Tricalcium silicate (TCS)-based materials produce calcium hydroxide as a byproduct of their hydration reaction. The present study investigated whether calcium ion release (CIR) affects their biological and antimicrobial properties when used as pulp protection materials. The effect of incorporation of micro-silica and calcium phosphate monobasic to radiopacified TCS-based materials was investigated. The commercial TCS-based Biodentine, Bio-C Pulpo, TotalFill Root Repair Material, TheraCal LC and a base/liner- ACTIVA BioACTIVE (Activa) were also evaluated. The hydration and CIR were monitored and correlated with biocompatibility and antimicrobial assessment of eluates. Overall, the additives altered the hydration and leaching profile of the prototype cements. The micro-silica inclusion resulted in a decreased long-term calcium hydroxide formation which was associated with neutralised cytotoxicity and antibacterial activity. Calcium phosphate did not alter the leaching profile, although a stronger antibacterial effect was induced. The commercial materials also had different CIR profiles. The water-based ones had higher CIR, and this was associated with stronger antimicrobial effect but not enhanced biological activity. Both TheraCal LC and Activa exhibited poor degree of conversion, low CIR, acceptable biocompatibility and moderate antibacterial activity. A positive correlation of CIR with antibacterial effectiveness was observed . No relation was shown between CIR and cytotoxicity . The additives modified the CIR. The antimicrobial properties were dependent on the CIR; the cytotoxicity of the materials was unaffected. Additionally, the use of MTA and hydraulic tricalcium silicate cements (TCS) for vital pulp therapy (VPT) has been found to increase the clinical success rates of direct and indirect pulp capping procedures considerably3. These hydraulic cements form calcium silicate hydrate (CSH) and calcium hydroxide (CH) in contact with water4. Release of calcium and hydroxyl ions promotes pulp tissue healing and mineralised tissue formation5 while the alkalinity contributes to microbial elimination6. To date, despite the well-established role of CH, the desirable dose-response release from pulp capping agents is unknown.Mineral trioxide aggregate has been developed and is thus considered to be the material of choice for root repair and surgical endodontic procedures7. Inert, non-inducing discoloration materials such as zirconium oxide (ZO) have now substituted the bismuth oxide radiopacifier8.The new generation of TCS-based materials contains modifications aimed at eliminating the limitations of MTA and enhancing its physicochemical properties. Accordingly, pure TCS has replaced Portland cement as evidenced in Biodentine  usage9, calcium phosphate (CP)10, silicon oxide (SO)11 or resin components6. Notably, CC is added to Biodentine serving as a nucleating agent12. The main rationale of CP-incorporation which is found in TotalFill  was for the enhancement of biological activity, based on the formation of mineral structures resembling bone13. In BioAggregate , SO is included to convert the CH to CSH in the longer term to strengthen the material, as initially exploited in the concrete industry14. A recently introduced pulp capping agent, Bio-C Pulpo  is also reported to contain SO15. Furthermore, the inclusion of light curable monomers, as in TheraCal LC 16, provides the ability to command cure the material and avoids problems with bonding composite resin to the underlying liner17.In the new generation materials, there are also a number of additives such as calcium carbonate (CC)19. Overall, it is evident that efforts directed toward the enhancement of physico-mechanical properties of the cements, might have an adverse effect on their chemical profile. The aim of the present study was to characterise a series of commercial and prototype TCS-based materials and elucidate a correlation path between alterations in the CH release and biological and antimicrobial properties following inclusion of CP, SO or resins. The null hypotheses tested were that different additives will not affect calcium ion release (CIR) and consequently, changes in calcium release will not alter cements’ biological and antimicrobial properties.The use of additives can alter the cement’s properties. Specifically, even though the beneficial effects of substitution of the Portland cement in MTA with pure calcium silicates and the use of inert radiopacifiers instead of bismuth oxide are indisputable, there is still a great controversy in the literature regarding the effect of further alterations. So far, addition of a second cementitious phase, namely calcium phosphate to TCS, inclusion of a resin matrix or incorporation of silicon oxide have been found to affect the hydration of materials and the calcium hydroxide released to various extentsBio-C Pulpo Biodentine TotalFill Root Repair Material Theracal ACTIVA BioACTIVE Base/Liner TCS  replaced with 30% ZO radiopacifier  (TCS/ZO)TCS/ZO with 15% CP replacement  in the cementitious phase (TCS-CP/ZO)TCS/ZO with 10 or 20% micro-silica  replacement of the cement .The following commercial and prototype materials were investigated:20 or photo-polymerisation for Theracal and Activa, they were immersed in 5 ml of Hank’s balanced salt solution  and incubated at 37 °C. Experiments were performed in triplicate and with three parallels for the biocompatibility and antibacterial assays.Replacements were performed by weight. Prototypes were mixed with distilled water. Materials were compacted inside round rubber moulds . Following initial setting time of hydraulic cements which was determined according to the American Society for Testing and Materials (ASTM) standards for setting time of hydraulic cements (ASTM C266-18)2. Decrease in the intensity of the 1640 cm−1 peak corresponding to –CH=CH2 stretching vibration was monitored. The peak that corresponds to the aromatic rings at 1608 cm−1 was used as an internal reference as it does not change in intensity during photo-polymerisation.A Fourier-transform infrared spectrometer  coupled with micro-attenuated total reflectance crystal  was used. Real-time DC of Theracal and Activa were determined by applying a 1 mm-thick sample onto the crystal and light curing for 180 s. The distance between the curing light  and the specimen was set at 1 mm. The wavelength of the curing light was 430–480 nm and the light intensity was 1,470 mW/cmFollowing a 28-d incubation period, pellets were retrieved, vacuum desiccated and characterised. For SEM and EDS, materials were embedded in resin , ground and polished  and imaged using SEM  in backscatter mode. EDS was performed in selected areas.For XRD analysis, pellets were crushed to a fine powder with an agate mortar and pestle. The diffractometer  with a CoKa radiation at 40 mA and 45 kV, rotated between 15–70° with a 0.02° 2θ step and a 0.6 s step time. DIFFRAC.EVA software  and International Centre for Diffraction Data database  served for phase identification.The immersion liquid was not changed at any point during the incubation period of pellets for all experiments conducted.The pH of eluates was measured 24 h post-immersion and weekly for 28-d using a pH meter .Pellets were weighed (0.0001 g precision) prior to immersion. One- and 28-d leachates were analysed using inductively coupled plasma-optical emission spectrometry .21. ATCC CCL-92 mouse 3T3 cells and primary human dental pulp cells (HDPCs) harvested from healthy root canals22 of two intact premolars of a 15-year old patient were used. Ethical approval was obtained from the Birmingham Community Healthcare NHS Foundation Trust .The effect of fresh, 1- and 28-d leachates on the metabolic activity of two cell types was assessed using the 3--2,5-diphenyltetrazolium bromide (MTT) assay2 flasks with 15 ml Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, supplemented with 1% penicillin/streptomycin, 2 mM glutamine with 10% heat-inactivated foetal bovine serum  (DMEM-10FBS) at 37 °C with 5% CO2 in a humidified atmosphere. Cells were subcultured at 70–80% confluency and seeded in 96-well plates at 10,000 for 3T3 cells and 6,000 for HDPCs in 100 μl DMEM-10FBS and incubated for 24 h. Passages 2–7 were used.Cells were cultured in 75 cmCytocompatibility of materials during extraction procedure and immediately after material manipulation and placement was investigated with a transwell system consisting of a 96-well receiver plate and an adjustable cell-culture plate with a porous membrane .Following the cell-seeding period, materials were placed in the wells of the receiver plate; 100 μl HBSS were transferred to each well and to empty wells for the blank control. Consequently, the cell-culture plate was placed upon the receiver plate. Following incubation for 24 h, the medium was aspirated and 100 μl MTT solution (0.5 mg/ml) was added to the wells. The MTT reagent was replaced with 100 μl dimethyl sulfoxide  after 4 h. Optical density (OD) was read in a microplate reader  at 570 nm. Results were expressed in respect to values of the blank control.in vitro cytotoxicity (ISO 10993-5:2009) by indirect contact were followed18. After incubation for 24 h, 100 μl of the test eluate (undiluted) or 4 serial two-fold dilutions were added to each culture. Pure HBSS served as a blank (control) group. The MTT assay was performed the following day, as previously described.The International Standards for Streptococcus mutans N3209 and Lactobacillus casei NCTC 16341 were cultured overnight  in Tryptic Soya Broth or Man, Rogosa & Sharpe broth  respectively. Bacterial cultures were standardised to OD 1.0 at 600 nm  and consequently diluted in fresh medium in a 1:4 ratio.The minimum inhibitory concentration (MIC) assay was performed in 96-well plates by adding 150 μl of each bacterial inoculum separately and an equal amount of the test leachate, either undiluted or following 4 serial two-fold dilutions in HBSS. Plates were