Among orthopedic surgery materials, poly (methyl methacrylate) (PMMA) is most commonly used for its excellent mechanical properties and rapid self-setting time. However, PMMA bone cement has been reported to cause thermal necrosis and to have poor bioactivity, which must be improved. In contrast, tricalcium silicate (TCS), the most significant component of Portland Cement and the most effective bone cement material, might not always meet the needs of the cement due to its poor mechanical properties and elevated pH levels during hydration. We hypothesize that the benefits of both PMMA and TCS can be harnessed by mixing them together in different proportions. This would represent a better solution for the issues faced when using them alone.We, therefore, prepared a novel organic-inorganic PMMA/TCS composite bone cement mixing PMMA and different amounts of TCS and tested its effect on the biophysical properties.in vitro study also revealed that the composite materials had higher cell viability than pure PMMA and TCS. Also, the in vivo study on animals indicated that the composite materials were more capable of forming bone, which further reinforced the biocompatibility of the proposed PMMA/TCS bone cement.The addition of 30% TCS reduced the exothermic temperature and pH variation during cement setting and hydration processes. However, the mechanical and handling properties of the bioactive PMMA/TCS composite were not affected. The By combining the advantages of each component, it could be possible to construct a more effective composite bone cement material. This would meet the needs of implantation material for orthopedic surgeries or a possible bone filler.  Scientific background on the subjectPoly (methylmethacrylate) (PMMA) is widely used for implant fixation in orthopedic and trauma surgery. However, it suffers from high exothermic reaction temperatures, low bioactivity, and monomer toxicity. Combining PMMA with inorganic fillers has shown improved properties. This study used tricalcium silicate (TCS) as an inorganic filler to develop a novel PMMA/TCS bone cement composite.What this study adds to the fieldOur study results suggest that the addition of TCS in the composite within the selected concentration range could induce bone regeneration and stabilize pH and exothermic temperature without compromising the mechanical and handling characteristics of PMMA bone cement. This could be a potential bone filler for various orthopedic surgeries.A bone tissue substitute is typically used to guide bone regeneration, leading to the development of artificial bone tissue substitutes such as bone cement. Clinically, the injection of bone cement using minimally invasive techniques has significant potential due to its advantages such as minimal secondary damage, flexibility in the operation process, and shorter hospital stays .In bone cement development, poly (methylmethacrylate) (PMMA)-based cement or its derivatives have been widely used for over 50 years to stabilize or fix the artificial joints or implants at the defect sites such as hip, wrist, shoulder, elbow and knee during various orthopedic and trauma surgeries ,3. PMMA As an alternative, inorganic cement with the significant components of calcium phosphate and calcium silicate, which have similar compositions to natural bones, was initially used to replace PMMA in the reconstruction of damaged bones . Through2) that forms during the hydration process of inorganic cement is likely to raise the pH of surrounding tissues [2 [Inorganic cement may have a limited mechanical property, particularly in terms of bending resistance and brittleness . Additio tissues ,17. Consssues [2 ,18. Unlissues [2 .2S), tricalcium silicate , tricalcium aluminate (C3A), and tetra calcium aluminoferrite (C4AF). Preparing this complicated composition is tedious and leads to diverse bioactive properties due to its various origins and preparation types [Among the inorganic fillers to PMMA bone cement, Portland Cement (P.C.) was considered an economical and viable alternative to other inorganic ingredients. P.C.-based materials have been used successfully for composite PMMA cement bioactivation . Howeveron types . Therefoon types ,26.This project aimed to develop a novel PMMA/TCS bone cement composite combining the desired properties of PMMA and TCS. The design of PMMA bone cement combined with TCS ceramics has not yet been described. The effect of TCS on PMMA bone cement's physicochemical and biological properties was studied systematically. Innovative composite bone cement was then developed based on the required properties of each component, such as a modified PMMA bone cement combining PMMA and TCS.We have applied two steps to prepare bone cement specimens. The detailed information on the preparation of raw materials for the bone cement can be seen in the The working and setting time was measured using Gillmore needles ,28. TCS −1 D695-91 to obtain the compressive strength. In this study, the compression was measured at a 20 mm/min speed until failure under room temperature. The compressive strength (MPa) was obtained from evaluating maximum stress after soaking in the deionized (DI) water for 7 and 14 days.According to ASTM, the complete set column specimens  was measured with Instron-1195 (USA), at a loading strain rate of 0.5 mm minλ = 0.15406 nm) operating at 40 mA and 40 kV. The diffraction data were collected in the range of 10–70° (2θ).The preliminary biocompatibility of material was evaluated through SBF immersion as prepared following Before and after soaking in SBF, the morphology differences in the PMMA/TCS bone cement samples were observed using a field-emission scanning electron microscope  at a potential of 20 kV. In addition, we have all samples prepared to be conductive by sputter-coating with platinum for morphological analysis.in vitro by immersion studies in SBF solution.For the pH measurement, groups of specimens were shaped into 10 mm in diameter and 5 mm in height [Tmax) indicates the highest temperature reached from the detectable maximum exothermic in the polymerization reaction [We have the exothermic temperature–time profiles during the solidification of specimens evaluated with a K-type thermocouple  connected to a temperature controller . PMMA/TCS bone cement samples with different weight ratios of TCS were molded in a PTFE mold . The temperatures were measured and noted every 30 s for 15 min at 24 °C. The peak temperature (The distribution of TCS additives in the PMMA matrix was verified using the micro-computed tomography technique (Micro-CT). PMMA/TCS bone cement with different TCS content was examined using a desk-top Micro-CT imager . The system was operated at an image resolution of 35 μm voxel with current and voltage maintained at 100 μA and 80 kV, respectively, and a 1.0 mm aluminum filter.2 incubator.We have the osteoblast-like MG-63 cell line from human osteosarcoma  applied to analyze the various PMMA/TCS bone cement combinations. Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P.S.) solution were purchased from Sigma–Aldrich (USA), and were used as the complete growth culture medium. The MG-63 cells were cultured in the growth culture medium at 37 °C and maintained in a 5% COA well-known MTT -2,5-Diphenyltetrazolium Bromide, Sigma) colorimetric assay was used to evaluate the cell viability of the PMMA/TCS bone cement samples with different weight ratios of TCS. This test was conducted following ISO 10993–5 . The det4) on the bone composites surface, we have the test conducted at 1, 3, 7, 14, and 21 days, accompanied by a culture medium changing thrice per week. Finally, we have the ALP production on the composite surfaces quantified with an ELISA reader at 405 nm.Considering the presence of alkaline phosphate (ALP) activity, the ALP T1016 kit  was equipped to determine the ALP production, a marker of osteogenesis. Following an early seeding of MG-63 cells  to evaluate the biocompatibility of the PMMA/TCS composite . Entire procedures were done at MacKay Memorial Hospital  with prior review and approval of the Institutional Animal Care and Use Committee (IACUC) (MMH-A-S-106-18). Experimental procedures were entirely performed under aseptic conditions to prevent the infection. We have two randomly selected groups with five rats in each one: (i) the control one was the blank group without any implantation, and (ii) the test group was the PMMA/TCS samples with TCS 30 implantation. The complete procedure can be seen in the section S4 in the supplementary data. Following the hematoxylin-eosin (HE) staining, the histological sections were analyzed using a Nikon Eclipse 50i upright microscope . Three wounds per group were conducted.To understand the in vitro experiments were repeated five times (n = 5), and the reuslts were represented as mean ± standard deviation. At a p-value <0.05, the obtained data with the Student's t-test were regarded as statistically significant.All the obtained data from the handling, compression, and in vitro bioactivity.The material must fulfil all the required characteristics that the bone cement should have. However, none of them is ideal, and there is still some limitation. In which case, we propose to have a bioactive bone cement composite prepared with TCS as additives and mixed with conventional PMMA to integrate the lowered cost and excellent bioactivity benefits of TCS with the high strength and fast setting properties of the pure PMMA. Therefore, we have the following results and discussions to investigate the PMMA/TCS composite characteristics, including their One of cement's most critical handling characteristics is its working and setting time, varied when their composition, mixing environment, and the presented additives were changed . In the As shown in p < 0.05) except the TCS 80, which showed similar compressive strength. The composite with 10% TCS produced a maximum strength value. However, the value was significantly reduced when the TCS composition was increased. The diminution phenomenon observed might be due to the discontinuous and disintegrated PMMA matrix after the addition of TCS, with the whole structure becoming porous, which causes a decline in compressive strength [The compressive strength change in the PMMA/TCS bone cement with different weight ratios of TCS (10%–80%) after 7 and 14 days of setting is represented in the strength . A similstrength .The minimum required compressive strength value of 70 MPa was defined in ISO-5833-2002 for the cured bone cement , and all3SiO5) with Barium sulfate (BaSO4) added as a radiopaque agent in the commercial PMMA bone powder. In addition, hydroxyapatite (HAp) and un-hydrated TCS were also observed. Moreover, the characteristic peak of HAp at 2θ = 32.72° appeared in each hybrid combination with almost similar intensity. Therefore, the precipitation of HAp observed might be the main reason for the bioactivity of bone cement [The components of our prepared PMMA/TCS cement were characterized using XRD. The diffraction patterns of the PMMA/TCS bone cement with a variation of TCS ratio (10%–30%) after immersing in SBF solution for seven days are shown in the e cement .Fig. 2(A2) is accompanied by the tricalcium silicate hydration and causes an elevated pH value up to 12 [up to 12 ,17.2 formed for the PMMA/TCS cement in the SBF environment. However, the pH values of living environments lower than 9.0 should be safe for the growth and proliferation of most human cells [A similar trend of pH variation was shown by the PMMA/TCS composite cement with a rapid pH increase in the first seven days but stabilized after that. At the same time, all the PMMA/TCS bone cement with different weight ratios of TCS (10%–30%) soaked in SBF showed a much lower pH than that of the pure TCS. Moreover, the stabilized pH values of composite cement increase as the weight percentage of the TCS in the PMMA increases, with the maximum pH value of 8.3 and 7.5 reached when the content of TCS is 30% and 10%, respectively, probably due to more of the Ca(OH)an cells .The SEM micrographs of PMMA/TCS cement surfaces are observed in n vitro bioactivity when immersed in SBF due to the induction of surface HAp formation [in vivo [Cement with PMMA alone is usually morphologically very dense, thus may not permit bone ingrowth. In contrast, TCS-based material may exhibit excellent iormation . Accordi[in vivo ,41 throu[in vivo . Most imFinally, the exothermic temperature of the cement samples was investigated in the temperature–time graph for change in temperature during the exothermic setting process of pure PMMA (0%) and PMMA/TCS bone cement composite samples with different TCS content involved . As shown in p < 0.05). The proliferation effect tends to increase with the increased TCS content, suggesting a positive impact of TCS on cell proliferation. Similar works were published that the cell proliferation and gene expression in osteoblast stimulated by the degradation of non-toxic ionic products from the calcium silicate bioactive glass [p < 0.05) than the PMMA/TCS bone cement samples, suggesting that a specific TCS concentration range is required to stimulate cell proliferation from the dissolution products of PMMA/TCS composite. Thus, the effective TCS concentration in promoting cell proliferation is another critical topic for further investigation.ve glass ,46. Howep < 0.05) after 1, 3, 7, 14, and 21 days in SBF. Also, at each measurement up to 21 days, the ALP concentration of the pure PMMA was significantly much lower (p < 0.001) than TCS 