obtained from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (DSMZ). Bacteria were routinely cultured using LB medium (The following foodborne isolates of reported . GenomesB medium  (Tritium600) of the overnight (ON) cultures in LB were measured and adjusted to OD600 2.0. Ten μl of each culture was spotted on Columbia agar base supplemented with 5% sheep blood (Tritium Microbiologie BV) and incubated at 37°C. The hemolytic activity of each strain was inspected after 24 and 48 hours (h) of incubation. Strains that caused lysis of the red blood cells exhibited a transparent clearing zone around the colonies, indicating the presence of the biosurfactant lichenysin.To screen strains for hemolytic activity, the Columbia blood agar method  was usedB. licheniformis strains to produce biosurfactants, the oil displacement method as described by B. licheniformis was dropped on the oil layer, and the diameter of displaced oil was measured.To assess the ability of B. licheniformis in LB was adjusted to OD600 0.05 in fresh LB medium and grown to OD600 ∼0.5 at 37°C. Then 2 μl was added to either 198 μl of LB medium, or LB medium containing 1 M NaCl, or LB containing 4% ethanol (v/v) in 96 well plates . Two different plates were incubated at 37 or 55°C in the Epoch2 microplate reader  in dual orbital shaking mode, and growth was followed for ∼20 h by measuring the OD600.An ON culture of 2, 5% CO2, 90 % N2. All strains were first cultured to an OD600 ∼ 0.5 aerobically, then 2 μl of cultures were added to 198 μl of anaerobic LB medium containing 0.2% of potassium nitrate (KNO3). Growth was followed by monitoring the OD600 for ∼20 h using the Epoch2 microplate reader (BioTek) inside the anaerobic chamber. Two μl of the blue fluorogenic dye Resazurin (1 g/l in distilled water)  was added to an additional well that contained (198 μl) cell culture and was used as a redox indicator. The color change from blue to pink and then to colorless indicated the absence of oxygen.For anaerobic growth, all lab materials and cultivation media were transferred into an anaerobic chamber (model #830 -ABE/OTA) , 24 h before the experiment. The gas mixture used was 5% HB. licheniformis food isolates B4123, B4094, and the type strain DSM13T were cultured either in LB or skimmed milk (Tritium Microbiologie BV) ON at 37°C, 220 rpm. The OD600 of ON cultures was measured and adjusted to OD600 0.05, diluted sixfold, resulting in around 102 colony-forming units (CFU) ml–1 in 100 ml of fresh prewarmed medium. Samples were collected at time point 0 (T0) and subsequently, cultures were incubated at 37°C, with continuous shaking (220 rpm) for 72 h. During the growth of these cultures, samples were collected to determine the CFUs in time and to determine the concentrations of lichenysin in the culture.Two selected In LB, samples were collected every 2 h for the first 24 h and for the first 28 h in skimmed milk to determine the CFUs in time. Lichenysin produced in the culture was determined at every 4 h in LB and skimmed milk, with additional selected time points in between the 4 h interval after cells reached the early stationary phase. Aliquots of 5 ml cell cultures were collected and frozen immediately at –20°C until used for lichenysin extraction.At each mentioned time point, 1 ml of the culture was 10-fold serially diluted using sterile phosphate-buffered saline (PBS), and appropriate dilutions were plated out either on LB agar or Plate count skimmed milk agar (PCMA) (Tritium Microbiologie BV) followed by incubation at 37°C ON and counting of the colonies. The CFU/ml at each time point for each strain was determined using the formula (Number of colonies × dilution factor) / volume spread on the plate. In addition, lichenysin concentrations in the cultures at each time point were analyzed and quantified (see sections “Lichenysin Extraction”).600 of ON cultures in skimmed milk, the milk was cleared by mixing 1 ml of ON culture with 9 ml of citrate milk clearing solution (2%) (Tritium Microbiologie BV). For growth in skimmed milk, two additional samples were collected at time points 26 and 28 h as all three strains grew slower in skimmed milk than in LB and cultures reached the stationary phase later than in LB. Incubation was performed at 37°C.To measure the OD10 CFU/ml for the strain B4094, B4123, and DSM13, respectively, in LB and skimmed milk from two independent experiments were calculated. Their respective specific growth rate (mu/μ) and lag time (lambda/λ) for each strain/medium were estimated using the Baranyi primary growth model  to an OD600 of 0.05, and cultures were grown to an OD600 of 0.5 by incubation at 37°C with agitation (200 rpm). Aliquots of 500 μl of the cultures that were grown in LB or skimmed milk were then spread evenly on the LB and skimmed milk agar plates containing 2% agar, respectively, and allowed to dry under the laminar flow hood. All plates were sealed with parafilm and incubated at 37°C for 10 days, after which biomass was collected from the agar surface using a sterile scraper. The collected biomass (150 mg) was weighed inside an empty soda-lime glass tube , and stored at –20°C until used for lichenysin extraction.To compare lichenysin produced in diverse environments (liquid broth versus solid surface), B4094, B4123, and DSM13g at room temperature. The liquid phase was transferred to a new glass tube. The cell pellet was subsequently resuspended in 2 ml of methanol, and the extraction process was repeated. Per sample, the liquid phases collected from the first and second extraction procedures were mixed and evaporated under N2 flow at 40°C in a heating block. The dry residue was resuspended in 500 μl of methanol and lichenysin was quantified in these samples.Lichenysin was extracted as described by Extraction of lichenysin from biomass was performed as described above for the liquid culture extractions. Three ml of pure methanol was added to 150 mg of cell biomass weighted in a soda-lime glass tube (DWK Life Sciences) and vortexed for 5 min until a homogenous mixture was obtained. The second extraction was performed using 1 ml of methanol. After evaporation, the dry lichenysin extract was resuspended in 150 μl of methanol before quantification.Qualitative and quantitative analyses of lichenysin A variants as extracted from either liquid cultures or biomass were performed using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) with high-resolution accurate mass spectroscopic (MS) detection. All chemicals and reagents used were HPLC grade (Merck). Surfactin  (Merck) and lichenysin ≥ 90.0% purity  were used as standards in the quantification.The instrumentation used for RP-HPLC was the Agilent 1260 SL system  consisting of a degasser, binary pump, thermostatted autosampler, and column compartment. Aliquots of 5 μl of lichenysin samples were injected onto a Kinetex C8 Minibore column  , and separation was performed at 50°C. The temperature of the autosampler was 10°C. Lichenysin A was eluted at a flow rate of 0.2 ml/min with a linear gradient of 0.10% formic acid in 40% water + 55 % acetonitrile + 5% tetrahydrofuran (mobile phase A) to 0.1% formic acid in 75% acetonitrile + 25% tetrahydrofuran (mobile phase B) in 20 min.