Schistosomiasis is the most important human helminth infection due to its impact on public health. The clinical manifestations are chronic and significantly decrease an individual’s quality of life. Infected individuals suffer from long-term organ pathologies including fibrosis which eventually leads to organ failure. The development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission.Schistosoma mansoni (Sm) cathepsin B, a peptidase involved in parasite feeding, as a prospective vaccine candidate. Our experimental formulation consisted of recombinant Sm-cathepsin B formulated in Montanide ISA 720 VG, a squalene based adjuvant containing a mannide mono-oleate emulsifier. Parasitological burden was assessed by determining adult worm, hepatic egg, and intestinal egg numbers in each mouse. Serum was used in ELISAs to evaluate production of antigen-specific antibodies, and isolated splenocytes were stimulated with the antigen for the analysis of cytokine secretion levels.Our group has chosen P = 0.0002, P < 0.0001, P = 0.0009, respectively). Immunizations with the vaccine elicited robust production of Sm-cathepsin B specific antibodies . Both antigen-specific IgG1 and IgG2c titers were observed, with the former having more elevated titers. Furthermore, splenocytes isolated from the immunized animals, compared to control animals, secreted higher levels of key Th1 cytokines, IFN-γ, IL-12, and TNF-α, as well as the Th2 cytokines IL-5 and IL-4 when stimulated with recombinant Sm-cathepsin B. The Th17 cytokine IL-17, the chemokine CCL5, and the growth factor GM-CSF were also significantly increased in the immunized animals compared to the controls.The Sm-cathepsin B and Montanide formulation conferred protection against a challenge infection by significantly reducing all forms of parasitological burdens. Worm burden, hepatic egg burden and intestinal egg burden were decreased by 60 %, 62 %, and 56 %, respectively in immunized animals compared to controls  contains supplementary material, which is available to authorized users. Schistosoma genus, is an important neglected tropical disease with a substantial impact on public health. Over 200 million people are infected with approximately half of these being school aged children [Schistosomiasis, caused by trematodes belonging to the children –3. It hachildren . This unchildren , 6. Treachildren . School-children . It has children . Furtherchildren –10. The children . Vaccinechildren , 12.Schistosoma mansoni (Sm) cathepsin B as a vaccine candidate. This cysteine peptidase, originally cloned and identified by Klinkert et al., [Our group has chosen to focus on  et al., , 14 is p et al., –19. The  et al., . The pre et al., –23. ThesAll animal procedures were conducted in accordance with Institutional Animal Care and Use Guidelines and were approved by the Animal Care and Use Committee at McGill University .Schistosoma mansoni cathepsin B protein was generated as described elsewhere [Schistosoma mansoni cathepsin B (Genbank accession number M21309.1) cDNA was synthesized by GenScript in pUC57 with modifications in order to be suitable for expression in the yeast Pichia pastoris [600 = 6). The cells were harvested by centrifugation at 3,000 × g for five minutes at room temperature. The pellet was resuspended in 200 ml induction media: buffered complex methanol medium . The culture was grown for three days at 28 °C with shaking (250 rpm). Every 24 h, methanol was added to a final concentration of 0.5 %. After the three days, the cells were harvested by centrifugation at 3,000 × g for five minutes at room temperature. The resulting expressed recombinant protein was purified using Ni-NTA affinity chromatography . Expression quality of recombinant Sm-cathepsin B was analyzed by Coomassie staining of polyacrylamide gel and western blot using antibodies directed against the His-tag  followed by a HRP-conjugated secondary antibody .The recombinant lsewhere . Brieflypastoris . The modpastoris . Sm-cathFemale 6–8 week old C57BL/6 mice were purchased from Charles River Laboratories . There were three immunization groups containing 10 mice each. The immunization groups were as follows: saline control mice were injected with 50 μl of phosphate buffered saline (PBS) . Adjuvant control mice received 50 μl of a 70 % volume formulation of Montanide ISA 720 VG . The experimental mice were immunized with a 50 μl solution of 20 μg recombinant Sm-cathepsin B in a 70 % volume formulation of Montanide ISA 720 VG. Each mouse was immunized intramuscularly at weeks 0, 3 and 6. The immunization timeline and the vaccine formulation (antigen: adjuvant ratio) were determined based on previous small scale immunization/challenge pilot studies (data not shown).Biomphalaria glabrata snails infected with the Schistosoma mansoni Puerto Rican strain were provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute . Cercariae, the infectious stage of the parasite, were collected from the shedding of the infected B. glabrata snails. Three weeks after the final immunization, all of the mice were challenged with 150 cercariae via tail exposure lasting one hour. The mice were sacrificed seven weeks post cercarial challenge. The adult worms were perfused from the hepatic portal system and counted manually [manually . The nummanually . The folBlood collection via saphenous bleed was performed on each mouse prior to each immunization as well as on the day of the cercarial challenge. Sera were obtained by allowing the collected blood to clot and then removing it by centrifugation. The humoral responses elicited by the immunizations were analyzed by enzyme-linked immunosorbent assay (ELISA) as described elsewhere . Briefly6 cells/well were seeded into 96-well plates. The cells were incubated at 37 °C in the presence of 2 μg/ml of recombinant Sm-cathepsin B. After 72 h, 100 μl of the supernatant was collected. The splenocyte supernatant was used to asses cytokine production by QUANSYS multiplex ELISA (16-plex)  following the manufacturer’s recommendations.The animals were sacrificed seven weeks post cercarial challenge. The spleens of each animal were collected and splenocytes were isolated as described elsewhere , 28. BriStatistical analysis was done using the Mann–Whitney test (nonparametric test) to compare different groups of data. Antibody and cytokine data are presented on semi-log plots showing geometric mean and 95 % confidence intervals. The software GraphPad Prism 5  was used. P values less than 0.05 were considered significant.The expression of the recombinant Sm-cathepsin B protein used for the immunizations was shown by both Coomassie staining of polyacrylamide gel  . There were no statistically significant difference in worm burdens between the two control groups (P = 0.1285).In order to determine the protective potential of the Sm-cathepsin B and Montanide ISA 720 VG formulation, an immunization regimen was tested using C57BL/6 mice as described above. The average worm number from the group that received the Sm-cathepsin B and Montanide ISA 720 VG formulation (16.4 ± 2.5 worms) was compared to the average from the adjuvant control group (41.1 ± 0.8 worms). Therefore, worm burden was reduced by 60.1 % in the mice that received the Sm-cathepsin B/adjuvant formulation compared to the adjuvant control mice (02) Fig. . CompareP < 0.0001) . There also seems to be a significant decrease in hepatic egg burden in the adjuvant control group compared to the saline group (P = 0.0185).Schistosomiasis pathology is linked to the parasite eggs becoming trapped in tissues. Hepatic and intestinal egg burdens were determined for both the experimental and control mouse groups. Comparisons between the adjuvant control group  and the Sm-cathepsin B/adjuvant formulation group  revealed a 61.5 % reduction in hepatic egg burden for the mice given the vaccine (01) Fig. . When coP = 0.0009) . There were no statistically significant difference in egg burdens between the two control groups (P = 0.4813). All of the results elucidating the protective potential of the Sm-cathepsin B formulation are summarized in Additional file Intestinal eggs were also enumerated from the digested organ using microscopy. The group containing the animals immunized with the Sm-cathepsin B/adjuvant formulation  had a 56.1 % reduction in intestinal egg burden compared to the group that received adjuvant only  (09) Fig. . CompareThe serum collected throughout the immunization regimen was used in order to analyze and compare the antibody production pattern between the three groups of mice used in this study. ELISAs were performed to determine antibody titers. In both the adjuvant control and saline control mice, there were no detectable levels of Sm-cathepsin B specific antibodies . In the Sm-cathepsin B/adjuvant formulation group, Sm-cathepsin B specific total IgG titers rose sharply after the first boost at week 3, reaching mean endpoint titers of 40,160 at week 6, and continued to rise until the challenge at week 9  Fig. . The serP > 0.05). There was an increase in Th1 cytokine production in the group that was immunized with Sm-cathepsin B with Montanide ISA 720 VG compared to the control groups. There was a significant increase in IFNγ secretion levels in the experimental group compared to the Montanide adjuvant control group (P = 0.0021) and the saline group (P < 0.0001)  and IL-12 (P = 0.0185) levels in the experimental group compared to the adjuvant control group . Th2 cytokine production was analyzed as well. There were statistically significant increases in IL-4 levels between the experimental and adjuvant groups (P = 0.0288) as well as between the experimental and the saline groups (P = 0.0147)  in the group immunized with the Sm-cathepsin B and Montanide ISA 720 VG formulation compared to the adjuvant alone control group . IL-17 levels were observed to gain insight into a possible Th17 involvement. Secretion levels of IL-17 were significantly increased in the experimental group compared to the adjuvant control group (P = 0.0433). However, this statistically significant increase was not seen when comparing the experimental group to the saline control group (P = 0.0892)  and GM-CSF levels (P = 0.0147) .Analysis of the cytokine panel using QUANSYS multiplex ELISA array revealed no significant differences in cytokine secretion levels between the two control groups (01) Fig. . There w47) Fig. . IL-5 seoup Fig. . This si92) Fig. . Other cSchistosoma mansoni antigens with the hopes of uncovering an optimal vaccine candidate. The committee’s goal was to find an antigen that could consistently induce 40 % protection or better [Due to its essential role in parasite growth and development and its continuous interplay with the host immune system, our group has focused on Sm-cathepsin B as a potential vaccine candidate. During the mid-1990s, the WHO Special Programme for Research and Training in Tropical Diseases (TDR/WHO) planned for the independent testing of various r better . Recentlr better . Howeverr better –33. FurtS. mansoni radiation attenuated (RA) cercariae vaccine has been essential to study immune effector mechanisms associated with protection as well as discovering potential parasite points of vulnerability. The RA vaccine is considered the gold standard for reproducible induced anti-worm immunity. Exposure to the attenuated cercariae can elicit significant protection levels against challenges with normal cercariae. Protection levels have been shown to range from 56 % all the way to 80 % when multiple vaccinations are performed in the mouse model [The se model –37. The Several studies have demonstrated that IgG antibodies play an important role in protection against schistosomiasis. The passive transfer of sera from chronically