30. The decrease in the activity of ALP seen after 14 days may be due to the upregulation of other genes for example osteocalcin, which made the expression of ALP to decline. Also, it is known that the ALP helps in the mineralization of the bone cells and we can see that the activity of ALP is higher in the 14th day, which proves that the cells are completely mineralized and after that the cells started to differentiate [The secreted ALP concentrations from co-cultured MG63 with pure PMMA and PMMA/TCS bone cement with different weight ratios of TCS after 1, 3, 7, 14, and 21 days were shown in rentiate . Moreoven vivo study.Considering the results obtained from handling time, compressive strength, pH, exothermic temperature and cell viability experiments, the higher concentration of TCS  mixed with PMMA provided the optimal and acceptable values for choosing it as a safe and appropriate bone cement composition for testing against animal in the following IIn vivo study in rat calvarial defect repair model were used for evaluating the clinical application of the PMMA/TCS composite for bone regeneration. Representative histological H&E was shown in in vivo results demonstrate the biocompatibility of the PMMA/TCS, especially at the interface and enhancement of bone like tissue growth after a more extended time. As a result, the TCS incorporated PMMA might improve the binding between in situ bone tissue and the bone cement, as TCS can facilitate strong osteocyte adhesion and differentiation. Furthermore, the unique bioactivity from this composite material design might provide much stable fixation after implantation.Based on these results, the implanted PMMA/TCS bone cement with the bone-like apatite layer in the ambient fluids might enhance bone regeneration. Unfortunately, we also encountered some limitations in this study, which we wish to address for better insights. For example, the molecular mechanisms of how the bone cement interacts with the bone-forming cells are still unknown, and further investigation is needed. The reason behind the higher cell viability exerted by pure PMMA extracts is also inconclusive. Additionally, the use of radiopaque materials such as Barium sulfate, which lack X-ray absorption properties, could adversely affect the mechanical properties of the proposed PMMA/TCS bone cement composite. Furthermore, we are still unsure as to whether the proposed PMMA/TCS composite will meet the requirements for use in clinical studies in the near future. Further studies into the safety of the composite are required before clinical studies can begin.In this study, a novel PMMA-based composite cement fabricated from the conventional PMMA mixed with TCS with different weight ratio was successfully prepared. TCS was selected due to its low cost and excellent bioactivity properties. However, MMA has high mechanical strength as well as fast setting properties. Combining both PMMA/TCS characteristics and the impact of TCS on their integrated properties should have investigated.in vivo animal study further demonstrates that the PMMA/TCS bone cement could enhance bone regeneration. Thus the novel PMMA based composite cement with the addition of TCS in the selected concentration range exhibiting the stabilized pH and the exothermic temperature values might be a potential bone filler for future orthopedic surgeries.The PMMA has rapid self-setting and high compressive strength but may cause thermal necrosis as the bone cement. Our developed bone cement suggests that TCS in the composite within the selected concentration range did not change the mechanical and handling characteristics of PMMA-based cement. On the other hand, the addition of TCS could induce bone-like apatite formation in SBF, making the composite cement bioactive. The apatite layer may further insulate the heat transfer and reduce the peak temperature observed during the cement setting process. Also, the dissolution extracts of composite could stimulate cell proliferation within the selected TCS concentration range. The The authors have declared that no conflicts of interest exists.


Hypertrophic pachymeningitis (HP) is a local or diffuse fibrous thickness of the dura mater of the brain or spinal cord, caused by infection or connective tissue disease. Headache is the most common clinical symptom, followed by various cranial nerve disorders such as visual impairment, diplopia, and hearing loss. HP can be classified into secondary and idiopathic. Here, we report a case of bilateral progressive profound sensorineural hearing loss diagnosed in a patient with idiopathic HP, where a cochlear implant was effectively used.The patient was a 77-year-old woman. Hearing loss gradually progressed bilaterally, and magnetic resonance imaging showed a space-occupying lesion with a continuous contrast enhancement in the bilateral internal auditory canals, and diffused dural thickening from the middle to the posterior cranial fossa.A trans-labyrinthine biopsy was conducted, and a definite diagnosis of idiopathic HP was made. Thickening of the dura mater in the bilateral internal auditory canals was thought to cause profound hearing loss.A cochlear implant was implemented 4 months after biopsy, and a favorable hearing response was obtained postoperatively.This is the first report of a cochlear implant in a patient with idiopathic HP. Cochlear implantation was considered a good treatment for profound hearing loss due to idiopathic HP, which provides a reference for patients to receive timely and correct treatment. This disease concept was first proposed by Charcot et al in the late 19th century and can be classified into secondary HP and idiopathic HP. The causes of secondary HP can be roughly divided into infectious causes, such as tuberculosis, fungi, and syphilis, and immune-mediated causes, such as an anti-neutrophil cytoplasmic antibody (ANCA)-related disease, IgG4-related disease, rheumatoid arthritis, sarcoidosis, and temporal arteritis. Although the frequency is low, tumor-based causes, such as dural metastasis of malignant tumors and malignant lymphomas, are also possible. Headache is the most common clinical symptom, followed by various cranial nerve disorders such as visual impairment, diplopia, and hearing loss. There are also reports of higher functional impairments such as disturbance of consciousness, convulsions, and memory impairment. A diagnosis of idiopathic HP is made if secondary diseases such as infections, autoimmune disease, or tumor-related causes are ruled out. Steroids are the most effective treatment for secondary HP.,3Hypertrophic pachymeningitis (HP) is a local or diffuse fibrous thickness of the dura mater of the brain or spinal cord, caused by infection or connective tissue disease.Here, we report a case of bilateral progressive profound sensorineural hearing loss diagnosed in a patient with idiopathic HP, where a cochlear implant was effectively used.The patient was a 77-year-old woman. She became aware of her tinnitus 6 years ago, but pure tone audiometry revealed a normal hearing level. The following year, she noticed a left-sided hearing loss, she was diagnosed with sudden hearing loss, and was treated with oral steroids, which improved her left hearing. However, her left-sided sensorineural hearing loss worsened 5 years later, and magnetic resonance imaging (MRI) revealed bilateral internal auditory canal-space-occupying lesions. The patient was referred to our department.The hearing level in pure tone audiometry was 30 dBHL on the right, and scaled out on the left Fig. A. The beGiven the risk of postoperative infection due to aplastic anemia, we did not conduct a biopsy of the inner ear canal and dura mater. The patient was introduced to a hearing aid for the right ear, and was followed up with audiometry and MRI periodically. Afterward, sporadic dizziness attacks began to appear, and hearing on the right side gradually worsened. Right sensorineural hearing loss progressed rapidly, and both sides were almost scaled out. To make a definite diagnosis, a biopsy was conducted using the trans-labyrinthine approach for the dural thickening and inner auditory canal-space occupying lesions on the left side.Intraoperative findings showed a slightly reddish dura mater of the inner auditory canal Fig. A, and a In the present case, the cause of hearing loss was thought to be due to the constriction of the cochlear nerve resulting from the dura thickening in the bilateral internal auditory canals. However, intracochlear calcification was already observed on CT before the biopsy Fig. , which wPostoperative cochlear implant threshold tests showed a favorable hearing response of 40 to 50 dB, and the hearing level has been maintained for 3 years.Informed consent has been obtained from the party concerned for publication of case reports.We experienced an idiopathic HP diagnosis following a biopsy accompanying bilateral progressive profound sensorineural hearing loss. Cochlear implantation was a useful method to overcome hearing loss in idiopathic HP. and 48% have vestibulocochlear neuropathy. Idiopathic HP is usually not associated with elevated angiotensin I-converting enzyme, myeloperoxidase anti-neutrophil cytoplasmic antibody, PR3-ANCA, rheumatoid factor, and antinuclear antibodies. Tests for Lyme disease, syphilis, tuberculosis, and fungi must be negative. Cerebrospinal fluid examinations by lumbar punctures should be negative for infection or malignancy. Idiopathic HP infers that the cause of HP is unknown. Although the pathogenic mechanism is largely unknown, it is generally thought that the inflammatory infiltrate consisting of B and T lymphocytes activates fibroblasts and induces collagen deposition, resulting in tissue hypertrophy and dural thickening. It has recently been suggested that a subset of this disease is IgG4-related diseases. Symptoms depend on the inflammatory lesion’s site and the adjacent neural structures’ compression.HP is a rare disease characterized by thickening of the dura mater, caused by various etiologies. Many patients are aware of chronic headaches at onset, Acoustic neuroma, a lesion of the 8th cranial nerve, is known to cause inner ear hearing loss due to tumor-induced ischemia and biochemical abnormalities in the inner ear fluid. Kawamura et al reported that a case of bilateral sensorineural hearing loss associated with HP in Cogan disease had a sense of pitch in the promontory test, and discussed that cochlear damage with a mechanism similar to that of acoustic neuroma might be the leading cause of hearing loss. Iwasaki et al found that HP patients with dizziness and hearing loss exhibited normal otoacoustic emissions and the presence of bilateral wave I prolongation in auditory brainstem response, therefore considering the possibility of inner ear hearing loss, and citing the obstruction of blood flow to the 8th cranial nerve due to dura mater thickening as a reason for this.In the present case, the sensorineural hearing loss was thought to be mainly caused by a retrolabyrinthine lesion, which causes auditory disturbances due to the entrapment of nerves in the inner auditory canal by the thickened dura mater. Furthermore, cochlear dysfunction was also considered a factor in hearing loss because intracochlear calcification was observed before surgery; therefore, favorable results were obtained with cochlear implantation. According to Bovo et al, HP-induced cochlear damage is due to compression of the inner ear artery and an autoimmune mechanism.,10Regarding cochlear implant surgery, inserting electrodes through the typical round window is difficult when there is intracochlear stenosis, as in the present case. Pasanisi et al and Kawamura et al used osteotomy and electrode insertion in patients with cochlear plantar rotation insufficiency with Cogan disease, and a similar approach was used in the present case.This is the first report of cochlear implantation for idiopathic HP. Cochlear implantation was an effective treatment for profound hearing loss in idiopathic HP. At present, no definitive treatment method has been shown to be effective for severe to profound hearing loss caused by idiopathic HP. Hearing aids are an option, but with severe to profound hearing loss the chances of getting a meaningful auditory response are unfortunately low. Cochlear implantation is expected to improve the quality of life of patients with hearing loss due to HP, as shown in this case.A limitation of this study is that it only reports one case. Therefore, whether cochlear implants will be effective in all cases of hearing loss due to idiopathic HP cannot be concluded. To this end, more cases need to be accumulated to determine the efficacy of cochlear implantation for hearing loss due to idiopathic HP.A patient with HP and bilateral internal auditory canal lesions presenting with bilateral progressive profound sensorineural hearing loss underwent a biopsy using the trans-labyrinthine method, and a definitive diagnosis of idiopathic HP was made. Cochlear implants can be helpful for profound hearing loss due to idiopathic HP.Conceptualization: Mitsuru Kitamura, Naoki Oishi.Data curation: Katsuyoshi Idei.Investigation: Marie N. Shimanuki, Makoto Hosoya, Nobuyoshi Tsuzuki, Natsuki Hasebe, Naoki Oishi.Methodology: Naoki Oishi.Project administration: Naoki Oishi.Supervision: Marie N Shimanuki, Makoto Hosoya, Nobuyoshi Tsuzuki, Takanori Nishiyama, Hiroyuki Ozawa, Naoki Oishi.Writing – original draft: Katsuyoshi Idei.Writing – review & editing: Mitsuru Kitamura, Naoki Oishi.