–1, nebulizer pressure 25 psi, capillary voltage 3500 V, and collision energies 25 psi.The mass spectrophotometer (Agilent Technologies) comprised a high-resolution accurate mass quadrupole time of flight G6530B, equipped with a dual Electrospray Ionization (ESI) source, operated in 2 GHz, in extended dynamic range mode. The ionization was performed in positive mode with reference mass correction. The fragmentor voltage was 150 V, gas temperature 350°C, drying gas flow 10 l minA calibration curve of lichenysin was made for external calibration based on lichenysin standards in acetonitrile  from a stock concentration of 5 mg/ml (Lipofabrik).x+ from the range of 100 to 2500 m/z. The most abundant ion representing lichenysin A in the standard was [M + H]+ 1021.6908 (m/z), similar to the description by The LCMS was operated in a full-scan mode to scan the parent ions (M + xH)via Reversed-Phase High-Performance Liquid Chromatography – Electro Spray Ionization Mass Spectrometry (RP-HPLC-ESI-MS). The remaining concentrated extract was entirely evaporated under a flow of nitrogen at 40°C in a heating block. The dry residue was resuspended in either 99% Dimethyl Sulfoxide (DMSO) or Dulbecco’s Modified Eagle Medium  without phenol red serum or antibiotics. Surfactin (purchased from Merck) was also used in cytotoxicity assays by dissolving 10 mg in 1 ml of distilled water (10 mM) (with an adjusted pH of ∼11.7 to obtain full solubility). Both lichenysin and surfactin were diluted to the desired concentration in DMEM medium without phenol red, serum and antibiotics before adding to the cells.To perform cytotoxicity assay, two batches of concentrated lichenysin were prepared from biomass grown on LB agar plates as described in section “Extraction of Lichenysin From Biomass Collected From Surface Growth on LB Agar and Skimmed Milk Agar.” Instead of using 150 mg biomass, lichenysin was extracted from 6.5 g of biomass from strain B4123, and dissolved in a final volume of 300 μl methanol. A small aliquot of 1 μl was taken to determine the concentration of the concentrated lichenysin extract 5 cells/ml (in 200 μl) in 96 wells-plates (Greiner). The 96 wells-plates were then incubated for 24 h at 37°C, under a gas atmosphere of 5% CO2 for cell adherence, and washed with PBS to remove unbound cells. The attached cells were exposed to different concentrations (0–200 μM) of surfactin [diluted with distilled water (pH 11.7) from a 10 mM stock] or lichenysin  for a period of 72 h.Human Embryonic Kidney (HEK293) cells were obtained from Invivogen , and Caco-2 human intestinal epithelial cells (ATCC HTB-37) were purchased from the American Type Culture Collection . Both cell lines were maintained in DMEM medium containing 10 % Fetal Bovine Serum (FBS) (Thermo Fischer Scientific) and 1 % penicillin/streptomycin (Merck) to attain a confluent growth. The grown HEK293 and Caco-2 cells were seeded and suspended at a final concentration of 2 × 10Pig ileum organoids were generated by the method described by The alamarBlue (Thermo Fischer Scientific) fluorometric assay was used to examine the cytotoxicity of lichenysin and surfactin toward two human cell lineages  and ileum organoids from pigs. Culturing medium DMEM with Alamar blue  and cells exposed to 10% DMSO were used to correct for background fluorescence and cell death, respectively. To assess cell viability or death upon exposure to surfactin or lichenysin, cells without treatment were used as a negative control. In the cytotoxicity assessment of surfactin and lichenysin toward pig ileum cells, cells treated with 1% DMSO instead of cells without treatment were used as a negative control because lichenysin was dissolved in DMSO instead of the DMEM medium.The alamarBlue assay was performed as described in The cell viability (%) was calculated by using the formula:s, the recorded fluorescence for cells in each well after the exposure to surfactin or lichenysin;Favg no cells control, the average fluorescence recorded for the no-cells control;Favg untreated cells, the average fluorescence recorded for the untreated cells;Favg 1% DMSO, the average fluorescence recorded for the cells treated with 1% DMSO.FB. licheniformis  and type strain DSM13T under various conditions are presented in via RP-HPLC-MS  of 11 isolates failed to grow, and B4094 showed the most growth.All 11 isolates showed comparable growth at 37°C in LB broth, except strain B4089 that grew more slowly. All isolates grew at 55°C in LB broth, a temperature close to their reported maximum growth limit of 58°C . Strainsensities . Growth T strain did not show hemolytic activity, while obvious clearing zones were formed on blood agar plates by the other eight strains. Strains B4089, B4092, B4094, B4124 showed weak hemolytic activity, strains B4125 and B4121 showed medium and strong hemolytic activity, respectively, and strain B4123 showed the strongest hemolytic activity  was found in 10 of 11 strains, based on analysis of the published genomes (lchAC (licC) and lchTE (licTE) but not lchAA (licA) and lchAB (licB). The presence of both genes was detected in this strain based on PCR analysis   . Based oT which was used as a reference strain.The growth and lichenysin production under different conditions was further assessed for three selected strains, namely: B4094 which showed robust growth in the presence of ethanol and NaCl, growth at 60°C, and produced heat-resistant spores ; B4123 wT was not hemolytic. DSM13T did not displace oil from water, but both B4094 and B4123 showed clear displacement activity (T to produce lichenysin (see previous section), these results indicate that B4094 and B4123 produce higher amounts of lichenysin than DSM13T, resulting in the observed hemolytic activity of erythrocytes and oil displacement.A direct comparison of the hemolytic activity of the strains and the growth under different conditions is presented in activity . In viewT , lag phase (λ), and the doubling time (Td) were calculated for each strain under different conditions  and the results are shown in 10 CFU/ml, each of the strains reached similar cell densities in a stationary phase of around 9 log10 CFU/ml within 12–14 h in LB   .