Streptococcus suis (S.suis) is an emerging zoonosis disease with a high prevalence in Southeast Asia. There are over 1,500 cases reported globally in which majority of cases are from Thailand followed by Vietnam. The disease leads to meningitis in human with sensorineural hearing loss (SNHL) as the most common complication suffered by the patients. Early diagnosis and treatment is important to prevent severe neurological complication. In this study, we aim to develop an easy-to-use risk score to promote early diagnosis and detection of S.suis in patients who potentially develop hearing loss.S.suis patient records in a tertiary hospital in Chiang Mai, Northern, Thailand was obtained. Univariate and multivariate logistic regressions were employed to develop a predictive model. The clinical risk score was constructed from the coefficients of significant predictors. Area under the receiver operator characteristic curve (AuROC) was identified to verify the model discriminative performance. Bootstrap technique with 1000-fold bootstrapping was used for internal validation.Data from a retrospective review of 13-year S. suis hearing loss were meningitis, raw pork consumption, and vertigo. The predictive score ranged from 0–4 and correctly classified 81.95% patients as being at risk of S.suis hearing loss. The model showed good power of prediction  and calibration .Among 133 patients, the incidence of hearing loss was 31.6% (n = 42). Significant predictors for S.suis hearing loss. We identified meningitis, raw pork consumption and vertigo as the main risk factors of S.suis hearing loss. Future studies are needed to optimize the developed scoring system and investigate its external validity before recommendation for use in clinical practice.To our best knowledge, this is the first risk scoring system development for  Streptococcus suis (S.suis) is a gram-positive alpha-hemolytic bacterium mainly in pigs which can causes serious infection in human with a high prevalence in Southeast Asia. There were over 1,500 S.suis cases reported worldwide, with the highest incidence in Thailand (> 600 cases) [S.suis infected patients would develop meningitis in which sensorineural hearing loss (SNHL) is the most common complication among most survivors [0 cases) . About turvivors . SNHL isurvivors , 4. ThisStreptococcus suis SNHL is poorly diagnosed because patients do not undergo audiometry screening until they complaint of severe hearing disorder. By then, the prognosis of the disease is severe with slim percentage of recovery. Prior animal experiments and electrophysiological studies in human revealed that the underlying mechanism of hearing loss is probably from bacterial invasion through cochlear aqueduct, which causes suppurative labyrinthitis [S.suis infection. This study aims to develop an easy-to-use risk score to promote early diagnosis and detection of S.suis infected patients who are prone to hearing loss in primary settings.inthitis , 6. Thisinthitis . SNHL waThe data was collected as a part of a retrospective cohort study over 13-year period at Chiang Mai University Hospital (CMUH) , a 1400-S. suis positive cases were identified based on the microbiology laboratory data available and hospital numbers (HNs) admitted from May 2005 to December 2018. S. suis cases were confirmed by blood or cerebrospinal fluid (CSF). All confirmed S.suis cases with available medical records were included in this analysis  culture with compatible clinical presentation. An audiogram was used to diagnose and monitor the degree of hearing ability. The degree of hearing loss was assessed by otorhinolaryngologists based on individual patients’ hearing thresholds in decibel (dB) at different frequencies. The level of hearing loss was classified into mild, moderate, severe and profound. Presbycusis or any pre-existing hearing loss would be excluded. Endocarditis and site of vegetation was assessed by echocardiography.The study was approved by the Research Ethics Committee, Faculty of Medicine, Chiang Mai University IRB no.010/2018) and Monash University Human Research Ethics Committee (MUHREC) (Project no.12225). Informed consent forms were not required in this retrospective review. The study reporting was in accordance to the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis (TRIPOD) statement [/2018 andS.suis hearing loss. Univariate analysis was performed to compare potential predictors between hearing loss and non-hearing loss patients. For continuous variables, Student’s t test was used in case of normal distribution whereas Mann-Whitney-Wilcoxon test was employed if the data is not normally distributed. Chi-square test or Fisher’s exact test was used for categorical variables. We performed both complete case analysis and multiple imputation using predictive mean matching with 20 iterations for missing Glasgow Coma Scale (GCS) values [S.suis hearing loss. Predictors that remained significant at p-value ≤ 0.10 would be included in the final parsimonious model.Demographics and clinical characteristics were initially analyzed descriptively. Variables are defined as risk factors associated with an increased risk of ) values . VariablS.suis hearing loss was illustrated. The risk stratification was done according to scores distribution into four quartile groups. The classification performances including sensitivity, specificity, positive (LR+) and negative likelihood ratios (LR-) were also calculated. A bootstrap with 1,000 replications technique was used for internal validation to correct for optimism [A clinical scoring system was developed by transforming regression coefficients into item scores. Each item score was generated by dividing with the smallest coefficient in the model and rounding the number to the closest integer . The modoptimism . All staS.suis infection were included in this analysis, majority were males (67.2%). S.suis infection in which 13 (9.77%) were mild SNHL, 7 (5.26%) were moderate SNHL, 4 (3.01%) were severe SNHL and 12 (9.02%) were profound SNHL. All SNHL still persisted based on audiometry upon discharge and latest follow-up visit up to December 2018.One hundred and thirty-three patients with S.suis hearing loss with raw pork consumption (p<0.001), valvular heart disease (VHD) (p = 0.009), alcoholic liver disease (ALD) (p = 0.093), acute meningitis (p<0.001), neck stiffness (p<0.001), infective endocarditis (IE) (p = 0.005), vomiting (p = 0.019), vertigo (p = 0.011) and lower level of serum creatinine (p = 0.018) and potassium (p = 0.002)  . Continuion data .S.suis hearing loss prediction   were included in the stepwise forward logistic regression. There were three predictors remained in the model at p-value ≤ 0.1 which were meningitis, raw pork consumption and vertigo. The final parsimonious model at significant level ≤ 0.1 which provided optimal the area under the receiver operating characteristic curve (AuROC) was selected. The scoring system indicated a good performance in 80–0.95) .S.suis hearing loss from non-hearing loss. The calibration plot and Hosmer-Lemeshow goodness-of-fit test indicated good model calibration , see The C statistics or AuROC was 0.86 (95% CI 0.78–0.93) suggesting acceptable discrimination from S. suis hearing loss increases as the score increases  and the original data (Dorigin) were 0.721 (95%CI 0.432–0.905) and 0.718 (95%CI 0.570–0.867) respectively. The average bias was -0.0022 (95%CI -0.002 to—0.002) indicating low bias and good calibration. The average C statistic from bootstraps was 0.860 (95%CI 0.716–0.953) with estimated bias of -0.001 (95%CI -0.001 to -0.001). The AuROC was 0.860 (95%CI 0.716–0.953).A bootstrap of 1000 sampling with replacement was performed for internal validation. The Somer’D coefficient which is an estimated correlation between the observed and predicted value of all boostrap data . Usually these data are available and do not require any invasive laboratory procedure. After evaluating its external validity, this simple scoring system might be useful to assess patients with S.suis hearing loss in hospital and primary care settings.To our best knowledge, this is the first risk scoring system development for S.suis infection in up to 50% of cases is assumed to be caused by the direct infection at the cochlea. The pathogen invasion through the cochlea aqueduct is believed to be from its exotoxin lytic action [S.suis infection. This study is the first attempt to develop a risk score for predicting S.suis hearing loss as a tool to support clinicians in managing the disease and reduce the impact from its sequelae. The strength of the study is a reasonably large sample size of S.suis patients compared to previous studies [S.suis as a ‘rare’ disease, the data from over 13-year period was captured to get a high sample size. With this regards, the treatment and management may have changed over the years. However, audiometry was routinely used to diagnose SNHL at this setting throughout the whole study period and S.suis infection is still generally susceptible to common antibiotics such as penicillin and ceftriaxone. Therefore, this might not have substantially affected the results of the study. In addition, there were few predictors included in the model in which history of raw pork consumption may be potentially to be affected by a recall bias. As our main purpose was to develop a simple clinical prediction tool which is easy to be applied in primary setting, having these predictors  which are available upon patients’ presentation should be practical to assist with initial detection of potential hearing loss cases. Raw pork consumption was known to be a potential factor in acquiring the disease whereas documentation on medical history of such cases was limited. However, we still could identify more than one-third of patients with documented medical history of raw pork consumption whereas there were many fewer cases with this medical history could be confirmed in the previous retrospective study at the same setting [S.suis patients for the purpose of external validation but we did not receive any response. According to the rule of thumb from a simulation study, the number of event per variable (EPV) of 10 or more was required to prevent bias in the regression coefficients [S.suis patients with hearing loss as convenience sample and there were three significant risk factors confirmed in the final model. Therefore, this should be considered to be sufficiently powered. Future studies to optimize such tool and investigation on its external validity should be mandated before implementation. However, robust statistical methods including the bootstrap of 1000 replications for internal validation were performed. The results demonstrated very close Somer’s D coefficients and very low bias between original and bootstrap data indicating good model calibration. The benefits of corticosteroids use in preventing SNHL from S.suis dexamethasone with antibiotics could not be determined in our study due to a relatively few number of patients received adjuvant corticosteroids with antibiotics. Apart from that most adjuvant corticosteroids were administered among those with moderate to profound SNHL after clinical features of hearing loss presentation. Finally, it should be noted that the participants included in the study were mainly from Northern, Thailand where traditional raw pork eating is practiced. This may limit generalizability in other settings especially where raw pork consumption is uncommon.The mechanism of SNHL which is a remarkable consequence from acquiring c action . Presencc action . From ou studies , 17. How setting . An exteficients . In our S.suis infection. Although this tool cannot replace clinical judgement, physicians can look upon these clinical characteristics in patients  for early detection of potential S.suis hearing loss cases and administrate immediate treatment to avoid long-term complications.In