incubated overnight and OD of wells was determined at 600 nm in a microplate reader.The IBM SPSS Statistics software version 24  was used. Prior to each statistical analysis, data were assessed for normality with the Shapiro–Wilk test. The majority of them followed the normal distribution and results were consequently processed using one-way ANOVA and Tukey post-hoc tests for multiple comparisons. Independent student’s t-tests were performed to compare leachates’ effects on cell cultures. Pearson correlation tests evaluated the relationship between mean values of calcium ion release and the respective biological and antimicrobial performance. The significance level was set at p = 0.050.The initial setting time of test materials following which they were immersed in the HBBS medium is presented in Table Both Activa and Theracal reached DC values below 30% during the 20-s photopolymerisation period indicated by the manufacturers. Theracal’s DC began to plateau beyond 90 s, while Activa’s DC plateaued at approximately 60 s Fig. .Figure 1The scanning electron micrographs and energy-dispersive spectroscopy (EDS) analyses are presented in the Supplementary file. Figure Both light curable materials Theracal and Activa exhibited several unhydrated cement particles throughout their resin matrix Fig. . Both maThe cement particles in the TCS/ZO showed a halo of hydration product. Incorporation of CP or mS altered the hydration kinetics. In the TCS-CP/ZO, the second cement type contained calcium, phosphorus and silicon and appeared lathlike. The mS-containing prototypes exhibited more advanced hydration. In the diffractograms Fig. , the proAll materials alkalinised significantly the HBSS solution (p < 0.001), except for Activa, which did not present any change in the 1-d (p = 1.000), 7-d (p = 1.000), 21-d (p = 0.717) and 28-d (p = 0.811) leachates, while it exhibited a significant decrease in the pH of the 14-d leachate (p < 0.014). Alkalinisation occurred from the first day in the hydraulic cements with no significant alterations in most materials Fig. . BiodentFigure Figures Transwell system. Totalfill was the only material that exhibited values of cell viability higher than 70% which is the threshold set by ISO 10993-5;200921 in both cell types. This was distinct to all the other commercial materials (p < 0.001). Theracal exhibited increased cytotoxicity in 3T3 cultures in comparison to the HDPCs (p = 0.004). Notably, in the HDPC cultures, Theracal reported higher MTT values than BCP, Biodentine and Activa (p < 0.001), which had minimal values in both cell types (p = 1.000).From analyses of the prototype materials, the TCS-mS20/ZO did not affect HDPC viability , reporting a better performance than the unmodified prototype cement (p = 0.049). All other materials exhibited a cytotoxic potential in both cell types . The TCS-mS10/ZO reported the lowest MTT values in the HDPCs .One- and 28-d leachates effect. For 1-d leachates, undiluted BCP, Totalfill, Activa and all the prototype materials reduced the metabolic activity of both cell types to different extents; undiluted Biodentine and Theracal were regarded as biocompatible (relative MTT activity > 70%), while in HDPC cultures, Biodentine reported the highest metabolic activity [p < 0.001 except for comparison with Theracal (p = 0.033)]. Totalfill exhibited increased cytotoxicity in HDPC cultures in comparison to the 3T3 ones (p < 0.001). The TCS/ZO presented a moderate decrease in MTT activity in HDPC cultures, marginally above 70%. All undiluted eluates of the modified prototype materials affected significantly more the metabolic activity in HDPC cultures in comparison to TCS/ZO .In the 28-d leachate exposed samples, all undiluted eluates were regarded as being cytotoxic (relative MTT activity < 70%) except for Theracal and Activa, which had an overall significantly better performance (p < 0.001) and reported also higher values for 3T3 cells (p = 0.010 and p < 0.001 respectively). Diluted leachates of all samples did not decrease the MTT values below the cytotoxicity margin. From the other commercial materials, Totalfill exhibited the lowest MTT values in both cell types . In the prototype materials, TCS-mS20/ZO exhibited better biocompatibility values in both cell types (p < 0.001). Values of the rest prototypes did not differ from the TCS/ZO (p = 1.000).S. mutans and L. casei . Notably, BCP reported higher inhibition than the other leachates (p < 0.001), except for Totalfill in the L. casei cultures (p = 0.997). BCP remained antibacterial even after 2 dilutions against S. mutans (p < 0.001). Biodentine and Theracal were antibacterial only against S. mutans (p < 0.001), while Activa was the only commercial material with no reported antibacterial effect (p = 1.000 for S. mutans and p = 0.471 in L. casei samples). From the prototype cements, 1-d eluates of TCS-CP/ZO reduced significantly the ODs of both S. mutans and L. casei (p < 0.001 and p = 0.002 respectively), while TCS-mS10/ZO had a demonstrable effect only against S. mutans (p < 0.001). TCS/ZO and TCS-mS20/ZO did not present any inhibitory effect (p = 1.000).Results from the MIC assay against test bacteria inoculums are presented in Fig. L. casei in pure eluates or after single-dilution (p < 0.001). Theracal remained antibacterial against S. mutans even after two dilutions (p < 0.001). From the prototype materials, TCS/ZO, TCS-CP/ZO and TCS-mS10/ZO were antibacterial against both species (p < 0.001), while TCS-mS20/ZO was effective only against S. mutans (p < 0.001).In the 28-d leachates, BCP exhibited the greatest inhibitory effect, reducing significantly the OD values of both bacterial inoculums in comparison with the positive control even after the third dilution (p < 0.001). All commercial materials inhibited the growth of both bacteria (p < 0.001) except for Activa, which was found antibacterial only against S. mutans and L. casei respectively). For cytotoxicity, even though a medium correlation coefficient was also reported in both cell types (0.3 < r < 0.49), overall this was non-significant (p = 0.150 for 3T3 cell cultures and p = 0.068 for HDPCs).A significant correlation between calcium ion release and antibacterial activity for both bacterial species studied was observed  are composed of a TCS cement phase and radiopacifier with various additives, which have been verified in the current study. All these materials are water-based. The prototype cements were created to assess the effect of the additives on calcium ion release and the associated biological and antimicrobial properties. These prototypes did not contain any setting accelerators or other additives for enhancement of physical properties in contrast to the commercial ones. Thus, a direct correlation between inclusion of specific additives and change in properties could be explored. Theracal also contains a TCS phase but in a resin matrix. Activa was studied due to the manufacturer’s claims of bioactivity. Additionally, assessing properties of the two resin-containing materials, Theracal and Activa in the same project assisted in the evaluation of modifications of the performance of a resin-modified glass ionomer by inclusion of a cementitious phase.23. On the contrary, a prolonged leaching period can result in high solubility values from the material and therefore potentially compromise the restoration’s sealing ability, while minimal solubility values can affect ion release25. Despite these well-described roles, a quantitative correlation path is yet to be determined and in vitro values do not reflect precisely the clinical scenario26.Calcium release has been correlated with the biological properties of hydraulic cements, as it stimulates the differentiation potential of dental pulp cells and facilitates mineralisation leading to deposition of a dentine-like barrier upon the surface of the pulp in the long-term5, thus, an association of the leaching profile with the biological performance was investigated, as well as the effect of compounds on CH release. The antimicrobial activity of pulp protection materials is desirable as bacteria can remain in the dentinal tubules following pulpal infections27. High alkalinity provides a bactericidal effect and therefore sustained alkalinisation is desirable28.Hydroxyl ions can trigger pulp tissue healing by inducing an initial necrotic layer29. Indirect biological and antimicrobial assays were performed to correlate the CH release with materials’ properties, without interference of materials’ surface30. Evaluation was performed following 1 or 28 d of immersion in HBSS to investigate the leaching profile of the hydraulic cements early during the hydration reaction and at a later stage where the final hardening and maximum physico-mechanical properties have been reached (28-d)4. Additionally, the immersion medium was not replaced at any point during the 28-d period since clinically the pulp capping agents are in contact with very little liquid in the application field. This resulted in leachate saturation as observed in the pH values. Biocompatibility was also assessed in a transwell system to investigate the leachates’ reactivity during the first hours of hydration.Characterisation analyses performed here provided precise information on the materials’ microstructural and elemental composition as well as their mineral phases. The extent of polymerisation was assessed for the resin-containing materials as leaching of potentially cytotoxic monomers can occur if these remain unpolymerised5. However, as the pH of most TCS-based materials appeared saturated, a correlation analysis with the pH would not provide clear conclusions. Overall, calcium ions leached from hydraulic cements derive mainly from dissolution of the calcium hydroxide by-product4. Therefore, an increase in calcium release is also indicative of higher hydroxyl ion diffusion as well.The present study showed that calcium ion release affects positively the antibacterial