Multi-institutional Assessment of Otitis Media Epidemiology Using Real-world Data.

OBJECTIVES: To determine rates and risk factors of pediatric otitis media (OM) using real-world electronic health record (PEDSnet) data from January 2009 through May 2021. STUDY DESIGN: Retrospective cohort study. SETTING: Seven pediatric academic health systems that participate in PEDSnet. METHODS: Children <6 months-old at time of first outpatient, Emergency Department, or inpatient visit were included and followed longitudinally. A time-to-event analysis was performed using a Cox proportional hazards model to estimate hazard ratios for OM incidence based on sociodemographic factors and specific health conditions. RESULTS: The PEDSnet cohort included 910,265 children, 54.3% male, mean age (months) 1.3 [standard deviation (SD) 1.6] and mean follow up (years) 4.3 (SD 3.2). By age 3 years, 39.6% of children had evidence of one OM episode. OM rates decreased following pneumococcal-13 vaccination (PCV-13) and the COVID-19 pandemic. Along with young age, non-Hispanic Black/African American or Hispanic race/ethnicity, public insurance, higher family income, and male sex had higher incidence rates. Health conditions that increased OM risk included cleft palate [adjusted hazard ratio (aHR) 4.0 [95% confidence interval (CI) 3.9-4.2], primary ciliary dyskinesia [aHR 2.5 (95% CI 1.8-3.5)], trisomy 21 [aHR 2.0 (95% CI 1.9-2.1)], atopic dermatitis [aHR 1.4 (95% CI 1.4-1.4)], and gastroesophageal reflux [aHR1.5 (95% CI 1.5-1.5)]. CONCLUSIONS: Approximately 20% of children by age 1 and 40% of children by age 3 years will have experienced an OM episode. OM rates decreased after PCV-13 and COVID-19. Children with abnormal ciliary function or craniofacial conditions, specifically cleft palate, carry the highest risk of OM.

The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

Spectral Resolution and Speech Perception in Cochlear Implanted School-Aged Children.

OBJECTIVE: Cochlear implantation of prelingually deaf infants provides auditory input sufficient to develop spoken language; however, outcomes remain variable. Inability to participate in speech perception testing limits testing device efficacy in young listeners. In postlingually implanted adults (aCI), speech perception correlates with spectral resolution an ability that relies independently on frequency resolution (FR) and spectral modulation sensitivity (SMS). The correlation of spectral resolution to speech perception is unknown in prelingually implanted children (cCI). In this study, FR and SMS were measured using a spectral ripple discrimination (SRD) task and were correlated with vowel and consonant identification. It was hypothesized that prelingually deaf cCI would show immature SMS relative to postlingually deaf aCI and that FR would correlate with speech identification. STUDY DESIGN: Cross-sectional study. SETTING: In-person, booth testing. METHODS: SRD was used to determine the highest spectral ripple density perceived at various modulation depths. FR and SMS were derived from spectral modulation transfer functions. Vowel and consonant identification was measured; SRD performance and speech identification were analyzed for correlation. RESULTS: Fifteen prelingually implanted cCI and 13 postlingually implanted aCI were included. FR and SMS were similar between cCI and aCI. Better FR was associated with better speech identification for most measures. CONCLUSION: Prelingually implanted cCI demonstrated adult-like FR and SMS; additionally, FR correlated with speech identification. FR may be a measure of CI efficacy in young listeners.

Genome-wide screens connect HD82 loss-of-function to purine analog resistance in African trypanosomes.

Nucleoside analogs have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono- and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1-related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.IMPORTANCEThere is substantial interest in developing nucleoside analogs as anti-parasitic agents. We used genome-scale genetic screening and discovered two proteins linked to purine analog resistance in African trypanosomes. Our screens also identified two nucleoside kinases required for pro-drug activation, further validating the approach. The top novel hit, HD82, is related to SAMHD1, a mammalian nuclear viral restriction factor. We validated HD82 and localized the protein to the trypanosome nucleus. HD82 appears to sensitize trypanosomes to nucleoside analogs by reducing native pools of nucleotides, providing insights into both nucleoside/nucleotide metabolism and nucleoside analog resistance in trypanosomatids.

An allele-selective inter-chromosomal protein bridge supports monogenic antigen expression in the African trypanosome.