conclusion, after external validation, our simple clinical risk score developed might be useful to aid clinicians in identifying patients who are likely to develop hearing loss from S.suis prevention, timely diagnosis and immediate antibiotic treatment before occurrence or sign of hearing loss symptoms are essential to prevent or reduce damage particularly SNHL caused by S.suis infection. Additionally, increased awareness among clinicians and microbiologists on S.suis infection as an emerging zoonosis is important to foster early disease detection, management, and prevention. Public awareness program and food safety campaign should also be considered for disease control and prevention.With an absence of vaccination for S1 TRIPOD Checklist(PDF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5′ donor splice site and predicted to cause exon skipping. Our results show that CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.Mutations in the  CDKL5; OMIM 300672, 300203) cause a broad spectrum of neuropsychiatric disorders that affect both genders and generally share the common features of severe intellectual disability and early drug-resistant epilepsy, emerging in the first months of life [CDKL5-deficiency disorder (CDD) and treatment is based on supportive therapy for the comorbidities, and on rehabilitation. Replacement gene and protein therapy could be an intriguing option, but this approach might be hampered by the need to preserve the finely regulated control of CDKL5 levels since CDKL5 is dosage sensitive, as suggested by the existence of a CDKL5 duplication syndrome [Mutations in the X-linked cyclin dependent kinase like gene ( of life . No curehttp://mecp2.chw.edu.au/cdkl5/cdkl5_variant_list_copy.php) and this fraction is largely underestimated, considering that missense changes can impair this process [In this peculiar scenario, correction approaches acting on pre-mRNA splicing would be of great interest for CDD since they would preserve the natural gene regulation and, even with the strongest correction effect, would not boost the expression above the physiological levels. It is worth noting that a relevant proportion ~15%) of gene variants associated with CDD are candidates to affect splicing , the RNA component of the key spliceosomal U1 ribonucleoprotein (U1snRNP) that mediates the 5′ splice site (5′ss) recognition and exon definition in the earliest splicing step . In addiCDKL5 splicing and demonstrated their association with CDKL5 deficiency. Most importantly, we developed U1snRNA variants able to rescue CDKL5 mRNA splicing, protein synthesis and function. Accordingly, the morphology of Cdkl5 null primary neurons is significantly ameliorated only when the expression of a CDKL5 splicing defective cDNA is combined with the presence of the therapeutic U1snRNA. To our knowledge, this is the first proof-of-concept of a splicing correction strategy applied to CDKL5.Here, we examined the molecular mechanisms underlying aberrant www.umd.be/HSF/) of the nucleotide changes annotated in the CDKL5 variation database (http://mecp2.chw.edu.au/cdkl5/cdkl5_variant_list_copy.php), we selected variants expected to affect splicing. Among them, as representative models, we focused on those changes that are predicted to weaken the authentic 5′ss of exons 3, 4, 7, and 9, which encode the key catalytic domain of CDKL5. Furthermore, since several disease-associated changes occur at the 5′ss of exon 16, this exon was also investigated  of CDKL5 protein levels as compared to wild-type lymphocytes B. This wCDKL5 splicing patterns by reverse transcriptase (RT)-PCR with oligonucleotides spanning exons 12–18 did not reveal aberrantly spliced transcripts in CDKL5 deficient lymphocytes but a remarkable reduction (~80%) of the correctly spliced transcripts  cloned in the exon-trapping pTB construct   A.While the expression of the c.744+1G>C mutant together with the compensatory U1snRNA favored the usage of an upstream exonic cryptic 5′ss, the ExSpeU1 induced the usage of a cryptic 5′ss located 8 nucleotides downstream the mutated 5′ss B, with pConcerning the c.99+1G>T, c.2376+1G>A and c.2376+1G>C variants, the co-expression of the corresponding compensatory U1snRNA was ineffective, whereas the ExSpeU1 variants led to the usage of cryptic 5′ss located 5 nucleotides downstream of the mutated 5′ss B. The coCDKL5 minigene variants with the compensatory U1snRNA or the ExSpeU1 revealed a complete splicing rescue as indicated by the exclusive presence of correct transcripts including exon 3 or 16 (CDKL5 deficiency (data not shown).Analogously, we challenged the c.99+5G>A and c.2376+5G>A mutants. The splicing analysis in cells co-expressing the  3 or 16 C. NotablTaken together these data show that U1snRNA variants are effective on mutations at +5 positions but not on variants at the conserved +1 nucleotide.CDKL5 minigene in which exon 3 and the surrounding introns are placed into the hCDKL5_1 full-length cDNA . Strikingly, a significant and dose-dependent increase of CDKL5 activity was also detected .To evaluate the impact of the splicing rescue on CDKL5 activity, we selected the c.99+5G>A mutation affecting the N-terminal catalytic domain as an informative model; we exploited a full-length splicing-competent gth cDNA A. As expnscripts B, lane 2nscripts C, lane 4nscripts B, lane 3nscripts C, lane 5detected C, as indp < 0.01; *** p < 0.001 by one-way ANOVA, followed by Tukey’s multiple comparison test)  D. The ExTaken together, these data prove that the splicing rescue mediated by the ExSpeU1 is paralleled by the restoration of proper protein biosynthesis and activity.CDKL5 could rescue typical phenotypes of mouse Cdkl5 null neurons [S3, we initially confirmed that the c.99+5G>A mutation induced exon skipping that can be rescued by the ExSpeU1 . Strikingly, co-transduction of the virus expressing the ExSpeU1S3 increased the number of intersections at any distance from the soma therefore exhibiting a strong rescuing effect (#p < 0.05 by two-way ANOVA followed by followed by Tukey’s multiple comparison test)  C.CDKL5 deficiency is a severe genetic condition with no cure. Conventional augmentative gene therapy could represent an intriguing option, but its development might be complicated by the need of a finely regulated CDKL5 expression, as suggested by the existence of a CDKL5 duplication syndrome [CDKL5 deficiency since they would preserve the physiological gene regulation. Among them, induction of ribosome read-through by aminoglycoside drugs is an attractive option, as recently demonstrated for mutations introducing premature termination codons (PTCs) and reported in ~15% of patients with CDKL5 deficiency [CDKL5 expression above the physiological levels, even in the presence of the strongest correction effect.At present, syndrome . In thisficiency . Howeverficiency ,14,17,24CDKL5 deficiency in the presence of the CDKL5 c.99+1G>T, c.99+5G>A, c.145+2T>C, c.463+1G>A, c.744+1G>C, c.2376+1G>C, c.2375+5G>A and c.2376+5G>C nucleotide variants. Moreover, since missense mutations can also impair splicing [CDKL5 deficiency. The molecular mechanisms underlying CDKL5 deficiency were also investigated in immortalized lymphocytes of a heterozygous female patient, carrying the splicing defective c.2376+5G>A allele. While these studies confirmed the CDKL5 splicing pattern observed in vitro, thus supporting the exploitation of the minigene approach for diagnostic purposes, they also highlighted the involvement of NMD, indicating the value of future studies aimed at investigating the therapeutic potential of inhibiting NMD for CDKL5 mutations affecting its terminal portions. Here, through the minigene expression approach, we investigated the molecular mechanisms leading to CDKL5 expression. Here, the delivery of both the compensatory U1snRNA or the second generation of U1snRNA variants, named Exon Specific U1snRNA (ExSpeU1), failed to restore exon definition in the presence of nucleotide changes occurring at position +1, in accordance with its crucial role within the 5′ss [The knowledge of the aberrant splicing mechanisms led us to explore the U1snRNA-mediated approach, a strategy that could maintain the fine regulated control of the 5′ss . On the CDKL5 exon 3 c.99+5G>A mutation and developed a splicing-competent full-length CDKL5 transgene. Moreover, since ExSpeU1s are predicted to possess higher exon specificity and reduced off-targets as compared to compensatory U1snRNAs [Cdkl5 null hippocampal neurons the U1s3-coding cassette as well as the full-length CDKL5 splicing-competent transgene. Rescue of CDKL5 was evident both at mRNA level and in the amelioration of dendritic morphology, evaluated by Sholl analysis. Considering that the recovery of Cdkl5 defective neuronal morphology is often considered a valid measurable outcome for the identification of novel therapeutic strategies, we find these results highly valuable.The majority (>99%) of introns are spliced out by the U2 spliceosome, with the canonical and highly conserved GT 5′ss  recognized by the 5′ tail of the U1snRNA. However, ~1% of U2-type introns have the GC dinucleotide at position +1/+2 of 5′ss and it is recognized that a higher complementarity with the U1snRNA is required to compensate the U to C substitution ,27. MoreU1snRNAs , only thU1snRNAs ,30, we vCDKL5 expression in the presence of different nucleotide changes impairing the splicing process. Moreover, these findings provide the rationale for an approach based on U1snRNA variants able to rescue the splicing defects at RNA and protein levels, and able to revert typical Cdkl5 null neuronal phenotypes. These results encourage further investigations in mouse models, generated to mimic CDKL5 splicing defects, to properly evaluate the therapeutic potential of transducing viral particles expressing an ExSpeU1 cassette. To our knowledge, these findings provide the first proof-of-concept of the U1snRNA-mediated rescue of CDKL5 splicing mutations and of nucleotide variants occurring within the non-canonical GC 5′ss.Overall, our in vitro and ex vivo data elucidate the molecular mechanisms underlying the defective The following antibodies were used for western blot and immunofluorescence experiments: mouse monoclonal anti-CDKL5 (for western blot) , rabbit polyclonal anti-CDKL5 (for immunofluorescence) , rabbit polyclonal anti-active Mitogen-Activated Protein Kinase , mouse monoclonal anti-α-tubulin , mouse monoclonal anti-GFP , mouse monoclonal anti-GAPDH .l-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich) at 37 °C with 5% CO2 in T75 flasks. When confluent, cells were washed with sterile 1× PBS (Sigma-Aldrich and passaged with 1× trypsin solution (Sigma-Aldrich), which was then inactivated with growth medium. Cells were centrifuged at 1200× g for 5 min and the cell pellet was disaggregated by pipetting in 1 mL of growth medium. Cells were seeded in 12-multiwell plates  for RT-PCR and western blot or in 24-multiwell plates for immunofluorescence.HEK293T and HeLa cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium) (Sigma-Aldrich) supplemented with 10% FBS , 1% 2 in 24-multiwell plates. Cells were seeded in 24-multiwell plates (Corning) for RT-PCR and western blot analyses .WT and c.2376+5G>A mutated Lymphoblastoid Cell Lines (LCLs) were maintained in RPMI medium-1640  (Sigma-Aldrich), supplemented with 15% FBS, 1% L-glutamine, 1% penicillin/streptomycin, 2.5% HEPES (Euroclone) and 0.1% nystatin (Sigma-Aldrich) at 37 °C with 5% COCdkl5−/y primary hippocampal neurons were prepared from E18 CD1 mouse embryos isolated from heterozygous females. Mouse genotype was determined by PCR protocol on genomic DNA purified from ear punches using the following primers: forward primer for the null allele 5′-ACGATAGAAATAGACGATCAACCC-3′; forward primer for the WT allele 5′-CCCAAGTATACCCCTTTCCA-3′; common reverse primer 5′-CTGTGACTAGGGGCTAGAGA-3′. Dissected tissues were washed in HBSS (Sigma-Aldrich) and then incubated at 37 °C for 15 min in HBSS containing 0.1% trypsin  and 0.05% DNase (Roche Diagnostics). The digestion was blocked with 10% FBS in DMEM and hippocampi were mechanically dissociated in 1 mL of dissecting medium with a sterile Pasteur pipette. Vital cells were counted using trypan blue and plated in 24-multiwell plates coated with poly-D-lysine (0.1 mg/mL) for RT-PCR or on poly-D-lysine coated coverslips  for immunofluorescence at a density of 70,000 neurons/well in 10% FBS in DMEM. After 2 h at 37 °C to allow cell attachment, the medium was replaced with Neurobasal (Life-Technologies), supplemented with 2% B27 (Life-Technologies), 1% glutamine and 0.5% penicillin/streptomycin.WT and WT and 2376+5G>A mutated LCLs were plated in 24-multiwell plates at a concentration of 70,000 cells/well and treated with 300 mg/mL of Cycloheximide (CHX) (Sigma-Aldrich), an inhibitor of NMD. After 4 h, cells were lysed in 300 µL Purezol reagent  for RNA purification. Then, 700 ng of isolated RNA were retrotranscribed using the iScript cDNA synthesis kit (Bio-Rad). Splicing pattern was analysed by RT-PCR using the following primers: a common forward primer, 5′-CAACAGCCTGCAACTCTTGT-3′; a reverse primer (exon 15–18), 5′-TCGGAATTGGGTACCATGGA-3′; a reverse primer (exon 16–18), 5′-GCTTTGGGTCTGCAGATCTG-3′. rRNA 18S was determined by the following primers: forward primer, 5′-GTAACCCGTTGAACCCCATT-3′ and reverse primer, 5′-CCATCCAATCGGTAGTAGCG-3′.CDKL5 (hCDKL5) exons 3, 4, 7, 9 or 16 and approximately 500 bp of each flanking intronic sequence were cloned into the pTB vector (pTBNde (min) was a gift from Prof. Franco Pagani, Addgene plasmid #15125) using the unique site NdeI. Splicing mutations were inserted using the Q5 Site Directed Mutagenesis kit .For the minigene splicing assay, human CDKL5 splicing competent cDNAs (pSC-CDKL5WT-pSC-CDKL599+5A) were generated by sequential cloning in the pGEM-T easy vector (Promega). The first fragment was generated by PCR on genomic DNA (gPCR) and it is composed of hCDKL5 Kozak sequence, followed by the ATG signal, exon 2 sequence and the initial part of intron 3, which bears the 5′ splicing donor site (5′ss). The amplicone was generated by using the following primers: forward, 5′-TGTATGCATCTCGAGAGTTTGTCTTCATGAAGATTCCT-3′ and reverse, 5′-TGTGAGCTCGAGACTCTGTCTTGAATGAATG-3′. PCR product was digested with NsiI/SacI and cloned in the pGEM-T easy vector. The second fragment was generated by PCR on the WT or c.99+5G>A mutated pTB exon 3 vector, by using the following primers: forward, 5′-TGTGAGCTCCGATCTGGACTACTGCAACC-3′ and reverse, 5′-TGTCATATGGTCATTATGAGGTGCACGGC-3′. This amplicone is composed of the terminal part of intron 3, comprising the 3′ splicing acceptor site (3′ss) and the branch point site (BS), followed by exon 3 and the initial part of intron 4, which carries the 5′ss. The PCR product was then digested with SacI/NdeI and cloned at the 3′ of the first fragment in the pGEM-T easy vector.The WT or the c.99+5G>A hCDKL5 vector [The third fragment comprises the terminal part of intron 4, bearing the 3′ss and BS, which was amplified by gPCR using the following primers: forward, 5′-TGTCATATGTTATCTCACTTTGGATTTGCAGTGG-3′ and reverse, 5′-ATCGCCACAATTTCATGTGTTTCC-3′. The sequence from exon 4 to exon 19 was amplified by PCR on the pEGFP-C1 h5 vector  exploitiHEK293T cells were transfected using Lipofectamine 3000  with 600 ng of WT or mutated pTB minigenes, together with 200 ng of GFP expressing plasmid (pEGFP-C1) to test transfection efficiency. U1snRNAs and ExSpeU1 expressing plasmids were transfected with a 1× molar ratio. The splicing pattern was analysed by RT-PCR with the primers α-2,3 Globin forward, 5′-CAACTTCAAGCTCCTAAGCCACTGC-3′ and Bra2 reverse, 5′-TAGGATCCGGTCACCAGGAAGTTGGTTAAATCA-3′.To test the effect of +5 splicing mutation, HEK293T cells were transfected using Lipofectamine 3000 with 600 ng of pSC-CDKL5WT or pSC-CDKL599+5A, together with a 1× or 2.5× molar ratio of U1snRNAs and ExSpeU1. The splicing pattern was analysed by RT-PCR with the primers forward, 5′-TGTATGCATCTCGAGAGTTTGTCTTCATGAAGATTCCT-3′ and reverse, 5′-CCGATACCATCTGGTGGCACG-3′.s3, the coding sequence of the U1s3 was cloned into a second-generation lentiviral plasmid backbone  using the BamHI-SalI restriction sites. On the other hand, the lentiviral plasmid expressing the full-length splicing competent CDKL5 transgene was generated by insertion of the CDKL5 transgene into the pLentilox backbone  through the XhoI-NotI restriction sites. To generate the lentiviral stock used to transduce hippocampal neurons, the pLV-U1s3 or pLenti-CDKL5 plasmids were co-transfected with the psPAX2 packaging and pMD2.G envelope plasmids into HEK293T cells. Virus supernatants were collected and lentiviral titer (2.67+05 vg/μL) was determined by fluorescence titering assay. Briefly, serial dilutions of lentiviral stock were used to infect 75,000 seeded cells. After 48 h post infection, cells were harvested, and the percentage of GFP-positive cells was evaluated by fluorescence-activated cell sorting (FACS) analysis.To create the lentiviral plasmid expressing the U1Cdkl5 null neurons were infected for immunofluorescence.Hippocampal neurons were infected at DIV1 with the lentivirus pLL SC-CDKL5WT, SC-CDKL599+5A alone or in combination with pLL ExSpeU1 and analyses conducted at DIV7. WT neurons were used for RT-PCR analysis, whereas Confluent HEK293T cells and LCLs were lysed in 120 µL of Laemmli Buffer. Following this, 20 µL of cell lysate were heated at 70 °C for 10 min and separated on a 10% SDS-PAGE. Proteins were blotted on a nitrocellulose membrane using a semidry transfer apparatus  and membranes incubated 1 h in blocking solution  and 5% non-fat dry milk). Membranes were incubated with primary antibody at the proper dilution: anti-phospho-TEY (1:2000 in 0.4% BSA in TBS-T), anti-α-tubulin , anti-GFP (1:1000 in 5% milk in TBS-T) and anti-GAPDH . After 3 washes in TBS-T, blots were incubated with the appropriate HRP-conjugated secondary antibody  and the immunocomplexes visualized by using the ECL substrate kit (GeneSpin) and the Uvitec system . Band density measurements were performed using the Uvitec software. To detect total CDKL5, phospho-TEY signal was stripped by incubating the membrane in the stripping solution  for 15 min at RT and washed in TBS-T for 20 min. The membrane was incubated in blocking solution for 1 h and then with the anti-CDKL5 primary antibody (1:1000 in 5% milk in TBS-T). The phosphorylation status of CDKL5 was calculated as a ratio between the phospho-TEY and the total isoform of CDKL5. CDKL5 levels were calculated by using, as internal control, GFP in HEK293T or GAPDH in LCLs. α-tubulin served as loading control.www.fiji.sc) in order to calculate total, nuclear and cytoplasmic fluorescence.HeLa cells were washed 3 times with 1X PBS for 5–10 min and then fixed with 4% ice-cold paraformaldehyde  for 15 min. After 3 washes in 1× PBS, cells were incubated for 5 h at RT in blocking solution . Cells were then incubated with anti-CDKL5 primary antibody (1:1000 in blocking solution) overnight at 4 °C and after 3 washes in 1× PBS, cells were incubated with the Alexa-fluor 568 donkey anti-rabbit secondary antibody (Life-Technologies) for 1 h at RT. After 3 washes in 1× PBS, DNA was counterstained with DAPI  for 5 min and coverslips were mounted with Fluoromount-G reagent (Life-Technologies). Images were acquired from at least 3 coverslips for each experimental group under a confocal fluorescence microscope  using an excitation wavelength of 568 nm with a 100× objective. Images were processed by Fiji software . Cells were then incubated for 1 h with the Alexa-fluor 488 anti-mouse secondary antibody (Life-Technologies) and, after 3 washes in 1× PBS, DNA was stained with DAPI (1:1000 in 1× PBS) for 5 min. Cells were washed 8–10 times with 1× PBS and coverslips were mounted with Fluoromount-G reagent. GFP-positive neurons were acquired under an epi-fluorescence microscope (Nikon), equipped with a 40× objective and neuronal arborisation was analysed using a Sholl Analysis plug-in distributed with Fiji software.p value < 0.05 was considered significant.Data are expressed as mean ± SEM and were analyzed using Graph Prism software 7.0. Data of CDKL5 expression and subcellular localization were analyzed by one-way ANOVA, whereas morphological results from Sholl analysis in neurons were analyzed by two-way ANOVA and multiple comparison was performed by Tukey’s post-hoc test. Repeated measures one-way AVOVA was used for the statistical analysis of cellular growth on LCLs cells and multiple comparison was performed by Sidak’s post-hoc test. A


Phosphene generation is an objective physical measure of potential transcranial alternating current stimulation (tACS) biological side effects. Interpretations from phosphene analysis can serve as a first step in understanding underlying mechanisms of tACS in healthy human subjects and assist validation of computational models.  This preliminary study introduces and tests methods to analyze predicted phosphene occurrence using computational head models constructed from tACS recipients against verbal testimonies of phosphene sensations. Predicted current densities in the eyes and the occipital lobe were also verified against previously published threshold values for phosphenes.  Six healthy subjects underwent 10 Hz tACS while being imaged in an MRI scanner. Two different electrode montages, T7-T8 and Fpz-Oz, were used. Subject ratings of phosphene experience were collected during tACS and compared against current density distributions predicted in eye and occipital lobe regions of interest (ROIs) determined for each subject. Calculated median current densities in each ROI were compared to minimum thresholds for phosphene generation.  All subjects reported phosphenes, and predicted median current densities in ROIs exceeded minimum thresholds for phosphenes found in the literature. Higher current densities in the eyes were consistently associated with decreased phosphene generation for the Fpz-Oz montage. There was an overall positive association between phosphene perceptions and current densities in the occipital lobe.  