Sinorhizobium meliloti BR-bodies promote fitness during host colonization.

Biomolecular condensates, such as the nucleoli or P-bodies, are non-membrane-bound assemblies of proteins and nucleic acids that facilitate specific cellular processes. Like eukaryotic P-bodies, the recently discovered bacterial ribonucleoprotein bodies (BR-bodies) organize the mRNA decay machinery, yet the similarities in molecular and cellular functions across species have been poorly explored. Here, we examine the functions of BR-bodies in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, which colonizes the roots of compatible legume plants. Assembly of BR-bodies into visible foci in S. meliloti cells requires the C-terminal intrinsically disordered region (IDR) of RNase E, and foci fusion is readily observed in vivo, suggesting they are liquid-like condensates that form via mRNA sequestration. Using Rif-seq to measure mRNA lifetimes, we found a global slowdown in mRNA decay in a mutant deficient in BR-bodies, indicating that compartmentalization of the degradation machinery promotes efficient mRNA turnover. While BR-bodies are constitutively present during exponential growth, the abundance of BR-bodies increases upon cell stress, whereby they promote stress resistance. Finally, using Medicago truncatula as host, we show that BR-bodies enhance competitiveness during colonization and appear to be required for effective symbiosis, as mutants without BR-bodies failed to stimulate plant growth. These results suggest that BR-bodies provide a fitness advantage for bacteria during infection, perhaps by enabling better resistance against the host immune response.

Sound Exposure Promotes Intratympanic Drug Delivery to the Inner Ear.

OBJECTIVE: To investigate the impact of sound exposure, with the resultant windows vibration on perilymphatic concentrations following intratympanic (IT) dexamethasone and gentamicin in an animal model. STUDY DESIGN: Animal model blinded study. SETTING: Animal facility of a tertiary medical center. METHODS: Bilateral IT dexamethasone or gentamicin was applied to 15 tested rats. Following injections, each rat was exposed for 3 minutes to free field 30 dB sound pressure level (SPL), 512 vHz noise, with 1 external auditory canal plugged (contralateral control). Following noise exposure, perilymph was obtained from both ears. Drug concentrations were measured using ultrahigh-performance liquid chromatography-mass spectrometer. RESULTS: For dexamethasone, the average (±SD) perilymphatic steroidal concentration was 0.417 µg/mL (±0.549) in the control ears versus 0.487 µg/mL (±0.636) in the sound-exposed ears (P = .008). The average (±SD) gentamicin perilymphatic concentration was 8.628 µg/mL (±2.549) in the sound-exposed ears, compared to 4.930 µg/mL (±0.668) in the contralateral control (nonsound exposed) ears. Sound exposure promoted steroidal and gentamicin diffusion to the inner ear by an averaged (±SD) factor of 1.431 and 1.730 (±0.291 and 0.339), respectively. CONCLUSION: Low-intensity noise (30 dB SPL) was found to enhance dexamethasone phosphate and gentamicin diffusion to the inner ear (by an averaged factor of ∼1.4 and 1.7, respectively) in a murine model.

Spatially Resolved Microglia/Macrophages in Recurrent Glioblastomas Overexpress Fatty Acid Metabolism and Phagocytic Genes.