–1) than in LB and entered the stationary phase approximately 10 h later. Final cell densities of around 9 log10 CFU/mL were reached after 22–24 h incubation  in LB at 20 h  and 5.4 μg/ml at a cell density of 8 log10 CFU/ml (strain B4123)  . Upon en0 CFU/ml .10 CFU/ml, with levels increasing to 8.1 μg/ml in the stationary stage (8.8 log10 CFU/ml) after 28 h of incubation , showing that cell concentrations in LB and skimmed milk did not change in cultures of B4094, B4123, and DSM13increase , S5.10 CFU/ml; (2) lichenysin production started when cells reached the late exponential phase in LB and skimmed milk; (3) lichenysin production was strain and medium-dependent at similar cell densities.It can be concluded from T upon growth in LB for 24 h and in skimmed milk for 28 h. An example of chromatograms with mass spectra from the extracts of the B4094 cultured cells in skimmed milk as retrieved at 28 h is shown in The concentrations of lichenysin as reported in the previous section consisted of the sum of lichenysin variants, all corresponding with lichenysin A variants. The molar masses of the detected variants are listed in T synthesized two other variants, namely, C11-lichenysin (979.6 Da) and C17-lichenysin (1063.7 Da). Lichenysin with the shortest hydrophobic tail (C11) was only detected in the case of the two food isolates (B4094 and B4123) grown in skimmed milk but not in LB. The relative abundance of lichenysin with the longest hydrophobic tail (C17) was higher in LB than in skimmed milk for all three strains . In addition, strains B4094, B4123 and DSM13 strains . The 14  strains , this maApproximately half of the main variants produced in LB consisted of C15-lichenysin, followed by C14 and smaller amounts of C13 > C16 > C12 . In skimT on agar surfaces was also quantified.To further test whether the medium composition and culture conditions affect the levels of lichenysin produced and the synthesis of different isoforms, the total lichenysin production of B4094, B4123, and DSM13T upon growth on LB agar and skimmed milk agar at 37 and 55°C, are presented in The amounts and variants of lichenysin as produced by strains B4094, B4123, and DSM13T, B4094, and B4123 yielded 25 μg lichenysin per g of wet biomass (μg/g), 267 μg/g, and 218 μg/g, respectively  for B4094, and ∼30 % less (168 μg/g) for B4123  was found to be produced at very low levels by all three strains at 37°C on LB agar but not on skimmed milk agar . The C18T, B4094, and B4123 in liquid broth using stagnant conditions. Results presented in Considering the above outcomes that culturing conditions can strongly influence the amount and type of lichenysin produced, lichenysin production was also evaluated upon culturing of DSM13B. subtilis derived surfactin as a control. The concentration of lichenysin and surfactin needed to reduce cell viability by 50% (IC50) is presented for different cell lines and organoids in 50 values of 16.6 μg/ml and 23.4 μg/ml, respectively  between strains. Some high producers in their study were associated with foodborne intoxications and they also classified strain ATCC14580 (=DSM13T) as a weak producer.Strain B4094 consistently produced more lichenysin than strain B4123 under all conditions tested, and both high producers synthesized much more lichenysin than DSM1310 CFU/ml. Production did not reach maximum levels until the stationary stage in both media. It is known that lichenysin production can be influenced by environmental factors, e.g., medium composition 2SO4, and NH4NO3] were present instead of organic nitrogen sources  were present  revealed that lichenysin was not detected at cell densities < 5 logposition , oxygen position , and theposition . In addie medium  and when present . LB contB. licheniformis is optimal at 35–45°C. Similarly, surfactin yield from B. subtilis was also influenced by temperature, with lower production at 45°C than at 35°C in Bacillus amyloliquefaciens was however lower at 25°C or 30°C than at 15°C  . This re at 15°C .In our study, cultures contained lichenysin A variants with different acyl chain lengths (C11–C18). The length of the fatty acid chain lengths may influence the toxicity of the compound; Previous studies on surfactin showed that C15 acyl chains had more efficient penetration strength into the cell membrane than its shorter counterparts C13 and C14-surfactin , and exh50) was 16.6 μg/ml for Caco- 2 human intestinal epithelial cells and 16.8 μg/ml for pig ileum organoids. These levels correspond with concentrations resulting in toxic effects on boar spermatozoa cells  . This indicates that lichenysin is more toxic to these cell types than surfactin. In the case of surfactin, the purchased compound largely contained C15 acyl chains (>99%). The lichenysin extracts used in the toxicity assays contained a mix of lichenysin variants, with C15-lichenysin being the most abundant  . For surB. licheniformis up to 5 log10 viable cells/ml in liquid foods are unlikely to pose a foodborne hazard related to the presence of lichenysin. However, at higher cell densities, a risk of foodborne intoxication may arise due to the production of the compound. Lichenysin levels produced in milk and LB were in the same range as the IC50 values of lichenysin for epithelial cells. The actual amounts to cause illness in vivo upon ingestion of foods will depend on various factors, most notably the amount of food ingested and the concentration of the lichenysin in the product. High producers are commonly found in foods: in the study by