These methods may have promise for predicting phosphene generation using data collected during in-scanner tACS sessions and may enable better understanding of phosphene origin. Additional empirical data in a larger cohort is required to fully test the robustness of the proposed methods. Future studies should include additional montages that could dissociate retinal and occipital stimulation. Since these pyramidal neurons were inactive during in vitro measurements, the threshold for depolarization of active pyramidal cells in vivo may be lower [Transcranial alternating current stimulation (tACS) is a noninvasive technique used to modulate brain function . Configube lower . A recenbe lower  reportedPhosphenes are light sensations occurring in the eyes that can be induced by external electrical stimuli . PreviouA local and “purely” cortical TMS application over the visual cortex was found to also induce phosphenes and, in fact, was used to measure the effects of tDCS on visual cortex excitability . This obOther reports have suggested that phosphene perception in tACS subjects, including those who undergo visual cortex stimulation, may actually originate in the retina. The ability of the retina to elicit phosphenes was first introduced by Brindley , where dThe occurrence of cortical phosphenes proposed by Kanai et al.  was lateTo date, there has been no study that investigates phosphene perception by directly comparing tACS subject testimonies and predictions derived from subject-specific models. Such a study would be particularly useful, since exact electrode locations and subject-specific head geometries are modeled, thus producing more precise predictions of current flow and likely phosphene experience. In this study, we propose methods to compare predicted phosphene occurrence and subject verbal testimonies of phosphene experience in tACS recipients. Predicted current density distributions in the eyes and the occipital lobe were calculated using computational head models derived from six tACS recipient MR image datasets. Predicted values were then compared to previously published thresholds for phosphene generation , 22, 23 All experimental procedures reported in this study followed protocols approved by the University of Florida (UF) and Arizona State University (ASU) Institutional Review Boards. Six healthy participants  received tACS-like treatment at 131 approximately 10 Hz as part of our previously reported magnetic resonance electrical impedance tomography (MREIT) current density and tissue conductivity imaging studies . High- r2 were encapsulated in saline-soaked sponges and secured on participant heads using an elastic bandage .Two electrode montages were used, namely, T7-T8 and Fpz-Oz, as shown in 3) over 160 sagittal slices, each of 1 mm thickness. High angular resolution diffusion-weighted imaging (HARDI) datasets were acquired over 6 gradient directions with low b-value (100 s/mm2) and a total of 64 gradient directions with high b-value (1000 s/mm2). Two sets of 6-direction DWI data were acquired, each having opposite phase encoding direction and were combined using the command topup in FSL [3 isotropic resolution. T1-weighted and HARDI datasets in each subject were coregistered prior to FEM construction and model tissue conductivity assignments.T1- and diffusion-weighted images of participants were acquired using a 3T MRI Phillips Achieva scanner housed at the Advanced Magnetic Resonance Imaging and Spectroscopy Facility, UF McKnight Brain Institute. The high-resolution 3D FLASH T1-weighted datasets were acquired with a 240 × 240 matrix size (voxel dimension of 1 mmp in FSL . The resCurrent administrations were performed following the MREIT-current density imaging (CDI) procedures described in our previous study . The MREVerbal testimonies of subject phosphene perceptions were recorded during MREIT imaging procedures, while subjects were still within the MR scanner with the room lights off in all sessions. All participants were asked to rate the intensity levels of phosphenes on a scale from 1 to 10, with 1 being no phosphenes and 10 being a white field. Averaged ratings were used if subjects underwent multiple stimulation procedures using the same montage.3 isotropic resolution using FreeSurfer . Details of the segmentation pipeline are contained in Indahlastari et al. [A realistic head model was constructed for each participant. Each model comprised ten different tissue types and nine different conductivities. Prior to segmentation, structural T1-weighted datasets were resampled to 256 × 256 × 256 1 mmi et al. . The eyei et al. .Segmented tissue masks were then processed following the modeling pipeline defined in our previous work . The resJinput) of 0.4 mA/m2 applied to anode electrodes , while cathode electrodes  were set to ground voltage. Voltage values throughout the entire head volume were solved for using COMSOL. Derived current density magnitudes were then interpolated onto a 256 × 256 × 256 mm grid using the MATLAB-COMSOL Livelink Interface (MLI) and masked to eye or occipital lobe ROIs. Median current densities in ROIs were computed in MATLAB  and compared to literature-sourced values for the minimum thresholds to induce phosphenes [Jmed) in each ROI to the input current density (Jinput) in each simulation case.The Laplace equation was solved on each model. Boundary conditions were specified as input normal current densities  and slopes were estimated using the npregress command in Stata .A nonparametric local-linear regression model was used to analyze correspondence between both raw and logged current density distributions in the eye, medial walls of the occipital lobe and the remainder of the occipital lobe, and subject ratings. An Epanechnikov kernel was used and 50 bootstrap replications were tested. Logged data were used because of the large dynamic range of the current density data . AdditioFinally, predicted electric field magnitudes were computed from median current densities observed in the OCC ROI divided by gray matter conductivity found in Overall, current density magnitudes in the eye ROI were about three times as large as in the OCC. Discrepancies in median current densities between anisotropic and isotropic models were within ±17% in the EYE and ± 30% in OCC ROI. Details of study results are contained in the following subsections.2 threshold presumed for phosphene generation [2 in the EYE and 68 mA/m2 in the OCC ROIs, respectively. Models of the Fpz-Oz montage had the largest median current densities in both ROIs. The smallest median current densities in the EYE and OCC ROIs were from models with the T7-T8 montage.neration . The larMedian current density values in ROIs, expressed as a percentage of input current density (%Js), are shown in 2 values found for T7-T8 models using both logged and unlogged data were over 0.4 for both isotropic and anisotropic model data. The model therefore suggested a large percentage of subject ratings was explained by current densities in eye and occipital lobes. Coefficients found in T7-T8 montage models suggested a stronger positive dependence of ratings on current densities in the eye than on occipital lobe current densities. Ratings for this montage also depended more on occipital medial wall current densities than on current densities in the remainder of the occipital lobe. For the Fpz-Oz montage, relationships between current densities in the eyes and occipital lobe and subject ratings were not as well correlated, with models showing lower calculated R2 values (0.38 for logged anisotropic model data and 0.33 for logged isotropic model data). For both isotropic and anisotropic models, there was a negative association between logged current densities in the eye and subject ratings and positive associations between current densities in the occipital lobe and ratings, as shown in In this study, we tested our proposed methods to compare predicted and observed phosphenes in six healthy tACS recipients. We expected that phosphenes would be experienced by all subjects, since tACS at 10 Hz has been repeatedly been shown to induce phosphenes even at low current intensities . All pre2 values (0.33–0.50) in all the four models suggesting a correlation between logged current density norms in eyes and occipital lobe compartments and phosphene intensity reports. The fact that predicted current densities in the eyes were negatively associated with phosphene observations may indicate that the eye response to phosphenes is saturated beyond a certain current intensity. We caution that these results are preliminary because of the limited dataset used. However, we did observe that nonparametric regression produced higher coefficients of regression (R2) values, but the same overall trends, as we found when performing linear regression on the logged data. We recommend that nonparametric statistical tests be used with a larger sample size to produce a more robust analysis because of the large ranges and likely nonlinear dependence of subject ratings on current density distributions.The Fpz-Oz montage had the highest phosphene ratings from subjects and the largest %Js in both ROIs, while the other montage ratings were lower, showing a broad agreement between model predictions and testimonies . An inteAll subjects in this study reported phosphene occurrence in their peripheral visual fields. The medial anterior wall of the visual cortex has been associated with the peripheral areas of the visual field . CoefficPrevious studies have suggested that tACS application via electrode locations near the eyes could elicit retinal phosphenes via current leakage from the scalp to the eyes , 19. TheNeuronal cell orientation can affect the likelihood of neuronal activation and may contribute to phosphene generation . Electriin vivo tACS current density imaging studies [We recognize limitations in computational models used in our study. Segmenting ROIs incorrectly, for instance, might alter predicted current density distributions in the ROIs. We computed the percentage differences between calculated median current densities in modified ROIs and reported values in  studies , 25. TheThe results presented in this study demonstrated our proposed methods of using computational modeling and verbal testimonies as useful to assess phosphene perception in tACS recipients. These methods should be implemented in a larger cohort to test the robustness of our statistical findings. In order to separate the possible relationship between electrode location and cortical or retinal phosphenes, future tACS studies should include electrode montages with both the anode and cathode electrodes located near the occipital cortex, or with both near the eyes. In addition, the association between the possible origins of phosphenes and the likelihood of neuron firing in that origin can be explored by computing additional parameters. For instance, calculating the radial components of current density entering the eye compartments and both the tangential and normal electric fields on the surface of the occipital cortex. Neuron firing may also depend on the input from the neighboring cells . For insThis study presented initial results from a novel approach that compared predicted modeled current densities and reported phosphene perceptions. Predicted current densities were assessed in two ROIs associated with possible phosphene origin: the eyes and the occipital lobe. As expected, all tACS recipients stimulated at 10 Hz reported phosphene generation. Predicted median current density values in ROIs exceeded current density thresholds previously reported for phosphene generation. There was not enough evidence acquired to separate retinal and cortical phosphenes. However, our initial findings suggested that all subjects experienced retinal phosphenes from receiving tACS using T7-T8 and Fpz-Oz montages. Further studies using our proposed methods may contribute in understanding the association between electrical stimulation and phosphene perception.


Background: Both hearing loss and mobility decline are well-known risk factors of cognitive impairment among older adults. However, the effects of the accumulation of these functional impairments are still unclear. Thus, the present study examined whether the interactive effects of hearing loss and poor gait performance contribute to cognitive impairments. Methods: Hearing loss and gait performance were assessed in 716 community-dwelling older adults at baseline. Pure-tone audiometry was conducted to determine hearing loss at 1  and 4 kHz . Poor gait performance was defined as the lowest quartile (fourth quartile) of age- and sex-appropriate mean gait velocity. Participants were then classified into four groups according to the presence of hearing loss and poor gait performance. Cognitive function was assessed using MMSE and MoCA at baseline and four years later. Results: Older adults who had either hearing loss (low or high tone) or poor gait performance showed lower MMSE and MoCA scores at baseline. Multiple regression models showed that hearing loss and poor gait performance at baseline were significantly associated with decreased cognitive function at follow-up. Among older adults with low tone hearing loss, absence of slow gait was not associated with decreased cognitive function. Conclusions: Our results indicate the possibility that hearing loss and slow gait synergistically increase the risk of cognitive impairment. The results also suggest that the effect of slow gait on cognition exceeds the effects of hearing loss, indicating the importance of maintaining mobility in late life.