infected or protected animals results in significantly reduced parasite burdens in the recipient animals –42. Stud+ T-cells with Th1 characteristics [S. mansoni vaccine candidates have shown an association between protection against parasite challenge and an increased IFN-ɣ production [It has long been a consensus that Th1 responses play a key role in protection against schistosomiasis. For instance, peripheral blood mononuclear cells isolated from individuals, who are considered naturally resistant to schistosomiasis, produce high levels of interferon-gamma (IFN-ɣ) when stimulated with schistosome antigen , 48. Moreristics , 45, 49.eristics , 45, 49.eristics . Both Thoduction –53. The oduction , 54. Theoduction . The CpGoduction . The Sm-oduction . IL-17 moduction  thus, deLevels of both GM-CSF and CCL5 were also significantly increased in the experimental animals compared to the control animals. GM-CSF is involved in macrophage recruitment whereas CCL5 is involved in the recruitment of T cells and granulocytes. Furthermore, CCL5 mediates proliferation and activation of natural killer (NK) cells, which play a role in schistosomiasis protection in elderly populations . IncreasThe results obtained from this study are promising and promote further testing of the vaccine candidate Sm-cathepsin B. In the future, the formulation can potentially be tested in a non-human primate model of schistosomiasis. These animals manifest a disease that is similar to that observed in humans, and the data from such a study may be more clinically applicable than data from a mouse study.In summary, the data presented in this manuscript demonstrated that immunizations with the formulation of Sm-cathepsin B in the presence of Montanide ISA 720 VG generated protection levels that are comparable to those obtained with the RA vaccine. This formulation elicited a robust production of antigen-specific antibodies and resulted in a favourable mixed Th1/Th2 response. In addition the formulation resulted in the increase of other cytokines such as IL-17 and GM-CSF. The mixed response elicited by the Sm-cathepsin B + Montanide formulation is likely the optimal immune scenario for the host. The essential parasite killing Th1 response was elicited (Th1) as well as the host-beneficial injury repair Th2 response (Th2). The present study along with our previous Sm-cathepsin B study  demonstr


Nonlinear physics plays an essential role in hearing. We demonstrate on a mesoscopic description level that during the evolutionary perfection of the hearing sensor, nonlinear physics led to the unique design of the cochlea observed in mammals, and that this design requests as a consequence the perception of pitch. Our insight challenges the view that mostly genetics is responsible for the uniformity of the construction of the mammalian hearing sensor. Our analysis also suggests that scaleable and non-scaleable arrangements of nonlinear sound detectors may be at the origin of the differences between hearing sensors in amniotic lineages. Nature provided our planet with an abundance of species. The question of how this abundance comes about has intrigued humans since early in their existence. In his treatise ‘On the Origin of Species’, Charles Darwin set forth in 1859 for a scientific explanationprestin is expressed, whereas audition is mediated in nonmammalian vertebrates and in insects by prestin-homologous proteinsDrosophilaDespite the high dimensionality of the space that must underly this optimization process, we observe in a number of instances an apparent convergence towards certain building principles, which is puzzling. The mammalian ear is one of these examples. After a long tradition of research on evolutionary linkage2345789101112While these genetical or physiological approaches have shed a fascinating light on how a major biological sense evolved and developed, they do not provide the arguments as to how this may have led to the sensory uniformity that we observe in particular within the mammalian family, despite evolutionary sensory specialization.Here, we investigate what role physics principles must have had in this process, and we do this exclusively at the level of a mesoscopic description . On this level, we will exhibit how physical principles constrain the solution space of optimal hearing sensors in such a way that for large frequency bands and sharp resolution hearing, a convergence towards the blueprint realized in the mammalian cochlea is highly likely to occur. From this, we will suggest that the close genetical relationship observed in the construction of the hearing sensors, while of interest in itself, should not be seen as the main origin of the phenomenon.Aedes aegypti performs ‘near-field’ hearing with a sensor that is tuned to the wingbeat frequency of femalesWe start by positing that sounds around a characteristic frequency are often of particular interest to the animal world . For spotting a predator, or a conspecific for reproduction, hearing a weak sound first among competitors is a substantial evolutionary advantage. In the simplest case, identifying one characteristic frequency will be important and might be sufficient. Insect hearing illustrates this at a fundamental level: The male mosquito For sound detection and perception, very faint input level sounds first need to be amplified actively202124Two prominent bifurcations2728A0 cos(2π f t) was included into the equation (A0 = 70 and f = 600 Hz). This term does not compromise the nature of the bifurcation and can be omitted for the following discussion. An enlightening understanding of the amplification dynamics can be provided by the behavior around zero displacement position x = 0, where the nonlinear damping term Pn(x) < 0 implies that energy is injected into the system, indicating active amplification , together with its first and second derivatives, are relatively small. This implies that for small receiver displacements, virtually no restoring force is present. By means of the negative damping term, the system is thus easily driven out to large amplitudes. The comparison between data and obtained trajectories reveals the close correspondence between the data and the model. After having determined the system equations for the fully self-sustained oscillatory state, we follow the system on the way back to below the bifurcation point , μm is still positive. Below, but close to the bifurcation point, where the antennal system usually operates, system-specific details are drowned out by the bifurcation properties. This implies that any such system, in particular Drosophila’s antenna equations, can be described in its essential features by the prototypical Hopf equationIn the insect case, evidence for a Hopf bifurcation underlying the amplification process is obtained as follows. Generally, biological small-signal amplifying systems rest below the bifurcation point to oscillation. The bifurcation point may, however, even be crossed under certain conditions, which can be used to infer the deeper nature of the active amplification process below the bifurcation. In the example of the Drosophila antennafication . Around on point . The recω close to the Hopf system’s characteristic frequency ω0, the system would oscillate at ω, at a considerable amplitude. The response shown in 31Comparison to the mammalian hearing system reveals that, from a fundamental dynamical systems view, insects and mammals share the generic function principles of the sensor. In the mammalian case, the nonlinear amplification is by electromotile outer hair cells embedded mechanically into the basilar membrane, working in the vicinity of, but below, a Hopf bifurcation. If stimulated by a signal of frequency The distinction of several frequencies, as emitted e.g. by a mate, predator, or prey, is of importance for survival and procreation in species that interact intricately with the world around. For a broad suite of frequencies, these hair cells must somehow embody a frequency tuning mechanism. The simplest solution on first view would be a construction by which each sensor inherently reacts to one specific frequency. In fact, the chordotonal organs and more specialized hearing organs that develop from them are found all over the insect bodyFor larger animals with an interest in a refined auditory environment, the natural solution is to locally concentrate the sensors. One complication, however, emerges: For nonlinear amplifiers, the superposition principle does not hold. Together with target frequencies, undesired interaction sound products are always generated (by amplifier interaction), which then are amplified by nearby amplifiers that have a characteristic frequency matching that of a combination tone . AmnioteThe simplest, relatively unsophisticated, arrangement of locally concentrated hearing sensors is found in the turtles and Tuatara, that probably still reflect the original stem reptile hearing systemLizards represent the next step of hearing sophistication. They show morphological gradients and variations of hair cells  frequencies across the device is needed. The single one-dimensional frequency arrangement first observed in the archosaurs27One of the driving organizational principles of Cortex is wiring optimizationWe now resume the discussion of how the mammals cope with the complexity cf.  that is From the classical signal processing dogma, undesired information should be filtered out as early as possible. Quite astonishingly, biological measurements and the corresponding models of the cochlea and cochlear nerve show that neither at the level of the cochlea nor higher up in the auditory pathway, does the mammalian auditory system make a noticeable effort to correct for the combination tones. Whereas filtering out at least some of the ‘artificial’ components seems perfectly possible for the biological system, this is just not how this system works32missing fundamental paradigm, Smoorenburg performed psycho-acoustical two-tone pitch-shift experiments. In these experimentsFrom other fields of physics , a common strategy for putting a grip on a complex phenomenon is to provide an overall ‘average’ characterization of the phenomenon. We now put forward that a similar effect could be the deeper nature of pitch perception. In the simple case of pure tone stimulations, pitch sensation coincides with the obvious physical properties of the stimulator. For slightly more complicated stimulations, the generated response develops, however, a profile of its own that departs substantially from the physical properties of the stimulating signal, due to characteristics that are rooted in the interaction among the nonlinear sensors. Such is the origin of the celebrated second pitch shift  investigfp has to be computed from the dominant peaks of the signal’s autocorrelation function in the cochlea  this happened. Combined approaches to the hearing system as an evolutionary prototype may finally shed light on the one fundamental question: What is the best computational framework for processing complex neural information? In light of the understanding that we have achieved regarding the first steps of the hearing pathway, such an expectation does not appear to be overly optimistic. If so, the physical principles underlying the hearing sensor evolution, would not only have provided us with (and have made us understand in a true sense) the blueprints for artificial hearing sensors of power and abilities that match the biological example, but would, moreover, reveal the fundamental principles followed by optimal signal processing.How to cite this article: Lorimer, T. et al. Mammalian cochlea as a physics guided evolution-optimized hearing sensor. Sci. Rep.5, 12492; doi: 10.1038/srep12492 (2015).