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.

Behavioral characterization of the cochlear amplifier lesion due to loss of function of stereocilin (STRC) in human subjects.

Loss of function of stereocilin (STRC) is the second most common cause of inherited hearing loss. The loss of the stereocilin protein, encoded by the STRC gene, induces the loss of connection between outer hair cells and tectorial membrane. This only affects the outer hair cells (OHCs) function, involving deficits of active cochlear frequency selectivity and amplifier functions despite preservation of normal inner hair cells. Better understanding of cochlear features associated with mutation of STRC will improve our knowledge of normal cochlear function, the pathophysiology of hearing impairment, and potentially enhance hearing aid and cochlear implant signal processing. Nine subjects with homozygous or compound heterozygous loss of function mutations in STRC were included, age 7-24 years. Temporal and spectral modulation perception were measured, characterized by spectral and temporal modulation transfer functions. Speech-in-noise perception was studied with spondee identification in adaptive steady-state noise and AzBio sentences with 0 and -5 dB SNR multitalker babble. Results were compared with normal hearing (NH) and cochlear implant (CI) listeners to place STRC-/- listeners' hearing capacity in context. Spectral ripple discrimination thresholds in the STRC-/- subjects were poorer than in NH listeners (p < 0.0001) but remained better than for CI listeners (p < 0.0001). Frequency resolution appeared impaired in the STRC-/- group compared to NH listeners but did not reach statistical significance (p = 0.06). Compared to NH listeners, amplitude modulation detection thresholds in the STRC-/- group did not reach significance (p=  0.06) but were better than in CI subjects (p < 0.0001). Temporal resolution in STRC-/- subjects was similar to NH (p = 0.98) but better than in CI listeners (p = 0.04). The spondee reception threshold in the STRC-/- group was worse than NH listeners (p = 0.0008) but better than CI listeners (p = 0.0001). For AzBio sentences, performance at 0 dB SNR was similar between the STRC-/- group and the NH group, 88 % and 97 % respectively. For -5 dB SNR, the STRC-/- performance was significantly poorer than NH, 40 % and 85 % respectively, yet much better than with CI who performed at 54 % at +5 dB SNR in children and 53 % at + 10 dB SNR in adults. To our knowledge, this is the first study of the psychoacoustic performance of human subjects lacking cochlear amplification but with normal inner hair cell function. Our data demonstrate preservation of temporal resolution and a trend to impaired frequency resolution in this group without reaching statistical significance. Speech-in-noise perception compared to NH listeners was impaired as well. All measures were better than those in CI listeners. It remains to be seen if hearing aid modifications, customized for the spectral deficits in STRC-/- listeners can improve speech understanding in noise. Since cochlear implants are also limited by deficient spectral selectivity, STRC-/- hearing may provide an upper bound on what could be obtained with better temporal coding in electrical stimulation.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.

Competition among variants is predictable and contributes to the antigenic variation dynamics of African trypanosomes.

Several persistent pathogens employ antigenic variation to continually evade mammalian host adaptive immune responses. African trypanosomes use variant surface glycoproteins (VSGs) for this purpose, transcribing one telomeric VSG expression-site at a time, and exploiting a reservoir of (sub)telomeric VSG templates to switch the active VSG. It has been known for over fifty years that new VSGs emerge in a predictable order in Trypanosoma brucei, and differential activation frequencies are now known to contribute to the hierarchy. Switching of approximately 0.01% of dividing cells to many new VSGs, in the absence of post-switching competition, suggests that VSGs are deployed in a highly profligate manner, however. Here, we report that switched trypanosomes do indeed compete, in a highly predictable manner that is dependent upon the activated VSG. We induced VSG gene recombination and switching in in vitro culture using CRISPR-Cas9 nuclease to target the active VSG. VSG dynamics, that were independent of host immune selection, were subsequently assessed using RNA-seq. Although trypanosomes activated VSGs from repressed expression-sites at relatively higher frequencies, the population of cells that activated minichromosomal VSGs subsequently displayed a competitive advantage and came to dominate. Furthermore, the advantage appeared to be more pronounced for longer VSGs. Differential growth of switched clones was also associated with wider differences, affecting transcripts involved in nucleolar function, translation, and energy metabolism. We conclude that antigenic variants compete, and that the population of cells that activates minichromosome derived VSGs displays a competitive advantage. Thus, competition among variants impacts antigenic variation dynamics in African trypanosomes and likely prolongs immune evasion with a limited set of antigens.