Mucormycosis is a life threatening fungal infection that occurs in immunocompromised patients.A 36-year-old Saudi male with known case of Insulin-Dependent Diabetes Mellitus (IDDM), who was presented with productive cough and diarrhea. Computed tomography (CT) revealed a disseminated fungal invasion of the lungs and spleen with invading the fundus of the stomach. An aggressive surgical approach and amphotericin B treatment resolved his condition.Disseminated Mucormycosis in one organ can spread hematogenously to other organs and can cause severe morbidity and mortality in high risk individuals. The diagnosis of mucormycosis is usually delayed, Antifungal therapy alone is insufficient for mucormycosis, and surgical debridement for all infected tissue is often required.Mucormycosis is a life threating infection, and usually the diagnosis late because of non-specific clinical, radiological feature and need biopsy for confirmation of the organism. The cornerstone for the management and increase the survival rate remain on rapid diagnosis, treatment of the underlying predisposing condition, and urgent surgical debridement. In tissue as well as in culture, these moulds form characteristic broad, non-septate or sparsely septate hyphae with right-angled branching.Mucormycosis was 1st described by Paulltaf in 1885 . Which cThe major risk factors predisposing individuals to the disease include prolonged or profound neutropenia, uncontrolled diabetic and metabolic acidosis, malnutrition, use of corticosteroids and treatment with the iron-chelating agent such as desferrioxamine. High-risk patient groups include hematopoieticstem cell transplant (HSCT) recipients, solid organ transplant (SOT) recipients, patients with hematological malignancies, patients with type I or II diabetes mellitus, burns patients, injection drug users and individuals with no apparent immunological defects .Mucormycosis infections occur following in halation, implantation, or ingestion of spores. The Mucormycosis infections are characterized by extensive angioinvasion that results in vessel thrombosis and subsequent tissue necrosis. Ischemic necrosis of infected tissues can prevent delivery of leukocytes and antifungal agents to the foci of infection. This angioinvasion likely contributes to the capacity of the organism to hematogenously disseminate to other target organs .Because mucormycosis is such an aggressive infection, an early diagnosis is essential for the successful management. The microscopic demonstration of Mucorales in clinical material taken from necrotic lesions or in sputum or Broncho alveolar lavage (BAL) fluid is more significant than their isolation in culture. If the management of mucormycosis is to be successful, treatment should be initiated as soon as the diagnosis is suspected, the underlying metabolic or immunological disorders that precipitated the infection must be corrected, infected necrotic tissue must be removed and an appropriate antifungal agent must be administered.Immunosuppressive and myelotoxic drugs should be reduced in dose or discontinued, provided this will not harm the patient. Iron chelation treatment with desferrioxamine should also be discontinued. Amphotericin B remains the most potent drug available for mucormycosis .2The work has been reported in line with the SCARE criteria .In November 2017, A 36 years old Saudi male with known case of IDDM, presented to the emergency department complained of 1 month history of diarrhea and cough. Diarrhea was watery with productive cough, yellowish in color, associated with shortness of breath and weight loss, no history of hemoptysis or abdominal pain, no contact with sick patient or using drug. No past surgical history. No significant family neither psychological history.On examination: Conscious oriented alert, not on respiratory distress, not pallor neither cyanosis, with lower limb edema grade 3, the patient was visibly underweight. Cardiovascular examination was unremarkable, Chest examination decrease air entry on left side with inspiratory crackle. Other systemic examinations were unremarkable.9/L,hgb7.1 g/dl, platelets 660 × 109/L, albumin 18 g/L, ESR 89 mm/h, CRP 74 mg/L and ECG was showing normal sinus rhythm. Chest x-ray revealed a cavity at the left side with pleural effusion , lungs (24%), and skin (19%). Dissemination developed in 23% of these cases. Although brain is a common site to spread the metastatic lesions may also be found in the liver, spleen, heart, and other organs. The overall mortality rate for the disease is 44% in diabetics, 35% in patients with no underlying conditions and 66% in patients with malignancies .Disseminated Mucormycosis in one organ can spread hematogenously to other organs and can cause severe morbidity and mortality in high risk individuals. Our patient was having uncontrolled diabetic for several years. Patients with diabetic ketoacidosis have elevated levels of available serum iron, likely due to release of iron from binding proteins in the presence of acidosis, elevated concentrations of glucose and iron enhanced surface GRP78 expression and resulting penetration through and damage of endothelial cells Mucorales in a receptor-dependent manner . AuthorsThe symptoms and evolution of disseminated mucormycosis vary widely, reflecting the host as well as the location and degree of vascular invasion and tissue infarction in the affected organs. The variable presentation of disseminated mucormycosis requires a high index of suspicion to enable early diagnosis.Patients with uncontrolled hyperglycemia, particularly those with ketoacidosis, are the most susceptible. .Mucormycosis may be the first manifestation in some patients with undiagnosed diabetes mellitus , but it Pulmonary mucormycosis is high (76%); it is even higher in severely immunosuppressed patients . May devIn rare circumstances, pulmonary mucormycosis can present as an endobronchial or tracheal lesion, especially in diabetics. Endobronchial mucormycosis can cause airway obstruction, resulting in lung collapse, and can lead to invasion of hilar blood vessels with subsequent massive hemoptysis .The most frequent findings include infiltration, consolidation, nodules, cavitation’s, atelectasis, effusion, posterior tracheal band thickening, hilar or mediastinal lymphadenopathy, and even normal findings ,20. A stAlthough our patient was having atypical GI symptoms, his histopathology was showing no fungal organism, we think that the inflammatory process in the spleen spread locally and invaded the fundus of the stomach.Gastrointestinal mucormycosis is uncommon and seldom diagnosed in living patients. In such cases, diagnosis is delayed, and the mortality rate is as high as 85% [Gastrointestinal mucormycosis can occur in any part of the alimentary system, but the stomach is most commonly affected, followed by the colon and ileum .The diagnosis of gastrointestinal mucormycosis is usually delayed, because its nonspecific presentation requires a high degree of suspicion, usually made by biopsy of the suspected area during surgery, endoscopy or by culturing gastric aspirates. The infection usually presents with an appendicular, cercal, or ileac mass or gastric perforation that may be associated with frequently massive upper gastrointestinal tract bleeding . In premGastrointestinal mucormycosis can also involve the liver, spleen, and pancreas. The fungus can invade bowel walls and blood vessels, resulting in bowel perforation, peritonitis, sepsis, and massive gastrointestinal hemorrhage, which is the most common cause of death .Antifungal therapy alone is insufficient for mucormycosis, and surgical debridement for all infected tissue is often required. Aside from the resistance of some fungal strains to amphotericin B, the pathogenesis of mucormycosis including angioinvasion, thrombosis, and tissue necrosis result in poor penetration of anti-infective agents to the site of infection . SurgicaAll of authors have no conflict of interest.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.In our institute, ethical approval is exempted, depend on acquired patient consent.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.All authors contributed to manuscript preparation, manuscript editing, manuscript review.We don’t need to register this work.The Guarantor is DR. Ashwag Alharbi.Not commissioned, externally peer reviewed.


We have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs.3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance.− cells within the tumor nests and the OCT4+ CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D.DAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4Our study demonstrated novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA. Colorectal cancer (CRC) is the third most common cancer worldwide, and the second major cause of cancer-related deaths in developed countries . In New The etiology and pathogenesis of CRC is complex  with envOver 90% of CRC are adenocarcinoma . The livThe cancer stem cell (CSC) concept of cancer proposes that a small subset of cells within the tumor - the CSCs, possess innate biological characteristics for self-renewal . CSCs ar+/NANOG+/KLF4+/c-Myc+/OCT4+ subpopulation and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− subpopulation confined to the peritumoral stroma (PTS), and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− subpopulation within the tumor nests (TNs) (+ cells within the PTS putatively representing the most primitive CSC subpopulation (We have previously identified and characterized three subpopulations of CSCs within liver metastasis from colon adenocarcinoma (LMCA): a SOX2ts (TNs) . We specpulation .The renin-angiotensin system (RAS) is known classically for its role in regulating blood pressure and fluid and electrolyte balance. There is increasing evidence indicating dysregulation of the RAS in malignancy and is associated with poor patient outcomes . The parCathepsin B, a cysteine protease, converts inactive pro-renin into active renin . CathepsN-glycosylated proenzyme of 39 kDa and is subsequently modified under acidic conditions to the single-chain form of mature enzyme of 29 kDa  in response to inflammatory and oncogenic stimuli .In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations within LMCA we have previously identified  using imThe same LMCA samples from nine male patients, aged 50–80  years  includedHematoxylin and eosin (H&E) staining was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients to confirm the presence and areas of TNs and the PTS, by an anatomical pathologist (HDB). These LMCA sections then underwent DAB IHC staining, as previously described , using pIF IHC staining was performed to determine co-expression of two proteins on three samples of LMCA from the original cohort of nine patients used for DAB IHC staining. Vectafluor Excel anti-mouse 488  and Alexa Fluor anti-rabbit 594  were utilized to detect the combinations. All IF IHC-stained slides were mounted in Vecta Shield Hardset mounting medium with 4′,6′-diamino-2-phenylindole (Vector Laboratories).Positive control tissues used for the primary antibodies were human placenta for cathepsin B; human breast carcinoma for cathepsin D; mouse bone marrow for cathepsin G; and human seminoma for OCT4, as previously described . A negatDAB IHC-stained slides were viewed using an Olympus BX53 microscope and photographed with an Olympus DP21 digital camera . IF IHC-stained slides were viewed and imaged using an Olympus FV1200 biological confocal laser-scanning microscope and processed with cellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).https://imagej.net/Welcome) on six fields of view of each DAB IHC-stained slide of the nine LMCA samples at 400x magnification. Fields of view were selected from regions that exhibited the highest density of staining. The proportion of cells within the TNs and the PTS stained positively in each field of view was calculated and the results were subjected to t-tests for related samples, using SPSS v.22 statistical package.Using the Olympus BX53 light microscope fitted with an Olympus DP21 digital camera, cell counting was performed using ImageJ , as previously described . Probes Total protein was extracted and precipitated from three snap-frozen LMCA samples of the original cohort of nine patients included for DAB IHC