effectiveness while it has a medium non-significant correlation with cytotoxicity. Therefore, an escalating reactivity was reported in leachates of TCS-based materials between the early and late stages of hydration. The relationship of the hydroxyl ions with biological properties and antimicrobial performance is also well-known14. Although BCP included a TCS phase, no crystalline CH was formed on hydration, however, the calcium ion release was the highest even at a relatively early stage. BCP’s 1-d leachate was reactive in terms of cytotoxicity and antibacterial performance due to its relatively high values of pH and calcium. In the transwell system, it induced cytotoxicity, providing an indication of the aggressive leaching taking place during setting reaction. The 28-d eluate exhibited increased cytotoxicity and antibacterial performance, as saturation of hydroxyl ions might have occurred.The BCP contains addition of SO, which is usually exploited to improve the cement’s mechanical properties19. Biodentine’s 1-d leachate appeared reactive while the TCS/ZO exhibited moderate cytotoxicity and no bacterial inhibition. Overall, the similar performance of these materials correlates with their alkalinisation potential. Despite being both alkaline, their pH increased after 14 d. A significant cytotoxic effect could be therefore triggered after the pH exceeds a critical level31. Interestingly, Biodentine behaved more aggressively in the transwell system, likely due to its accelerated hydration. Besides the additives that have been tested in the current work, the commercial materials also contain calcium chloride as setting accelerator, calcium carbonate which serves as a nucleating agent or water-soluble polymers which reduce the powder to liquid ratio. This alters their chemical profile in different extents.Biodentine exhibited peaks for CH and demonstrated an overall enhanced hydration and calcium leaching profile in comparison with the chemically relevant TCS/ZO due to the presence of calcium chloride and CC in the formerTotalfill exhibited a CP phase and an enhanced leaching profile which correlated with an escalating cytotoxic potential and stable antibacterial performance. Conversely, it appeared biocompatible in the transwell assay. It was observed that Totalfill remained unset during the assay period Table  and ther32. Consequently, leaching was compromised33 and eluates were less reactive. The moderate alkalinisation effect following resin inclusion in TCS resulted in better biocompatibility values. However, the aggressive performance observed in the transwell assay might derive from differences in the extraction conditions which affected leaching and monomer release. Reports in the literature regarding Theracal’s biocompatibility are also controversial35. Interestingly, an escalating antimicrobial potential was observed against S. mutans, which suggests that high alkalinity is not the only factor responsible for its antimicrobial activity. Elution of unreacted monomers might contribute to the antibacterial effect36.The resin modification in Theracal induced significant alterations in its hydration37. For the materials tested, their shade might have compromised curing, not allowing adequate light transmission38. As a result, a significant amount of unreacted monomers is present in the materials following restoration.Theracal and Activa exhibited poor DC during the indicated light curing period (20 s). Under ideal light curing conditions as the ones established in the current experimental setup, the DC of a resin ranges between 50–70%40, albeit the manufacturer stating that Activa does not contain Bis-GMA or TEGDMA monomers41, which have been reported as cytotoxic43. A direct correlation between the amount of fluoride release and cytotoxicity of the glass ionomer based materials has been also suggested44 and should be further investigated assessing leaching of other elements in the formulation of Activa. The 28-d leachate was biocompatible, indicating that no long-term degradation of monomers occurs. In terms of antibacterial effectiveness, Activa’s inert performance was in accordance with its leaching profile.Activa presented the typical microstructure of a glass ionomer with negligible calcium release and no alkalinisation potential. One-d and fresh eluates exhibited cytotoxicity possibly due to monomers leaching, as it has been reported previously for materials of similar composition18, no significant effect is reported with lower incorporation.In the prototypes, inclusion of micro-silica resulted in a plateau in calcium release at early stages and depletion of CH, especially in the TCS-mS20/ZO, which resulted in moderate reactivity. Leaching of the CP-containing material was not altered, albeit an enhanced initial antibacterial effect was observed. As high amounts of CP-inclusion compromise leachingInclusion of additives to tricalcium silicate-based materials changed their properties. Commercial water-based hydraulic cements such as BCP, Biodentine and Totalfill exhibit increased calcium release, antibacterial activity and effect in cell metabolic activity as elution occurs throughout time. This chemical profile is compromised following a resin modification, as in Theracal, while is totally absent in a resin-based glass ionomer cement (Activa). Incorporation of silicon oxide in hydraulic cements plateaus calcium release, while inclusion of calcium phosphate in 15% does not affect significantly the calcium leaching and alkalinisation potential. Overall, calcium release is positively correlated with enhanced antimicrobial performance, while cytotoxicity is mainly affected by the alkalinity. Alterations in the traditional formulation of a radiopacified hydraulic cement in an effort to improve physical properties or handling characteristics, should be investigated as incorporation of additives may diminish the chemical, biological and antimicrobial characteristics.Figure S1


Objectives: The aims of the current study were: (1) to compare growth trajectories of speech recognition in noise for children with normal hearing (CNH) and children who are hard of hearing (CHH) and (2) to determine the effects of auditory access, vocabulary size, and working memory on growth trajectories of speech recognition in noise in CHH.Design: Participants included 290 children enrolled in a longitudinal study. Children received a comprehensive battery of measures annually, including speech recognition in noise, vocabulary, and working memory. We collected measures of unaided and aided hearing and daily hearing aid (HA) use to quantify aided auditory experience . We used a longitudinal regression framework to examine the trajectories of speech recognition in noise in CNH and CHH. To determine factors that were associated with growth trajectories for CHH, we used a longitudinal regression model in which the dependent variable was speech recognition in noise scores, and the independent variables were grade, maternal education level, age at confirmation of hearing loss, vocabulary scores, working memory scores, and HA dosage.Results: We found a significant effect of grade and hearing status. Older children and CNH showed stronger speech recognition in noise scores compared to younger children and CHH. The growth trajectories for both groups were parallel over time. For CHH, older age, stronger vocabulary skills, and greater average HA dosage supported speech recognition in noise.Conclusion: The current study is among the first to compare developmental growth rates in speech recognition for CHH and CNH. CHH demonstrated persistent deficits in speech recognition in noise out to age 11, with no evidence of convergence or divergence between groups. These trends highlight the need to provide support for children with all degrees of hearing loss in the academic setting as they transition into secondary grades. The results also elucidate factors that influence growth trajectories for speech recognition in noise for children; stronger vocabulary skills and higher HA dosage supported speech recognition in degraded situations. This knowledge helps us to develop a more comprehensive model of spoken word recognition in children. Every year, approximately three in 1,000 children are born with a significant hearing loss . ChildreGiven their reduced access to spectral and temporal cues in the speech signal, as well as reduced binaural processing, it is not surprising that listening in noise is a challenge for CHH. In addition to limited longitudinal data, previous large-scale studies of speech recognition in children with hearing loss have focused primarily on children with congenital, severe-profound hearing loss who use CIs . CHH areIn environments with degraded signals (either due to poor acoustics or reduced hearing levels), listeners rely on higher level cognitive and linguistic skills to interpret information about the input . AccordiIn addition to exploring the role of working memory capacity, the ELU model also predicts that language abilities will influence the ability to recognize degraded speech . PerformA third relevant factor to consider when examining sources of variance in speech recognition in noise is auditory access, particularly because CHH show large individual differences in this variable . AuditorAn alternative to relying on PTA is to examine audibility levels, as measured by the Speech Intelligibility Index (SII). SII is a measurement that describes the proportion of speech accessible to the listener, with or without HAs. It accounts for the configuration of hearing loss, differences in ear canal size, and amplification characteristics of HAs. Studies have shown an association between SII and speech recognition in CHH ; howeverA third way to examine auditory access is to consider individual differences in the amount of daily HA use. Only a few studies have looked at hours of HA use as a predictor variable of speech recognition in noise. To better understand