Current neuroscientific models describe the functional neural architecture of visual working memory (VWM) as an interaction of the frontal-parietal control network and more posterior areas in the ventral visual stream (Jonides et al.,  Working memory is a limited-capacity system for the maintenance and manipulation of information in service of ongoing tasks. The classic model of working memory ; instead, we will examine whether recent findings from neuroscientific studies of early VWM development can be fit into the adult model above.This conceptualization of WM is grounded in an extensive body of neuroscientific research, the majority of which has been conducted with human adults  or EEG. In this task, an object is hidden at one of two locations and the infant is allowed to manually search for it. Once the infant repeatedly succeeds at one location, the object is then hidden at the other location. In the looking-based version of this task, looking times to the two locations are contrasted.In one of the first studies to measure regional blood-flow changes in infants using fNIRS, Baird et al.  found thDuring the infancy period, functional connectivity of the VWM network appears to become less diffuse with age. Cuevas et al.  found anResting-state fMRI (rs-fMRI) has been used to identify functional connections between brain regions in the absence of any task. This latter aspect makes this method particularly attractive for studies of early development, as infants can be scanned during sleep. In a pioneering study, Alcauter et al.  tracked The involvement of posterior cortical areas in infant VWM has primarily been examined using more modern behavioral paradigms, such as Violation-of-Expectation (VoE), in conjunction with fNIRS, or EEG. Using fNIRS, Wilcox and colleagues found that the anterior temporal cortex showed consistent activation when infants noticed a change in the features of an object that they held in mind when it reappeared from behind an occluder . Studies by Wilcox, Kaufman and their colleagues found storage-related VWM activity in the temporal and occipital cortices as well, which may mirror similar findings in adults in the ventral visual stream  conducted with this notoriously challenging age range is primarily due to practical limitations: Preschool-age children require special experimental designs as they are rarely willing to participate for an extended time, and they often do not follow verbal instructions reliably. One notable limitation of three of the four fNIRS studies reviewed below is that hemodynamic responses were measured only in the frontal areas  have multiple connections to the medial temporal lobe  task .In addition to hippocampal lesions, Weiss et al.  found thThese findings suggest that the MTL gives rise to the development of PFC-associated VWM skills, such as manipulation and interference resolution, likely through reciprocal neuroanatomical connections . We also see some evidence for the functional reorganization of the network during the early life period: for example, a shift away from the salience network from infancy to adulthood . Studies on VWM mechanisms in early childhood have not recorded from these posterior areas, so our understanding of how these areas support VWM in this age range is, at the moment, limited.kA defining characteristic of VWM is its limited capacity. This functional characteristic can serve as a signature to identify VWM storage-related mechanisms: In these structures, activity is expected to gradually increase with the number of to-be-remembered items, and then remain constant when capacity limit has been reached .k), and measures neural activity as a function of capacity, in order to identify storage-related mechanisms.Quantifies capacity behaviorally  in the near future. Thus, a crucial open question for future studies is how neural activity in the VWM network changes in children under 3 years of age using this task. As well, future studies with preschool-age children that record from posterior cortices  awarded to ZK.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.


Horizontal gene transfer (HGT) or lateral gene transfer (LGT) involves the transmission of genetic material between distinct evolutionary lineages and can be an important source of biological innovation. For instance, the acquisition of foreign genes can allow recipient organisms to adapt to new lifestyles or to exploit a novel ecological niche, such as a host environment. HGT has long been recognised as an important factor contributing to the evolution of prokaryotic lineages especially in connection to the evolution of pathogencity ,2. HowevOomycetes are eukaryotic microbes that generally grow filamentously and feed osmotrophically by secreting enzymes into the external environment, breaking down complex molecules, and importing nutrients into the cell . These fThraustotheca clavata, that obtain nutrients from decaying matter, to parasites of plants and animals such as Phythophthora infestans and Saprolegnia parasitica, respectively )Phytophthora lineage, which includes plant parasitic oomycetes that establish hemibiotrophic interactions with their host plants  or Saprolegniales spp., which include nonpathogenic saprobes and opportunistic pathogens of plants and animals that obtain nutrients via saprotrophy and/or necrotrophy .,13.PhytoPhytophthora spp. (with a mean of 4.37 gene copies per genome per HGT) compared to the seven Pythium spp. that feed by saprotrophy and necrotrophy , fun, fun12])Oomycetes and fungi are distantly related, but they exhibit similarities in their osmotrophic feeding strategies, life cycles, and filamentous growth characteristics. Many of these characteristics, specifically those associated with the process of infecting plant tissue, were thought to be the product of convergent evolution ,20. Yet,Phytophthora spp. Treatment of Phytophthora spp. infections of plants has been hampered historically with limited transferability of antifungal pesticides [Taken together, this body of work shows that HGT has played an important role in the evolution of the oomycetes. Although the picture is somewhat biased by the focus of the published studies, this work suggests HGT has been particularly important in the evolution of hemibiotrophic plant pathogenic traits of sticides . UnderstS1 TableRepresentative protein IDs are also provided for each HGT gene family.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableThe total number of paralogues and the percentage of paralogues that were predicted to encode a secreted protein are displayed for each HGT gene family. The total number of HGT genes, mean rates of duplication, and percent of total ORFs that have been horizontally acquired are displayed for each oomycete species considered in the analysis.(XLSX)Click here for additional data file.


Lichtheimia ramosa is an infrequent opportunistic infection that can potentially be angioinvasive when affecting inmunocompromised hosts. We present a fatal case of mucormycosis, affecting a 56-year-old male with diabetes mellitus and siderosis, initially admitted to our hospital due to an H1N1 infection. The subject's clinical condition worsened and he finally died because of a necrotizing bilateral pneumonia with disseminated mycotic thromboses due to Lichtheimia ramosa, which is an emerging Mucoralean fungus. This is an infrequent case because of the extent to which it affected a subject without overt immunocompromise. This case underlines the importance of an early premortem diagnosis and treatment in order to prevent rapid progression of this disease, as well as the need of considering mucormycosis when facing subjects with multiple emboli and fever unresponsive to usual antimicrobials.Mucormycosis due to A 56-year-old man was admitted with a one-week history of nonproductive cough, myalgias, dyspnoea, and fever (39°C). The subject was a former smoker, with a daily intake of 104 g of alcohol. His past medical history included well-controlled type 2 diabetes mellitus and siderosis, which had been diagnosed by bronchoscopy after finding a mild interstitial pattern in the CT. Lung function was normal at all times and the patient was never treated with oral corticosteroids before hospital admission. He also suffered from left renal agenesis. A chest radiograph showed a bilateral pneumonia and empirical treatment with meropenem and levofloxacin was started. Oseltamivir was also started empirically, given that this subject was admitted during later winter and at high risk of suffering an infection caused by the H1N1 influenza virus.Worsening of the subject's clinical condition prompted the performance of a computed tomography (CT) 5 days after admission, which confirmed the diagnosis of pneumonia and incidentally found an acute pulmonary bilateral thromboembolism Figures . Low mol Streptococcus pyogenes, the antibiotic therapy was switched to meropenem and cotrimoxazole. Transbronchial biopsies showed nonspecific acute alveolar damage.A bronchoscopy was performed on the 7th day after admission and due to positive sputum cultures forAfter 9 days of progressive recovery, the subject's clinical condition worsened again, with fever, high acute phase reactants, leukocytosis, odynophagia, and persistent neck pain. Antibiotics were switched again to meropenem and linezolid and a neck-CT plus a neck ultrasonography were performed, with findings suggestive of a de Quervain subacute thyroiditis . High-doAnother thoracic CT was performed 18 days after admission, showing multiple cavitary nodules and masses in both lungs , as wellVoriconazole was empirically started for a presumed fungal infection, and both transthoracic and transesophageal echocardiograms were negative for endocarditis. Serologic tests for HIV and B and C hepatitis were negative, as were autoimmune markers such as ANA and ANCA. A bone marrow biopsy was also performed but did not show any sign of hematologic malignancy.On the third day of established kidney dysfunction, haemodialysis was started. On the 25th day after admission, the subject's respiratory condition worsened again, requiring invasive ventilation. On the 26th day after admission, the subject suffered from neurologic worsening, consisting of a decline of consciousness, with a GCS of 7 and a left unreactive pupil. A cranial CT showed a left frontal hematoma with mass effect , prompti Lichtheimia ramosa, with mycotic thrombotic embolisms in the lungs , kidney, brain, thyroid, heart, liver, spleen, mediastinal lymph nodes, bladder, ureter, and small bowel.Necropsy results demonstrated invasive mucormycosis due to Mucor, Rhizopus, or Lichtheimia (previously known as Absidia). It manifests in a variety of syndromes, affecting mainly immunocompromised subjects  and those with diabetes mellitus, iron overload, and under treatment with glucocorticoids and it can also be manifested by cutaneous and subcutaneous disease in some trauma patients without known immunocompromise. These fungi are ubiquitous, and both rhino-orbital-cerebral and pulmonary mucormycosis are acquired by the inhalation of their spores. They are potentially angioinvasive; thus the disseminated disease is characterized by vascular invasion and thrombosis. Despite their low virulence, Lichtheimia species are currently regarded as emerging pathogens among Mucoralean fungi [Mucormycosis is an opportunistic invasive infection caused by fungi of the order Mucorales, likean fungi .We are presenting a fatal case of invasive mucormycosis in a subject where the main underlying condition was diabetes mellitus, although it was well-controlled, and without traces of ketoacidosis. Some authors  have pre Lichtheimia, although this species of Mucoral fungi should be regarded as an emerging pathogen according to recent reviews [It is possible that our subject had a healthcare associated mucormycosis after having been admitted at our hospital for an H1N1 infection