Evidence suggests that sensory impairment is linked to dementia; however, the role of social network and leisure activity in this relationship is unclear.Examine the association of hearing and visual impairment with dementia, and whether a rich social network and leisure activity moderates this association.n = 2,579) were followed up for up for a median of 10 years (interquartile range = 6). Visual impairment was assessed with a reading acuity test, and hearing impairment was ascertained via self-report and medical records. Dementia was diagnosed following international criteria. Data on social network and leisure activity was collected via self-report. Hazard ratios (HRs) of dementia risk were derived from Cox regression models.Dementia-free older adults from the Swedish National Study on Aging and Care in Kungsholmen . Compared to participants with no sensory impairment and a moderate-to-rich social network, those with dual impairment and low social network or leisure activity had higher dementia risk , whereas participants with dual impairment with a moderate-to-rich social network or leisure activity did not have significantly higher dementia risk .A richer social network and participation in stimulating activities may mitigate the higher dementia risk in older adults with dual impairment in vision and hearing. Impairments in hearing and vision are frequent in older adults, with an estimated 50% of individuals over 60 years reporting hearing or vision loss and 11.3% of those over the age of 80 reporting having both, referred to as dual impairment . These iSeveral prospective studies have shown that hearing and visual impairments in older adults increase the risk of cognitive impairment and dementia independently, with some inconsistent results . The majGrowing evidence suggests that engagement in mentally, physically, or socially stimulating leisure activities is associated with lower dementia risk in older adults . FurtherIn the present study, we aimed to examine the association of hearing and vision impairment with dementia, and to explore whether a rich social network and engagement in leisure activities may attenuate dementia risk related to sensory impairment.n = 310), Parkinson’s disease (n = 24), schizophrenia (n = 13) or developmental disorders (n = 3), and missing information on hearing (n = 40), or vision (n = 55) assessments at baseline. We further excluded 339 individuals who failed to participate in at least one follow-up. Thus, 2579 participants were included in the current study  is an ongoing prospective population-based study , includint study . During nt study The ethical committee at Karolinska Institutet and the regional ethical review board in Stockholm approved all parts of SNAC-K, including linkage with registries. All participants or their next-of-kin (in case of cognitively impaired participants) provided written informed consent.The Jaeger eye chart was used to assess near visual acuity at a normal reading distance. If the participant typically wears eyeglasses or contact lenses full-time, they were instructed to wear them during the test. The “J” value of the smallest block of text the participant could read was recorded . Each eye was tested separately and the average of the two values was used. A J1 value corresponds to a 20/20  . Hence, a) Yes, but uses hearing aid; b) Yes, but uses no aid; and c) No. Moreover, medical records from the national patient registry (NPR) which covers all inpatient and outpatient specialist care in Sweden, were obtained through linkage and the following ICD codes were included in hearing loss/deafness: H80 Otosclerosis; H90 Conductive and sensorineural hearing loss; H911 Presbycusis; H913 Deaf mutism, not elsewhere classified; H919 Hearing loss, unspecified; Q16 Congenital malformations of ear causing impairment of hearing; Z453 Adjustment and management of implanted hearing device; Z461 Fitting and adjustment of hearing aid; Z962 Presence of otological and audiological implants; and Z974 Presence of external hearing-aid. We categorized participants as hearing impaired if they either reported self-reported hearing impairment or had a hearing loss/deafness ICD codes as described above.This was assessed during the nurse interview at baseline based on self-reported information on hearing ability. The participants were asked “Do you have trouble hearing?” with possible answers Dual impairment was defined as having both vision impairment and hearing impairment.During the baseline nurse interview, participants were asked about their engagement in 26 leisure activities over the past 12 months . These aAn index of late-life social network was created on the basis of network size  and perceived social support   17]. Th. Th17]. Dementia was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-IV-TR (DSM-IV-TR) criteria, using a validated three-step procedure. Two independent physicians carried out preliminary clinical diagnoses by reviewing the participant’s neurological, cognitive, and physical status. In case of disagreement an external neurologist made the final diagnosis . PhysiciAPOE) genotyping was performed using MALDI-TOF analysis on the Sequenom MassARRAY platform ) or additive  and/or baseline MMSE < 27 (n = 197). Estimates remained similar to those from the main analyses but were no longer significant, most likely due to loss of power  dual sensory impairment in vision and hearing was associated with higher risk of dementia, whereas single sensory impairment was not, and 2) the increased risk for dementia in people with dual sensory impairment may be attenuated by high leisure activity or a rich social network.Several longitudinal population-based studies have reported a higher risk for dementia in individuals with either single or multiple sensory impairments . Our finAlthough a growing body of evidence suggests that sensory impairments may be linked to a higher risk of dementia and cognitive decline, not much has been done to explore the several hypotheses that attempt to explain this relationship. The complex intersection of processing sensory information and cognitive function makes it difficult to tease apart the mechanisms mediating the association between sensory impairment and dementia. One theory is that a decline in sensory function is part of the normal aging process and may therefore decline in concordance with cognitive function . HoweverParticipating in leisure activities and having a rich social life has consistently been related to better cognitive outcomes in older adults . IsolatiUntangling the relationship between sensory impairment, dementia, and participation in leisure activity and social engagement is complex as several non-mutually exclusive explanations may exist. For instance, individuals with sensory impairment may have worse health in general and increased risk of mortality , preventThis study has several strengths, including its longitudinal design with an extended period of observation and a very high rate of participant involvement. Additionally, it has a relatively substantial sample size and incorporates medical diagnoses made by physicians, data from national registries, and information on dementia status obtained from death certificates or medical records at the time of hospital discharge for participants who died during the follow-up. Moreover, we had detailed questionnaire data on social and lifestyle measures, allowing the possibility to assess the joint effects of leisure activities and social network on sensory impairment-related dementia risk. Furthermore, we performed sensitivity analyses to address the potential reverse causation by excluding participants with dementia during the first 3 years of follow up, and the findings were similar. However, some limitations need to be acknowledged. Our measure of hearing impairment was based on self-report and linkage with medical records, which may have resulted in underreporting of impairment. Vision impairment was based on a measure which only tested visual acuity and may therefore not have been sensitive to other forms of visual impairment. Such misclassifications may have biased our results toward the null, or toward more conservative estimates. Finally, the generalizability of the findings may be limited. The study population consisted of highly educated and relatively healthy older adults. This may have affected the magnitude of our results toward an underestimation.In conclusion, our study demonstrated that dual sensory impairment is associated with higher risk of dementia, and that this risk might be buffered by having a rich social network and engaging in leisure activities. Our results highlight the need for future behavioral interventions that integrate mental, social, and physical aspects of lifestyle to investigate how and to what extent dementia can be prevented in people with sensory impairments. Our findings also call for a better awareness, monitoring and control of sensory impairments by professional and informal care providers of older people.Click here for additional data file.