B. licheniformis, depending on the strain present, the composition of the food and conditions during processing and storage in the chain. In solid foods, the distribution of the organism in the product may be inhomogeneous with ‘hot spots’ of high cell concentrations and lichenysin production. In confirmed foodborne intoxication cases due to consumption of solid product contaminated with B. licheniformis, high levels of the organism were observed   .B. licheniformis can be actively introduced into the food chain . This is usually done in the form of spores. From a food processing point of view, it is important that the selected strains do not possess genetic elements that enable the production of extremely heat-resistant spores  are inactivated. This stresses the importance of the control of this organism throughout the entire production chain, by inactivation of viable cells and spores in ingredients and prevention of outgrowth using appropriate preservation systems and/or low-temperature control.From a food safety point of view, the production of lichenysin must be prevented, requiring a thorough assessment of surfactant production capacity. Once lichenysin is produced in food upon the growth of The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Ethics Committee of Wageningen University and Research. Written informed consent was obtained from the owners for the participation of their animals in this study.B. licheniformis strains, hemolysis test, and anaerobic growth experiments. BF-C performed the alamarBlue cytotoxicity assay and took microscopic pictures. KY, TA, and MW-B discussed the experimental design and results. JW, TA, and MW-B reviewed the manuscript. All authors contributed to the article and approved the submitted version.KY designed and conducted the majority of the experiments, analyzed the results, and wrote the manuscript. MP performed the screening of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The use of statins in type 2 diabetic patients significantly reduced the risk of SNHL or tinnitus by 24.8%  after controlling for confounders. Similar results were found for the association between statin use and SNHL . The protective effects of statins against SNHL were consistent regardless of age and sex. The use of statins for type 2 diabetic patients was significantly associated with a reduced risk of SNHL, regardless of age and sex. Further studies are needed, especially large cohort studies, to evaluate the long-term protective effects of statins.Statins have emerged as protective agents against sensorineural hearing loss (SNHL) associated with dyslipidemia, but the effects of statins on SNHL are not consistent. The purpose of this study was to investigate the association between statin use and the risk of SNHL using a hospital cohort. This nested case-control study included type 2 diabetic patients over the age of 18 years without a history of hearing loss. Of these, 1379 patients newly diagnosed with SNHL or tinnitus were classified as cases, and 5512 patients matched to the cases based on age, sex, and index year were classified as controls. Chi-squared tests were used to compare categorical variables between the two groups. Odds ratios (ORs) and adjusted odds ratios (AOR) were calculated from univariate and multivariable unconditional logistic regression analyses, respectively. There was a significant difference in the prevalence of statin use between the cases and controls (53.7% vs. 61.2%, respectively;  Hearing loss is one of the least visible but most uncomfortable diseases. According to the World Health Organization (WHO), over 5% of the world’s population (432 million adults and 34 million children) has hearing loss and over 900 million people will develop hearing loss by 2050 . GloballAmong the types of hearing loss, sensorineural hearing loss (SNHL) is a condition involving damaged hair cells and the auditory nerve, which is distinguished from conductive hearing loss . SeveralSNHL is loss of hearing with damage to the inner ear, whereas tinnitus is an auditory sensation in the absence of external stimulus ,8. HeariStatins are widely used as cholesterol-lowering agents through competitive 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibition. Due to the strong evidence for statins as a treatment option for dyslipidemia, they have emerged as protective agents of hearing associated with dyslipidemia ,18. Seve2, total cholesterol 240 mg/dL or higher, chronic kidney disease, causative diseases of hearing loss, and ototoxic medication use were significant factors for the risk of SNHL or tinnitus.Among 50,129 patients who were eligible for this study, 35 patients were excluded due to presbycusis, deafness, or conductive hearing loss during the study period . Of 50,0p < 0.001). When analyzed by statin type, most statins were associated with reduced risk of SNHL or tinnitus, except for fluvastatin and pravastatin. The reductions in the risk of SNHL or tinnitus were 26.2% (95% CI 13.9–36.8%), 36.8% (95% CI 18.0–51.3%), 24.2% (95% CI 9.3–36.6%), and 33.2% (95% CI 16.2–46.7%) for atorvastatin, pitavastatin, rosuvastatin, and simvastatin, respectively.The results of univariate and multivariable analyses are shown in p > 0.05).2, GFR less than 30 mL/min/1.73 m2, chronic kidney disease, causative diseases for hearing loss, and ototoxic medication use were significant factors for SNHL .To construct the study cohort, we identified all outpatients aged at least 18 years with a diagnosis of type 2 DM (ICD-10: E11) between 1 January 2015 and 31 December 2019. Individuals diagnosed with all types of hearing loss and tinnitus before the study period (from 1 January 1997 to 31 December 2014) were excluded. To distinguish from other types of hearing loss, based on the most likely mechanisms of SNHL or tinnitus related to statins, patients newly diagnosed with presbycusis (ICD-10: H91.1), deafness (ICD-10: H91.3), or conductive hearing loss  during the study period (between 1 January 2015 and 31 December 2019) were also excluded.Among our study cohort, cases were defined as all patients newly diagnosed with SNHL  or tinnitus (ICD-10: H93.1) during the study period. We assigned the first visit for the diagnosis of SNHL or tinnitus as the index date for the case. Among the remaining study cohort, controls