staining, separated by SDS-PAGE and transferred to a PVDF membrane using previously described methods . DetectiTM F96 MicroWellTM black polystyrene plate  using the Varioskan Flash plate reader . Tonsil and denatured tonsil tissue lysates were used as appropriate positive and negative controls, respectively. All experiments were performed in duplicates with averages taken.Enzymatic activities of cathepsin B and cathepsin D were determined in snap-frozen LMCA samples from the same three patients used in WB, using enzymatic activity assay kits for cathepsin B  and cathepsin D , as previously described . Fluoresarrows) and some cells within the PTS (arrowheads). Granular cytoplasmic staining of cathepsin D (arrows) and some cells within the PTS (arrowheads). Cytoplasmic expression of cathepsin G (arrowheads). Interestingly there was minimal staining for cathepsin B . DAB IHC staining showed cytoplasmic expression of cathepsin B , brown bhepsin D , brown whepsin G , brown whepsin B , brown, hepsin B , brown ahepsin B , brown oPositive controls for cathepsin B , brown, arrows) and the OCT4+ cells within the PTS (arrowheads). Similarly, cathepsin D was expressed by the OCT4− cells within the TNs (arrows) and the OCT4+ CSCs within the PTS (arrowheads). This was in contrast to cathepsin G which was localized to a few cells within the PTS (arrows), separate from the OCT4+ subpopulation (arrowheads). Images of the individual stains are presented in IF IHC staining was performed on three representative LMCA samples from the original cohort of nine patients used for DAB IHC staining, to determine the expression of cathepsins B, D and G in relation to the CSC subpopulations, previously identified. Interestingly, cathepsin B was expressed by cells within the TNs . There was no significant difference between cells stained positively within the TNs and those within the PTS for cathepsin D (p = 0.589) and cathepsin G (p = 0.927).Cell counting on DAB IHC-stained slides for cathepsins B, D and G  in the np = 0.345). However, the mean level of expression of cathepsin B (p = 0.028) and the mean level of expression of cathepsin D (p = 0.028) were significant greater than that of cathepsin G.NanoString mRNA expression analysis confirmed the presence of mRNA transcripts for cathepsin B, D and G relative to the housekeeping gene GUSB in all six snap-frozen LMCA samples . StatistWB of the three snap-frozen LMCA samples demonstrated the presence of bands at the expected molecular weight for cathepsin B  and cathTo determine the functional activity of both cathepsin B and cathepsin D, which were detected by WB, we performed enzymatic activity analysis on the same three snap-frozen LMCA tissue samples, for both cathepsins. All the LMCA samples demonstrated enzymatic activity of cathepsin B and cathepsin D relative to that of the positive and negative control tissues . This pr+ cells that have been isolated from primary colon cancer and liver metastases, have been shown to be capable of initiating tumor growth in immunodeficient mice  and c-MYC play a significant role in triggering pluripotency in somatic cells, and they have been used to identify CSC subpopulations within various types of cancer . CD44, Lent mice .The self-renewing capacity of CSCs and their persistence despite conventional therapy offers a possible explanation for tumor recurrence and metastasis . CSCs ar+ CSC subpopulation within the PTS of the LMCA samples we have examined. However, cathepsin G was localized to a separate OCT4− subpopulation.Our previous characterization of three putative CSC subpopulations within LMCA led us to propose the existence of a hierarchy of CSCs within this tumor . InteresIn vitro and in vivo studies have demonstrated the involvement of the RAS in proliferative signaling, evasion of growth suppressors, resistance of cell death, angiogenesis, reprogramming of energy metabolism, inflammation, cell migration, invasion and metastasis  with written informed consent from all subjects in accordance with the Declaration of Helsinki.TI and ST formulated the study hypothesis. TI, SW and ST designed the study. SW, SM, HB, ST and TI interpreted the DAB and IF IHC data. SW, ST and TI interpreted the NanoString mRNA data. SM performed cell counting on DAB IHC stained slides. BVS performed WB and enzymatic activity assays. RM conducted statistical analysis and interpreted the results. SM, ST and TI drafted the manuscript. All authors commented on and approved the manuscript.TI and STT are inventors of the PCT patent applications Cancer Diagnosis and Therapy (No. PCT/NZ2015/050108), and Cancer Therapeutic (PCT/NZ2018/050006). 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.


Methadone is a long-acting opioid receptor agonist. Reported adverse effects of methadone include constipation, respiratory depression, dizziness, nausea, vomiting, itching, sweating, rhabdomyolysis, QT prolongation, and orthostatic hypotension. Hearing loss has been rarely reported following methadone use, and when reported, long term follow-up is rare.Herein we report a case of methadone poisoning with rhabdomyolysis, acute kidney injury, and persistent hearing loss documented by a 2 year follow up.The patient was a 34 years old male who presented with a reduced level of consciousness and acute hearing loss after suicidal ingestion of 40 mg of methadone while experiencing family-related stresses. He had no prior history of methadone use, abuse, or addiction.Initial laboratory testing was significant for a serum creatinine concentration of 4.1 mg/dl, a mixed metabolic and respiratory acidosis, thrombocytopenia, abnormal hepatic transaminases, and coagulation tests. The patient then developed severe rhabdomyolysis. Also, audiometry showed a bilateral sensorineural hearing loss. The patient required hemodialysis for 11 days while his metabolic abnormalities gradually resolved. However, his hearing loss was persistent, as demonstrated by 2 years of follow up.Our patient simultaneously had kidney failure, rhabdomyolysis, and permanent hearing loss following methadone poisoning. Although rare, ototoxicity and permanent hearing loss may happen in cases of methadone poisoning. While opioid-induced hearing loss is uncommon, methadone toxicity should be taken into account for any previously healthy patient presenting with acute hearing loss with or without rhabdomyolysis. Methadone is a synthetic μ-opioid receptor agonist used in the treatment both of pain and opioid dependence –4. In thRapidly progressive sensorineural hearing loss has been rarely reported in opioid analgesic users, including those using methadone –8. The mA 34-year-old male presented to the Emergency Department with a reduced level of consciousness and hearing loss 21 h after ingesting a 40 mg methadone tablet in a suicide attempt in front of his family after a serious conflict with his father. The family denied any history of a psychiatric disorder or prior methadone use. Prehospital emergency service administered 0.4 mg of naloxone due to apneustic breathing and miotic pupils, which resulted in a marked improvement in the patient’s breathing. He and his family denied any specific drug abuse history. He smoked cigarettes but did not use alcohol. Before this suicide attempt, he was healthy without any significant past medical history and did not have any known auditory deficits.370 C, oxygen saturation while receiving 8 l oxygen per minute by nasal cannula 89%. At the time of admission to the ED and he complained of bilateral hearing loss. His urine was noted to be dark, and a rapid urine toxicology screen test was positive for methadone but was negative for amphetamines, barbiturates, benzodiazepines, cocaine, morphine, phencyclidine, and tetrahydrocannabinol. Blood acetaminophen, salicylate, and ethanol assays were negative.On admission to the Emergency Department (ED) the patient was lethargic, had miotic pupils and the following vital signs: blood pressure 110/70 mmHg, heart rate 110 beats per minute, respiratory rate 16 breaths per minute, temperature 3 , aspartate aminotransferase (AST) 1417 U/L, alanine aminotransferase (ALT) 1125 U/L, BUN 85 mg/dL, Cr 4.1 mg/dL, international normalized ratio (INR) 2.24, and Platelet count of 66,115 /mmInitial treatment consisted of the administration of intravenous fluids and sodium bicarbonate. Twelve hours post-admission, the patient was transferred to the Intensive Care Unit (ICU), where he received a naloxone infusion for 3 days.On admission to the ICU, SPO2, while using an oxygen mask at 8 l/minute was 97%, and his arterial blood gas assay showed: pH 7.33, PCO2 43.1 mmHg, and serum HCO3 23 mmol/l.On the following day, he was alert and breathing normally with a leukocytosis of 14,116 cells/microliter (mcl) (Table By the third day of admission, the patient complained of myalgia and had a urine output of approximately 100 ml/24 h, a BUN of170 mg/dL, serum creatinine of 7.1 mg/dL, and a serum uric acid 11 mg/dL. Non-heparinized hemodialysis was initiated. Kidney sonography was suggestive of parenchymal kidney disease and acute tubular necrosis. He required hemodialysis for 11 days.During 10 days of admission to the ICU, his hepatic transaminases and CPK gradually normalized (Table The patient was transferred to the internal medicine ward on day 11. During the 5 days of hospital stay in the internal ward, the patient’s hearing loss continued despite the normalization of his laboratory studies. He was discharged after 15 days of hospitalization. After 2 years of follow-up, his hearing loss persisted.Here we report a case of methadone poisoning following a suicide attempt with a 40-mg tablet of methadone who exhibited rhabdomyolysis, acute renal failure, and chronic bilateral sensorineural hearing loss. Unlike most prior cases, our patient’s hearing loss persisted despite the normalization of his renal function and resolution of his metabolic abnormalities.Sudden sensorineural hearing loss has several potential causes, including infections, autoimmune diseases, trauma, vascular disorders, and neoplastic diseases, but most cases are idiopathic . Many ofOtotoxicity is a known rare complication of opioid use. In 1979, Mulch et al. reported the first case of opioid-induced hearing loss, which was associated with hydrocodone abuse. Since then, several case reports have described both reversible and irreversible bilateral hearing loss following hydrocodone/acetaminophen , oxymorpIn 2009, the first case of bilateral hearing loss due to methadone was reported in a 37-year-old man who overdosed on 75 mg methadone tablets . Thier pSubsequently, a number of patients have been reported to develop hearing loss after methadone use , 24, 25.The mechanism through which opioids cause hearing loss has not been adequately explained. Among the theories that have been offered for this complication is the contamination of the drug with unknown materials. However, this theory is more likely for heroin-induced sensorineural hearing loss, since methadone is usually produced in pharmaceutical laboratories .Another theorized mechanism for opioid-associated hearing loss is hypoperfusion of the vestibulocochlear system secondary to opioid-induced vasospasm or vasculitis. The origin of this theory is the observation of cerebral infarcts following heroin used due to the resulting vasculitis , 22. HowOur patient had metabolic acidosis, rhabdomyolysis, and acute renal failure requiring hemodialysis. Rhabdomyolysis is a serious syndrome caused by the breakdown of skeletal muscle fibers followed by leakage of muscle contents into the patient’s circulation . The cauSurprisingly, rhabdomyolysis and acute kidney injury were in the first day after ingestion in our case. Rapid onset of rhabdomyolysis and acute kidney injury following methadone poisoning has rarely reported , 32. LikMethadone poisoning may induce rhabdomyolysis, acute kidney injury, and permanent hearing loss. Although most ototoxicity due to methadone poisoning is manifested as transient hearing loss, permanent hearing loss may rarely occur. While opioid-induced hearing loss is uncommon, methadone toxicity should be taken into account for any previously healthy patient presenting with acute hearing loss, with or without rhabdomyolysis. The relationship, if any, between rhabdomyolysis and hearing loss, warrants further study.