with a secondary pneumonia, although we did not search for any particular portal of entry and, in any case, Mucoralean fungi are ubiquitous. In this review , 22% of This case report shows that seemingly inmunocompetent individuals can develop fatal fungal infections. The necropsy showed thrombotic mycosis in the lungs, kidney, brain, thyroid, heart, spleen, liver, lymph nodes, bladder, ureter, and small bowel. The two typical presentations are rhino-orbital-cerebral and pulmonary disease , and onc Lichtheimia ramosa are similar to those of an invasive aspergillosis. It is important to think in mucormycosis as well as in aspergillosis, as the antifungal agents appropriate for each infection are different [In subjects who develop features suggestive of widespread thrombosis and infarction and a fever unresponsive to antibacterial antibiotics, fungal infections (including mucormycosis) should be considered in order to make an early diagnosis . In addiifferent . Lichtheimia ramosa and other mucormycosis. Lichtheimia is usually sensitive to posaconazole, which is possibly the preferred oral therapy. Amphotericin B is the most active drug against Lichtheimia species, with a geometric mean in vitro of 0.07 mg/liter at 24 h, while posaconazole had the highest activity among the azoles (geometric mean of 0.34 mg/liter at 24 h) [Amphotericin B is the preferred empiric treatment forat 24 h) . Treatmeat 24 h) .Lichtheimia ramosa) and the fatality of the fungal infection in a seemingly inmunocompetent host. It summarises the difficulty of making an early diagnosis and the importance of considering mucormycosis when subjects develop widespread thrombosis and fever unresponsive to other antimicrobials.In conclusion, the case we present represents an infrequent entity because of the causal species (Consider mucormycosis when multiple emboli and fever unresponsive to usual antimicrobial are present.Early premortem diagnosis in immunocompromised patients diminishes morbidity and mortality.When should a mucormycosis be suspected? Lichtheimia ramosa?How can we manage an infection due toWhen should a mucormycosis be suspected? Mucormycosis is an opportunistic infection which can be invasive when affecting immunocompromised hosts, causing vascular invasion and thromboses. Although some authors have presented cases demonstrating that mucormycosis can occur in healthy individuals without any contributory factors, even well-controlled diabetes may represent a risk factor for Mucoralean infections. Lichtheimia ramosa? The clinical course and the imaging of invasive Lichtheimia ramosa are similar to those of invasive aspergillosis. However, antifungal agents appropriate for each infection are different. Amphotericin B is the preferred empiric treatment for Lichtheimia ramosa and other mucormycoses. Lichtheimia is usually sensitive to posaconazole, which is possibly the preferred oral therapy. Treatment of mucormycosis involves a combination of empirical antifungal treatment and surgical debridement of involved tissues.How can we manage an infection due to


Recent research has revealed an association between hearing impairment and dementia. The objective of this study is to determine the effect of hearing impairment on dementia incidence in a longitudinal study, and whether ear, nose, and throat (ENT) specialist care, care level, institutionalization, or depression mediates or moderates this pathway. The present study used a longitudinal sample of 154,783 persons aged 65 and older from claims data of the largest German health insurer; containing 14,602 incident dementia diagnoses between 2006 and 2010. Dementia and hearing impairment diagnoses were defined according to International Classification of Diseases, Tenth Revision, codes. We used a Kaplan Meier estimator and performed Cox proportional hazard models to explore the effect of hearing impairment on dementia incidence, controlling for ENT specialist care, care level, institutionalization, and depression. Gender, age, and comorbidities were controlled for as potential confounders. Patients with bilateral  and side-unspecified  hearing impairment had higher risks of dementia incidence than patients without hearing impairment. We found no significant effect for unilateral hearing impairment and other diseases of the ear. The effect of hearing impairment was only partly mediated through ENT specialist utilization. Significant interaction between hearing impairment and specialist care, care level, and institutionalization, respectively, indicated moderating effects. We discuss possible explanations for these effects. This study underlines the importance of the association between hearing impairment and dementia. Preserving hearing ability may maintain social participation and may reduce the burden associated with dementia. The particular impact of hearing aid use should be the subject of further investigations, as it offers potential intervention on the pathway to dementia. The prevention and treatment of dementia has become a public priority in the course of demographic ageing. Although studies suggest that trends in medical, lifestyle, and societal risk factors of dementia may have led to a reduction in the prevalence of dementia , there sWhile no cure of dementia is available at present, the future number of dementia cases may be altered by disease-modifying interventions to prevent or slow down the progression of the disease . EmpiricBoth diseases have a high relevance for society. The prevalence of dementia increases exponentially with age and doubles every five to six years, from about 2% at age 65 to 30–40% between ages 90 to 100 –15 and aThere is no common definition or measure of hearing impairment and hearing loss. This makes it difficult to compare prevalence estimations . The mosCommon to both may be social withdrawal, reduced perception, repeated questioning, impaired short-term or reduced working memory, word finding disorders or communication disorders, as well as problems in following a conversation , 30–34.Possible aspects that may explain the association between dementia and hearing impairment have been summarized by Kilimann et al. . First, Given the knowledge of the associations between hearing impairment and cognitive decline as well as dementia, Dawes et al.  formulatAfter establishing a regional association between hearing impairment and dementia in our previous study , the aimWe used data of the AOK (Allgemeine Ortskrankenkasse), which is the largest health insurance company in Germany. About one third of the German population is insured through the AOK and the proportion increases with age . A randoData access was legally approved by the WIdO. The study is based on anonymized administrative claims data that never involved patients directly. Individual patients cannot be identified and the analyses presented do not affect patients whose anonymized records were used.th Revision (ICD-10): G30, G31.0, G31.82, G23.1, F00, F01, F02, F03, and F05.1. Due to the character of health claims data as a secondary data source, it was not possible to validate dementia diagnoses in face-to-face examinations. In order to avoid a potential overestimation of the true number of cases we applied a validation procedure ; bilateral hearing impairment ; unspecified hearing impairment without information on laterality ; all other diseases of the ear and mastoid process, including all diagnoses not related to hearing impairment. In the case of simultaneously occurring diagnoses we took only one of them into account in the following priority order from high to low: bilateral hearing impairment; unilateral hearing impairment; unspecified hearing impairment; all other diseases of the ear and mastoid process.We took four key variables into account as potential mediators or moderators. We controlled whether the patient ever received treatment by an ENT doctor during the observation period, but only in the presence of at least one of the above-mentioned hearing impairment diagnoses. We controlled whether the person lived in a nursing home, whether the person ever had a diagnosis of depression during the observation period (ICD-10 codes F32 and F33), and for care level. According to the care level, individuals are entitled to benefits or services from the German statutory long-term care insurance. After passing an assessment mainly based on impairments in activities of daily living, applicants are assigned to one of three care levels . We then addressed a series of co-morbidities: diabetes mellitus (ICD-10 codes E10–E14); hypertension ; hypercholesterolemia (E78.0); cerebrovascular disease ; cancer ; diseases of the heart ; chronic lung diseases ; rheumatism ; paralysis ; injuries to the lower extremities ; blindness (H54). A variable in the model indicates whether a person received no, one, two, three, four, five, six, or seven or more of these diagnoses during the observation period. We also took into account a tinnitus diagnosis (H93.1). Demographic information contained gender and five-year age-groups beginning at age 65.We applied methods of event history analysis to analyze the risk of incident dementia. We used the Kaplan-Meier estimator to measure the fraction of subjects living without a dementia diagnosis for a certain amount of time after the year 2006. Cox proportional hazard models were conducted to analyze the determinants for the risk of dementia incidence. Persons were followed until death or censoring by leaving AOK insurance coverage or death. The censoring time was set in the middle of the last observed quarter, and deaths were assumed to have been in the middle of the month of death. We examined direct associations between hearing impairment and dementia incidence in models adjusted for age, gender, co-morbidities, and with and without the four potential mediators or moderators. A mediating effect appears when the following criteria are met: First, hearing impairment affects the risk of dementia incidence. Second, the potential mediator affects dementia onset in a model that controls for hearing impairment. Third, the coefficients of hearing impairment in the latter model decreases when controlled for the potential mediator. A moderating effect is explored by the interaction effect of hearing impairment and the variable of interest.The 154,783 persons contributed 669,524 person-years and 14,602 incident dementia diagnoses during the period 2006 to 2010 . The oveKaplan-Meier estimators for hearing impairment and each key variable on its own (results not shown) revealed that persons with bilateral hearing impairment experienced a faster incident dementia diagnosis compared to all other persons. Persons who received treatment by an ENT doctor experienced a faster incident dementia diagnosis compared to persons without such treatment. The higher the care level the faster the transition to dementia and the same was true for those living in a nursing home. Depression was also associated with a faster transition to dementia. The combined information of hearing impairment and the key variables, respectively, provides a more particular view of the relationship Figs –4. BilatHazard ratios estimated by multivariate Cox proportional hazard models are presented in In interaction effects models, we found significant main effects for hearing impairment, particularly for bilateral and unspecified hearing impairment which increase the risk of dementia. We found a significant main effect for treatment by an ENT doctor which lowers the risk of dementia, while the main effects of care level, nursing home, and depression are associated with an increased risk. The statistically significant interaction effect below one  for those with bilateral hearing impairment and professional treatment implies a comparatively lower dementia risk of the professionally treated as compared to those without ENT treatment . There wHearing impairment increases the risk of