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei.

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.

Predictors of Time Spent in Special Education Among Students With Severe-to-Profound Hearing Loss.

OBJECTIVE: Determine if students with severe-to-profound hearing loss with cochlear implants (CIs) mainstream (transition to general education) more than students with hearing amplification at the population level. STUDY DESIGN: Cross-sectional secondary analysis of data from the National Center of Education Statistics. SETTING: Special education (SpEd) students in the United States who had severe to profound "hearing impairment" and were 6 to 16 years old at enrollment from 2000 to 2001. METHODS: We weighted the data to produce national estimates, performed multiple imputations for missingness, and built a multivariate linear regression model, which was cross-validated with a multivariate Poisson regression model. We used a theory-based approach to model-building using a directed acyclic graph to identify the minimally sufficient adjustment set of variables, which included school district urbanicity, student's age when they started SpEd, other disabilities, home language, and caregiver education. RESULTS: We identified 7267 students with CIs and 28,794 students with hearing amplification. CI users mainstreamed more than peers using hearing amplification during secondary school (40.29% less daily time in special education, p = .004) but not during primary school (9.19% less daily time in SpEd, p = .155). Additional significant predictors of mainstreaming varied between the primary and secondary school cohorts and included school district urbanicity and the student's age when they started SpEd. CONCLUSION: CI status predicts daily time spent in SpEd among a secondary school cohort. These findings do not establish causation. The National Center of Education Statistics should consider linking to clinical databases in future studies.

Association of Genetic Diagnoses for Childhood-Onset Hearing Loss With Cochlear Implant Outcomes.

Importance: In the US, most childhood-onset bilateral sensorineural hearing loss is genetic, with more than 120 genes and thousands of different alleles known. Primary treatments are hearing aids and cochlear implants. Genetic diagnosis can inform progression of hearing loss, indicate potential syndromic features, and suggest best timing for individualized treatment. Objective: To identify the genetic causes of childhood-onset hearing loss and characterize severity, progression, and cochlear implant success associated with genotype in a single large clinical cohort. Design, Setting, and Participants: This cross-sectional analysis (genomics) and retrospective cohort analysis (audiological measures) were conducted from 2019 to 2022 at the otolaryngology and audiology clinics of Seattle Children's Hospital and the University of Washington and included 449 children from 406 families with bilateral sensorineural hearing loss with an onset younger than 18 years. Data were analyzed between January and June 2022. Main Outcomes and Measures: Genetic diagnoses based on genomic sequencing and structural variant analysis of the DNA of participants; severity and progression of hearing loss as measured by audiologic testing; and cochlear implant success as measured by pediatric and adult speech perception tests. Hearing thresholds and speech perception scores were evaluated with respect to age at implant, months since implant, and genotype using a multivariate analysis of variance and covariance. Results: Of 406 participants, 208 (51%) were female, 17 (4%) were African/African American, 32 (8%) were East Asian, 219 (54%) were European, 53 (13%) were Latino/Admixed American, and 16 (4%) were South Asian. Genomic analysis yielded genetic diagnoses for 210 of 406 families (52%), including 55 of 82 multiplex families (67%) and 155 of 324 singleton families (48%). Rates of genetic diagnosis were similar for children of all ancestries. Causal variants occurred in 43 different genes, with each child (with 1 exception) having causative variant(s) in only 1 gene. Hearing loss severity, affected frequencies, and progression varied by gene and, for some genes, by genotype within gene. For children with causative mutations in MYO6, OTOA, SLC26A4, TMPRSS3, or severe loss-of-function variants in GJB2, hearing loss was progressive, with losses of more than 10 dB per decade. For all children with cochlear implants, outcomes of adult speech perception tests were greater than preimplanted levels. Yet the degree of success varied substantially by genotype. Adjusting for age at implant and interval since implant, speech perception was highest for children with hearing loss due to MITF or TMPRSS3. Conclusions and Relevance: The results of this cross-sectional study suggest that genetic diagnosis is now sufficiently advanced to enable its integration into precision medical care for childhood-onset hearing loss.

