Chronic suppurative otitis media (CSOM) is one of the most common infectious diseases of the middle ear especially affecting children, leading to delay in language development and communication. ...Although Staphylococcus aureus is the most common pathogen associated with CSOM, its interaction with middle ear epithelial cells is not well known. In the present study, we observed that otopathogenic S. aureus has the ability to invade human middle ear epithelial cells (HMEECs) in a dose and time dependent manner. Scanning electron microscopy demonstrated time dependent increase in the number of S. aureus on the surface of HMEECs. We observed that otopathogenic S. aureus primarily employs a cholesterol dependent pathway to colonize HMEECs. In agreement with these findings, confocal microscopy showed that S. aureus colocalized with lipid rafts in HMEECs. The results of the present study provide new insights into the pathogenesis of S. aureus induced CSOM. The availability of in vitro cell culture model will pave the way to develop novel effective treatment modalities for CSOM beyond antibiotic therapy.
gene encodes unconventional myosin VIIA, which, when mutated, causes a phenotypic spectrum ranging from recessive hearing loss DFNB2 to deaf-blindness, Usher Type 1B (USH1B).
mutations are reported ...in nine DFNB2 families to date, none from sub-Saharan Africa.In DNA, from a cohort of 94 individuals representing 92 families from the Limpopo province of South Africa, eight
variations were detected among 10 individuals. Family studies identified homozygous and compound heterozygous mutations in 17 individuals out of 32 available family members. Four mutations were novel, p.Gly329Asp, p.Arg373His, p.Tyr1780Ser, and p.Pro2126Leufs*5. Two variations, p.Ser617Pro and p.Thr381Met, previously listed as of uncertain significance (ClinVar), were confirmed to be pathogenic. The identified mutations are predicted to interfere with the conformational properties of myosin VIIA through interruption or abrogation of multiple interactions between the mutant and neighbouring residues. Specifically, p.Pro2126Leufs*5, is predicted to abolish the critical site for the interactions between the tail and the motor domain essential for the autoregulation, leaving a non-functional, unregulated protein that causes hearing loss. We have identified
as a possible key deafness gene among indigenous sub-Saharan Africans. The spectrum of
mutations in this South African population points to DFNB2 as a specific entity that may occur in a homozygous or in a compound heterozygous state.
Abstract
Background
Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral ...vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas.
Methods
Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns.
Results
Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance.
Conclusion
MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.
Background. Waardenburg syndrome (WS) is one of the most common forms of syndromic deafness with heterogeneity of loci and alleles and variable expressivity of clinical features. Methods. The ...technology of single-nucleotide variants (SNV) and copy number variation (CNV) detection was developed to investigate the genotype spectrum of WS in a Chinese population. Results. Ninety WS patients and 24 additional family members were recruited for the study. Fourteen mutations had not been previously reported, including c.808C>G, c.117C>A, c.152T>G, c.803G>T, c.793-3T >G, and c.801delT on PAX3; c.642_650delAAG on MITF; c.122G>T and c.127C>T on SOX10; c.230C>G and c.365C>T on SNAI2; and c.481A>G, c.1018C>G, and c.1015C>T on EDNRB. Three CNVs were de novo and first reported in our study. Five EDNRB variants were associated with WS type 1 in the heterozygous state for the first time, with a detection rate of 22.2%. Freckles occur only in WS type 2. Yellow hair, amblyopia, congenital ptosis, narrow palpebral fissures, and pigmentation spots are rare and unique symptoms in WS patients from China. Conclusions. EDNRB should be considered as another prevalent pathogenic gene in WS type 1. Our study expanded the genotype and phenotype spectrum of WS, and diagnostic next-generation sequencing is promising for WS.