the impact of auditory access on listening in noise, we propose to conceptualize the auditory experience of CHH as a combination of unaided hearing, aided SII, and amount of HA use . Our pasIn summary, no studies have compared developmental trajectories in speech recognition in noise between CNH and CHH. This paper describes results from a longitudinal study in which speech recognition in noise measures were collected on an annual basis in school-age CNH and CHH. The aims of this study were to: (1) compare the growth rates for speech recognition in noise for CNH and CHH, (2) determine whether CHH and CNH show similar growth rates over time, and (3) identify the auditory, cognitive, and linguistic factors that are associated with individual differences in growth rates for speech recognition in noise for CHH. It is expected that this knowledge will provide us with further insight into the everyday functional listening skills of children with and without hearing loss.n = 199; CNH, n = 92) who were enrolled in a multicenter, longitudinal study on outcomes of children with mild to severe hearing loss, Outcomes of School-Age Children who are Hard of Hearing (OSACHH). The primary recruitment sites were the University of Iowa, Boys Town National Research Hospital, and University of North Carolina-Chapel Hill. Some of the children from the Iowa and Boys Town test sites also participated in a second longitudinal project that was conducted during the same time period as OSACHH. This second project was called Complex Listening in School-Age Hard of Hearing Children.Participants included 290 children  = −1.61, p = 0.11]. Demographic information, including audiologic data for the CHH, is provided in CHH had a permanent bilateral hearing loss with a better-ear four-frequency PTA in the mild to moderately severe range. One hundred seventy-nine children had a sensorineural or mixed hearing loss, 15 had a conductive hearing loss, and two had auditory neuropathy spectrum disorder. Three children did not have the type of hearing loss reported. Both CHH and CNH used spoken English as the primary communication mode and had no major vision, motor, or cognitive impairments. CNH and CHH were matched by age. There was no significant between-group difference in maternal education level .During each test visit, the caregiver completed a questionnaire related to daily HA use (available at: Aided Better-ear SII − (24 − Daily HA Use hours)Unaided Better-ear SII. The number of hours of daily HA use is weighted by aided SII (access to speech with HAs). If SII = 1, the child has full access to the speech spectrum for that number of hours throughout the day. The amount of time the child does not wear HAs during the day, weighted by unaided SII (access to speech without HAs), is then subtracted from the hours of use weighted by aided SII. A smaller value indicates lower HA dosage and a greater value indicates higher HA dosage.To measure the combined effects of HA use and audibility levels (aided and unaided), we calculated a variable termed “HA dosage.” This metric can be conceptualized as how much daily access a child receives from HAs. HA dosage combines the number of hours of daily HA use with aided and unaided hearing into one weighted measure of how much auditory access a child experiences during the dayWe administered the BKB-SIN test  at each Test protocols were developed to be appropriate for children utilizing spoken English in first through fourth grade. Test protocols varied depending on the year of testing. First and third grade test batteries were the same, and second and fourth grade test batteries were the same.viated Scale of Intelligence-2  and must determine if it is true or false. After hearing a set of two sentences, the participant repeats back the last word of each sentence in the order that he/she heard them. This task is also administered using a span procedure; if the participant accurately identifies the last words of the sentences in the correct order, the span increases by one sentence. The correlation for Listening Recall and Odd One Out raw scores for participants in first and third grade was 0.61. Raw scores were transformed into z-scores and averaged together to form a composite score.At second and fourth grade, we administered the Listening Recall and Odd One Out tasks. In addition, we administered Backward Digit span, a working memory span measure in which the participant hears a series of numbers and is instructed to verbally repeat them back in reverse order. The correlation between raw scores for Backward Digit Span and Listening Recall was 0.50, the correlation for Backward Digit Span and Odd One Out was 0.52, and the correlation for Listening Recall and Odd One Out was 0.52 for participants in second and fourth grade. The raw scores of the three variables were transformed into z-scores and averaged together to compute a composite working memory score at second and fourth grade.Our first two research questions evaluated the growth trajectories of the BKB-SIN SNR-50 scores for CHH and CNH, and whether the two groups showed similarities or differences in their rate of growth. To address these research questions, we constructed a longitudinal regression model. The fixed effects in the regression model were grade ; hearing status ; and an interaction between grade and hearing status. To account for the correlation due to repeated measures, we included a correlation structure on the residuals. The Akaike Information Criterion AIC;  was usedThe third research question examined which factors were associated with individual differences in growth rate for speech recognition in noise for only CHH. To construct this analysis, we used a linear regression model with a heterogeneous compound symmetric error structure to account for correlation between grades and unequal variances between grades. The dependent variable was again growth rate on BKB-SIN SNR-50 scores. The fixed effects were grade, maternal education level, age at confirmation of hearing loss, vocabulary composite z-scores, and working memory composite z-scores. Maternal education level was coded as ordinal levels . We also included average HA dosage across visits and change in HA dosage as separate fixed effects because HA dosage is a time-varying covariate. Change in HA dosage is calculated as each participant’s HA dosage at a given visit subtracted from the average HA dosage across visits. These separate variables allowed us to determine whether the average levels of HA dosage across visits or change in HA dosage were associated with growth rate.F = 23.78, p < 0.0001. Each older grade had a lower SNR-50 compared to younger grades (see t(284) = 8.19, p < 0.001. The interaction between grade and hearing status was not statistically significant, F = 1.18, p = 0.3154. This lack of an interaction is evident in We found a significant main effect for grade, ades see . There wF = 6.04, p = 0.0006], vocabulary composite z-scores , and average HA dosage  were significantly associated with rates of growth in BKB-SIN SNR-50 scores. Maternal education level , age at confirmation of hearing loss , working memory composite z-scores , and change in HA dosage  were not significant predictors. Stronger vocabulary skills . Further, we only tested participants up to 11 years of age, which is still a period of early adolescence. While the current data trends suggest that CNH and CHH show parallel rates of development in speech recognition in noise, it is possible that we may see differences in growth trajectories past 11 years , particuWe also note that the inclusionary and exclusionary criteria for this study resulted in a homogeneous cohort of children from English-speaking backgrounds with no additional motor or cognitive deficits. Thus, the current results may not generalize to linguistically diverse populations or children with hearing loss who have additional disabilities. We excluded children with profound hearing loss because we were interested in the impact of hearing loss in the mild to severe range. It is possible that we would have seen a stronger impact of age at confirmation of hearing loss if children who are deaf had been included in the sample. We did not control for the type of hearing loss because our goal was to recruit as many children with permanent hearing loss as possible; however, the majority of children presented with sensorineural hearing loss. We acknowledge that the consequences of sensorineural and conductive hearing loss can impact speech recognition in noise differently, but our limited number of children with conductive hearing loss prevents us from analyzing these children as a separate group.A final limitation is that we restricted our speech in noise measure to the BKB-SIN test, which uses a four-talker babble as the competing signal. Other studies have shown that informational masking is increased as the number of competing talkers is decreased , CNH demThe current study established longitudinal growth trajectories of speech recognition in noise for school-age CHH and CNH. As a group, CHH demonstrated deficits in speech recognition in noise. These deficits do not appear to converge toward or diverge from CNH, as the growth rates were parallel for the CHH and CNH. These findings also helped us identify the underlying mechanisms that drive growth in speech recognition, with stronger vocabulary and higher HA dosage supporting speech recognition in degraded situations.The datasets generated for this study are available on request to the corresponding author.EW and RM conceived of the presented idea. EW took the lead in writing the manuscript. CS and RM contributed to the interpretation of the result and the final version of the manuscript. JO performed the analytic and statistical calculations and data visualization. All authors provided critical feedback and helped shape the research, analysis, and manuscript.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.