Spectrotemporal Modulation Discrimination in Infants With Normal Hearing.

OBJECTIVES: Spectral resolution correlates with speech understanding in post-lingually deafened adults with cochlear implants (CIs) and is proposed as a non-linguistic measure of device efficacy in implanted infants. However, spectral resolution develops gradually through adolescence regardless of hearing status. Spectral resolution relies on two different factors that mature at markedly different rates: Resolution of ripple peaks (frequency resolution) matures during infancy whereas sensitivity to across-spectrum intensity modulation (spectral modulation sensitivity) matures by age 12. Investigation of spectral resolution as a clinical measure for implanted infants requires understanding how each factor develops and constrains speech understanding with a CI. This study addresses the limitations of the present literature. First, the paucity of relevant data requires replication and generalization across measures of spectral resolution. Second, criticism that previously used measures of spectral resolution may reflect non-spectral cues needs to be addressed. Third, rigorous behavioral measurement of spectral resolution in individual infants is limited by attrition. To address these limitations, we measured discrimination of spectrally modulated, or rippled, sounds at two modulation depths in normal hearing (NH) infants and adults. Non-spectral cues were limited by constructing stimuli with spectral envelopes that change in phase across time. Pilot testing suggested that dynamic spectral envelope stimuli appeared to hold infants' attention and lengthen habituation time relative to previously used static ripple stimuli. A post-hoc condition was added to ensure that the stimulus noise carrier was not obscuring age differences in spectral resolution. The degree of improvement in discrimination at higher ripple depth represents spectral frequency resolution independent of the overall threshold. It was hypothesized that adults would have better thresholds than infants but both groups would show similar effects of modulation depth. DESIGN: Participants were 53 6- to 7-month-old infants and 23 adults with NH with no risk factors for hearing loss who passed bilateral otoacoustic emissions screening. Stimuli were created from complexes with 33- or 100-tones per octave, amplitude-modulated across frequency and time with constant 5 Hz envelope phase-drift and spectral ripple density from 1 to 20 ripples per octave (RPO). An observer-based, single-interval procedure measured the highest RPO (1 to 19) a listener could discriminate from a 20 RPO stimulus. Age-group and stimulus pure-tone complex were between-subjects variables whereas modulation depth (10 or 20 dB) was within-subjects. Linear-mixed model analysis was used to test for the significance of the main effects and interactions. RESULTS: All adults and 94% of infants provided ripple density thresholds at both modulation depths. The upper range of threshold approached 17 RPO with the 100-tones/octave carrier and 20 dB depth condition. As expected, mean threshold was significantly better with the 100-tones/octave compared with the 33-tones/octave complex, better in adults than in infants, and better at 20 dB than 10 dB modulation depth. None of the interactions reached significance, suggesting that the effect of modulation depth on the threshold was not different for infants or adults. CONCLUSIONS: Spectral ripple discrimination can be measured in infants with minimal listener attrition using dynamic ripple stimuli. Results are consistent with previous findings that spectral resolution is immature in infancy due to immature spectral modulation sensitivity rather than frequency resolution.

Sensitivity to Deaf Culture Among Otolaryngology and Audiology Trainees.

OBJECTIVE: The Deaf community is an ethnolinguistic minority group. Low sensitivity to Deaf culture contributes to health disparities among Deaf patients. This study determines the level of sensitivity to Deaf culture among otolaryngology-head and neck surgery (OHNS) and audiology trainees. METHODS: Cross-sectional survey study of OHNS and audiology trainees from 10 large US institutions. Trainees were queried on their exposure to and comfort with Deaf patients and their education on, attitude toward, and awareness and knowledge of Deaf culture. Sensitivity to Deaf culture was operationalized as awareness and knowledge of Deaf culture. These were assessed using a 35-item instrument that was previously developed using a d/Deaf community-based participatory approach to research. We used T-tests to compare the sample to previous samples of medical students with training in Deaf culture (MS-TDCs) and general practitioners (GPs). RESULTS: There were 91 completed surveys (response rate 44.5%). Almost all were aware of Deaf culture (97.8%). The mean knowledge score was 55.0% (standard deviation (SD) 13.4%), which was significantly higher than that for GPs at 43.0% (SD 15.0%) (95% confidence interval 8.1%, 15.8%, P < .0001) but significantly lower than that for MS-TDCs at 69.0% (SD 13.0%)(CI -20.3%, -7.6%, P < .0001). Knowledge scores were comparable for OHNS and audiology trainees (P = .09). CONCLUSION: This sample of OHNS and audiology trainees was more sensitive to Deaf culture than GPs but less sensitive than MS-TDCs. Developing specialty-specific education may be warranted. LEVEL OF EVIDENCE: 4.