Maintenance of activities of daily living (ADL) during acute hospitalization is an important treatment goal, especially for elderly inpatients with diseases that often leave disabilities, such as cerebral infarction. However, studies assessing risk-adjusted ADL changes are limited. In this study, we developed and calculated a hospital standardized ADL ratio (HSAR) using Japanese administrative claims data to measure the quality of hospitalization care for patients with cerebral infarction.This study was designed as a retrospective observational study using the Japanese administrative claim data from 2012 to 2019. The data of all hospital admissions with a primary diagnosis of cerebral infarction  were used. The HSAR was defined as the ratio of the observed number of ADL maintenance patients to the expected number of ADL maintenance patients multiplied by 100, and ratio of ADL maintenance patients was risk-adjusted using multivariable logistic regression analyses. The c-statistic was used to evaluate the predictive accuracy of the logistic models. Changes in HSARs in each consecutive period were assessed using Spearman’s correlation coefficient.A total of 36,401 patients from 22 hospitals were included in this study. All variables used in the analyses were associated with ADL maintenance, and evaluations using the HSAR model showed predictive ability with c-statistics .The findings indicated a need to support hospitals with a low HSAR because hospitals with high/low HSAR were likely to produce the same results in the subsequent periods. HSAR can be used as a new quality indicator of in-hospital care and may contribute to the assessment and improvement of the quality of care.The online version contains supplementary material available at 10.1186/s12877-023-03957-4. In the past few decades, developed countries have faced similar challenges related to the quality and efficacy of care for rapidly aging populations, including an increasing prevalence of chronic illnesses, rising healthcare expenditure, and maldistribution of medical resources. The increasing demand for healthcare services in super-aged society requires overall optimization of effective use of healthcare resources. A reduction in the length of hospital stay (LOS) among patients is one aspect of this strategy. In Japan, which has a super-aged society, the shift from hospital care to community care, including shortening of the LOS, has been promoted as a health policy based on the Community-based Integrated Care System –5.It is important to discharge elderly patients admitted for acute diseases in a condition that allows them to be independent. However, policymakers and physicians were concerned that policies targeting shortening of the LOS and reduction of readmission might have unintended consequences that adversely affect patient care, potentially leading to increased deterioration in the patient’s condition after discharge . For insCerebral infarction is a common disease among the elderly and is responsible for a decline in ADL, particularly manifesting in the form of motor, somatosensory, language, visual, attention, and memory deficits . In JapaOptimal care during hospitalization may reduce worsening of patient outcomes. However, adjustment of patient risks is required to compare and evaluate hospitals with different capacities and functions and ascertain whether each hospital’s care brought standard outcomes. In this regard, risk-adjusted tools or quality indicators, such as the standardized mortality ratio, were used worldwide . Some ofThis study aimed to develop and evaluate the hospital-level risk-adjusted ADL maintenance during hospitalization for elderly patients with cerebral infarction.This retrospective observational study was conducted using DPC/PDPS data. We developed methods for determining the risk-adjusted patients’ risk of a decline in ADL during hospitalization on the basis of previous studies and calculated the HSAR using these methods , 23. In Patient data were eligible for enrollment in this study if the patients were at least 65 years of age and had presented to the emergency room with cerebral infarction in fiscal years 2012–2019. In risk adjustment, it is difficult to cover more than one disease. On DPC/PDPS system, cerebral infarction existed as a single claim category and had a severity variable. Since the main objective of this study was to develop a new indicator, a single disease was first included. Only hospitals that provided data for all years from 2012 to 2019 and had treated at least 100 elderly patients with cerebral infarction in each period  were included.We constructed two HSAR models: an HSAR model using all-period data and the other model using data for each period separately. All inpatients with a primary diagnosis of cerebral infarction were identified using the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) code I63. The HSAR was defined as the ratio of the observed number of patients showing ADL maintenance aggregated at the hospital level to the expected number of patients showing ADL maintenance aggregated at the hospital level multiplied by 100. The Barthel Index (BI) was used for ADL evaluations. Cerebral infarction is likely to cause multiple disabilities associated with declining ADL; therefore, we thought that the BI was appropriate because it is an international index used in hospitals and nursing homes to comprehensively assess ADL. In the BI, ten variables describing ADL and mobility are scored, with a higher number reflecting greater ability to function independently following hospital discharge –28. The Multivariable logistic regression analysis was performed to predict the chances of ADL maintenance for each patient with patient-level risk factors (covariates). Coefficients derived from the logistic regression analysis were used to calculate the probability of ADL maintenance. The sum of the predicted probabilities of ADL maintenance  indicated the total number of patients expected to show ADL maintenance in each hospital. The ratio of the observed number of patients showing ADL maintenance to the expected number of patients showing ADL maintenance served as the standardized ratio for the hospital of interest. An HSAR above 100 indicated that the ADL maintenance ratio was higher than the overall average.We calculated hospital performance as an outcome during the study period while considering patient-level case-mix variables to account for the patient’s state at admission. First, we considered the differences in the characteristics between patients showing ADL maintenance and those showing a decline in ADL. Second, in the logistic regression analysis, the data included eight variables: Age, Sex, Surgery during hospitalization, Charlson Comorbidity Index (CCI), Pre-stroke Rankin Scale (PRS) score, Japan Coma Scale (JCS) score, Timing of stroke symptom, and Rresidence before admission. Among these risk-adjusted variables, Age, Sex, Surgery, PRS score, and CCI have been used for risk adjustment in previous studies , 29, andP-value < 0.01 was considered statistically significant. In the logistic regression analyses, we obtained the odds ratio and 95% confidence interval (CI), and control variables with two-sided P-value < 0.05 were considered to be statistically significant. All statistical analyses were performed using IBM SPSS version 27.To evaluate the predictive accuracy of the logistic models, we used the c-statistic, which was derived by calculating the proportion of concordant pairs. A c-statistic value of 0.5 indicates that the model is no better than random chance in predicting ADL maintenance, and a value of 1.0 indicates perfect discrimination. In this study, Spearman’s correlation coefficient analyses were used to evaluate the relationships between the HSARs for each period because the small sample size did not allow for a normal distribution of the HSAR. As an exploratory analysis, a sensitivity analyses were conducted, excluding the patients with BI score < 60 at admission and the discharged to another hospital, respectively, which could affect the results. For confirmation of risk-adjusted control variables, a two-sided From April 2012 to March 2020, 36,401 patients  at 22 hospitals were included in this investigation. The demographic characteristics of the patients are presented in Table Chi-square tests and Mann-Whitney U tests showed that all risk-adjusted variables were associated with differences in the changes in ADL (ADL maintenance or ADL decline) Table . Table 2Table In this retrospective observational study performed with administrative claims data, the HSAR for cerebral infarction, which was a patient-risk-adjusted indicator with high predictivity, could capture the quality of hospital care. Therefore, we considered the HSAR to be a useful quality indicator to measure the care during acute hospitalization when considering the post-hospitalization life of the elderly. In Japan, the DPC/PDPS is a standard reimbursement system for acute-care hospitals; therefore, the HSAR methods can be widely applied. The results of this study showed that HSAR-based evaluations provide a robust model, and HSAR can be used as a relatively simple tool by hospitals.Among individual control variables, aging is well known to contribute to the aggravation of cerebral infarction and decline in ADL. The result of chi-square tests and Mann-Whitney U tests for the relationship between changes in ADL and the variables showed significance. After adjusting for patient risk, some hospitals were found to have lower HSAR scores. Since the aim of this study was to identify hospitals that did not meet the standard outcomes for which they should intervene, the HSAR allowed for relative quality assessment and possible improvement on the basis of the case-mix. In this observational study, the confounding variables could not be adjusted; however, the variables used in the regression analysis were considered appropriate. For the risk-adjusted method, we used logistic regression analysis and the case-mix approach as used in previous studies, which were also effective in this study. In the correlation analyses for HSARs, we found that hospitals with high/low HSAR have tend to produce similar results in the following period. Thus, the results suggested that the HSAR for cerebral infarction identified the characteristics of hospital care for ADL maintenance. Notably, we found that hospitals with high/low HSAR were likely to produce the same results in the following period. This result suggested that the HSAR for elderly patients with cerebral infarction was a stable indicator and that the HSAR of the hospitals was influenced by underlying factors. We thought that it is necessary to investigate the characteristics of hospitals with good HSAR so that those with poor HSAR can get beneficial information in improving the ADL care.Our study included a large sample size to develop risk-adjusted methods for quality indicator. To the best of our knowledge, this is the first large-scale study to calculate the  HSAR using DPC/PDPS data. Importantly, the results for the c-statistics showed predictive ability and the HSAR model was highly robust. However, this study had some limitations. First, the hospitals assessed in this study may not be representative of all hospitals in Japan because they voluntarily participated in the benchmarking project. In addition, the information on hospitals was anonymized, so the bed size and function of hospitals could not be used as a variable, and additional discussion of the results was not possible. We plan a validation study using larger-scale DPC/PDPS data in Japan as a future study. Second, other risk factors for deterioration of ADL, such as medical history or socioeconomic status were not considered in this study –39. The The results of this study indicated the need for research on the care system for the maintenance of ADL. To improve care during hospitalization, the findings from a good care system of hospitals with HSAR over 100 should be used to evaluate the care in hospitals with HSAR less than 100, and hospitals and policymakers should evaluate the quality of hospital care management, including rehabilitation . In a suIn conclusion, the study of the HSAR as an indicator to support hospital management, including the trends in one's own hospital, may lead to improve the quality of care. In this study, the HSARs for cerebral infarction could be calculated using administrative claims data in Japan. As a new quality indicator, HSARs showed variation among hospitals with comparable case-mixes and might contribute to the development of more useful set of quality indicators for hospitals to improve their quality of care.Additional file 1: Table S1. Demographic characteristics in each period.Additional file 2: Table S2. Variables in the logistic regression analysis in each period.Additional file 3: Table S3. Results of sensitivity analyses.Additional file 4: Table S4. Correlation of HSAR between in each period.Additional file 5: Figure S1. Variation of the expected number of ADL maintenance patients and the actual number of ADL maintenance patients.Additional file 6: Figure S2. Changes of HSAR by fiscal year in each hospital.