Mucormycosis is a rare, potentially fatal and opportunistic infection caused by fungi belonging to the order Mucorales. Rhinocerebral, gastrointestinal, pulmonary, cutaneous and disseminated are the different forms of mucormycosis. Rhinocerebral mucormycosis is the most common type and presents as a highly destructive infection in immunocompromised hosts, especially in patients with poorly controlled diabetes. The infection originates in the nasal mucosa owing to fungal inoculation and then spreads to the paranasal sinuses, orbits, orbital apex, cavernous sinuses and brain. Our patient was a 36-year-old female with poorly controlled diabetes who presented with orbital symptoms and signs, with very subtle involvement of the sinuses. She had stenosis of the entire left internal carotid artery, with multiple small infarcts in the left frontal and parietal lobes. She incidentally had tooth caries tooth with a periapical cyst and an oroantral fistula. Ours was a histopathologically proven case of rhino-orbito-cerebral mucormycosis. The surgeon suspected left paranasal sinus pathology with orbital extension and requested a CT scan of the paranasal sinuses and an MRI of the orbits with screening of the brain.A 36-year-old female presented with gradually increasing left eye swelling, progressive loss of vision and left-sided facial pain for 2 months. A few days prior to the peaking of symptoms, she had symptoms of cold. She was a known diabetic and hypertensive. On examination , there wCT scan of the paranasal sinuses  demonstrT1 weighted images . The preseptal lesion did not show any restriction of diffusion. Multiple small acute infarcts were seen in the left corona radiata and parietal lobe with an apparent diffusion coefficient value similar to the orbital lesions (0.407 × 10–3 mm2 s–1).Diffusion-weighted images  showed rSusceptibility weighted imaging  demonstrT1 weighted images  belonging to the class of Phycomycetes. They are recognized microscopically as broad, non-septate and obtuse-angle branching hyphae. They are ubiquitous and normally saprophytic in humans. Inoculation occurs when spores reach the nasal cavity during inhalation. They can produce fatal disease in persons with immunosuppression.Paltauf2Five clinical forms of mucormycosis are recognized: rhino-orbito-cerebral, gastrointestinal, pulmonary, cutaneous and disseminated. The rhinocerebral form is the most common and is seen mostly in patients with uncontrolled diabetes.3 It spreads to the brain via the orbital apex, orbital vessels or the cribriform plate.4 In our case, there was involvement of the left orbit along the medial wall, the preseptal space and the orbital apex, with subtle involvement of the left ethmoid sinus.In the rhinocerebral form, the infection originates in the nasal cavity and then spreads to the paranasal sinuses. From the sinuses, it spreads to the medial orbit and the orbital apex through the nasolacrimal duct and the medial orbital wall. Spread of infection from the sinuses to the orbit is facilitated by the thinness of the lamina papyracea, congenital dehiscence in the medial wall and fenestrations in the medial wall by arteries and veins.5 In our case, there was involvement of the cavernous sinus, stenosis of the left internal carotid artery and a small peripherally enhancing lesion just above the cavernous portion of the internal carotid artery. Internal carotid artery occlusion has a high mortality rate.6 From the cavernous sinus, the infection can spread to the brain parenchyma or the cranial nerves such as the fifth and the seventh. Inferiorly, the infection can spread to the skull base, including the pterygopalatine fossa, as in our case. Brain parenchyma involvement may be in the form of infarcts owing to vascular thrombosis, mycotic emboli and frontal lobe abscesses.Intracranial spread to the cavernous sinus may result in haemorrhage or thrombosis of the internal carotid artery, or thrombosis of the cavernous sinus.6 Thrombosis may be due to endothelial damage from hyphal invasion or growth within the lumen.The pathophysiology of mucormycosis is related to the invasion of the walls of the vessels, especially the arteries. Hyphae could be demonstrated in the walls of the internal carotid arteries in all the cases in a study by Lowe and Hudson.7 Differential diagnosis for orbital cellulitis and sinusitis in patients with immunosuppression are: mucormycosis and other fungal infections, paranasal sinus malignancy, bacterial cellulitis, carotid cavernous fistula and thrombosis, inflammatory pseudotumor and Graves’ disease.8 Rhinocerebral mucormycosis should be considered in the differential diagnosis of patients presenting with symptoms of unilateral cranial nerve involvement, as in Garcin syndrome.9The common presenting symptoms are headache, low-grade fever, facial swelling, sinusitis, orbital apex syndrome and cranial palsies from cavernous sinus involvement. Visual loss occurs much earier in rhinocerebral mucormycosis than in bacterial cavernous sinus thrombosis.11 Maxillary, ethmoid, frontal and sphenoid sinuses are involved in decreasing order of frequency.10 Our case demonstrated mucosal thickening in the maxillary sinus without any nodularity, hyperdensity or air–fluid levels. Orbital images in our case demonstrated thickening and lateral displacement of the medial rectus muscle, which is characteristic of orbital invasion from disease in the ethmoid sinuses.11 Lack of enhancement of the ophthalmic and internal carotid arteries or the superior ophthalmic vein can occur owing to vasculitis and thrombosis.12CT scan findings described in sinonasal mucormycosis include soft tissue opacification of the sinuses with hyperdense material, nodular mucosal thickening and absence of fluid levels.MRI has a very important role in the diagnosis of rhinocerebral mucormycosis, especially in the early detection of orbital and intracranial complications.T1 and T2 weighted images, with majority of the lesions showing isointense signal relative to brain in T1 weighted images.13 The signal intensity in T2 weighted images has been reported to be more variable, with 20% showing hyperintense signal and the rest of the lesions showing either hypointense or isointense signal.13Sinus contents can show variable MR signal characteristics on 9 Diffusion restriction may be the earliest indicator of optic nerve infarction on MRI.14Diffusion-weighted image sequences may demonstrate restriction of diffusion in the lesions owing to infarction.T1 weighted images better demonstrate the intracranial spread by recognizing meningeal enhancement, invasion of the cavernous sinus or the internal carotid artery, and vascular complications such as ischaemia.15 The enhancement pattern may be variable.8 Rhinocerebral mucormycosis should be suspected when there is a lack of enhancement of the mucosa of the sinus owing to invasion of smaller vessels supplying the mucosa by the hyphae.9 Non-enhancement of the involved turbinate is called the “black turbinate sign”.9Contrast-enhanced In our case, there were multiple peripherally enhancing lesions in the orbit with a small intracranial extension. Diffuse enhancement was seen in the retro-orbital space, optic nerve, along the intracranial portion of the internal carotid artery and the cavernous sinus. Subtle enhancement was seen in the left ethmoid sinus in continuity with enhancement along the medial orbital wall. The entire internal carotid artery showed moderate stenosis.Prognosis is very poor and mortality is very high in rhinocerebral mucormycosis with intracranial extension.We want to emphasize that MRI is the most important imaging modality in making an early diagnosis and detecting complications. CT scan is complimentary to MRI and is helpful in detecting any bony defects. Histopathological identification of broad, aseptate, obtuse-angle branching hyphal elements in the background of inflammatory necrotic tissue is confirmatory. Our case is unique as there was extensive orbital involvement, stenosis of the entire internal carotid artery and very subtle involvement of the ethmoid sinus. Although biopsy from the ethmoid sinus did not reveal any fungal elements, we presume that it was owing to a lack of representative sample. Our case is also unique because of the presence of tooth caries, with periapical cyst and oroantral fistula. We are not sure whether these are incidental findings or related to the spread of mucormycosis.Mucor, Rhizopus and Absida) that are ubiquitous and normally saprophytic in humans but can produce fatal disease in persons with immunosuppression. The causative fungi are recognized by viewing broad, aseptate, obtuse-angle branching hyphal elements.Mucormycosis is caused by fungal organisms belonging to the family of Mucoraceae (Five major types of mucormycosis are recognized: rhinocerebral, gastrointestinal, pulmonary, cutaneous and disseminated. The rhinocerebral form is the most common and is seen mostly in patients with uncontrolled diabetes.In the rhino-orbito-cerebral form, the infection originates in the nasal cavity and spreads to the paranasal sinuses, orbits and then intracranially, with cerebral or cranial nerve involvement. Intracranial spread to the cavernous sinus may result in haemorrhage or thrombosis of the internal carotid artery, or thrombosis of the cavernous sinus.The pathophysiology of mucormycosis is related to the invasion of the walls of the vessels, especially the arteries.The common presenting symptoms are headache, low-grade fever, facial swelling, sinusitis, orbital apex syndrome, visual loss, cranial palsies from cavernous sinus involvement and focal neurological defects owing to cerebral involvement.The differential diagnoses for orbital cellulitis and sinusitis in patients with immunosuppression are: mucormycosis and other fungal infections, paranasal sinus malignancy, bacterial cellulitis, carotid cavernous fistula and thrombosis, inflammatory pseudotumour and Graves’ disease.CT scan findings in sinonasal mucormycosis are soft tissue opacification of the sinuses with hyperdense material, nodular mucosal thickening and an absence of fluid levels.The maxillary, ethmoid, frontal and sphenoid sinuses are involved in decreasing order of frequency.T1 and T2 weighted images, variable contrast enhancement and restriction of diffusion. Contrast-enhanced T1 weighted images are helpful in delineating the orbital and intracranial spread.MRI has a very important role in the diagnosis of rhinocerebral mucormycosis, especially in the early detection of orbital and intracranial spread and their complications. Lesions can show variable MR signal characteristics on Prognosis is very poor and mortality is very high in rhino-orbito-cerebral mucormycosis, especially with intracranial extension.Informed consent was obtained and is held on record.