A comprehensive understanding about the pathogenesis of otitis media (OM), one of the most common pediatric diseases, has the potential to alleviate a substantial disease burden across the globe. ...Advancements in genetic and bioinformatic detection methods, as well as a growing interest in the microbiome, has enhanced the capability of researchers to investigate the interplay between host genes, host microbiome, invading bacteria, and resulting OM susceptibility. Early studies deciphering the role of genetics in OM susceptibility assessed the heritability of the phenotype in twin and triplet studies, followed by linkage studies, candidate gene approaches, and genome-wide association studies that have helped in the identification of specific loci. With the advancements in techniques, various chromosomal regions and genes such as
,
,
,
, and others have been implicated in predisposition to OM, yet questions still remain as to whether these implicated genes truly play a causative role in OM and to what extent. Meanwhile, 16S ribosomal RNA (rRNA) sequencing, microbial quantitative trait loci (mbQTL), and microbial genome-wide association studies (mGWAS) have mapped the microbiome of upper airways sites and therefore helped in enabling a more detailed study of interactions between host polymorphisms and host microbiome composition. Variants of specific genes conferring increased OM susceptibility, such as
, have also been shown to influence the microbial composition of the outer and middle ear in patients with OM, suggesting their role as mediators of disease. These interactions appear to impact the colonization of known otopathogens (
,
, and
), as well as
,
,
,
, and
populations that have also been implicated in OM pathogenesis. Meanwhile, studies demonstrating an increased abundance of
and
in healthy patients compared to those with OM suggest a protective role for these bacteria, thereby introducing potential avenues for future probiotic treatment. Incorporating insights from these genetic, microbiome, and host-pathogen studies will allow for a more robust, comprehensive understanding of OM pathogenesis that can ultimately facilitate in the development of exciting new treatment modalities.
The success of the Fontan procedure for congenital single ventricle anatomy has resulted in adult patients with Fontan physiology requiring anaesthesia for cardiac and non-cardiac procedures. We ...present the perioperative management of a patient with Fontan physiology who underwent electrophysiological study with radiofrequency ablation for atrial tachycardia under general anaesthesia. Good communication between the multidisciplinary teams, a detailed understanding of the patient’s complex cardiac anatomy and physiology, as well as the ability to recognise and manage perioperative complications all play a vital role for a successful outcome.
The unparalleled heterogeneity in genetic causes of hearing loss along with remarkable differences in prevalence of causative variants among ethnic groups makes single gene tests technically ...inefficient. Although hundreds of genes have been reported to be associated with nonsyndromic hearing loss (NSHL), GJB2, GJB6, SLC26A4, and mitochondrial (mt) MT-RNR1 and MTTS are the major contributors. In order to provide a faster, more comprehensive and cost effective assay, we constructed a DNA fluidic array, CapitalBioMiamiOtoArray, for the detection of sequence variants in five genes that are common in most populations of European descent. They consist of c.35delG, p.W44C, p.L90P, c.167delT (GJB2); 309kb deletion (GJB6); p.L236P, p.T416P (SLC26A4); and m.1555A>G, m.7444G>A (mtDNA). We have validated our hearing loss array by analyzing a total of 160 DNAs samples. Our results show 100% concordance between the fluidic array biochip-based approach and the established Sanger sequencing method, thus proving its robustness and reliability at a relatively low cost.
Spontaneous haemothorax complicating the treatment of pulmonary embolism is rare and potentially fatal. We describe a patient with pulmonary embolism and severe pleuritic pain who developed a ...life-threatening haemothorax 10 days later while on rivaroxaban therapy. This case highlights the fact that severe pleuritic pain associated with pulmonary embolism may indicate subclinical infarction of tissue near the visceral pleura with an increased risk of pleural effusion and the subsequent development of a haemothorax. It is important to recognise such danger signs warranting closer attention, especially since the increased use of direct oral anticoagulants has facilitated ambulatory care and this complication may manifest in the outpatient setting.
Spontaneous haemothorax may occur in the first 2 weeks after a patient starts anticoagulation.Severe pleuritic pain in a patient with pulmonary embolism may indicate subclinical infarction near the visceral pleura with an increased risk of pleural effusion and the subsequent development of a spontaneous haemothorax.Patients with severe pain and pleural effusion should be monitored closely, especially if they are outpatients, even though initial radiological findings are not significant.
The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely ...heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found
Slc22a4
transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.
The COVID-19 pandemic is an unprecedented crisis that has taken the world by storm, and it has taken an especially immense toll on the healthcare sector. Although much effort has been made to make ...changes in key areas such as clinical practice, national policy and research, we believe that it is just as important to evaluate the impact of the crisis on postgraduate medical training. To this end, we would like to share our experience within an anaesthesiology residency programme in Singapore, the SingHealth Anaesthesiology Residency Programme, which we hope will benefit other training programmes and anyone involved in postgraduate medical education as a whole. Key challenges identified include restrictions on teaching events, difficulties in completing core posting requirements, changes in clinical workload, postponement of examinations, exposure risk (particularly in relation to aerosol-generating procedures) and psychological burden. Strategies that have been implemented to tackle these challenges are also described, including the use of online platforms, modifications to posting and promotion requirements, manpower adjustments, provision of protective equipment and training in infectious disease protocols. Ultimately, we believe that a supportive work environment is essential to ensure the wellbeing of residents in times of crisis.
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