Bone conduction devices (BCDs) are widely used in the treatment of conductive hearing loss (CHL), but their applications on unilateral CHL (UCHL) patients remain controversial. To evaluate the effects of BCDs in UCHL, a systematic search was undertaken until May 2023 following the PRISMA guidelines. Among the 391 references, 21 studies met the inclusion criteria and were ultimately selected for review. Data on hearing thresholds, speech recognition, sound localization, and subjective questionnaire outcomes were collected and summarized. Moderate hearing threshold improvements were found in UCHL patients aided with BCDs. Their speech recognition abilities improved significantly. However, sound localization results showed wide individual variations. According to subjective questionnaires, BCDs had an overall positive influence on the daily life of UCHL patients, although several unfavorable experiences were reported by some of them. We concluded that the positive audiological benefits and subjective questionnaire results have made BCDs a credible intervention for UCHL patients. Before final implantations, UCHL patients should first go through a period of time when they were fitted with non-implantable BCDs as a trial. Conductive hearing loss (CHL) occurs when sound waves cannot reach the inner ear due to defects of sound pathways in the outer and/or middle ear. Patients with unilateral conductive hearing loss (UCHL), i.e., CHL in one ear, suffer from hearing problems caused by the head shadow effect and sound localization troubles. ® ), transcutaneous passive BCDs  and the Sophono device ) and transcutaneous active BCDs with an implanted actuator , Osia® ) ) OR (BCHAs[Title/Abstract])) OR (bone conduction hearing[Title/Abstract])) OR (bone conduction hearing aids[Title/Abstract])) OR (BAHA[Title/Abstract])) AND ). Web of Science : ) OR TS = (single sided deafness)) AND (TS = (BCHAs) OR TS = (BAHA)). This study is conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  and the Studies were selected for this review if they conform to the following criteria: (1) the study subjects were patients with unilateral conductive or mixed hearing loss (2) the study subjects received bone-conduction hearing aids (BCHAs)/BCDs as intervention (3) the study reported outcomes of audiological measures and/or questionnaires (4) the study design was retrospective or prospective. Studies were particularly excluded if they: (1) Only included SSD or other unilateral sensorineural hearing loss, (2) Only included patients with bilateral hearing loss, (3) Used air conduction or cartilage conduction hearing aids, (4) Did not meet the inclusion criteria. There was no restriction on language. After primary search, duplicates were removed before the title and abstract review was undertaken. The studies were selected based on the inclusion/exclusion criteria.Two independent reviewers extracted the information separately. The included study types were all clinical trials, either retrospective or prospective. Before final inclusion, all the studies underwent quality assessments using the Agency for Healthcare Research and Quality (AHRQ) scale. Disagreements on study selection or quality assessment were resolved through a full discussion with a third reviewer. The outcomes of interest were hearing thresholds, speech recognition, sound localization, and subjective questionnaires. Other data points of interest included: study design, number and mean age of subjects, etiology of deafness, and types of BCDs. In studies that also examined non-criterion-meeting subjects , we only collected information on the UCHL subjects.The primary search yielded 398 results, an additional 49 studies were hand-searched and added. Fifty-six duplicates were removed, and 244 studies were removed for not being original studies. Afterwards, 147 studies remained for title and abstract review, and 72 studies were excluded. Then we full-text reviewed the rest 75 studies, and 54 of them were excluded based on the inclusion and exclusion criteria. Ultimately, 21 studies were decided suitable to be included in this systemic review see . Table 1In all of the studies, the unaided and aided conditions were compared through prospective or retrospective analysis. Three of the studies recruited normal-hearing as control groups ,7,8, whiThe studies were done in the UK , USA 2121, CanadThe BCDs used are Bone-anchored hearing aids (BAHA) ,18,20,21Sound field hearing thresholds (pure tone average (PTA) gain), speech recognition (sometimes described as speech discrimination or speech perception), and sound localization test results are valuable audiological outcomes that reflect speech-identification and localization abilities. Only 16 out of the 21 included studies presented these audiological outcomes. Here we collected the available audiological data of the studies and summarized the outcomes in ® Attract, Brotto et al. [Of the 21 studies included in our analysis, 10 tested pure tone audiograms and sound field hearing thresholds. Overall, the aided hearing thresholds show great improvement when compared with unaided. For sound field hearing threshold tests, most studies had speakers placed 1 m distance in front of the subject’s head. Warble tones at frequencies of 0.5, 1, 2, and 4 kHz were presented. With the normal ear blocked, the mean tone thresholds of the above frequencies, i.e., the PTA, were usually measured. When aided with BAHA, Wazen et al. [o et al.  reportedo et al.  reportedo et al. , and froo et al. , 58 dB to et al. , respecto et al. , 51.9 ± o et al. , and 53.o et al. , 51.36 ±o et al. .50) and word recognition score (WRS) were used to represent the speech recognition ability. A decrease in SNR represents an improvement in speech recognition ability. Some studies tested noise from various angles, while some studies tested with one fixed noise speaker. Generally, speech recognition in quiet improved in at least a portion of the subjects tested [Of the 21 studies, 10 investigated speech recognition (some described as speech discrimination or speech perception) in quiet and in noise. The setups for testing speech recognition were similar to that for testing sound field hearing thresholds, except that speech signals were played. Speech tests were consistent with the subjects’ native language. Speech reception threshold (SRT), speech-to-noise ratio (SNR) at which the patient score was 50%  is calculated as an outcome in 5 of the studies ,19,20,22General improvements in sound localization were found in five studies ,20,22,25Various scales were developed to evaluate the auditory behavioral, and daily-use information of different BCDs. 15 of the 21 studies used subjective questionnaires to evaluate the behavioral and developmental outcomes of UCHL patients. The detailed information of the studies was reviewed and summarized in p < 0.001) [Abbreviated Profile of Hearing-Aid Benefit (APHAB)  is a 24-< 0.001) . The outThe Glasgow Benefit Inventory (GBI) ,33 and tp = 0.046) for adults aided with BAHA. In the children’s version of SSQ, the group showed a total mean score of 6.6 with the BAHA. Hirth et al. [p = 0.0313). Kuthubutheen et al. [p < 0.05). Liu et al. [The Speech, Spatial, and Qualities of Hearing Scale (SSQ)  aims to h et al.  also repn et al.  reportedIOI-HA refers to the International Outcome Inventory for Hearing Aids. Seven items are included: use, benefit, residual activity limitation, satisfaction, residual participation, impact on others, and quality of life. Scores above three indicate the success of the hearing aid fitting compared to the unaided situation. All of the studies that involved IOI-HA presented a mean score above three in all seven items ,8,26.A specific 20-question subjective questionnaire for ADHEAR users was created to assess the effectiveness and ease of use of the device in subjects’ daily life during the testing period. Liu et al. retrospectively collected the subjective satisfaction of 13 subjects with UCHL who received ADHEAR, and they reported an average daily use time of 7.2 h, and 85% of them considered ADHEAR a valuable hearing aid in total . KuthubuTwo studies ,24 contap < 0.001) in comparison to the UCHL group .Studies that involved the Chung and Stephens’ questionnaire  reportedp < 0.025). This result also supported the opinions stated in the studies of Hol et al. [Differences were found between congenital UCHL and acquired UCHL patients, especially in sound localization tests. Subjects with congenital UCHL somehow already have good test results unaided ,20,41, tl et al. , Kunst el et al. , and Snil et al. , but witFor studies that included both unilateral and bilateral CHL groups, the audiological beneficial effects were much more significant in the bilateral CHL groups. This was reasonable because the UCHL patients did not experience as much inconvenience as bilateral CHL patients before BCD treatments. In the case of UCHL patients, the improvement of living quality and hearing abilities in real-life situations seemed to be of more importance. To decide whether BCDs provide UCHL patients with actual audiological benefits, unmasked hearing thresholds are very valuable indicators. The study of Polonenko et al.  revealedTwelve different types of questionnaires were involved in the selected studies. Only a few studies adopted the same questionnaire and therefore the sample size was relatively small. In spite of this, questionnaire outcomes showed that patients have an overall high satisfaction rate and optimistic attitudes towards the BCDs.BCD applications come with the potential cost of convenience, comfort, money, and appearance. These are the aspects we should value more when applying BCDs and receiving feedback from patients.Comparative works on audiological and subjective outcomes among different BCDs were also available in some of the literature. Hol et al. , DenoyelChildren with unilateral hearing loss often have worse language and speech performance than their peers . ChildreDue to the wide individual differences of UCHL patients when fitted with BCDs, we highly recommend that all UCHL patients who wish to use implantable BCDs should first go through a period of time when they were fitted with non-implantable BCDs as a trial. If the audiological benefits and satisfaction were promising, then implantations could be carried out successively.BCDs deliver an overall benefit to patients with UCHL. It is important that UCHL patients receive BCDs to rehabilitate binaural hearing. Children with congenital UCHL should be treated early to reach proper speech-language development and auditory cortex development. Adults with UCHL can also use BCDs to aid their hearing and localizing abilities. Due to the wide individual variations, we recommend a period of trials on headband BCDs to help decide whether long-term usage or implantations are necessary. The type of BCD should be selected according to individual conditions.