Increasing access to cochlear implants within the resource-constrained South African context calls for careful investigation of all factors that might influence benefit from this technological advancement.The aim of this study was to investigate preservation of hearing following cochlear implant surgery and whether a relationship existed between the post-operative hearing findings and certain factors.Within a quantitative paradigm, a retrospective data review design was adopted where a sample consisting of audiological records from 60 observations and surgical records from two cochlear implant units in South Africa was investigated. These records were selected using purposive sampling and consisted of records from participants ranging from 6 to 59 years. Comparative analysis of unaided audiological test results was pre- and post-operatively performed, where all paitents were implanted with cochlear devices. Factors documented to have a possible influence on post-operative outcomes were examined in an attempt to establish relationships that may exist. Findings were analysed by means of both inferential and descriptive statistics.The findings indicated 92% success rate in preservation of residual hearing. There was a direct correlation between surgical techniques, as well as cochlear implant type and the successful hearing findings, in the absence of surgical complications. Other factors explored did not have any negative effect on the hearing findings.The study findings suggest improved surgical outcomes with enhanced surgical techniques and advanced technology, with a clear negative impact of intraoperative complications on the outcomes. Evidence of improved speech perception because of technological advances in cochlear implantation and improvements in surgical techniques exists  were women, with the mean age being 30.8 years. All participant records had to meet specific inclusion criteria. Participation criteria used in South Africa include cochlear implant fitting, residual preoperative hearing thresholds, less than 60% for sentence recognition in the ear to be implanted , electrode type, electrode array insertion, electrode array depth, surgical technique and intraoperative complications.The researcher collected additional data, which included the following:The change in hearing thresholds from preoperative to post-operative hearing threshold levels was classified for each participant, with a change of 0–10 dB depicting complete preservation of residual hearing, a change greater than 10 dB indicating partial preservation and no response post-surgery indicating complete loss of residual hearing. These findings are published by Gautschi-Mills et al. , and thePre- and post-operative unaided thresholds were presented in order to compare them. For inferential statistics, the 95% confidence level was used throughout. General patterns or trends were examined to determine whether certain relationships existed between the post-operative hearing thresholds and the following:degree of preoperative hearingduration between surgery and unaided post-operative hearing testingelectrode type.X2 test was used to assess the relationships between categorical variables.The Ethical clearance was obtained prior to the commencement of the study (Protocol number: M111037), with permission from all relevant authorities. Furthermore, this research adhered to the Helsinki Declaration of 1975, as revised in 2008.Findings from the study of which this current study formed a part, revealed that preoperatively participants in the overall sample presented with severe to profound range of hearing loss, with some degree of low-frequency residual hearing with poorer hearing in the high frequencies  and 2 G.These findings are consistent with the tonotopicity of the cochlea, where high frequencies are more susceptible to damage than lower frequencies .Current findings are inconsistent with those by Balkany et al.  who founDespite the fact that there was a correlation between the degree of preoperative hearing threshold levels and post-operative hearing in Balkany et al.’s  study, tAs depicted in An analysis of the relationship between the hearing findings and aetiology revealed that the effect of aetiology on change in hearing thresholds was not significant for any of the frequencies. The fact that aetiologies in the present study did not have any effect on the hearing findings is consistent with the findings of Skarzynski et al.  who founThe mean duration of hearing loss prior to surgery was established to determine the relationship between the duration of the hearing loss before surgery and the post-operative hearing findings.The study findings indicated that the mean duration of hearing loss prior to surgery was 22 years and 9 months (s.d. = ± 3.1) overall. The effect of duration of hearing loss was not significant for any of the frequencies. Thus, there was no significant difference in terms of post-operative hearing threshold levels between those implantees who had a longer duration of preoperative hearing loss and those implantees who experienced hearing loss for a shorter period of time. This is contrary to what Lenarz et al.  found, tThe researcher examined the mean time between surgery and first post-operative hearing test to ascertain whether this had had any influence on the post-operative hearing threshold levels. This was used as a covariate in the analysis of the results in order to prevent the findings being affected by any extraneous variables. The mean time between surgery and first post-operative hearing test was 24.7 months (s.d. = ±9.0) (range 0.5–159 months).Findings on the relationship between the hearing findings and the duration between surgery and unaided post-operative hearing testing indicated that this time was not significant. This means that the duration between surgery and unaided post-operative hearing testing did not affect the preservation of residual hearing post-operatively. This is a notable finding from this study where the upper extreme was 13 years and 4 months. These findings seem contrary to previously published research where a number of patients lost their residual hearing at different times post-surgery, with 30% of the sample experiencing a low-frequency threshold drop of more than 30 dB over time . In this study, the electrode type that was used most after the two categories mentioned above  was the CI24R. However, there were only five observations for the CI24R. There were even fewer observations for other electrode types that included CI24M, Nucleus CI24(ST) and Nucleus CI422. Statistical analysis could not be performed on these electrode types as there was not sufficient data because the number of observations was too small.As shown in The effect of electrode type on change in hearing thresholds was not significant for any of the frequencies, with the exception of 2000 Hz: the mean change in hearing thresholds was 5.9 dB higher for other versus Nucleus Freedom (NF). In other words, the electrode type did not have a negative effect on the post-operative hearing threshold levels , with the exception of that at 2000 Hz. Given that there was no trend with respect to the other frequencies, this could have been a spurious result.Although the discrepancy of 5.9 dB between the NF and other was only at one frequency, namely 2000 Hz, and the difference was minimal, this was clinically relevant for speech understanding.The current findings are consistent with those of Cosetti et al.  who founThe large majority see  of the pThe Contour Advance electrode is the only perimodiolar array in the industry and is pre-curved for complete insertion, with Softip that enables atraumatic insertion  at both centres combined; therefore, it can be inferred that this technique did not negatively influence the hearing findings. O’Connell, Hunter and Wanna  assert aIn the current study , the resThere was only one case of electrode array insertion depth with partial insertion, possibly because of obstructions within the cochlea, such as cochlear otosclerosis, cochlear anomalies and so on  dB lower for Contour on stylet (Contour) versus AOS, and the mean change in hearing thresholds was 7.9 (6.8) dB higher for other versus AOS. As this discrepancy was limited to one frequency (2000 Hz), and as the change in hearing threshold levels was minimal, this was not statistically significant.From the surgical notes of the surgeons, the researcher investigated surgical complications to determine the possible influence on residual hearing and whether they affected the conservation of functional hearing post-surgery. These are reported in the Gautschi-Mills et al.  study, wAccording to Clark, Tong and Martin  and BalkWhen summarising the relationship between the hearing findings and the various factors listed above, it became evident that the variables investigated had little or no negative effect in general on the preservation of residual hearing, with the exception of surgical complications  means that the current findings show a snapshot at one point in time post-surgery without establishing whether the preserved residual hearing thresholds remained stable over time. Thirdly, although the current sample size was deemed large enough, the fact that it was conducted in only two cochlear implant units in South Africa limits the generalisability of the findings to the broader population in South Africa. Despite these limitations, the findings have implications for clinical assessment and management, as well as for surgical management of cochlear implant candidates. Preoperative counselling of cochlear implant candidates with residual hearing may benefit from current evidence as candidates may be informed about the possible benefits and risks involved. Future cochlear implant candidates with residual hearing and those candidates for EAS may therefore benefit from these findings as these may assist them in the decision-making process. The conservation of residual hearing post-surgery has many advantages for future cochlear implant candidates with preoperative residual hearing and for those implantees with preserved post-operative residual hearing. These cochlear implant candidates and recipients could then become candidates for EAS, which, in turn, has many hearing benefits, such as improved speech perception in noise and musical acuity. Evidence of the negative impact of intraoperative complications on preservation of residual hearing found in the current study raises implications for surgeons involved in cochlear implantation.For replication and generalisation of the current findings, as well as opportunity for diversity in the various variables explored, it is recommended that this study should be conducted on a broader population at a number of different cochlear implant centres across South Africa, with larger sample sizes, in order to obtain data that could be generalised to an even broader population in South Africa.