dementia. The effect is partly independent of treatment by an ENT doctor, care level, institutionalization, depression, and co-morbidities. This is not only in line with our previous study  and otheOur focus is on hearing impairment, and according to the International Classification of Diseases we also take laterality as well as other diseases of the ear and mastoid process not related to hearing impairment into account. As expected we found significant effects for bilateral hearing impairment, i.e. an increased risk for dementia incidence. We also found an increased risk of dementia for persons with unspecified hearing impairment. We suppose that this effect is driven by underlying bilateral hearing impairment although the laterality is unspecified by the diagnosing physician. Controlling for all information in the full model Model 5, , we founIn order to assess actual hearing ability, it is necessary to use formal audiometry measures, such as pure tone threshold average, speech discrimination scores, or word recognition scores . Whilst We adjusted for a series of information to obtain potential mediating or moderating effects on the pathway between hearing impairment and dementia. One important aspect in the analysis of hearing impairment and dementia is the use of hearing aids. Hearing aids may delay or prevent the occurrence of dementia by improving the ability of hearing. Studies found a positive association of hearing aid use and cognition . UnfortuWe included care level to account for the general frailty and vulnerability of older persons and found an increased risk for dementia with higher care level. Findings on the association between frailty and dementia are somewhat inconsistent with regard to type of dementia. Gray et al.  found evOur results reveal that living in a nursing home had a direct effect on the transition to dementia but does not decrease the effect size of hearing impairment. Thus, institutionalization does not work as a mediator in our study. With regard to the significant interaction effect below one for the individuals with bilateral hearing impairment and who were institutionalized, institutionalization rather works as a moderator in our study. We suggest that individuals living in a nursing home benefit from being under daily observation, and that an occurring or existing hearing impairment is more likely to be noticed and treated, subsequently leading to a decreased risk for dementia.Another important aspect is depression. Depression is associated with hearing impairment ; it is bA high number of co-morbidities increased the risk of dementia incidence. The indicator used in our study includes several diseases known as risk factors for cognitive decline and dementia, e.g. vascular diseases –64, cereAlthough we controlled for a series of relevant information, there remained a direct effect of hearing impairment on dementia. Several possible mechanisms may explain this effect. First, a common neurodegenerative process may induce hearing impairment as well as cognitive impairment through decreased functionality of the auditory system  and of tWe have to consider potential limitations associated with the data. Other studies analyzed the specific impact of hearing impairment on Alzheimer’s disease or dementia of the Alzheimer’s type , 75, 76.Health claims data are primarily relevant for cost reimbursement and cost calculation, which leads to two issues. First, only those diagnoses leading to treatment are relevant for the purposes of cost calculation. Thus, a patient’s cognitive impairment might not be documented if no further treatment is given. This could be particularly true for mild cases of dementia and cognitive impairment. The incidence of dementia will certainly be biased to higher ages when the symptoms of the disease become more obvious . Second,The strength of this study is the large longitudinal sample with more than 150,000 persons including the institutionalized. Excluding the institutionalized population would lead to a bias, as dementia incidence is four times higher among those living in nursing homes or assisted living compared to those elder who do not live in such facilities . SimilarHearing impairment increases the risk of dementia incidence, underlining the importance of good hearing ability. A screening for hearing impairment should be considered in case of cognitive impairment , and a wS1 TableData source: Claims data AOK 2006–2010. IR: Incidence rate per 100 PY. LCI: 95% lover confidence interval. UCI: 95% upper confidence interval.(DOCX)Click here for additional data file.S2 TableS2 Table presents the effects of gender, age, comorbidities, tinnitus, treatment by an ENT doctor, care level, nursing home, and depression, respectively, not shown in Tables (DOCX)Click here for additional data file.


Understanding underlying pathological mechanisms is prerequisite for a sensible design of protective therapies against hearing loss. The triad of age-related, noise-generated, and drug-induced hearing loss displays intriguing similarities in some cellular responses of cochlear sensory cells such as a potential involvement of reactive oxygen species (ROS) and apoptotic and necrotic cell death. On the other hand, detailed studies have revealed that molecular pathways are considerably complex and, importantly, it has become clear that pharmacological protection successful against one form of hearing loss will not necessarily protect against another. This review will summarize pathological and pathophysiological features of age-related hearing impairment (ARHI) in human and animal models and address selected aspects of the commonality (or lack thereof) of cellular responses in ARHI to drugs and noise. Acquired hearing loss is the most common hearing impairment in modern societies. Not all of its causes, however, are of modern origin. Of the major contributing entities, presbycusis or age-related hearing impairment (ARHI) has been recognized by physicians and societies since ancient times. The environmental overload with damaging noise levels, beginning with the industrial revolution, and the introduction of ototoxic antibiotics and anti-cancer agents in the last century added to the spectrum of hazards to the auditory system and established the current triad of drug-induced, noise-induced and age-related hearing losses.Medical science and research over the last centuries have given us an excellent knowledge of inner ear pathologies and their functional consequences. Recent advances have added insights into molecular pathways, their physiological control mechanisms, and their modulation by environmental and genetic influences. Intriguing biochemical and molecular parallels began to surface, leading to speculation of a common underlying mechanism of acquired hearing loss and the prospect for a unified pharmacological protection. However, details about the individuality of cell death and homeostatic pathways and differentiated responses to attempted protection soon dispelled this notion. This review will focus on ARHI and compare its biochemical and molecular characteristics to ototoxic hair cell death and noise trauma. While the overall pattern of presbycusis is also shaped by changes in central auditory pathways, we will limit our considerations to the cochlea.ARHI is the most prevalent form of hearing loss in humans. It is characterized by decreased hearing sensitivity, decreased ability to understand speech in a noisy environment, slowed central processing of acoustic stimuli, and impaired sound localization  is affected in both genders by 60–69 years  gene at the ahl locus predisposing them to accelerated hearing loss, distinct from the late-life age-related changes in other mouse strains and in human. Indeed, no convincing evidence yet exists associating variants in human CDH23 with ARHI. For example, a recent study of 310 aging participants with poor hearing vs. 308 participants with good hearing found no association of a common CDH23 variant with presbycusis .Animal models that reflect a sensorineural ARHI with loss of outer and, later, inner hair cells include aging rats and mice. CBA/J mice as well as the CBA/CaJ and CAST strains present little evidence of hearing loss until 18 months of age, i.e., long into their lifespan, when they begin to show hair cell loss and elevated auditory thresholds , compound action potential and distortion-product otoacoustic emission  than in younger (15–38 years old) subjects  are produced in every cell by enzymatic reactions and mitochondrial respiratory mechanisms. However, only their excess—either via overproduction or insufficient detoxification—will create oxidative stress and potentially trigger cell death. A well-maintained redox balance is important for the cochlea as for any other tissue. Transgenic mice with deficiencies in antioxidant systems, lacking the enzyme copper/zinc superoxide dismutase, lost hair cells and auditory neurons to a greater extent than their normal littermates  for the 13 proteins that it encodes are more likely to occur and persist in a lifetime than mutations in nuclear DNA. As a consequence, mitochondrial function may be compromised and pathology can become evident, varying with cell type and the type of mutation  and old (18–26-month-old) CBA/J mice with hearing deficits. The middle-aged mice also bore nuclei with necrotic features, which were absent from old mice  are a family of kinases that include c-jun NH2-terminal kinases (JNK), p38 MAPKs, and other extracellular signal-regulated kinases. Immunohistochemical analyses in the cochlea of aging CBA/J mice demonstrated increased phosphorylation  of JNK and p38 MAPK in outer hair cells  for the last 3 years of their lives . To what extend these two effects can be distinguished is an interesting theoretical question but of no practical consequence. Diet is an easily modifiable factor and any delay in ARHI would have important implications for public health. Unfortunately, there is no simple remedy.2), magnesium, and lycopene (a carotenoid antioxidant). Similarly, an epidemiological study of 1,910 Korean subjects aged 50–80 years compared their hearing thresholds with self-reported intake of dietary supplements , and niacin (vitamin B3) positively correlated with better thresholds. A survey of a general population in the USA aged 20–69 years  likewise found that diets high in vitamin C, beta-carotene, and magnesium were correlated with better hearing  and vitamin B12  with low serum levels of folic acid  state are confounding factors that are capable of influencing the success of therapeutic interventions. Furthermore, subtle forms of auditory damage may escape simple audiometric tests and a final verdict on success or failure of protective strategies may have to await more sophisticated audiological assessments.There is another unresolved question. While a short-term administration of antioxidants or vitamins might be safe for drug and noise exposure, the hazards of a life-long regimen of nutritional supplements are unknown. Studies in animals as well as clinical trials have warned of a potentially increased risk of cancer and mortality associated with prolonged consumption of antioxidants or vitamins. Specifically, meta-analyses of clinical trials singled out beta-carotene, vitamin A, and vitamin E as associated with increased overall mortality (Miller et al., When we compare the mechanisms of drug, noise, and age-related hearing loss, there appear to be more similarities between drug- and noise-induced hearing loss than between those two and age Table . One of Looking ahead, regeneration of human hair cells seems decades away but other approaches may provide new directions. Some aspects of ARHI may relate to the phenomenon of cochlear synaptopathy, causing “hidden hearing loss” by the loss of auditory nerve fiber/hair cell connections (Kujawa and Liberman, 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.