BACKGROUND: Glioblastoma (GBM) tumors are rich in tumor-associated microglia/macrophages. Changes associated with treatment in this specific cell population are poorly understood. Therefore, we studied changes in gene expression of tumor-associated microglia/macrophages (Iba1+) cells in de novo versus recurrent GBMs. METHODS: NanoString GeoMx® Digital Spatial Transcriptomic Profiling of microglia/macrophages (Iba1+) and glial cells (Gfap+) cells identified on tumor sections was performed on paired de novo and recurrent samples obtained from three IDH-wildtype GBM patients. The impact of differentially expressed genes on patient survival was evaluated using publicly available data. RESULTS: Unsupervised analyses of the NanoString GeoMx® Digital Spatial Profiling data revealed clustering based on the transcriptomic data from Iba1+ and Gfap+ cells. As expected, conventional differential gene expression and enrichment analyses revealed upregulation of immune-function-related genes in Iba1+ cells compared to Gfap+ cells. A focused differential gene expression analysis revealed upregulation of phagocytosis and fatty acid/lipid metabolism genes in Iba1+ cells in recurrent GBM samples compared to de novo GBM samples. Importantly, of these genes, the lipid metabolism gene PLD3 consistently correlated with survival in multiple different publicly available datasets. CONCLUSION: Tumor-associated microglia/macrophages in recurrent GBM overexpress genes involved in fatty acid/lipid metabolism. Further investigation is needed to fully delineate the role of PLD phospholipases in GBM progression.

Balloon dilation of the eustachian tube using endovascular balloon under local anesthesia-a case series and systematic literature review.

Objective: To report a novel technique in Balloon Dilation of Eustachian Tube (BDET) using an endovascular balloon (EVB), in a prospective cohort. The results are compared with reported outcomes using standard balloons. Methods: Demographic information and clinical parameters were collected prospectively fora series of patients with obstructive eustachian tube dysfunction (OETD). Balloon dilation Eustachian tuboplasty was performed under local anesthesia in a tertiary referral center, using the EVB. Systematic literature review was used for comparison, using Medline via "PubMed", "Embase", and "Web of Science". Results: Eight OETD candidates (12 ears) were enrolled; 5 males and 3 females. Average age was 48 (range -23 to 63) years. The most common presenting symptom was aural fullness (9/12), followed by ear pressure (7/12), hearing loss (5/12) and tinnitus (4/12). Otoscopically, tympanic membrane retraction was evident in 10/12 ears, the majority of which was class II-Sade classification. Pre-operative tympanogram was type B and C in 7 and 5 ears, respectively. All BDETs were performed without complications. Post-operative tympanometry was A in 8/12 ears. Post-operatively, Eustachian Tube Dysfunction Questionnaire-7 results reduced to within normal limits (average score ≤3) in 11/12 ears (p = 0.0014). The systematic literature review included 6 papers (193 patients, 262 ETs) with comparable results, most also with little adverse effects. Conclusion: BDET using an EVB is a safe and effective option for OETD. It is well tolerated under local anesthesia in properly selected individuals. The reduced procedural cost may be an important factor in certain healthcare jurisdictions.

Guided Online Mindfulness-Based Intervention for Community Residents with Emotional Distress during the COVID-19 Pandemic.

This study aimed to assess the efficacy of a guided online mindfulness-based intervention (iMBI) for community residents experiencing emotional distress during the COVID-19 pandemic in Hong Kong. In a randomized controlled trial, 64 participants were recruited from collaborating community-based mental health service units in Hong Kong and assigned to either the treatment (n = 32) or control (n = 32) groups. The treatment group received a guided iMBI consisting of 16 online modules, weekly telephone counseling, and two half-day online workshops on mindfulness practice. In contrast, the waitlist control group did not receive any intervention during the initial stage. Using a 2 (two groups) × time (pre versus post) repeated measures linear mixed model and one-way analysis of variance, authors demonstrated that the treatment group experienced a significantly larger reduction in anxiety and depressive symptoms with a large effect size compared with the control group. Additionally, the treatment group showed a significantly greater improvement in mindfulness with a moderate effect size. The findings support the effectiveness of guided iMBI for community residents experiencing emotional distress during the COVID-19 pandemic in Hong Kong.

Homeodomain-only protein suppresses proliferation and contributes to differentiation- and age-related reduced CD8+ T cell expansion.