were randomly matched to cases at a ratio of 4:1 by age, sex, and index year. For cases, the index date was the first diagnosis date; for controls, the index date was the first outpatient visiting date in the matched index year.Statin was the main exposure in this study. Statins used in the hospital included atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Patients were considered exposed to statin if they had ever been prescribed any statins  code: C01AA, C10BX, or C10BA) within one year before the index date.The following data were collected from electronic medical records: patient age, sex, BMI, comorbidities, concurrent medications, and laboratory data. Laboratory data included serum cholesterol, LDL-C, high-density lipoprotein-cholesterol (HDL-C), triglycerides, and hemoglobin A1c (HbA1c).Based on the findings of previous studies on risk factors for hearing loss ,55, we cFor ototoxic medication ,56, we ip-value < 0.05. Subgroup analyses by sex and age were carried out to evaluate the influence of sex and age on the results. For the secondary analysis, we repeated the primary analysis after restricting cases to those with SNHL only. All statistical analyses were performed with SAS 9.4 . A p-value < 0.05 was considered statistically significant.For the primary analysis, we investigated the effects of statins on SNHL or tinnitus. Chi-squared tests were used to compare categorical variables between two groups. ORs and adjusted odds ratios (AOR) were calculated from univariate and multivariable unconditional logistic regression analyses, respectively. To adjust the confounding factors, multivariable analysis with backward elimination was performed including the factors with a This study detected a potential association between statin use and SNHL after adjusting for relevant confounders. The results showed that the use of statins for type 2 DM patients was significantly associated with a reduced risk of SNHL, regardless of age and sex. Further studies are needed, especially large cohort studies, to evaluate the long-term protective effects of statins.


As a side effect, in case of reduced auditory input, such as permanent hearing loss or frequency specific deprivation, this mechanism may eventually lead to the perception of phantom sounds like tinnitus or the Zwicker tone illusion. Using computational modeling, the biological plausibility of this processing principle was already demonstrated. Here, we provide experimental results that further support the stochastic resonance model of auditory perception. In particular, Mongolian gerbils were exposed to moderate intensity, non-damaging long-term notched noise, which mimics hearing loss for frequencies within the notch. Remarkably, the animals developed significantly increased sensitivity, i.e. improved hearing thresholds, for the frequency centered within the notch, but not for frequencies outside the notch. In addition, most animals treated with the new paradigm showed identical behavioral signs of phantom sound perception (tinnitus) as animals with acoustic trauma induced tinnitus. In contrast, animals treated with broadband noise as a control condition did not show any significant threshold change, nor behavioral signs of phantom sound perception.Recently, it was proposed that a processing principle called  However, until recently the underlying neural processes remained rather elusive.Whether listening to nearly inaudible quiet whispering or enjoying a loud rock concert, the human auditory system has a remarkable ability to adapt to changing sound pressure levels covering several orders of magnitude from the absolute threshold of hearing to the threshold of pain and beyond. Several studies even demonstrate—under certain conditions—the healthy auditory system's ability to further improve sensitivity even below the absolute threshold of hearingadaptive stochastic resonance5 is exploited by the auditory system in order to continuously maintain optimal sensitivity even to highly variable sound pressure levels and changing statistics of the acoustic environment7.In recent studies, we argued that a processing principle called 9. In the last decades, stochastic resonance has been found in a vast number of different organisms and biological systems11. In particular, in neuroscience stochastic resonance helps to explain how nervous systems robustly operate in noisy environments12.The term stochastic resonance refers to a phenomenon, where a signal of arbitrary kind, which is too weak for a certain sensor for being detected, can be made detectable by adding a random signal, i.e. noise, of appropriate intensity to the sensor input6. There, auditory input from the cochlea converges with projections from the somatosensory system13. Since this somatosensory input is largely uncorrelated with the auditory signals, we argued that these somatosensory projections serve as a random neuronal signal, i.e. neuronal noise, which is necessary for stochastic resonance to work7. The intensity of the neuronal noise in the healthy and impaired auditory system is continuously adjusted, depending on the statistics of the auditory input. In case of reduced auditory input for instance, the internal neuronal noise would be upregulated, i.e. somatosensory projections dis-inhibited, which results in increased sensitivity by means of stochastic resonance, thereby enhancing information transmission from the cochlea to the central auditory system. This assumption is supported by empirical findings that somatosensory projections to the cochlear nucleus are actually upregulated after unilateral deafness16.According to our model, stochastic resonance is a major processing principle of the auditory system, and most probably takes place in the dorsal cochlear nucleus17, we were able to demonstrate that noise even enhances the information flux in such small neural systems, a phenomenon for which the term “recurrence resonance” has been coined18. By constructing a hybrid computational model based on deep neural networks, we further demonstrated the biological plausibility of the proposed processing principle, and demonstrated in a simulation of the impaired auditory system that stochastic resonance may even improve, i.e. partly restore, speech recognition after hearing loss19. According to our