Escherichia coli has two of them, and the histidine kinase BtsS is a high-affinity sensor for extracellular pyruvate that together with the response regulator BtsR activates the expression of btsT, which encodes a high-affinity pyruvate transporter. However, the molecular mechanism of how pyruvate binding triggers a response is still unclear. Here, we experimentally show that BtsS consists of seven transmembrane helices, with the N-terminus exposed on the periplasmic side. Using a site-directed mutagenesis-based screening assay, Arg72, Arg99, Cys110, and Ser113, all located on the periplasmic side in three transmembrane helices, were identified as critical for the binding of pyruvate to BtsS, a finding that was further confirmed by molecular dynamics simulation studies. In addition, Mn2+-dependent autophosphorylation of wild-type BtsS was demonstrated for the first time, and individual replacement of the four amino acids affected this process as well as dimerization and consequently btsT expression. This study demonstrates how binding of a metabolite to the membrane-integrated sensor domain triggers signaling in the cytoplasm.LytS/LytTR-type histidine kinase/response regulator systems regulate crucial host-specific mechanisms during infection of human or plant hosts. E. coli and identified the pyruvate binding site within the membrane-spanning domains. It is a small cavity, and pyruvate forms interactions with the side chains of Arg72, Arg99, Cys110, and Ser113 located in transmembrane helices III, IV, and V, respectively. Our results can serve as a starting point to convert BtsS into a sensor for structurally similar ligands such as lactate, which can be used as biosensor in medicine.Here, we studied the LytS-type histidine kinase BtsS of  Escherichia coli, there are two LytS/LytTR-type systems, namely, BtsS/BtsR (Two-component systems (TCSs) in most cases consist of a membrane-anchored histidine kinase (HK) and a cognate cytoplasmic response regulator (RR) . All histsS/BtsR , 5 and YtsS/BtsR    receptor protein (CRP) (CRP-cAMP) for expression (btsT is regulated by carbon storage regulator A (CsrA) (btsT transcription is under positive feedback regulation by the YpdA/YpdB-system and its target gene product YhjX (BtsS consists of an input domain containing the membrane-integrated 5TMR_LYT domain pfam07694) and the cytosolic GAF domain, and the DHp and CA domains , 11. In  and the ter BtsT  Fig. 1)Kd = 58.A (CsrA) . In addiuct YhjX . The molE. coli has two different LytS/LytTR-type systems for pyruvate sensing (BtsS/BtsR and PyrS/PyrR) and three pyruvate transporters   and two pyruvate transporters (BtsT and CstA) (Vibrio campbellii has one pyruvate sensing system (BtsS/BtsR) and only one pyruvate transporter (BtsU) (It should be noted that nd CstA) , whereasnd CstA) , and ther (BtsU) . These br (BtsU) –18.E. coli is a sensor with the N-terminus located on the periplasmic side. We found that Arg72, Arg99, Cys110, and Ser113 are crucial for the binding of pyruvate to BtsS and subsequent autophosphorylation, and consequently btsT expression. For the first time, we demonstrated the autophosphorylation activity of BtsS, and this LytS-type histidine kinase was found to prefer Mn2+-ATP instead of Mg2+-ATP.Here, we showed that the LytS-type histidine kinase BtsS of BtsS is a LytS-type histidine kinase. The characteristic feature of all members of the LytS-type family is the 5TMR-LYT domain suggesting that BtsS is anchored with at least five transmembrane helices in the membrane. However, the first 36 amino acids of the receptor, which form at least another transmembrane helix, do not belong to this domain . Prediction of transmembrane helices based on hydrophobicity using three software tools  or cytoplasmically localized  maltose-binding protein (MBP) . Unlike E. coli MG1655 the E. coli mutant MM39 has a deletion of the malE gene and cannot grow with maltose as the sole carbon source (C source)  or the hybrid MalEp-BtsS, respectively. Both strains were able to grow with maltose as a C source, indicating functional active MalE. In contrast, E. coli strain MM39 transformed with pMAL-c2X or pMALc-btsS encoding MalE without leader peptide  or the hybrid MalEc-BtsS, respectively, was unable to grow on maltose-containing plates  . We then source) . Subsequg plates .in vivo. We assumed that only the hybrid protein with the N-terminus of BtsS on the correct side of the membrane would result in a correct membrane topology and a functional active receptor. The MBP-BtsS constructs were tested together with the response regulator BtsR for complementation of our reporter strain lacking the btsS/R genes and expressing a btsT promoter-luciferase fusion (PbtsT::luxCDABE). Strains were cultivated in minimal medium with pyruvate (5 mM) and succinate (15 mM) as C source, a condition that induces btsT expression  and that pyruvate binds to the extracellular side of the membrane-spanning domain  and size (Gln) or even to have the opposite charge (Glu). Arg72 and Arg99 could not be replaced by any other amino acid because all corresponding BtsS variants prevented nduction . These ain vitro . Therefore, we did not investigate these four amino acids further.In contrast, replacement of Lys26, Arg39, Arg131, and Arg192 with positively charged amino acids and other amino acids was tolerated, and the corresponding BtsS variants were able to respond to pyruvate . ConsistbtsT induction, whereas the BtsS variants C110A, S113A, and T114A were unable to do so  allowed pyruvate-dependent to do so . We thene active . All varin vivo activity of the BtsS variants was thus far based on the response of the complemented reporter strain to pyruvate (5 mM) as a C source. To test for a possible change in the affinity of the BtsS variants for pyruvate, we tested the pyruvate-dependent btsT induction in two additional in vivo assays. First, strains were grown in M9 minimal medium with different pyruvate concentrations (5–50 mM), while the total carbon concentration (50 mM) was kept constant by adding succinate. Whereas we found concentration-dependent btsT expression for the wild-type BtsS, BtsS-R72A, BtsS-R99A, BtsS-C110A, and BtsS-S113A did not induce btsT expression under any condition (E. coli cells exposed to very low pyruvate concentrations (10 µM to 1 mM) induce btsT in a concentration-dependent manner. We used this protocol and found not only an approximately 100-fold lower response of the BtsS-R72A, BtsS-R99A, BtsS-C110A, and BtsS-S113A variants compared with wild type, but also a slight shift to higher pyruvate concentrations   and the signal intensity of the whole area (outer circle). We used membrane vesicles prepared from the E. coli ∆btsSR∆ypdABC mutant after overproduction of BtsS (WT) or BtsS-R72A, BtsS-R99A, BtsS-C110A, BtsS-S113A, and incubated equal amounts of protein with increasing concentrations of pyruvate, each containing 15 µM radiolabeled 14C-pyruvate. We then dropped the mixture onto a nitrocellulose membrane   was subtracted for all concentrations and dK values were calculated. For wild-type BtsS, a dK value of 67.3 ± 10.6 µM was determined, consistent with previous measurements  , which win vitro . In thismembrane . Migratiund (EV) . Dark in± 8.0 µM . These rBased on the experimental information, we assume that pyruvate binds near the residues identified by mutagenesis. From structural modeling and MD-based positioning of the pyruvate in the N-terminal domain , we founUpon perception of a stimulus, histidine kinases generally transduce the information into an intracellular signal by initiating a phosphorylation cascade . In our 2+-ATP, only very weak autophosphorylation was detectable  and BtsS-R192A  retained the concentration-dependent response to pyruvate.We then asked whether pyruvate binding stimulates autophosphorylation activity. To test this, the autokinase activity of wild-type BtsS and all pyruvate-insensitive BtsS variants was determined in the presence of different pyruvate concentrations. For wild type, we found a 1.6-fold increase of the initial rate in the presence of 25 µM pyruvate. With 50 and 100 µM pyruvate, the initial rate was also increased ; Fig. S4in vivo. Dimerization is a prerequisite for all histidine kinases to mediate autophosphorylation by ATP either in trans or in cis  input domain that receives signals  and functioned as a pyruvate sensor  (Escherichia coli (PDB: 1MZO)   . In the B: 1MZO) , pyruvat5, 4JX6) , 42. For5, 4JX6) . Cys110 btsT in vivo   . The Kdsduction . Clustalsduction  . Plasmid DNAs were isolated using a HiYield plasmid mini kit (Sued Laborbedarf). DNA fragments were purified from agarose gels using a HiYield PCR cleanup and gel extraction kit (Sued Laborbedarf). Restriction enzymes and other DNA-modifying enzymes were purchased from New England BioLabs and used according to the manufacturer’s directions.All E. coli MM39 was transformed with plasmid pMAL-p2X, pMAL-c2X, pMALp-btsS, or pMALc-btsS, and then plated on LB agar plates with streptomycin (50 µg/mL) and ampicillin (100 µg/mL). In parallel, E. coli MG1655 and MM39 were streaked on LB agar plates as controls. Subsequently, individual colonies were re-streaked on M9 minimal medium agar plates supplemented with 0.1% (wt/vol) maltose as sole C source and incubated at 37°C for 36 h (for 36 h .in vivo activity of BtsS/R was measured by monitoring the expression of btsT (E. coli MG1655ΔbtsSR was co-transformed with plasmids pBBR-btsT-lux and pBAD24 (negative control) or pBAD24-btsS/R (positive control) or pBAD24-pMALp-btsS/R or pBAD24-pMALc-btsS/R. Overnight cultures in LB medium were used to inoculate M9 minimal medium containing 5 mM pyruvate, L-lactate, or succinate, respectively, supplementary with 15 mM succinate and incubated in a plate reader  at 37°C. Both OD600 and luminescence were determined continuously. Luminescence was determined as relative light units (RLU counts s−1) related to the OD600.The  of btsT . E. coliE. coli MG1655ΔbtsSR was co-transformed with plasmids pBAD24-btsS/R and pBBR-btsT-lux or pBAD24-btsS/R variants and pBBR-btsT-lux. After overnight incubation, a colony was inoculated in 5 mL LB medium with the appropriate antibiotics (ampicillin and gentamycin) and cells were grown at 37°C until OD600 reached 0.5. Cells were transferred to M9 minimal medium supplemented with 5 mM pyruvate or lactate or succinate  to give a starting optical density at 600 nm (OD600) of 0.05 and incubated at 37°C with constant agitation. Growth (OD600) and luminescence were measured at 10 min intervals in a luminescence reader . Using the same protocol, different concentrations of pyruvate  were tested as the C source, with the total C concentration kept constant at 50 mM in each case by addition of succinate. Alternatively, overnight grown cells were inoculated in 0.1× LB medium and after 1 h different concentrations of pyruvate were added (−1) per OD600. Flag-tagged BtsS or variants were detected by western blot analysis using an anti-Flag antibody (Thermo Fisher) and a secondary anti-mouse antibody conjugated to alkaline phosphatase (Thermo Fisher).E. coli TKR2000 transformed with plasmid pBAD24-btsS-6His or variants was cultivated in KML medium  supplemented with ampicillin (100 µg/mL). At an OD600 of 0.5, 0.2% (wt/vol) arabinose was added, and cells were grown at 18°C overnight. Cells were harvested by low-speed centrifugation, and membrane vesicles were prepared as described (2 or 5 mM MnCl2 and 2 mM dithiothreitol) at 25°C. Phosphorylation was initiated by the addition of 20 µM [γ-32P]ATP  (2 for 5 min.escribed . Membraniotrend) . At diffiotrend) . All samin vivo using the bacterial adenylate cyclase two-hybrid assay (BACTH)  at 37°C, and used to inoculate 0.1× LB medium (nutrient limitation)  . E. coliitation)  supplemeer units . BtsS hyin vitro as previously described  were mixed with 15 µM radiolabeled (3-14C) pyruvate  and incubated for 20 min at room temperature. Triplicate 5 µL