Previous studies investigating speech perception in noise have typically been conducted with static masker positions. The aim of this study was to investigate the effect of spatial separation of source and masker  in a moving masker setup and to evaluate the impact of adaptive beamforming in comparison with fixed directional microphones in cochlear implant (CI) users.0N0 and in a moving masker setup (S0Nmove) in 12 normal hearing participants and 14 CI users . Speech processor settings were a moderately directional microphone, a fixed beamformer, or an adaptive beamformer. The moving noise source was generated by means of wave field synthesis and was smoothly moved in a shape of a half-circle from one ear to the contralateral ear. Noise was presented in either of two conditions: continuous or modulated.Speech reception thresholds (SRT) were measured in S0Nmove setup were significantly improved compared to the S0N0 setup for both the normal hearing control group and the bilateral group in continuous noise, and for the control group in modulated noise. There was no effect of subject group. A significant effect of directional sensitivity was found in the S0Nmove setup. In the bilateral group, the adaptive beamformer achieved lower SRTs than the fixed beamformer setting. Adaptive beamforming improved SRT in both CI user groups substantially by about 3 dB  and 8 dB  depending on masker type.SRTs in the SCI users showed SRM that was comparable to normal hearing subjects. In listening situations of everyday life with spatial separation of source and masker, directional microphones significantly improved speech perception with individual improvements of up to 15 dB SNR. Users of bilateral speech processors with both directional microphones obtained the highest benefit. Regularly, background noise is present which compromises speech perception even in normal hearing listeners. Depending on the environment, acoustical properties of noise differ in terms of spectral shape and temporal envelope. Additionally, noise masker position may vary during a conversation. Binaural hearing enables localization of sound sources and perceptual separation of speech sources from irrelevant noise . SpatialRecipients of cochlear implants (CIs) in both ears or with bimodal fitting  can benefit from bilateral hearing to improve speech perception in interfering noise. They also show a benefit for different masker and target positions –6. BesidOver the last decades, improved stimulation strategies ,12 and nAs demonstrated in In summary, the aims of the present study were (1) to investigate SRM in a moving masker setup with different temporal envelopes of masker in normal hearing subjects and CI listeners, and (2) to evaluate the impact of adaptive beamforming compared to fixed directional microphones on speech perception in moving noise.12 normal hearing participants  and 14 CI users  took part in the measurements. In the CI group, 7 of the participants were bilateral CI users (mean age 49.9 ± 14.3 years) and 7 were bimodal CI users (mean age 49.6 ± 19.1 years) with a hearing aid in the contralateral ear. All CI patients were users of Cochlear CP810 speech processors and were tested in best aided condition. Demographical data of CI users is shown in The study was approved by the local institutional review board . Subjects gave their written consent of participation and received financial compensation for their participation.The individual clinical standard MAP with ACE strategy  was usedStandard: A moderately directional microphone, with about 5 dB attenuation at 180°. This moderate amount of directionality mimics the natural ear.Fixed (ZOOM): Narrower beam towards the front than standard with about 5 dB attenuation at 180° compared to 0° with maximal attenuation of about 17 dB at 120°. Polar plots of standard and fixed directionality of the CP810 speech processor are shown in  = 14.21, p < 0.001) and noise type  with an interaction of subject group and noise condition . A Tukey post hoc analysis confirmed lower SRTs in setting adaptive compared to fixed (p = 0.015) and standard (p < 0.001) setting. Post hoc analysis showed significantly lower SRTs in the bilateral group in continuous noise for adaptive compared to fixed  and to standard  settings. SRT in the adaptive setting was found to be significantly lower than the standard setting in modulated noise . No differences between directional sensitivity settings were found in the bimodal group, probably generated by large between subject variability.Individual and mean speech reception thresholds of CI users in Sstandard and fixed or adaptive settings was calculated. The results are illustrated as SRT improvement in fixed setting and -0.4 dB to 15.1 dB in the adaptive setting compared to standard.Individual subjects SRTs were found to be highly independent of directional sensitivity settings . This isadaptive compared to the fixed setting in the bimodal group  and in the bilateral group  in continuous noise. A comparison between bilateral and bimodal group in the adaptive setting revealed significantly higher improvement in the bilateral group  whereas for fixed setting only a tendency of higher improvement was observed.Significantly higher improvement of SRT was observed in the In modulated noise, significant differences between different directional sensitivity settings and subject groups were absent.0N+90 or S0N-90 setups. The present study examined speech reception thresholds and SRM in a continuously moving noise source setup with cochlear implant users.Spatial release from masking (SRM) in cochlear implant patients was investigated in previous studies solely in fixed loudspeaker setups  with meThe first part of the discussion in the following compares SRM in moving noise setup to results from studies with a fixed masker position. Furthermore, the effect of the two different masker types and the contribution of binaural cues in cochlear implant users are discussed.The second part of discussion describes the potential impact of adaptive beamforming on speech perception in cochlear implant users.In listeners with normal hearing, the presence of short temporal gaps in the envelope of masking noise leads to strong SRT improvement. Compared to continuous masking, the beneficial effect amounts to about 7 dB with modulated masking noise . The expThe beneficial effect of a co-located fluctuating masker in normal hearing subjects was reaffirmed in the present study group (8.7 dB). The glimpsing effect was 2 dB lower with moving masker (6.7 dB) compared to the static masker condition.0N0 and S0Nmove setups. The reason for the absence of the glimpsing effect in bilateral CI users is still unclear.Most hearing-impaired patients suffer from deteriorated gap listening abilities manifested in higher thresholds, which presumably do originate from slow rates of recovery . The abs0N0 setup). The large variability in SRT in modulated masker condition observed in the bimodal group is mainly explained by the large variations seen in the individual pure tone audiograms . Since this correlation was absent in continuous masker condition, potentially a residual glimpsing effect is responsible for this effect observed in the bimodal listener group.Concerning the bimodal subject group, limited aided hearing in the contralateral ear may enhance individual performance due to a residual glimpsing effect. Compared with the bilateral CI subject group, bimodal listeners showed a tendency to benefit from acoustic hearing in the contralateral ear . In the0N90 and 1.4 dB for S0N180 in normal hearing subjects applying a continuous masker of a spectral shape resembling the speech signal. Vom Hoevel and Platte [0N90 but a higher SRM of 4 dB in S0N180. In the present study, mean SRM in the normal hearing group was 4.3 dB for continuous noise. This result is in line with Plomp and Mimpen and vom Hoevel and Platte since an SRM lower than in S0N90 but higher than in S0N180 setup would be expected in the moving masker setup.Plomp and Mimpen  reportedd Platte  reportedfm = 4 Hz and a modulation of m = 62%. This setting is comparable to the modulated masker used in the present study. As outlined in the results section of the present study, the SRM difference between continuous and modulated noise was 2 dB. This result is in line with Carhart et al. who identified a difference of 1.7 dB between white noise and modulated white noise (S0N90 speaker setup). The reduced SRM in modulated noise compared to a continuous masker could be explained by the “glimpsing effect” [0N0 setup of about 8 dB, the effect of source separation on speech perception is lower compared to continuous noise conditions.Carhart et al.  measured effect” . Since tSpatial release from masking in CI users was investigated in several previous studies with fixed masker positions. In the following, studies with maskers at the sides of or behind the listener are compared with the results of the present study.0N0 vs. S0N-90 and S0N+90) in a group of bilateral CI patients  in continuous noise. Van Hoesel and Tyler [standard (everyday preprocessing condition), which was used in the present study. Therefore, SRM in S0N90 is expected to be about 1.5 dB higher in this population of CI users with a moderately directional microphone compared to speech processors with omnidirectional microphone.Schleich et al.  reportednd Tyler  reportedAccording to previous studies concerned with SRM in fixed masker position, lower SRTs compared with co-located masker measurements were expected due to head shadow and squelch effect in the moving masker paradigm. In the present study, the impact of head shadow on interaural level differences was not constant over the presentation of the moving masker.At a masker position of 180°, head shadowing is absent. At this position, interaural time differences and interaural level differences of speech and masker are expected to be close to zero and only summation effects potentially improve speech perception. However, in the present study, the actual duration of presentation of the masker at a location of about 180° was below 100 ms. Therefore, substantial summation effects were unlikely in such a short time period.adaptive beamforming algorithm substantially decreased SRTs in both patient groups compared to the standard microphone directionality by 3.0 dB  and 7.8 dB  in the continuous noise condition. Even in the modulated noise condition, a large effect was present . In everyday listening situations, the occurrence of modulated noise is more frequently expected. Therefore, it can be expected that the current implementation of the beamforming algorithm will provide additional benefit compared to standard directional sensitivity in everyday life challenging listening situations.The standard and adaptive beamforming conditions, mean individual improvement in the bilateral group is about twice as high as in the bimodal group for both continuous noise (3.0 vs. 7.8 dB) and modulated noise (3.3 vs. 6.6 dB). The binaural combination of adaptive beamforming seems to double SRT improvement. It is obvious that bimodal patients receive only half of the beneficial effect, since in the moving noise source setup the support of beamforming is only present for half of the speech stimulus sentence. In contrast, wearing bilateral adaptive beamformers ensured noise reduction during the whole sentence presentation when the noise source travels