model, the increased internal neuronal noise, i.e. upregulated somatosensory input to the auditory system, corresponds to the observed permanent increase of spontaneous firing rates within the auditory system in case of chronic hearing loss6. This neural hyperactivity in turn, was found to be correlated with subjective tinnitus in many studies. Our model provides a unified mechanistic explanation of how hearing loss, phantom perceptions like tinnitus or the Zwicker tone illusion20, and neural hyperactivity are related to each other21, namely that these phantom perceptions are side effects of the physiological neuronal mechanism to (partly) restore hearing thresholds. Therefore, our model provides a mechanistic explanation why patients with hearing loss and with tinnitus on average have better hearing thresholds than patients with hearing loss and without tinnitus22, i.e. why auditory sensitivity is enhanced in tinnitus subjects compared with non-tinnitus subjects23. In addition, our model also provides a mechanistic explanation why during the Zwicker tone sensation, auditory sensitivity for tone pulses at frequencies adjacent to the Zwicker tone are improved by up to 13 dB3. Tinnitus and Zwicker tone correspond to the perception of the increased neural noise from the somatosensory system, and this noise helps to increase auditory sensitivity by means of stochastic resonance.In neural network simulations, in particular within 3-neuron motifs4 fits well into our model. It shows that it is not the source of the modulating noise but the spectral information within the auditory system is the important factor for hearing threshold improvement.Also, work from other groups, that found that not only internal neuronal noise but also external acoustic stimulation with spectral fitted noise stimuli can improve hearing in healthy subjects even beyond the absolute threshold of hearing24.Recently, all these implications of our model even lead us to a novel treatment strategy for tinnitus: we already demonstrated in a pilot study, how the presentation of external, spectrally matched, near-threshold acoustic noise, may significantly decrease tinnitus perception by replacing internal neural noisesimulated hearing loss through long-term noise exposure with notched noise at moderate, non-damaging sound intensities. This paradigm provides the possibility to simulate transient hearing loss and complete recovery from hearing loss without changing the peripheral or central auditory system permanently, e.g. sound trauma induced cochlear damage.In order to provide further evidence for the hypothesis that adaptive stochastic resonance plays a major role in the auditory system and especially in phantom sound perception, we developed a novel animal experimental paradigm: 25. Therefore, it seems possible to simulate a certain hearing loss by exposure with sound or noise where the mean  power of the frequency band of desired hearing loss is decreased (= dipped) compared to adjacent frequencies. Hence, the otherwise flat power spectrum has a dip that mimics hearing loss. From the perspective of the auditory system, it should make no difference whether certain frequency channels receive less input due to cochlear damage or due to changed statistics of the acoustic environment. After the end of long-term acoustic noise exposure, animals' acoustic environment has again a flat frequency power spectrum on longer time scales, which, analogously to the above argument, corresponds to a complete restoration of hearing. Thus, the post-noise exposure phase of the paradigm corresponds to a complete restoration of hearing, or a simulated treatment with an ideal hearing aid, respectively.In nature, the standard acoustic environment, i.e. the temporally averaged frequency power spectrum, is broad and flat, i.e. all frequencies have statistically similar mean sound pressure levels—at least on time scales of days to weeks, and at least in the frequency range best perceivable by animals and humansnoise group and frequency; F-statistics given there). Neither in the factor noise group  have been treated with the new paradigm , and additional 11 animals with the control paradigm . The relative mean threshold changes immediately after treatment are summarized in Fig. In contrast, hearing thresholds for 1 and 4 kHz (where the spectrum was flat) did not change significantly  showed a complete removal of behavioral signs of tinnitus Fig. A, whereaise Fig. D.Figure Interestingly, the animals that developed behavioral signs of tinnitus during notched noise exposure can be divided into two groups: a broadband tinnitus group, where more than two frequencies are affected (n = 5), and tonal tinnitus group, where only one or two frequencies are affected (n = 4). In the tonal tinnitus group, three of the four animals show behavioral signs of tinnitus exactly where expected, namely at 2 kHz. However, why either broadband tinnitus or tonal tinnitus develops during notched noise exposure remains unclear and may be part of further studies.21) and theoretical studies19 indicates that the auditory system actually exploits stochastic resonance and actively tunes the intensity of the required neuronal noise by adjusting the somatosensory input to the dorsal cochlear nucleus. By that, stochastic resonance contributes crucially to the auditory system’s ability to adapt to changing sound pressure levels.We presented a novel experimental paradigm to simulate transient hearing loss, which in turn induced improvement of hearing thresholds and perception of phantom sounds when hearing was normal again. Both effects are in line with our hypothesis that stochastic resonance plays a major role in the auditory system. The converging evidence from numerous empirical  on hearing threshold changes and induction of phantom sounds may be investigated.Furthermore, the presented results are similar to ear plugging studies, where healthy human subjects are provided with ear plugs for 2 weeks, mimicking hearing loss. These subjects perceive a transient tinnitus after removal of ear plugs31, whereas in humans the corresponding duration is up to 20 s32. Thus, 2 weeks of notched noise exposure in rodents correspond to a much longer treatment with ear plugs in humans than just 2 weeks; this may also explain the large