Serratia marcescens and demonstrates a wide array of biological activities and applications. Derivatives of prodiginine have since been characterized in the marine γ-proteobacterium, Pseudoalteromonas. Although biosynthetic gene clusters involved in prodiginine synthesis display homology among genera, there is an evident structural difference in the resulting metabolites. This review will summarize prodiginine biosynthesis, bioactivity, and gene regulation in Pseudoalteromonas in comparison to the previously characterized species of Serratia, discuss the ecological contributions of Pseudoalteromonas in the marine microbiome and their eukaryotic hosts, and consider the importance of modern functional genomics and classic DNA manipulation to understand the overall prodiginine biosynthesis pathway.The Prodiginine family consists of primarily red-pigmented tripyrrole secondary metabolites that were first characterized in the Gram-negative bacterial species  Serratia marcescens. It consisted of a straight alkyl chain substituent and was named prodigiosin  prodigiosin   . PigP wagulators  Further gulators . Indepengulators . Due to  species .Pseudoalteromonas remains elusive. To date, more than 200 Pseudoalteromonas genomes are sequenced and available on genome databases. Of those 200+ genomes, 9 genomes are associated with a prodiginine-producing species and only 3 belong to a tambjamine-producing species.Although extensive studies were done to determine the bioactivity and chemical structure of these prodiginine analogs, the means of biological synthesis in Pseudoalteromonas, their bioactivity, and their biosynthesis. These ideas may provide further insight into the role prodiginines play in Pseudoalteromonas, their hosts, and the marine microbiome.This review will focus on prodiginine classes that were characterized in Pseudoalteromonas bacterial strains synthesize prodiginine analogs including prodigiosin, cycloprodigiosin, and 2-(p-hydroxybenzyl) prodigiosin (n-amyl-pyrrole (MAP) and 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC). These analogs demonstrate antibacterial, anticancer, and immunosuppressive bioactivity and each analog exhibits its own specific bioactive properties. The specificity among these microbial pigments emphasizes their potential biomedical application.digiosin . These pSerratiamarcescens and later characterized in Pseudoalteromonas rubra  and is composed of genes pigB, pigD, and pigE. First, PigD, in the presence of coenzyme thiamine pyrophosphate (TPP), catalyzes the addition of pyruvate (2) to 2-octenal (1). The formation of 3-acetyloctanal (3) occurs with the release of CO2, followed by the transfer of an amino group to the aldehyde by PigE, and cyclization to form H2MAP (4). PigB catalyzes further oxidation to form MAP (5)  .pigA, pigF-J, pigL, and pigM). 4′-phosphopantetheinyl transferase (PigL) activates peptidyl carrier protein (PCP) domain of PigG from apo to holo form through the addition of the 4′-phosphopantetheinyl group. PigI and ATP catalyze the transfer of the L-prolyl group of L-proline (6) to the thiol group of phosphopantetheine to form a L-prolyl-S-PCP intermediate (7). PigA oxidizes the intermediate to pyrrolyl-2-carboxyl-S-PCP (8). This proceeds with a polyketide-type chain extension, which starts with the transfer of the pyrrole-2-carboxyl group from PigG to the cysteine active site of PigJ, generating a pyrrole-2-carboxyl thioester (9). Separately, the ACP domains of PigH are phosphopantetheinylated, providing binding sites for the malonyl group of malonyl-CoA (10). The bound malonyl (11) then undergoes decarboxylation, resulting in a subsequent condensation with the pyrrole-2-carboxyl thioester attached to PigJ and forms pyrrolyl-β-ketothioester on PigH (12). PigH catalyzes another decarboxylation between serine and pyrrolyl-β-ketothioester to generate 4-hydroxy-2,2′-bipyrrole-5-methanol (HBM) (13) (14). The final step of MBC (15) biosynthesis involves the methylation of the HBC hydroxyl group by methyltransferase (PigF) and oxidoreductase (PigN) (The common pathway in most prodiginine-producing microorganisms is MBC biosynthesis. This pathway is composed of seven genes (BM) (13) . The alce (PigN) .5) and MBC (15) are synthesized, PigC uses ATP to facilitate the terminal condensation of these two pyrroles to form prodigiosin (16). PigC has shown sequence similarity to an ATP-binding and phosphoryl transfer domains at the N- and C- terminus, respectively   . Furtherl blooms .Trypanosoma cruzi which causes Chagas disease  (22) is a red-pigmented secondary metabolite that was isolated from the marine bacteria P. denitrificans and P. rubra (5).Recently, prodiginine classes were characterized in the marine bacterium genus P. rubra . The keyStreptomyces, redG and mcpG encode oxygenases that cyclize undecylprodiginine to form butyl-meta-cycloheptylprodiginine and metacycloprodigiosin, respectively (pig biosynthetic gene cluster (22).Cyclic prodiginines and their respective oxygenase for cyclization were identified. In ectively . Recent  cluster . This enIt is still unclear what sort of advantages, if any, come with prodigiosin cyclization . WhetherPlasmodium berghei cells than chloroquine and its derivatives  prodigiosin (HBPG) (23) was isolated from the marine bacterium P. rubra (23) and prodigiosin (16) is the para-addition of phenol to the C-10 carbon of pyrrole ring A of MBC (15), suggesting variations to the MBC pathway and homologs in the MAP pathway and terminal condensation step. Currently, there is no literature describing HBPG biosynthesis. Difficulties in characterization arise when biosynthetic genes are not clustered together, like that of the cPrG pathway. As shown by 2-, E. coli, and C. albicans  and begins with dodecenoic acid (17). Until recently, afaA was thought to initiate the enamine biosynthesis (17) (18) undergoes oxidation by dehydrogenase (TamT), introducing a π-bond to C-3 carbon of the fatty acyl side chain (19). Reductase/aminotransferase (TamH) facilitates the reduction of the CoA-ester (19) and transamination to dodec-3-en-1-amine (20). TamQ facilitates the condensation of MBC (15) and enamine (20) to form tambjamine (21) (Novel to the ynthesis . Howeversis (17) . The actine (21) .S. aureus, E. coli, C. albicans, and Malassezia furfur  responsible for enamine biosynthesis in P. tunicata showed 40% identity to an aminotransferase (pigE) responsible for MAP biosynthesis in Serratia and the other two Pseudoalteromonas species (With current breakthroughs in functional genomics, one can now identify homologous prodiginine biosynthetic gene clusters within pathways . Prelimiaged 54% . Althoughomology . However species .P. rubra, play a key role in both regulating and biosynthesizing prodiginine analogs. However, PRUB680 showed low sequence homology to redG in Streptomyces at 46% (tamA was responsible for the initiation of enamine biosynthesis, further feeding into tambjamine biosynthesis. That is not to say that afaA does not still play a role in tambjamine biosynthesis.As previously discussed, there are limitations to relying solely on functional genomics. Genes outside of the primary gene cluster, as demonstrated with PRUB680 in s at 46% . AlthougPseudoalteromonas’ genome and explore their numerous biological pathways. Pseudoalteromonas genome can be manipulated via Tn10 random mutagenesis, however, this method requires screening hundreds to thousands of mutants before isolating one that disrupts the gene of interest. P. rubra’s genome via suicide and expression shuttle vectors, respectively. Recently, Pseudoalteromonas phages and prophages belonging to the myoviridae, podoviridae, and siphoviridae families have been isolated and sequenced several instances Pseudoalteromonas.Several molecular tools have been used to manipulate Pseudoalteromonas species such as their ecological and overall roles within the marine microbiome.In conclusion, a more holistic approach to understanding bioactivity and gene regulation will lay the foundation for answering overarching questions concerning pigmented FS-K and JA contributed to the conception of the focus for the study. FS-K contributed to the compilation of all sections, figure and table design, and wrote the first draft of the manuscript. CI performed the compilation of biological activity, ecological and evolutionary significance sections. KH contributed to the biosynthetic pathways and biological activity sections. All authors contributed to revision, read and approved the submitted version of the manuscript.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.