around the back of the CI listener.Comparing standard setting (“everyday”) and fixed (“ZOOM”) is the angle of minimal sensitivity at 120°, and the level of attenuation at this direction. The adaptive (“BEAM”) setting applied different frontal directionality compared to fixed setting. Therefore, it remains difficult to distinguish whether the amount of SRT improvement was due to the ability of “BEAM” to adapt to the noise source or by generally enhanced frontal directionality. However, the results of the present study clearly showed the benefit of microphone directionality for speech perception in free field conditions.The three different microphone sensitivity settings applied a different amount of directional sensitivity to the front. The difference between 0Nmove setup, the adaptive setting on the side opposite to the noise could employ the microphone input signal to form a better reference signal. Kokkinakis and Loizou [0N90 setup with two synchronized BEAM algorithms compared to independently running speech processors. Buechner et al. [rd order beamformer (two synchronized Phonak Ambra hearing aids connected to the CI via auxiliary input) in a quasi-diffuse noise field S0N±70, ±135,180.One limitation in today’s CI speech processors and most hearing aids is the independent operation of each device. It is expected that bilateral CI users will benefit from synchronization of both CI speech processors. With bilaterally synchronized processors in Sd Loizou  already r et al.  reported0N90 setup. Differing from the present study where two omnidirectional microphones were applied, beamforming was implemented using the combination of directional and omnidirectional microphone.The performance of adaptive beamforming algorithms was previously reported by Wouters and Vanden Berghe , and SprWouters and Vanden Berghe reported a mean improvement in SRT (lists of numbers) of 9.5 dB in speech-weighted noise and 10 dB for ICRA noise compared to the standard directional microphone. These results were obtained by feeding the preprocessed stimuli recorded via BTE processor positioned at the artificial ear of a dummy head into the auxiliary input of a clinical speech processor.adaptive setting “BEAM” implemented in the Cochlear Freedom speech processor (predecessor of the CP810 device). SRT improvement measured in 5 unilateral CI users for the Dutch/Flemish LIST sentences [Spriet et al. investigated the entences  was repoMean improvement in the aforementioned studies with adaptive beamforming was higher than in both CI subject groups of the present study. This difference may be explained by the position of the noise source, which was fixed at 90° in those studies. Furthermore, patients were tested monaurally and the noise source was located at the implanted ear. In contrast to monaural test conditions in the aforementioned studies, all subjects in the present study were binaural listeners (CI+HA or CI+CI). Binaurally aided subjects can benefit from head shadowing during the moving noise masker presentation (except for 180°), independent from the setting of directional sensitivity. Thus, there is less room for improvement from using beamforming algorithms when comparing binaural and unilateral listening.Spatial release from masking (SRM) was present in hearing-impaired listeners using cochlear implants either bimodally or bilaterally. A semi-circular motion of a masker in the back during speech presentation from zero degrees azimuth decreased SRM compared to a fixed masker position at 90°. The amount of benefit obtained from SRM (about 3 to 4.5 dB) was comparable between bimodal and bilateral CI users as well as normal hearing subjects.The results of the present study demonstrated the impact of advanced signal processing applied in CI processor devices in listening situations with spatial separation of source and noise masker. Compared to fixed microphone sensitivity, adaptive beamforming yielded improvements of up to 15 dB SNR especially in moving masker setups. Bilaterally worn CI devices employing adaptive beamforming obtained the largest improvement. However, beamforming algorithms are known to work best in anechoic conditions. Therefore, further investigations of everyday listening situations in reverberant rooms are of interest. A combination of beamforming with other preprocessing (e.g. noise reduction) could potentially improve speech perception, especially in reverberant conditions.As demonstrated by Rader et al. , temporaIn the future, the implementation of binaural signal processing strategies with synchronized adaptive beamforming could further improve speech perception in complex noise environments.S1 Table(XLS)Click here for additional data file.


Star, Cyp11a1, Cyp17a1, and Hsd3b2) were observed in the cocultured TCs. Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs. This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development. Androgen secreted by theca cells (TCs) can initiate follicle development and ovulation and provide precursor materials for estrogen synthesis. Therefore, studies on ovarian microenvironment will not only lead to better understanding of the steroidogenesis but also have clinical significance for ovarian endocrine abnormalities such as hyperandrogenism in polycystic ovary syndrome (PCOS). This study applied the Transwell coculture model to investigate if the interaction between granulosa and theca cells may affect androgen production in theca cells. Concentrations of testosterone and androstenedione in the spent medium were measured by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The results show that the coculture with granulosa cells (GCs) increases steroidogenesis in TCs. In addition, testosterone and androstenedione productions in response to LH stimulation were also increased in the coculture model. Significantly increased mRNA expressions of steroidogenic enzymes ( Oocyte, granulosa cells (GCs), and theca cells (TCs) are the main components of follicles, and the functions and interactions among them play crucial role(s) in steroidogenesis, follicular development, and atresia . Oocyte- Cyp17 mRNA and protein expression in rats [Androgen is mainly secreted from ovary and adrenal gland. In ovaries, the steroidogenesis is accomplished by both the granulosa cells and theca cells. The synthesis of various steroids is determined by cell-specific expression of a series of steroidogenic enzymes. The transportation of cholesterol into the mitochondria is facilitated by STAR which regulates the production of androgens. It is established that the androgen-secreting theca cells express P450Scc (CYP11A1), P450C17 (CYP17), and HSD3B2, all of which are key enzymes essential for androgen biosynthesis. In hyperandrogenemia patients, the circulatory testosterone and androstenedione are primarily produced by follicular TCs of ovary . Althoug in rats , 10 and In vitro studies have shown that, as a key feature of TCs, the increment of androgen production was driven by LH in a dose-dependent manner [t manner , 11, 12.t manner . There at manner . SpecifiThe objective of this study is to explore the functional interactions between GCs and TCs and their involvement in steroid metabolism. We applied the Transwell coculture model to determine the effects of GCs on the steroidogenic function of TCs in the mouse preantral follicles by measuring the change of androgen concentration in response to LH stimulation. Results of the study will provide basic information on the significance of cell interactions for steroidogenesis in ovary. ad libitum. All animal management procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.Immature Female ICR mice for research (21 days old) were obtained from Lab Animal Center of Weifang Medical University. Mice were housed under controlled conditions with a daily lighting (12 h light and 12 h darkness). Food and water were providedμg/mL streptomycin. Ovaries were cleaned of any surrounding tissue and repeatedly punctured with 29 G needles to release GCs. The remaining ovarian tissues were incubated in Medium 199 supplemented with 4 mg/mL collagenase I , 10 μg/mL DNase I , and 0.1% BSA for 60 min at 37°C following three times of washing with Medium 199. Then the TCs were placed in Medium 199 containing 0.1% BSA, 1% FBS, 1% insulin-transferrin-selenium (ITS) , 100 IU/mL of penicillin, and 100 μg/mL streptomycin. After overnight incubation, the wells were washed with Medium 199 to remove unattached cells.TCs were isolated from mice aged 21 days and cultured following published protocols with minor modifications , 16. Briμg/mL streptomycin. After overnight incubation, the wells were washed with DMEM/F12 medium to remove unattached cells. Cellular morphology was observed under the microscope.For GCs collection, the released cells from the above steps were placed in prewarmed medium of DMEM/F12. Following three times of washing, the GCs were transferred to the conditioned DMEM/F12 medium containing 0.1% BSA, 10% FBS, 100 IU/mL penicillin, and 100 TCs were seeded on coverslip following washing with Medium 199 to remove unattached cells after overnight incubation. For the identification of TCs and GCs, a CYP17A1 antibody, a marker enzyme specifically expressed in TC, was used to verify the purity and identify the cells. Icy acetone was added and a coverslip was placed at −20°C for 10 minutes. After washing and permeabilization with PBS containing 0.5% Triton X-100, the cells were blocked with goat serum  for 1 h in a humidified chamber at 37°C. Subsequently, slides were incubated with CYP17A1 antibody  or FSHR antibody  overnight at 4°C. Slides were incubated with secondary fluorescent FITC  for 1 h in a humidified chamber at 37°C. After washing and nuclear staining with Hoechst for 10 seconds, the staining was stopped. The slides were seal-capped with Debico and observed under a fluorescence microscope.μm pore size, 24 mm diameter; Corning, NY) inner membrane at a density of 3 × 105 and cultured with 1 mL DMEM/F12 supplemented with 0.1% BSA and antibiotics. After transfer into 6-well plate, TCs were seeded at a density of 1 × 105 with Medium 199 supplemented with 0.1% BSA, 1% ITS, and antibiotics. For androgen assay and quantitative real-time PCR analyses, cells were cultured in serum-free medium for 72 h.GCs were seeded in Transwell  and culture continued for 48 h. Then the medium was collected for subsequent measurement.The spent medium was collected at 24 h, 48 h, and 72 h. After centrifugation at 2500 ×g at 4°C for 15 minutes, the supernatant was stored at −80°C for androgen measurement. The concentrations of testosterone and androstenedione were determined using radioimmunoassay (RIA) kit  and enzyme linked immunosorbent assay (ELISA) kit , respectively, according to manufacturer's protocols. Star, Cyp11a1, Cyp17a1, Hsd3b2, and LHR expression were listed in TCs were harvested and RNA was extracted using Trizol reagents . RNA was measured with spectrometry for OD260/280 and cDNAs were prepared with PrimeScript Reverse Transcription Kit  according to manufacturer's recommendations. Real-time PCR was performed with StepOne Plus Real-Time PCR System  using SYBR Premix Ex Taq II . Primers were designed for each gene using the Primer Premier 5.0 software . The designation and sequences of specific PCR primers ofμL cDNA, 10 pmol primer, and 1 μL SYBR Green Master Mix  in a total volume of 20 μL. GAPDH mRNA was measured and the results served as internal control. PCR conditions were initial denaturation at 95°C for 1 min followed by 40 cycles of amplification: denaturation at 95°C for 15 sec, annealing at 60°C for 1 min, and extension at 72°C for 3 min. Melting curve analysis was performed to confirm the specificity of amplification. The difference of CT between steroidogenic enzymes and GAPDH was defined as ΔCT. The difference of ΔCT between experimental and control groups was defined as ΔΔCT. The relative quantity was determined by 2−ΔΔCT [Each sample was assayed in duplicate using 1 y 2−ΔΔCT .t-test was used, and for multiple comparisons, one-way ANOVA (SPSS 16.0) was used. P < 0.05 was considered as reaching a statistically significant level.All data were expressed as mean ± SEM from at least three independent experiments. For comparison of quantitative data of two groups, Student's After overnight incubation, the wells coated with TCs were washed with medium to remove unattached cells. The cellular morphology and confluence of TCs were observed under the microscope. Typically, the attached cells looked thin and dendritic, indicating their TC origin. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. The purity of TCs was further verified with immunofluorescence staining using a TC-specific marker Cytochrome P450, family 17, subfamily A, polypeptide 1 , and the GC-specific marker Follicle Stimulating Hormone Receptor (FSHR) for reverse identification. The results in P < 0.05,  in vitro conditions lacking the supports from other cell types. In the spent medium of GCs cultured alone, the levels of testosterone ( in vitro culture (P > 0.05). Taken together, these results indicated that GCs could promote TCs production of testosterone and androstenedione.Concentrations of testosterone and androstenedione in the spent medium were measured at 24 h, 48 h, and 72 h using RIA and ELISA, respectively. When cocultured with GCs, TCs produced an increased amount of testosterone and androstenedione than TCs cultured alone . Thus, increasing expression of the genes involved with androgen synthesis appeared to account for the increased androgen production. These results indicated that paracrine factors secreted from GCs can pass the Transwell insert membrane and regulate the transcription of steroidogenic enzymes.To investigate the regulatory pathway leading to the increased androgen production, quantitative real-time PCR was performed to determine the expression of genes involved in androgen synthesis LHR mRNA expression of TCs in the cocultured model manifested no difference compared with TCs cultured alone at 24 h  in TCs and then subsequently aromatized in GCs . Based oParrott and Skinner reported that GCs could promote ovarian interstitial cells recruitment to form theca cell layers around follicles and that inhibin secreted from GCs could induce maturation of the dominant follicle and the production of androstenedione . These f LHR mRNA expression in TCs. The presence of GCs led to a significant increase on LHR mRNA expression of TCs at 48 h but manifested no difference compared with TCs cultured alone at 24 h and 72 h most likely due to the suboptimal in vitro culture conditions, for example, the lack of support by LH and other hormones.To determine the effect of GCs on androgen productions of TCs in response to LH, acquisition of LH responsiveness in preantral follicular TCs from the mouse ovary was evaluated. In this study, we found that the cocultured GCs could enhance the capacity of LH responsiveness on increasing Star, Cyp11a1, Cyp17a1, and Hsd3b2 [ Star, Cyp11a1, Cyp17a1, and Hsd3b2, therefore inducing androgen synthesis in TCs.It is generally accepted that LH responsiveness and responses to LH on androgen production are two well-established functions of TCs in ovary –27. By cd Hsd3b2 . Previoud Hsd3b2 , 11, 12.d Hsd3b2 , 30. Rosd Hsd3b2  found thd Hsd3b2 . In our d Hsd3b2 , 31, 32. in vitro cocultured model including GCs and TCs from mouse preantral follicles recapitulates some native ovary function. However, it is unable to completely represent in vivo microenvironment of whole follicles. Furthermore, it is not yet known what effects TCs-derived factors may exert on GCs and oocytes. Therefore, the function and mechanism of TCs exerted in the ovary as well as the interaction between TCs with GCs and oocytes deserve further exploration in vivo.As an important component of a follicle, TCs exert certain effects on steroidogenesis, follicular development, and atresia. In the present study, theIn conclusion, the present results demonstrated the key role of granulosa cells for the steroid production in theca cells. The Transwell coculture model has provided a technical basis for studying some features of interactions between follicular cell groups under different pathophysiological conditions such as that of hyperandrogenemia.


Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail. Cysteine cathepsins are members of the papain-like cysteine peptidase family . DespiteAll the cathepsins share the same structural scaffold, also called the papain-like fold. The structure consists of two subdomains which have been termed the L- and R-domains referring to their position when the molecule is shown in the standard orientation . The actZymogen activation is one of the major means of regulations of cathepsin activity. All the cathepsins are namely synthesized as inactive zymogens and activated in the acidic milieu of the endolysosomal vesicles. The molecular mechanism of their activation was puzzling for a long time. The critical information came from the combination of structural studies of procathepsins B, K, and L, which showed that the propeptide runs through the active site of cathepsins in the opposite direction of the substrate, thus excluding the cleavage of the propeptide in the molecule without enormous and energetically unfavorable structural movements of the propeptide –19, therThe other major regulators of cysteine cathepsins are macromolecular inhibitors that bind into the active site and thereby prevent association of the peptidase with its substrate. They belong to several distinct families including the cystatins, the thyropins, and the serpins that can, in addition to serine proteases, also inhibit several cathepsins , 22–25.Glycosaminoglycans (GAGs) are heteropolysaccharides composed of repeating disaccharide units with a high negative charge. This is a result of the presence of multiple carboxyl groups and sulfate substitutions. Most of GAGs are sulfated, including chondroitin sulfates (CS), keratan sulfate (KS), dermatan sulfate (DS), heparan sulfate (HS), and heparin, whereas hyaluronan (HA) is the only nonsulfated GAG. In recent years GAGs have been emerging as important regulators of cysteine cathepsins with diverse effects on their targets. Traditionally, cysteine cathepsins had been viewed as lysosomal proteases and, like other lysosomal enzymes, had been known to be inhibited by intralysosomal GAGs in the resting lysosome . Today,  T. congolense parasite homologue congopain [The regulation of papain-like cysteine peptidases by GAGs was first described for cathepsin L , 29. In ongopain , suggestongopain . Neverthongopain , 34–36.The majority of information about the molecular mechanism of GAG-assisted cathepsin activation came from a study by Caglič et al. using human cathepsin B as a model . As showThe other important role of GAGs in the regulation of cathepsin activity came from the studies on papain, the archetypal representative of the family . This moOf all papain-like peptidases, cathepsin K has been established as the cathepsin most tightly linked to glycosaminoglycans. It was originally identified as a protease expressed predominantly in osteoclasts  and its kcat/Km). In a later study, keratan sulfate (KS) and C6S were found to have a stimulatory effect on cathepsin K similar to that of C4S, whereas heparin and HS had a limited effect on the collagenolytic activity of cathepsin K [The interaction of cathepsin K with different glycosaminoglycans has been the subject of in-depth investigation. Though several aspects of these interactions remain elusive, accumulated data suggest that the interactions are heterogeneous and diverse and probably involve multiple binding sites on the enzyme. Chondroitin-4-sulfate (C4S) was initially identified as the GAG with the most dramatic effect on cathepsin K  while thhepsin K . Early ehepsin K ; howeverhepsin K . Nonethehepsin K .Km [The kinetic mechanism of the effect of CS, DS, and heparin (HP) on cathepsin K was also investigated in detail at physiological plasma pH of 7.4. Under these conditions, CS and DS were characterized as nonessential activators with a predominant effect on the affinity for the substrate Km . DS was Km . IntrinsKm .The crystal structure of cathepsin K and C4S revealed the structural basis for the interaction . The binThe kinetic behavior of DS was analogous to that of CS: it was hence proposed that it interacts with cathepsin K in the same manner as CS . HeparinApart from cathepsin K, two other human papain-like peptidases, cathepsins S and B, have been shown to be regulated by glycosaminoglycans in their mature forms , 57. Catβ-secretase that produces amyloid β peptides in secretory vesicles of neuronal chromaffin cells [Cathepsin B is unique among cysteine cathepsins for being both an endopeptidase and a peptidyl dipeptidase, depending on the conformation of the occluding loop, a cathepsin B-specific structure that provides a pH-specific switch between both activities . In the in cells . Howeverin cells  and the in cells .Binding of HP or HS has been shown to increase the stability of the otherwise unstable enzyme at alkaline pH (8.0), while slightly reducing the activity of the enzyme along the entire pH profile of the enzyme . ComputaInterestingly, papain has also been shown to interact with GAGs. Despite bearing no physiological relevance, these interactions point to evolutionarily conserved mechanisms of regulation within the family. HP inhibited papain by a hyperbolic mixed mechanism and affected its conformation . A class Trypanosoma brucei, has been found to be allosterically modulated by HS. The effect of HS in this study was subtle and it had the ability to reverse substrate inhibition by a small dipeptide substrate (Z-Phe-Arg-AMC) [ Trypanosoma cruzi. In this case, HS was an activator of the peptidase that caused a significant (up to 6-fold) increase in the activity of the peptidase measured with a synthetic substrate. Moreover, HS increased the release of kinin from high molecular weight kininogen by cruzipain in vitro as well as by living trypomastigotes and reduced the inhibitory properties of kininogen towards cruzipain [ Leishmania mexicana [In addition, a few examples of proteases from protozoan parasites have been described that interact with GAGs, suggesting the possibility of their influence on host-parasite interactions. The cathepsin L homolog brucipain, a crucial virulence factor of the protozoan parasiteArg-AMC) . A stronruzipain . Similarmexicana . In thismexicana . Altoget per se as well as the viability and cell count of osteoclasts and osteoblasts would remain undisturbed.Cathepsin K currently represents the most attractive drug target among the cathepsins, although cathepsin S is also a relevant target in diseases associated with elevated immune response, such as bronchial asthma and psoriasis , 27. Sev