Mucormycosis involves the central nervous system by direct extension from infected paranasal sinuses or hematogenous dissemination from the lungs. Incidence rates of this rare disease seem to be rising, with a shift from the rhino-orbital-cerebral syndrome typical of patients with diabetes mellitus and ketoacidosis, to disseminated disease in patients with hematological malignancies. We present our current understanding of the pathobiology, clinical features, and diagnostic and treatment strategies of cerebral mucormycosis. Despite advances in imaging and the availability of novel drugs, cerebral mucormycosis continues to be associated with high rates of death and disability. Emerging molecular diagnostics, advances in experimental systems and the establishment of large patient registries are key components of ongoing efforts to provide a timely diagnosis and effective treatment to patients with cerebral mucormycosis. Mucormycosis is the second most frequent invasive mold disease after aspergillosis ,2,3, witInvolvement of the central nervous system (CNS) represents one of the most severe manifestations of mucormycosis, and often determines the survival and functional outcome of the patient ,9,14. Current estimates of the frequency of central nervous system (CNS) mucormycosis are largely based on published case reports and small series , which aThe frequency of CNS mucormycosis is highly dependent on the population studied  8,18,1919,20. InPatients with diabetes mellitus are at risk for rhino-orbital-cerebral mucormycosis (ROCM), specifically in the context of uncontrolled glycemia and diabetic ketoacidosis (DKA) . Of 337 Unlike the situation in developed countries, where malignancy has emerged as the main risk factor for mucormycosis (see below), uncontrolled diabetes mellitus continues to be the dominant driver of disease in developing nations ,24,25. MMalignancy, particularly hematological, has replaced diabetes mellitus as the most frequent risk factor associated with mucormycosis of all kinds in developed countries ,9,22,26.Aspergillus spp. [Mucormycosis of the CNS seems to be less common among patients with malignancy and recipients of stem cell transplantation, as compared to those with diabetes mellitus . Only splus spp. . Pagano lus spp. . In a relus spp. . Intereslus spp.  retrospePneumocystis jirovecii pneumonia were reported [The Bruton tyrosine kinase (Btk) inhibitor ibrutinib, used in the treatment of mantle cell lymphoma and chronic lymphocytic leukemia, has been associated with invasive fungal diseases with frequent involvement of the CNS. Specifically, intracerebral invasive aspergillosis and, to a lesser extent, cryptococcosis and reported ,37,38,39reported ,41,42. Oreported . The rolreported . Thus, tMucormycosis after trauma typically involves soft tissues of the limbs, and less frequently the abdominal or thoracic wall, head and neck . DissemiInjectable drug use is associated with a unique form of isolated cerebral mucormycosis , presumaSignificant progress has been made in deciphering the virulence factors of Mucorales, as well as some of the molecular events that mediate endothelial cell invasion and damage ,49,50,51The primary route of infection is by inhalation of sporangiospores, the asexual spores of Mucorales, leading to invasive sinopulmonary infection in susceptible individuals. From there, CNS penetration occurs either by hematogenous spread or by direct cranial penetration from the paranasal sinuses. Different host innate defenses have specific roles in containing inhaled sporangiospores and preventing their germination and propagation. Dormant sporangiospores inhaled into the paranasal sinuses and airways are resistant to killing by phagocytes of both immunocompetent and immunocompromised hosts . GerminaInvolvement of the CNS occurs most frequently (70%) due to contiguous spread from the paranasal sinuses and orbits . The remAutopsy studies show that for all three routes, Mucorales enter the brain by way of the cerebral vascular system . Fungal Rhizopus arrhizus spores and hyphae adhere to endothelial cells and damage them [R. arrhizus and other Mucorales, and is required for endocytosis and subsequent endothelial cell damage [R. arrhizus infection [Because angioinvasion is a hallmark of both local tissue destruction and hematogenous spread in mucormycosis , attentiage them . Glucoseage them . GRP78 ol damage . Interesl damage . In vivonfection . These fnfection .Cryptococcus neoformans underscore the full complexity of microvascular host defense and evasion mechanisms employed by the fungus to enter the brain [C. neoformans uses various strategies to traverse the blood–brain barrier, among them a “Trojan horse” mechanism where the fungus hijacks host phagocytes and endocytic signaling pathways and uses them as vehicles [Specific mechanisms of neuroinvasion by Mucorales have not yet been described. Lessons learned from the neuroinvasive Basidiomycete he brain . For exavehicles . WhetherAcquisition of iron from the environment represents an important Mucorales virulence factor, but direct evidence for its role in CNS invasion is lacking. Iron acquisition is mediated through the production of high affinity iron permeases and siderophores . In the Rhizopus species, principally R. arrhizus, are the most frequent species causing microbiologically-confirmed mucormycosis in the majority of case series [Mucor spp and Lichtheimia (formerly Absidia) spp. [Rhizopus, Mucor and Lichtheimia account for 70% to 80% of reported mucormycosis cases [Cunninghamella bertholletiae, Rhizomucor spp., Syncephalastrum racemosum, Saksanea vasiformis, and Apophysomyces elegans [Rhizopus spp. are the most frequent species recovered from patients with all forms of CNS mucormycosis, including ROCM, disseminated mucormycosis and primary CNS infection in people who inject drugs [C. bertholletiae has a strong predilection for patients with cancer and neutropenia, causing respiratory infection with early angioinvasion and hematogenous dissemination [C. bertholletiae infection, 10 (48%) had CNS disease [Rhizopus, Mucor, and Lichtheimia) and C. bertholletiae, those caused by Apophysomyces elegans appear to occur primarily in immunocompetent patients [Lichtheimia spp., Apophysomyces elegans and Saksanea vasiformis are strongly associated with post-traumatic mucormycosis, and have only rarely been implicated in CNS disease [A. elegans, is reported almost exclusively from India and China [e series ,65. Othe elegans ,66. Rhiz disease ,66,67,68nd China ,24,66,69nd China .Patients with CNS mucormycosis may either present with neurological deficit without clinically evident extracranial disease, as in the case of isolated cerebral mucormycosis, or more commonly with rhino-orbito-cerebral or pulmonary disease that progresses to involve the CNS. These scenarios are discussed below with their respective radiographic findings.Mucormycosis of the sinuses involves the turbinates and paranasal sinuses initially. At this stage, symptoms may be indistinguishable from those of more common causes of sinusitis, and include sinus pain, congestion, mouth or facial pain, hyposmia and anosmia ,70. On eSinus mucormycosis is seen on computed tomography (CT) and magnetic resonance imaging (MRI) as nonspecific nodular mucosal thickening. Inflammatory soft tissue infiltration frequently extends to subcutaneous facial tissue and to the infratemporal and temporal fossae . Non-enhSon et al. compared clinical features of patients with ROCM and bacterial orbital cellulitis, and found that mucormycosis presented more frequently with extraocular muscle limitation and sinus mucosal thickening on CT imaging, and less frequently with eyelid swelling . HoweverThe three most frequent imaging findings in intracranial mucormycosis are cavernous sinus thrombosis, brain infarction and internal carotid artery occlusion ,78. BraiPulmonary mucormycosis typically presents in patients with hematologic malignancies and prolonged profound neutropenia as persistent or recurrent fever, cough, and dyspnea, with or without concomitant sinusitis ,80. PleuAspergillus spp. [Early differentiation between invasive pulmonary aspergillosis and pulmonary mucormycosis, before dissemination to the CNS, is a clinical challenge that has important therapeutic implications. Chamilos et al. reviewed contemporaneous patients with cancer and pulmonary aspergillosis or mucormycosis, and found that concomitant sinusitis, receipt of voriconazole as antifungal prophylaxis, and the presence on chest CT of ≥10 pulmonary nodules or pleural effusion were significantly associated with a diagnosis of mucormycosis . The prelus spp. . The useThe typical presentation of this syndrome is the recent onset of altered mental status, headache, hemiplegia and dysarthria in a person with a history of intravenous drug injection . Fever iAspergillus spp. are the most frequently recovered organisms, followed by Mucorales [Intracranial fungal granuloma is a distinct clinical entity, with most cases to date reported from India ,90. Abouucorales ,90. CT aucorales . The froucorales ,90. Diabucorales . Patientucorales ,90.Aspergillus species. The non-septate hyphae of Mucorales are susceptible to damage from shear stress [There are currently no clinically available circulating biomarkers of mucormycosis . Therefor stress , and thur stress .Because a brain biopsy is highly invasive and may lead to neurological deficits, the diagnosis of CNS mucormycosis is frequently made indirectly by identifying the pathogen in the sinuses or lungs. Patients with suspected ROCM should be examined by an Ear, Nose and Throat (ENT) surgeon experienced in the diagnosis and treatment of this disease. Fiberoptic examination of the nasal cavity and septum may reveal ischemic or necrotic mucosal lesions, which should be biopsied. Flexible bronchoscopy and bronchoalveolar lavage (BAL) or CT-guided lung biopsy are used to diagnose pulmonary mucormycosis .Several culture-independent technologies for the diagnosis of mucormycosis from tissue specimens or blood are being developed. A monoclonal antibody (2DA6) against alpha 1,6 linked mannose, which is conserved among the Zygomycota and Ascomycota phyla, appears promising for the detection of a broad range of pathogenic fungi, including Mucorales . 