N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects.Trichosanthin (TCS) is an RNA  Trichosanthes kirilowii Maximowicz (Tian Hua Fen), which has been used clinically as an abortifacient drug in early and mid-gestation for centuries. Apart from inducing midterm abortion [Trichosanthin (TCS) is extracted from the root tuber of the Chinese medicinal herb abortion , TCS disabortion ,3 and anabortion ,5 activiabortion ,7,8,9,10N-glycosidic bond at adenine-4324 of 28 S rRNA [The precursor of TCS is a 27 kDa protein consisting of 289 aa. The active form of TCS is obtained after the N-terminal 23 aa signal peptide and C-terminal 19 aa peptide are removed . TCS bel8 S rRNA . This ha8 S rRNA ,14.+CD25+ regulatory T cells [TCS manifests attractive pharmacological properties for its anti-tumor ,15,16,17 T cells ,28 and t T cells ,28. In tThe crystal structure shows that TCS consists of two domains . The larTCS was identified to bind the ribosomal stalk P proteins and L10a ,33. In eThe NMR structure study of the stalk protein complex of P1/P2 heterodimer and biochemical analyses demonstrated that the flexible C-terminal tails of eukaryotic ribosome stalk can form an arm-like structure that extend with a radius up to ~125 Å ,44,45. TTaken together, the structural and biochemical observations suggest that RIPs and eEF2 may compete for binding to the flexible C-terminal tail of ribosomal stalk P protein. Therefore, we proposed that eukaryote-specific RIPs may hijack the elongation-factor recruiting function of ribosomal stalk to access to the α-SRL .It is difficult for TCS to enter cells because the protein lacks a lectin-binding domain found in ricin. It is found that both receptor-mediated endocytosis and non-specific entry may be involved in the cell entry route of TCS  Figure . TCS couTCS has a broad spectrum of biological and pharmacological activities, including induction of abortion, inhibition of tumor cell proliferation via render protein synthesis, anti-viral activity, effective against a variety of tumors and immunomodulatory activity. These have been reviewed previously . Here, w+ cells in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex [Previous studies reported that TCS selectively inhibits the replication of HIV virus type 1 (HIV-1) in both acutely infected T-lymphoblastoid cells and chronically infected macrophages in vitro . Phase I complex ,66. It i complex . Intrigu complex . Combine complex . The pen complex . It was  complex .Trichosanthes kirilowii has a long history of clinical use for treating hepatitis B virus (HBV) in China. As the main component in the aqueous extract of Trichosanthis Radix, TCS is found responsible for the anti-HBV effect [The plant V effect . TCS hasV effect . HoweverHerpes simplex virus (HSV) is responsible for a broad range of human diseases . Type 2 Previous studies have indicated that TCS exerts a selective and high toxicity for a broad range of tumor cells ,77 in viTCS is known to induce apoptosis of various tumor cell lines. Previous reviews have summarized that TCS could regulate various signaling pathways to determinate the tumor cell fates and induce apoptosis of tumor cells ,91, incl(1) Inhibition of Tumor Cell ProliferationTCS may activate the JNK/MAPK signaling pathway to inhibit the cell viability and proliferation of human epithelial type 2 (HEp-2) and AMC-HN-8 human laryngeal epidermoid carcinoma cells . In HeLa(2) Induction of Intrinsic and Extrinsic Apoptosis in Tumor Cells2+) and ROS production in HeLa [TCS treatment could induce elevation of intracellular calcium ions  Regulation of Apoptosis-Associated GenesRecent reports showed that TCS could inhibit DNA methyltransferase 1 (DNMT1) and restore the expression of methylation-silenced tumor suppressor genes in adenomatous polyposis coli (APC) and tumor suppressor gene in lung cancer 1 (TSLC1) . TCS act(4) Regulation of CytoskeletonTCS can induce specific changes, such as depolymerizing microfilaments (MF) and ring-shaped microtubules (MT) structure in cytoskeleton configuration in apoptotic HeLa cells  Figure . MF rearN-glycosidase activity [Recent studies have demonstrated that TCS exerts significant anti-tumor effect on human gastric cancer MKN-45 cells via up-regulation of the autophagy protein 5 (Atg5), and conversion of the autophagosome marker LC3 I to LC3 II, then activates NF-kB/p53 pathway, thereby inducing the generation of reactive oxygen species (ROS) to induce gastric cancer cell autophagy . Howeveractivity ,106.+CD25+ regulatory T cells, thus prolonging survival duration and preventing graft-versus-host disease in the mice model [A previous study showed that TCS is a potent immunosuppressive protein which could affect humoral immunity through regulating the ratio of immunoregulatory T lymphocytes cells ,107 and ce model . It has ce model . These rTo promote the potential use of TCS as a therapeutic agent, there are attempts to use different approaches to modify TCS as summarized in a previous review . MonocloTCS possesses a number of biological activities, including anti-virus, anti-tumor and immune-regulatory functions. Existing reports showed that the anti-tumor properties and the mechanism of TCS vary in different tumor cell lines, including inhibition of viability and proliferation, induction of expression of apoptosis-related genes, regulation of cytoskeleton structure and activation of multiple intrinsic and extrinsic apoptosis/autophagy pathways of tumor cells. Also, TCS exploits different antivirus approaches toward various viruses. Although the mechanism of action of TCS has not yet been solved, there are ongoing efforts to further modify TCS to promote its pharmacological properties and explore the potential medicinal applications of TCS in cancer immunotherapy. TCS may also have potential therapeutic value for transplantation rejection and other inflammatory diseases for its immunomodulatory effects. Further research on the mechanism and the pharmacological application of TCS will not only increase the translational values of this important protein, but the effort may also be extended to RIPs found in other plants, bacteria and mushrooms.


Inheritance of a deafness-causing genotype does not necessarily mean that a person will have profound hearing loss. The presence of a modifying gene can change the effect of the deafness genotype. As an example, mutations found commonly among the deaf are at times found among their normal hearing relatives [1]. The implication is that there are genes or gene products whose interactions allow the normal physiological function of the inner ear despite a mutation that would normally disrupt the process. Deafness is not the first disorder in which modifiers can change the expected outcome, nor will it be the last, but it is very unusual for the outcome to be so dramatically changed.