aliquots were spotted onto a nitrocellulose membrane, dried, and radioactivity was visualized using a PhosphorImager. Quantification of the areas of radioactive signal intensities was performed using ImageJ. The fraction bound (FB) was calculated using the equation FB = (Iinner − Ibackground)/Itotal  were added to the mixture. Competition efficiency (CE) was calculated as follows: CE = (FB(NC) − FB(pyr))/FB(NC). FB(NC) is calculated from the sample without cold pyruvate (NC = no competition), and FB(pyr) are all values calculated for the samples containing a mixture of 14C-pyruvate and different concentrations of cold pyruvate. For all FB values, the value of the negative control (empty vector) was subtracted. The concentration of membrane vesicles used in this experiment was 25 mg/mL, which contain approximately 7% BtsS-6His or variants.The differential radial capillary action of ligand assay (DRaCALA)  was usedescribed . E. coli/Itotal . To deteE. coli) with bound pyruvate in the PDB: 1MZO entry. This known structure was used to first manually place pyruvate in the putative-binding pocket in the BtsS model  and to derive upper bound distance restraints between pyruvate and the side chain functional groups of the four critical residues. The modeling and pyruvate docking continued by first performing an energy minimization  to remove sterical overlap in the start structure using the Amber20 molecular modeling package (−1Å−2) except for the residues in close vicinity of the bound pyruvate. In addition, the simulations included the distance restraints between pyruvate and the four critical residues in the binding region. After a step-wise heating (0.5 ns), MD simulations were continued for 10 ns and resulted in a stable binding geometry  that was again energy minimized for 2,500 steps to obtain a structural model of the complex.A structural model of a BtsS dimer was generated using AlphaFold2  and the  package . In the


A hallmark of photoreceptor degenerations is progressive, aberrant remodeling of the surviving retinal neurons and glia following photoreceptor loss. The exact relationship between neurons and glia remodeling in this late stage of retinal degeneration, however, is unclear. This study assessed this by examining Müller cell dysfunction via glutamine synthetase immunoreactivity and its spatial association with retinal neuron subpopulations through various cell markers.n = minimum 5 per age) and control heterozygous rd1 mice retinae  were isolated, fixed and cryosectioned. Fluorescent immunolabeling of glutamine synthetase was performed and retinal areas quantified as having low glutamine synthetase immunoreactivity if proportion of labeled pixels in an area was less than two standard deviations of the mean of the total retina. Other Müller cell markers such as Sox9 and Glial fibrillary acidic protein along with neuronal cell markers Calbindin, Calretinin, recoverin, Protein kinase C-α, Glutamic acid decarboxylase 67, and Islet-1 were then quantified within areas of low and normal synthetase immunoreactivity.Aged Rd1 mice retinae . Immunoreactivity of other Müller cell markers, however, were unaffected suggesting Müller cells were still present in these low glutamine synthetase immunoreactive regions. Glutamine synthetase immunoreactivity loss affected specific neuronal populations: Type 2, Type 8 cone, and rod bipolar cells, as well as AII amacrine cells based on reduced recoverin, protein kinase Ca and parvalbumin immunoreactivity, respectively. The number of cell nuclei within regions of low glutamine synthetase immunoreactivity was also reduced suggesting possible neuronal loss rather than reduced cell marker immunoreactivity.These findings further support a strong interplay between glia-neuronal alterations in late-stage degeneration and highlight a need for future studies and consideration in intervention development. Retinitis Pigmentosa (RP) is the most common inherited retinal degeneration affecting one in 4,000 individuals . It resuAn added complexity is that inner retinal changes are dependent on disease stage. For example, Glial remodeling also exhibits separate, time dependent phases of change. For example, early degeneration is associated with loss of Müller cell processes and hyperexpression of glial fibrillary acidic protein (GFAP) while late degeneration is associated with the converse, Müller cell hypertrophy and glial seal formation . MetabolA quantitative time course of Müller cell dysfunction (based on loss of glutamine synthetase immunoreactivity) is currently unknown. However, qualitatively data from n = minimum 5 per age). The control mice (C57Bl/6) were examined at the oldest age only . Animals were maintained on a 12 h light/dark cycle and had access to standard mouse chow and water ad libitum. The experimental protocols in this study were approved by the University of Melbourne and UNSW Sydney Animal Ethics committees.Rd1 mice (on a C57Bl/6 background)  were stuMouse retinae were processed and immunostained as described previously , 2017. B°C with primary antibodies at the dilutions specified in For immunostaining, retinal sections were blocked for 60 min with 6% (v/v) goat serum, 1% (w/v) bovine serum albumin, 0.1% (v/v) Triton-X then incubated overnight at 4All samples that were stained for the same antibody were processed at the same time with no difference in antibody batch number to address possible issues from histological preparation. To decrease effect of eccentricity all samples were taken within 500 μm of the central retina from temporal to nasal. Images were taken within a week of staining with fixed laser settings for DAPI, AlexaFluor488, and AlexaFluor594 labeling to decrease errors due to laser variability.rd1 at P150, P300, and P536, in confocal images. Immunolabeling of cell markers was assessed from microscope images in a using a sliding window analysis in ImageJ 1 and a custom MATLAB®  script that utilized the Image Processing Toolbox.A systematic approach was developed to assess quantitatively the area specific differences in immunoreactivity within and between areas of the retina in the control mice at P536 and The window width was set to the pixel equivalent of 25 μm, translated from the scale bar on each image, and only pixels within the region of the neural retina were counted in the window. The neural retina in each window was defined as the pixels contained between the inner limiting membrane to the outermost part of the neural retina. Due to degeneration in rd1 mice, this outer border was manually delineated by user tracing of the retina within the image. As window width was constant, total pixel count within a given window was a function of retinal thickness.PV by the criterionTo quantify an immunolabeled cell marker within a given window, images were separated into RGB channels and then individual pixels (P) were thresholded based on their respective pixel values, PT is the binary thresholded pixel value and MIPV is the mean pixel value of the image for their respective channel and immunolabel. In short, individual pixels were considered above threshold if their value was greater than twice that of the relevant mean pixel value. The positive pixel ratio, PPratio of each window for each channel or immunolabel was then determined bywhere Pn is the total number of pixels in the window counted as neural retina. In short, the positive pixel ratio revealed the proportion of pixels above threshold to the total number of pixels of the retina within the window, thus normalizing the positive pixel count to the thickness of the neural retina. The analysis was repeated across 25 μm windows across the whole length of the retina for each immunolabel in each microscope image using a window step size of 5 μm. In total, sliding window analysis was conducted on 37 images spanning 24,005 μm retinal length for control mice (n = 5 at P536), 90 images spanning 57,540 μm retinal length for rd1 mice at P150 (n = 5), 80 images spanning 51,050 μm retinal length for rd1 mice at P300 (n = 4) and 102 images spanning 58,435 μm of retina for rd1 mice at P536 (n = 6).where norm) or low (GSlow) utilizing the mean and standard deviations obtained from the control using the formulaAfter analyzing the GS immunolabeling for all 24,005 μm of control retinae, the GS labeling of an Area (A) of rd1 retina was defined as either normal  retina (see Where Pectively . In shortina see .n = minimum 5 per age) was stained with DAPI for nuclear labeling. All positive pixel ratios, except GS, were normalized to the mean positive pixel ratios of normal GS areas within each retinal image to allow consistent, relative assessment of differences in other immunolabels between areas of normal vs. low GS areas within a retinal image. For the majority of cell markers, only retina at P536 were assessed as this was the only age that demonstrated numerous and long areas of low glutamine synthetase immunoreactivity that could be reliably quantified.A total of 24,675 μm of rd1 retina , with an α of 0.05. For t-tests with multiple numbers of tests the α was adjusted to the Bonferroni adjusted α (αB) of 0.0125 and 0.0167, when the number of tests were 4 and 3, respectively. For multiple groups a Tukey-Kramer post-hoc test was performed. Statistical analyses were performed using MATLAB®  v9.9.0.1570001 (R2020b) and the Statistics and Machine Learning Toolbox v12.0.All variables are expressed as mean ± standard error. Data was analysed using the two-sample t-test, all p < 0.0001, αB = 0.0125; t-test, p > 0.0125, αB = 0.0125; Tukey-Kramer, p > 0.05), though some regional thickening was qualitatively observed in low GS regions . The standard deviation for the Positive Pixel ratio of GS in the control was 0.565. For the rd1 at P150, P300, and P536 it was 0.0666, 0.0537, and 0.0400, respectively, such that raw variability was similar for all groups.Glutamine Synthetase immunoreactivity was not significantly different between the rd1 P150 and control retinae  and Sox9. Despite the observation of numerous areas of low GS immunoreactivity in rd1 retina at P536 , GFAP imt-test, p = 0.20; t-test, p = 0.52; Similarly, Sox9 immunoreactivity was present throughout the rd1 retina at P536 and in areas where normal GS immunoreactivity was present, Sox9 was assessed in normal and low GS areas . When quGlutamine synthetase contributes to a major function of Müller cells: the clearance of extracellular glutamate to maintain retinal neurons and their microenvironment . Thus, lt-test, p < 0.01; normal, 2.73 ± 0.31 Recoverin positive cells/100 μm; GSlow, 1.57 ± 0.30 Recoverin positive cells/100 μm, t-test, p < 0.05). Similarly, PKC-α immunoreactivity was significantly decreased by 13% in areas of low versus normal GS immunoreactivity in the rd1 retina , protein kinase C-alpha  and Islet-1 . Recoverin immunoreactivity was significantly decreased by 30% in areas of low GS immunoreactivity compared to normal GS immunoreactivity  is lost in the rd1 retina as discrete regions that increased in both size and number as a function of age. This loss is not likely due to Müller cell loss as GFAP immunoreactivity was unaltered. Specific loss of neural macromolecular markers pertaining to specific amacrine and bipolar cell populations occurred in areas of low GS immunoreactivity and this was likely, in part, due to neuronal death based on decreased DAPI labeling. These data shed light on glia-neuronal alterations in late-stage degeneration and could provide insight for interventions to combat them.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by University of Melbourne and University of New South Wales Animal Ethics Committees.LN-S performed the tissue preparation, immunostaining, and imaging. HR performed the quantification, programming, and statistical analysis. EF supplied the mice. MS provided study funding in part. All authors contributed to the manuscript and approved the submitted version.