difference of more than 11 dB threshold shift between the treated and control group. Hence, one might hypothesize that, if humans would be treated with ear plugs for a longer time, then the tinnitus sensation may become chronic in some subjects as well. This would indicate that the duration of ear plugging or notched noise exposure correlates with the ratio of subjects or rodents developing chronic tinnitus. This is an interesting question to be addressed—at least on the level of rodents—in a follow-up study.It is worth mentioning that in ear plugging studies, no subject reported tinnitus 2 weeks after the end of ear plugging, which seems to be in contrast with the here presented results. However, we speculate that this has something to do with the fact that neural processing and homeostatic plasticity in rodents takes place on shorter time scales than in humans. For instance the maximum duration of the Mongolian gerbil’s auditory sensory memory for random waveforms is about 80 msFurthermore, one may argue that, since thresholds obtained with sensitive auditory brainstem response (ABR) methods are in most animals approximately 10–20 dB above neuronal or behavioral data, the measured responses do not necessarily reflect threshold sensitivity, but rather some nonlinear interaction of a larger number of neurons, which makes it hard to interpret in terms of sensitivity. To decide this question, a replication of the presented study could be performed, yet with the difference that hearing thresholds are estimate with some behavioral paradigm, or cortical recordings, instead of ABR measurements.As mentioned in the results the animals develop either broadband or tonal tinnitus, which does not seem to correlate with either being transient Fig. , group A24. Finally, we note that our paradigm is an important step towards reducing the health burden on laboratory animals, since instead of applying a permanent noise trauma in order to induce tinnitus, the hearing loss can now be simulated.The clinical relevance of our study is underlined by the fact that recently, the implications of our model lead us to a novel treatment strategy for tinnitus: we already demonstrated in a pilot study, how the presentation of external, spectrally matched, near-threshold acoustic noise, may significantly decrease tinnitus perceptionMeriones unguiculatus, male, 12 weeks old) were housed in standard animal racks  in groups of two animals per cage with free access to water and food at 20–24 °C room temperature under 12/12 h dark/light cycle. The use and care of animals was approved by the state of Bavaria . All methods were carried out in accordance with relevant guidelines and regulations. Furthermore, the study was carried out in compliance with the ARRIVE guidelines. Gerbils were purchased from Charles River Laboratories Inc. .Twenty-five Mongolian gerbils  spectrum is maximal (20 dB) and decreases sigmoidally (in order to avoid sharp edges in the spectrum) with increasing distance to the center. At the borders (1.4 and 2.8 kHz) the power-difference is zero, i.e. the power is identical to the rest of the power spectrum. For the control group non-dipped (= flat) white noise was used.34 was used to assess the potential existence of a tinnitus percept. Animals were tested before (pre), immediately after (acute), 1 week and 2 weeks after long-term noise exposure.Exposure to 2 kHz dipped (n = 14)/flat (n = 11) noise at low sound pressure levels (50 dB SPL) took 2 weeks for each animal. The notched noise had a dipped spectral envelope. This new paradigm simulates both hearing loss (dip in frequency spectrum) and potential subsequent treatment with an ideal hearing aid or therapy that fully restores hearing abilities. Hearing thresholds for frequencies of 1, 2 and 4 kHz were measured using ABR in all animals before and immediately after long-term noise exposure. The GPIAS paradigm35. Pure tone stimuli of different frequencies  were generated by a custom-made python program and presented at different, pseudo-randomized intensities ranging from 90 to 0 dB SPL in 5 dB steps. Stimulation was free-field to the measured ear  at a time via a speaker (Sinus Live NEO) corrected for its frequency transfer function to be flat within ± 1 dB at a distance of approximately 30 mm from the animal's pinna while the contra-lateral ear was tamped with an ear plug. To compensate for speaker artifacts stimuli were presented as double trials consisting of two 6 ms stimuli  of the same amplitude but opposite phase, separated by 100 ms of silence. 120 double trials of each combination of intensity and frequency were presented pseudo-randomly at an inter-stimulus interval of 500 ms.In order to assess the animal's hearing thresholds, ABR were recorded using a custom made setup36.For the measurements the Mongolian gerbils were kept under deep anesthesia. Anesthesia was induced by an initial dose of 0.3 ml of a ketamine-xylacin-mixture , and maintained by continuous application of that mixture at a rate of 0.2–0.3 ml/h by a syringe pump. As has been demonstrated previously, such ketamine–xylazine anesthesia has only little effect on ABR signals compared to awake animalsDuring measurements, animals were placed on a heating pad at 37 °C. Data were recorded using three silver wire electrodes positioned subcutaneously, one for grounding at the back of the animals, one reference electrode at the forehead and the measuring electrode infra-auricular overlying the bulla. The potential difference between the reference and measuring electrode was amplified by a low noise amplifier . Note that for further analysis the amplified signal was used, that is, amplitudes are given in mV whereas the actual neuronal signals were in µV range. The output signal of the amplifier was digitalized and recorded by an analog–digital converter card  with a sampling rate of 20 kHz and synchronized with the stimulation via the trigger signal of the stimulation computer. Raw data of 120 double trials per sound level for one stimulus frequency were averaged. Finally, these averaged responses of the two single, phase inverted stimuli within one double trial were averaged to eliminate stimulus artifacts. From these averaged, artifact-corrected data the root mean square (RMS) values within a time window from 0 to 10 ms