Anti-trypanosomatid drug discovery: progress and challenges.

Leishmaniasis (visceral and cutaneous), Chagas disease and human African trypanosomiasis cause substantial death and morbidity, particularly in low- and middle-income countries. Although the situation has improved for human African trypanosomiasis, there remains an urgent need for new medicines to treat leishmaniasis and Chagas disease; the clinical development pipeline is particularly sparse for Chagas disease. In this Review, we describe recent advances in our understanding of the biology of the causative pathogens, particularly from the drug discovery perspective, and we explore the progress that has been made in the development of new drug candidates and the identification of promising molecular targets. We also explore the challenges in developing new clinical candidates and discuss potential solutions to overcome such hurdles.

Beyond Laryngeal Clefts: Interarytenoid Injection Augmentation to Predict Success of Suture Augmentation in Children.

OBJECTIVE: To assess the efficacy of interarytenoid injection augmentation (IAIA) and the ability of IAIA to predict response to interarytenoid suture augmentation (IASA) based on diet advancement on video fluoroscopic swallow studies (VFSS). METHODS: Retrospective cohort analysis of patients with persistent pharyngeal dysphagia at a tertiary children's hospital with VFSS pre- and post-IAIA were included between March 2011 and June 2019. RESULTS: Median age of the 229 patients was 2.2 years (5.8 months-19 years). Interarytenoid mucosal height (IAMH) was found to be above the false vocal folds in 112 patients (53.4%) and at true vocal folds in 10 (4.9%) patients. On VFSS post-IAIA, 95 (41.5%) patients were successfully advanced in recommended diet consistency, 115 (50.2%) were stable, and 19 (8.3%) needed thicker consistency. Paired t-tests on pre- and post-operative consistency scores showed significant improvement, p-value of <0.0001, 95% confidence interval (CI; 0.50-0.85). Poisson regression found no covariates with significant association with improvement on IAIA. For IASA patients, 35/60 (58.3%) improved on post-op VFSS. Paired t-tests on pre- and post-operative consistency scores showed significant improvement, p-value of <0.0001, 95% CI (0.63-1.33). Positive predictive value for IAIA predicting response to IASA was 77% with positive likelihood ratio of 2.3. The response to IAIA versus no response to IAIA likelihood ratios were found to have a statistically significant difference (p < 0.05). CONCLUSIONS: Our study suggests IAIA yields objective improvement in swallow function on VFSS in nearly half of our patients and may be a reliable diagnostic tool to predict response to IASA in patients with persistent pharyngeal dysphagia with or without a laryngeal cleft. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1749-1756, 2023.

Genome-scale RNA interference profiling of Trypanosoma brucei cell cycle progression defects.

Trypanosomatids, which include major pathogens of humans and livestock, are flagellated protozoa for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle defects in Trypanosoma brucei. We induced massive parallel knockdown, sorted the perturbed population using high-throughput flow cytometry, deep-sequenced RNAi-targets from each stage and digitally reconstructed cell cycle profiles at a genomic scale; also enabling data visualisation using an online tool ( https://tryp-cycle.pages.dev/ ). Analysis of several hundred genes that impact cell cycle progression reveals >100 flagellar component knockdowns linked to genome endoreduplication, evidence for metabolic control of the G1-S transition, surface antigen regulatory mRNA-binding protein knockdowns linked to G2M accumulation, and a putative nucleoredoxin required for both mitochondrial genome segregation and for mitosis. The outputs provide comprehensive functional genomic evidence for the known and novel machineries, pathways and regulators that coordinate trypanosome cell cycle progression.

Charge-based interactions through peptide position 4 drive diversity of antigen presentation by human leukocyte antigen class I molecules.

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.