Dear Editor,Compared with traditional technologies affecting gene expression, changing DNA sequences of target genes is one of the most outstanding characters of CRISPR . Single-guide RNAs (sgRNAs) guiding endonuclease Cas to target sites is a crucial step of CRISPR-Cas system for changing DNA sequences. An ideal sgRNA should only bind to the target gene. However, similar sequences of non-target sites can also be recognized leading to off-target effects . How to Compared with previous tools , CRISPR-GE (http://skl.scau.edu.cn/targetdesign/), CRISPR-P (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR), E-CRISP (http://www.e-crisp.org/E-CRISP/index.html), and MultiTargeter (http://multicrispr.net/basic_input.html) to design sgRNAs. After that, the five sgRNAs of each gene with the highest scores were collected to construct the testing database. Comparing the sgRNA-design results of CIDP with the testing database, we found that most of sgRNAs designed by CIDP can be found in the testing database (87.4%, In order to evaluate design results of CIDP, we randomly selected 1000 genes of e 87.4%, . Moreovee 87.4%, . These rCIDP utilizes genomic sequences to build the database containing short segments whose sequences are unique across the whole genome. When designing sgRNAs, CIDP will extract candidate sgRNAs from gene sequences based on the selected PAM model, and then filter these sgRNAs by using the built database. Furthermore, CIDP will align the remaining sgRNAs to genome sequences to identify potential off-target sites and calculate off-target rates. Therefore, we believe that the sgRNAs designed by CIDP should be highly reliable, whereas, no matter the building or aligning process, the two operations were all performed at the level of genome. In general, a large amount of computer memory is required for supporting genome-level operations. This is an important reason why the tools performing genome-level operations can only be run on Linux servers , 11. HowIn order to solve the efficiency problem of CIDP as much as we could, we developed a Linux version of CIDP that can be run on Linux servers. Moreover, the built database can be reused. Therefore, it is not required for users to build the database repeatedly. The CRISPR system is a common technology for wet experiments, and a species may be studied for several years by some wet labs. In this situation, the built database can also be reused by researchers in the same lab. It is truly hard to finish genome-level jobs under the condition of limited memory with high efficiency. Increasing CIDP efficiency is an important task for us in the future. In the next version, we will try to design functions in a multi-threaded or multi-processed manner which may elevate the design efficiency, even though these manners still require amount of memory support.Web_Material_uhad092Click here for additional data file.


Mucorales and associated with high mortality rates. Rhino-orbito-cerebral localization usually occurs in individuals with uncontrolled diabetes mellitus. We report the case of a 41-year-old male, with previously undiagnosed diabetes, who presented with unilateral facial extensive black eschar and signs of diabetic ketoacidosis. Cerebral magnetic resonance imaging showed left pansinusitis, left craniofacial edematous infiltrate, and left proptosis. A left internal temporal abscess was identified at an early pre-suppurative stage. Magnetic resonance angiography revealed total occlusion of the left intracranial internal carotid artery. A histopathological study of nasal mucosa biopsy suggested mucormycosis. According to the clinical presentation and the radiological and histopathological findings, rhino-orbito-cerebral mucormycosis was presumed. Culture of nasal, ocular, and skin lesion specimens grew Rhizomucorsp. and confirmed the diagnosis. The patient was treated with systemic liposomal amphotericin B. He died of multiple organ failure before surgical debridement was possible as he was in critical condition requiring stabilization before surgical treatment.Mucormycosis is a rare opportunistic fungal infection caused by  Mucorales [Mucormycosis is a deadly emerging infection, caused by fungi belonging to the order ucorales ,2. Theseucorales . It is wucorales . This diucorales . Hence, ucorales .Rhino-orbito-cerebral mucormycosis (ROCM) is more frequently recognized in poorly controlled diabetes mellitus and associated with high mortality rates above 50% . Herein,2 at 93%. The patient presented signs of diabetic ketoacidosis . Other biologic abnormalities included anemia (hemoglobin: 86 g/L) elevated white blood cells (27970/mm3 without blasts), elevated C-reactive protein (371 mg/L), creatinine (144 µmol/L), and hyperkalemia (6.9 mmol/L). Serum albumin was low (1.9 g/dL), and both gamma glutamyl transferase and lactate dehydrogenase levels were high. Serological tests for viral Hepatitis B, C and HIV 1,2 were negative.A 41-year-old man was referred to the intensive care unit (ICU) with extensive necrotic facial cellulitis and widespread edema. Nasal mucosa's black eschar had spread to the left cheek, the left orbit, the upper lip, and the oral cavity  showed left pansinusitis, left craniofacial edematous infiltrate, and left proptosis Figure . In the Histopathological study of nasal mucosa biopsy revealed chronic pyogranulomatous inflammation with non-septate, non-pigmented broad ribbon-like hyphae suggesting mucormycosis.Mucorales infection . Direct microscopic examination of the samples in 10% potassium hydroxide (KOH) mount and then May Grunwald Giemsa (MGG) staining found hyaline, non-septate irregular hyphae with branching at 90° angle, consistent with n Figure . The cul. Figure  and confDuring hospitalization in the ICU, among other measures of care, the patient received insulin therapy and blood transfusion. His blood glycemia was successfully controlled. He was treated with liposomal amphotericin B (lAMB) (5mg/kg/24H). He died due to multiple organ failure after four days of antifungal therapy, before surgical debridement was possible as he was in critical condition requiring stabilization prior to coordinated surgical multidisciplinary care.Mucorales, usually found in soil, and decomposing organic material. Eleven genera and thirty-eight distinct species of Mucorales are implicated in mucormycosis [Rhizopus, Mucor, and Rhizomucor are responsible for the majority of mucormycosis, other clinically important species include Actinomucor, Apophysomyces, Cunninghamella, Lichtheimia, Saksenaea, and Syncephalastrum [Mucormycosis is a potentially fatal emerging infection, caused by fungi belonging to the order rmycosis . While Ralastrum .Mucormycosis occurs mostly in hematology, solid organ transplant, and diabetic patients with hyperglycemia and ketoacidosis, although it can also develop in immunocompetent individuals after a trauma or burn . Our patROCM is the most prevalent presentation in diabetic patients, especially those with poorly controlled diabetes mellitus ,3. SevenImaging is essential for figuring out the extent of disease and directing medical and surgical treatment. Thus, cranial computed tomography or MRI is strongly recommended to determine if sinusitis is present in diabetic patients with facial pain, signs of sinusitis, proptosis, ophthalmoplegia, or amaurosis . In our Early clinical recognition and a multidisciplinary approach are mandatory for the successful management of mucormycosis. However, where the clinical presentation or radiological characteristics are suggestive, urgent tissue histopathology and culture are required .Aspergillus spp. for Mucorales, especially when fungus hyphae are distorted or scarce [Rizomucor sp. All clinical, radiological, and pathological findings were consistent with ROCM in a diabetes mellitus patient with ketoacidosis.The fundamentals in diagnosing mucormycosis include direct microscopy, culture, and histopathology of various clinical samples. An essential diagnostic tool is tissue identification, which differentiates the presence of the fungus as a pathogen in the specimen from its presence as a contaminant in culture . It can r scarce . Howeverr scarce . In our Rhizopus arrhizus following amphotericin B therapy. The minimal inhibitory concentration for itraconazole in this particular species is low [Except for amphotericin B (AMB), mucorales exhibit intrinsic resistance to the majority of antifungals used in clinical settings, giving medical professionals few therapeutic options . The uses is low .Regular monitoring of the renal functions, electrolyte balance, and diabetes mellitus status is required. The two main issues that are often seen during intravenous AMB therapy and need to be dealt with daily are hypokalemia and acute kidney injury . Prompt Rhino-orbito-cerebral mucormycosis is a rare, fulminant, and invasive fungal infection, associated with significant morbidity and mortality in diabetic patients. This difficult-to-diagnose disease is also difficult to treat. Early diagnosis, prompt institution of lAMB therapy, and urgent aggressive and repetitive surgical debridement associated with control of underlying predisposing factors are mandatory for successful management and improved prognosis.


A microfissure near the round window niche is an anatomical structure that communicates between middle ear and the ampulla of the posterior semicircular canal. It has been suggested that the microfissure can cause inner ear symptoms; however, the etiology has not yet been confirmed clinically. We report, to our knowledge, the first case of microfissure with complaint of hearing loss and vertigo and improvement in hearing after surgical sealing of the microfissure. A 50-year-old man complained of hearing disturbance, tinnitus with flowing-water sound in the left ear, and a floating sensation upon pushing the left tragus. He had moderate sensorineural hearing loss (43.3 dB) in the left ear for 3 days. His hearing worsened and he complained of severe vertigo. An exploratory tympanotomy was performed 8 days after onset. A microfissure and accumulation of clear fluid in the floor of the round window niche were detected, and leakage point was packed with connective tissue. One month after surgery, his hearing (20.0 dB) and disequilibrium had improved. The inner ear symptoms improved after the surgery in this case, suggesting that the microfissure might have caused the symptoms. Perilymphatic fistula (PLF) is a distinctive otological disorder. It involves an abnormal opening between the inner ear and the external surface of the labyrinthine capsule, allowing perilymph to leak and causing specific disruptions in hearing, balance, or both . There aThe two recognized sites for PLFs from anatomical microfissure are the fissula ante fenestram at the oval window , the usuA 50-year-old man presented to our clinic with a 3-day history of hearing disturbance and tinnitus with flowing-water sounds in the left ear. The symptoms did not have an instantaneous onset, but were slowly progressive. He did not remember any antecedent event related to barotrauma. He had no history of trauma, ear disease or ear surgery. Additionally, he described a feeling of floating when he pressed the tragus of his left ear. An audiogram showed moderate sensorineural hearing loss (43.3 dB) in the left ear . He did In regard to the pathogenesis of the round window fissure, Okano et al.  found itAs shown in During surgery it was challenging to determine whether the fluid observed was perilymph or exudate. Because the fluid was not tested for cochlin tomoprotein , we cannPLF commonly links trauma, barotrauma, ear disease or ear surgery , 4. The The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.TSe: Conceptualization, Data curation, Writing – original draft. AK: Data curation, Writing – original draft. YK: Data curation, Writing – original draft. MA: Data curation, Writing – original draft. TSh: Writing – review & editing. MK: Writing – review & editing.The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.