after stimulus onset were calculated to obtain a measure of ABR amplitude for each stimulus intensity presented.35).Hearing thresholds were estimated by fitting the ABR root-mean-square amplitudes to different stimulus intensities with a hard sigmoid function. In combination with subsampling, this universally applicable procedure provides a robust threshold estimation as well as an accurate uncertainty estimate. Furthermore, this method has no systematic dependence on the noise and does not even require samples in the full dynamic range of the sensory system  which was positioned at a distance of 10 cm in front of two loudspeakers, one for the moderate background noise and one for the startle stimulus. The startle response was measured by a sensor platform with three integrated acceleration sensors . Stimulus generation and data acquisition used custom-made software . As startle amplitudes tend to be higher for the first few trials, the first five startle stimuli were presented before the beginning of each measurement to rule out strong habituation effects. All animals were then subjected to continuous band pass filtered 60 dB SPL loud background noise  with medium frequencies of 1–8 kHz in ½ octave steps and a bandwidth of ± ¼ octave. The mean duration of the background noise before the startle noise burst was 10 ± 2.5 s, it ended at the beginning of the startle noise burst. The startle white noise burst  was presented either 50 ms after a 50 ms long silent gap  in the background noise, or it was presented without any gap. For each background frequency, 20 repetitions with and without gap were presented in randomized order. Every animal was tested four times, the first time in a naïve condition before treatment, and the other times immediately after, 1 week and 2 weeks after being taken out of the long-term noise exposure. The whole experimental session did not last longer than 30 min.Putative perception of phantom sounds was assessed with the gap pre-pulse inhibition of the acoustic startle reflex (GPIAS) paradigm34. Briefly, as the measured response amplitudes are log-normally distributed, acquired amplitudes were logarithmized. Then we exploited the full combinatorial power of all log-normalized response amplitudes to obtain the distributions of the prepulse inhibition (PPI) before and after treatment of the animals. From these distributions, we calculated the change of the behavioral response. Positive values indicate a stronger effect of the gap in the post compared to the pre condition. Negative values indicate less effect of the gap after treatment, i.e., a stronger startle response despite the present gap, which indicates a “filling” of the gap by a tinnitus percept in that frequency range. Additionally, these now normally distributed data were analyzed using parametrical Student’s t-tests to find significant changes of the PPI change (ΔPPI) which can be used to define the strength of a possible tinnitus percept.Data were analyzed using the method described inDue to experimental constrains, only 11 out of the 14 animals with 2 kHz dipped noise and 4 out of 11 control animals with flat noise exposure could be tested with this paradigm.noise group and stimulation frequency. Post-hoc tests were performed with Tukey tests, already correcting for multiple comparisons. Additionally the threshold shifts at the different stimulation frequencies of both noise exposure groups were tested against zero (= no shift) by single sided t-tests, Bonferroni corrected for multiple comparisons. These tests were undertaken to rule out, that the control animals did show an “inverse” effect compared to the 2 kHz dipped noise exposure group. As mentioned above, the changes in GPIAS responses have been analyzed with a custom made Python 3.6 software described in34. The reason for using t-tests (corrected for multiple comparisons) here is the at least partial independency of each tested frequency relative to the other and the reduction of perquisites required for the statistics. We wanted to know, at which frequency exactly a possible tinnitus percept emerged and not only if it is somewhere in the data .Threshold shifts have been calculated by subtracting the pre exposure ABR threshold from the post exposure ABR threshold at each tested frequency for each individual. By this, a negative threshold shift corresponds to a better hearing after noise exposure at a given frequency. The threshold shifts have been analyzed by a two-factorial ANOVA using Statistica 8  with the factors


Currently the world is witnessing an explosion in mucormycosis in India among COVID-19 patients with an official count of 28,252 cases as of 06/07/2021. Thus, novel therapeutic modalities are needed. We previously reported on a mouse monoclonal antibody (C2) targeting CotH invasins being protective against mucormycosis. Here, we humanized C2 MAb and assessed its efficacy Rhizopus delemar-induced injury to A549 alveolar epithelial and primary human endothelial cells and for enhancing human neutrophil killing of the fungus in vitro. C2 and the humanized clones were also compared for their ability to protect neutropenic mice from mucormycosis induced by R. delemar or Mucor cicrinelloides with and without antifungal therapy. The C2 (IgG1) paratopes of the heavy chain and light chain were grafted on the most suitable human IgG1 with back mutations in the paratopes needed to restore binding of humanized clones to CotH3 (by biolayer interferometry using Gator). Clones were compared to C2 in their ability to prevent vs. ~50 nM for C2). One humanized clone (VX01) doubled the ability of neutrophils to kill R. delemar and resulted in ~50% reduction in host cell damage. A single low dose of VX01 (30 µg) given 24 h post infection resulted in comparable survival of 60-70% in mice infected intratracheally with either R. delemar or M. cicrinelloides vs. placebo mice . Importantly, VX01 acted synergistically in protecting mice when combined with liposomal amphotericin B or posaconazole in a severe model of mucormycosis with treatment starting 48 h post infection . GLP-tissue cross reactivity studies of VX01 showed favorable safety profiles. Three humanized clones showed 10-fold enhanced binding affinity to CotH3 protein  Ashraf S. Ibrahim, PhD, Vitalex Biosciences (Shareholder)