2DA6 waSuccessful treatment of mucormycosis requires surgical debridement of infected tissue, prompt institution of effective antifungal treatment, and correction of the underlying metabolic and immune derangement . TimelinSurgical debridement is considered a mainstay of mucormycosis treatment, and was associated with improved survival in several case series ,9,18. InDebridement of infected brain tissue is associated with severe morbidity and uncertain benefits. In a report of the Italian Epidemiological Surveillance of Infections in Hematological Diseases (SEIFEM) group, surgical resection was undertaken less frequently for patients with hematological malignancy and CNS Invasive Fungal Disease (IFD) (10%) than for those with IFD of other sites (33%) . This isDecisions regarding the surgical approach, its goals and extent require careful discussion among neurosurgeons, infectious diseases specialists, radiologists, patients and their families . Acceptel-AmB) is based on clinical experience and in vitro activity of this agent against the majority of Mucorales isolates. However, amphotericin B achieves low brain tissue concentrations and is associated with nephrotoxicity at high doses. Combining l-AmB with an extended-spectrum azole, either posaconazole or isavuconazole, may increase the likelihood of achieving effective drug concentrations in the brain, but supporting clinical data for such combinations is lacking. Specifically, retrospective analyses of monotherapy versus combination therapy for mucormycosis in patients with hematological malignancies showed no difference in outcomes between treatment strategies [l-AmB may be switched to maintenance treatment with the less toxic azoles, posaconazole or isavuconazole. The optimal duration of treatment for CNS mucormycosis is unknown. Factors that should be weighed when deciding on the duration of treatment are the extent of surgical debridement performed and the immune state of the patient [There are no prospective controlled clinical trials on antifungal drugs for the treatment CNS mucormycosis. In general, antifungals used to treat CNS mycoses must meet several requirements: 1. In vitro, preferably cidal activity against the infecting fungal strain; 2. Adequate penetration of the blood–brain barrier resulting in tissue drug concentrations that reliably exceed the MIC; 3. A favorable safety profile, allowing treatment for extended time periods. Unfortunately, no single drug for mucormycosis meets all of the above requirements. Initial treatment with liposomal amphotericin B  improved survival whereas amphotericin B deoxycholate (1 mg/kg/day) had no significant effect on morality [R. oryzae from the lungs more effectively than does l-AmB [Amphotericin B is considered the treatment of choice for mucormycosis, although never assessed in a prospective controlled clinical trial. The liposomal formulation is preferred, as the high doses of amphotericin B deoxycholate (AmBd) required to achieve a fungicidal effect against morality . Amphotees l-AmB . Converses l-AmB . l-AmB dose was ≥5 mg/kg thrice weekly (see below) [l-AmB are not more effective than standard dosing (≥5 mg/kg/d), and are associated with high rates of nephrotoxicity. Similar findings were noted in an analyses of treatment outcomes in two patient registries [Clinical data is mostly retrospective. In an analysis of patients with mucormycosis and hematologic malignancies, delayed initiation of treatment with amphotericin B (≥6 days after diagnosis) was independently associated with increased mortality . The lare below) . Overallgistries .l-AmB or AmBd [Amphotericin B crosses the blood–brain barrier poorly, and is detectable at low concentrations in the brain parenchyma of patients treated with either  or AmBd ,114. Cer or AmBd . Althoug or AmBd , this dr or AmBd ,117. Int or AmBd . The lim or AmBd .There is limited information on the CNS penetration of isavuconazole, but emerging animal and human data indicate that it is comparable to that of voriconazole. The brain/plasma concentration ratio was 1.8 after a single dose, and close to 1 after repeated dosing ,121. TheCSF concentrations of posaconazole are highly variable in treated patients, with CSF to plasma ratios ranging from 2.3 to <0.01 ,124,125.Mucorales spp. Data on the use of in vitro susceptibility testing results (MIC) to guide treatment decisions are very limited. If the infecting strain is grown in culture, antifungal susceptibility testing may be performed using standardized broth microdilution methods. Susceptibility breakpoints have not been established for Mucorales spp. in vitro, and synergized with l-AmB to improve the survival of mice with experimental mucormycosis [l-AmB. Treatment with deferasirox resulted in rapid clinical and radiological resolution of cerebellar disease [l-AmB and deferasirox was compared with l-AmB and placebo in a multicenter, randomized blinded clinical trial (the DEFEAT Mucor study) [p = 0.1; 90-day mortality, 82% versus 22%, p = 0.01) [Given the critical role played by iron acquisition in the virulence of Mucorales spp, strategies to limit the availability of iron have been explored as adjunctive therapy in the treatment of mucormycosis. Deferasirox, an iron chelator that is not utilized by Mucorales as a xenosiderophore, was found to have cidal activity against rmycosis ,130. A rrmycosis . The pat disease . Treatmer study) . Twenty  = 0.01) . Imbalan = 0.01) .p = 0.02). However, HBO was not a significant predictor of survival in the multivariate analysis [Hyperbaric oxygen  has transient fungistatic activity in vitro ,134, andanalysis . In sum,p = 0.02) [While lacking intrinsic activity against Mucorales, echinocandins have synergistic activity with AmBd and L-AmB against Mucorales, both in vitro and in experimental murine mucormycosis . In a re = 0.02) . These f = 0.02) . Given tSeveral unmet needs remain for the treatment of mucormycosis in general and for CNS mucormycosis in particular . Despite


Age-related hearing loss (HL), defined by a pure-tone average (PTA) >25 decibels (dB) has been associated with depressive symptoms. We aimed to assess whether this association is present when hearing is better than the arbitrary, but widely-used, 25 dB threshold. The sampled population was the multicentered Hispanic Community Health Study . Cross-sectional data from 2008-2011 were available. Hearing was measured with pure tone audiometry. Clinically-significant depressive symptoms (CSDS) were defined by a score ≥10 on the 10-item Center for Epidemiologic Studies Depression Scale (CESD-10). Participants’ mean age was 58.3 years . Among those with classically-defined normal hearing (PTA ≤25 dB), a 10 dB increase in HL was associated with 1.26 times the odds  of CSDS, adjusting for age, gender, education, vascular disease, and hearing aid use . Results held even for a stricter HL cutpoint of 15 dB. Among subjects with strictly normal hearing (PTA ≤15 dB), a 10 dB increase in HL was associated with 1.47  times the odds of CSDS, adjusting for confounders (p<0.01). Results also held when defining CSDS by an alternative CESD-10 score ≥16. In conclusion, increasing hearing thresholds were independently associated with CSDS among adults with subclinical HL (PTA ≤25 dB). Studies investigating whether treating HL can prevent late life depression should consider a lower threshold for defining HL.


Hearing loss or hearing impairment is a clinically and genetically heterogeneous disorder. More than 117 genes were discovered to date in hereditary, nonsyndromic hearing loss (NSHL). Identifying novel gene variants and their frequency in specific populations is valuable for public health and potentially for genetic screening of NSHL.To identify the gene variants underlying NSHL in a Pakistani cohort.GJB2, the gene with the highest incidence of variants in other populations with NSHL. We found known homozygous as well as compound heterozygous GJB variants in 15 individuals. Next, we used targeted next generation sequencing (TNGS) for the remaining 25 individuals and identified 20 different variants in 14 genes .A cohort of 40 school‐aged children with NSHL was initially screened for variants in We described common and novel variants in 15 genes in a Pakistani cohort of NSHL. To the Editor:http://hereditaryhearingloss.org/)  gene which is the most commonly mutated gene in recessive NSHL  technology using the Ion Torrent platform for a panel of 63 genes implicated in hearing loss as described previously . Ten were novel such as two homozygous missense variant in MYO15A c.[9518‐2A > G];p.[N/A], c.3944G > A;p.Gly1315Glu, one novel variant c.199T > G; p.Tyr67Asp in LHFPL5 in two unrelated individuals, one novel variant c.2519G > A; p.Trp840* in ESPN, one in KCNQ4 c.1288G > A; p.Glu430Lys, one in ESRRB c521G > A; p.Arg174His, one in DFNB59 c.147T > A; p.Tyr49*, one in GIPC3 c.759C > G; p.Ser253Arg, one in TMC1 c.662A > G; p.Tyr221Cys, and one novel variant in LRTOMT c.154C > T; p.Arg52Trp , Sorting Intolerant from Tolerant (SIFT) (http://sift.jcvi.org/), PROVEAN (http://www.provean.jcvi.org) Mutation Taster (http://www.mutationtaster.org/), Human Splicing Finder (HSF) (http://www.umd.be/HSF) and CADD (https://cadd.gs.washington.edu)  province, Pakistan. A clinical questionnaire was used to collect medical history and rule out the history of other diseases potentially affecting hearing and environmental factors such as antibiotic use, excessive noise exposure, and infection that could cause hearing loss. Detailed physical examination did not reveal other abnormal findings than hearing loss. Palpable goiters and other dysmorphic features were not observed in any of the participants with hearing loss; their hearing was ranging from severely impaired to deaf, listed in Figure http://www.ncbi.nlm.nih.gov/SNP/),Exome Variant Server (http://evs.gs.washington.edu/EVS/), GnomAD (https://gnomad.broadinstitute.org), Human gene mutation database , 1,000 Genomes (http://www.1000genomes.org/), and Exome aggregation consortium (ExAC) (http://www.exac.broadinstitute.org). Finally, for the interpretation of variants, the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines were used , 5  of 40 patients had known variants in SLC26A4 c.679G > C; p.Ala227Pro, 3 of 40 had known variants in MYO7A c.1258A > T; p.Lys420*, c.4838delA; p.Asp1613Valfs*32, 2 of 40 had known variants in GRXCR1 c.784C > T; p.Arg262*, 2 of 40 had known variants in TMPRSS3 c.727G > A; p.Gly243Arg, c.1219T > C; p.Cys407Arg, 2 of 40 had known variants in WFS1 c.2338G > A; p.Gly780Ser, c.2590G > A; p.Glu864Lys, 1 of 40 had known variant in ESPN c.2519G > A; p.Trp840*,1  of 40 had known variant in LRTMOT c.154C > T; p.Arg52Trp and were already known to be associated with syndromic or NSHL  had SLC26A4, and (3/40) had MYO7A variants responsible for NSHL. We found the incidence of GJB2 variants is higher compared to other populations. While this would argue for screening for GJB2 variants in the first place, we found a comparably high incidence for SLC26A4 and MYO7A variants together. Future research could specifically take advantage of using TNGS in the Pakistani cohort to capture a larger proportion of NSHL cases of genetic origin. We expect our study will enhance public awareness toward this hearing loss trouble and genetic counseling significance for the affected families and their members. This study expanded a spectrum of disease causing variants in genes involved in causing hearing loss.In our patient cohort we found 15/40 patients had The URLs for data presented herein are as follows:http://www.1000genomes.org/1,000 Genomes: http://evs.gs.washington.edu/EVS/Exome Variant Server: http://exac.broadinstitue.org/ExAC: http://www.ncbi.nlm.nih.gov/SNP/dbSNP: http://www.omim.org/OMIM: http://www.biobase-international.com/products/hgmdHGMD: http://sift.jcvi.org/SIFT: http://genetics.bwh.harvard.edu/pph2/Polyphen2: http://www.broadinstitute.org/igv/IGV: http://annovar.openbioinformatics.org/en/latest/ANNOVAR: http://www.mutationtaster.org/Mutation Taster: http://www.omim.org/OMIM: http://genome.ucsc.eduUCSC Genome Browser: The authors declare no conflicts of interest.