T cell activation is a tightly controlled process involving both positive and negative regulators. The precise mechanisms governing the negative regulators in T cell proliferation remain incompletely understood. Here, we report that homeodomain-only protein (HOPX), a homeodomain-containing protein, and its most abundant isoform HOPXb, negatively regulate activation-induced proliferation of human T cells. We found that HOPX expression progressively increased from naïve (TN) to central memory (TCM) to effector memory (TEM) cells, with a notable upregulation following in vitro stimulation. Overexpression of HOPXb leads to a reduction in TN cell proliferation while HOPX knockdown promotes proliferation of TN and TEM cells. Furthermore, we demonstrated that HOPX binds to promoters and exerts repressive effects on the expression of MYC and NR4A1, two positive regulators known to promote T cell proliferation. Importantly, our findings suggest aging is associated with increased HOPX expression, and that knockdown of HOPX enhances the proliferation of CD8+ T cells in older adults. Our findings provide compelling evidence that HOPX serves as a negative regulator of T cell activation and plays a pivotal role in T cell differentiation and in age-related-reduction in T cell proliferation.

A multitask approach for automated detection and segmentation of thyroid nodules in ultrasound images.

An increase in the incidence and diagnosis of thyroid nodules and thyroid cancer underscores the need for a better approach to nodule detection and risk stratification in ultrasound (US) images that can reduce healthcare costs, patient discomfort, and unnecessary invasive procedures. However, variability in ultrasound technique and interpretation makes the diagnostic process partially subjective. Therefore, an automated approach that detects and segments nodules could improve performance on downstream tasks, such as risk stratification. Ultrasound studies were acquired from 280 patients at UCLA Health, totaling 9888 images, and annotated by collaborating radiologists. Current deep learning architectures for segmentation are typically semi-automated because they are evaluated solely on images known to have nodules and do not assess ability to identify suspicious images. However, the proposed multitask approach both detects suspicious images and segments potential nodules; this allows for a clinically translatable model that aptly parallels the workflow for thyroid nodule assessment. The multitask approach is centered on an anomaly detection (AD) module that can be integrated with any UNet architecture variant to improve image-level nodule detection. Of the evaluated multitask models, a UNet with a ImageNet pretrained encoder and AD achieved the highest F1 score of 0.839 and image-wide Dice similarity coefficient of 0.808 on the hold-out test set. Furthermore, models were evaluated on two external validations datasets to demonstrate generalizability and robustness to data variability. Ultimately, the proposed architecture is an automated multitask method that expands on previous methods by successfully both detecting and segmenting nodules in ultrasound.

Biophysical Control of the Glioblastoma Immunosuppressive Microenvironment: Opportunities for Immunotherapy.

GBM is the most aggressive and common form of primary brain cancer with a dismal prognosis. Current GBM treatments have not improved patient survival, due to the propensity for tumor cell adaptation and immune evasion, leading to a persistent progression of the disease. In recent years, the tumor microenvironment (TME) has been identified as a critical regulator of these pro-tumorigenic changes, providing a complex array of biomolecular and biophysical signals that facilitate evasion strategies by modulating tumor cells, stromal cells, and immune populations. Efforts to unravel these complex TME interactions are necessary to improve GBM therapy. Immunotherapy is a promising treatment strategy that utilizes a patient's own immune system for tumor eradication and has exhibited exciting results in many cancer types; however, the highly immunosuppressive interactions between the immune cell populations and the GBM TME continue to present challenges. In order to elucidate these interactions, novel bioengineering models are being employed to decipher the mechanisms of immunologically "cold" GBMs. Additionally, these data are being leveraged to develop cell engineering strategies to bolster immunotherapy efficacy. This review presents an in-depth analysis of the biophysical interactions of the GBM TME and immune cell populations as well as the systems used to elucidate the underlying immunosuppressive mechanisms for improving current therapies.

MicroRNA-29a: a novel target for non-operative management of symptomatic lumbar spinal stenosis.

PURPOSE: Lumbar spinal stenosis (LSS) is the most common reason for spinal surgery in patients over the age of 65, and there are few effective non-surgical treatments. Therefore, the development of novel treatment or preventative modalities to decrease overall cost and morbidity associated with LSS is an urgent matter. The cause of LSS is multifactorial; however, a significant contributor is ligamentum flavum hypertrophy (LFH) which causes mechanical compression of the cauda equina or nerve roots. We assessed the role of a novel target, microRNA-29a (miR-29a), in LFH and investigated the potential for using miR-29a as a therapeutic means to combat LSS. METHODS: Ligamentum flavum (LF) tissue was collected from patients undergoing decompressive surgery for LSS and assessed for levels of miR-29a and pro-fibrotic protein expression. LF cell cultures were then transfected with either miR-29a over-expressor (agonist) or inhibitor (antagonist). The effects of over-expression and under-expression of miR-29a on expression of pro-fibrotic proteins was assessed. RESULTS: We demonstrated that LF at stenotic levels had a loss of miR-29a expression. This was associated with greater LF tissue thickness and higher mRNA levels of collagen I and III. We also demonstrated that miR29-a plays a direct role in the regulation of collagen gene expression in ligamentum flavum. Specifically, agents that increase miR-29a may attenuate LFH, while those that decrease miR-29a promote fibrosis and LFH. CONCLUSION: This study demonstrates that miR-29a may potentially be used to treat LFH and provides groundwork to initiate the development of a therapeutic product for LSS.

Evaluation of the Effect of Lorlatinib on CYP2B6, CYP2C9, UGT, and P-Glycoprotein Substrates in Patients with Advanced Non-Small Cell Lung Cancer.

BACKGROUND AND OBJECTIVE: Lorlatinib is a tyrosine kinase inhibitor approved for the treatment of advanced anaplastic lymphoma kinase-positive non-small cell lung cancer. This study assessed the effect of steady-state lorlatinib on the metabolic enzymes cytochrome P450 (CYP) 2B6, CYP2C9, and uridine 5'-diphospho-glucuronosyltransferase (UGT) and the P-glycoprotein (P-gp) transporter. METHODS: Thirty-two patients received a single oral dose of a probe drug on Day - 2 to determine the pharmacokinetics of the probe drug alone. Starting on Day 1, patients received 100 mg oral lorlatinib daily. On Day 15, a single oral dose of the probe drug was administered concurrently with lorlatinib. Pharmacokinetic parameters for these probe substrates were assessed. RESULTS: Plasma exposures of all probe substrates were reduced by lorlatinib compared with the probe alone. The greatest reduction in area under the plasma concentration-time curve from time zero to infinity (AUC∞) and maximum (peak) plasma drug concentration (Cmax) (67% and 63% decrease, respectively) was observed with the P-gp probe substrate fexofenadine. Lorlatinib coadministration also decreased the AUC∞ and Cmax of bupropion (CYP2B6 probe substrate) by 25% and 27%, tolbutamide (CYP2C9 probe substrate) by 43% and 15%, and acetaminophen (UGT probe substrate) by 45% and 28%, respectively. CONCLUSIONS: Lorlatinib is a net moderate inducer of P-gp and a weak inducer of CYP2B6, CYP2C9, and UGT after steady state is achieved with daily dosing. Medications that are P-gp substrates with a narrow therapeutic window should be avoided in patients taking lorlatinib; no dose modifications are needed with substrates of CYP2B6, CYP2C9, or UGT. CLINICALTRIALS: gov: NCT01970865.

Virtual Reality Simulation for the Middle Cranial Fossa Approach: A Validation Study.

BACKGROUND AND OBJECTIVES: Virtual reality (VR) surgical rehearsal is an educational tool that exists in a safe environment. Validation is necessary to establish the educational value of this platform. The middle cranial fossa (MCF) is ideal for simulation because trainees have limited exposure to this approach and it has considerable complication risk. Our objectives were to assess the face, content, and construct validities of an MCF VR simulation, as well as the change in performance across serial simulations. METHODS: Using high-resolution volumetric data sets of human cadavers, the authors generated a high-fidelity visual and haptic rendering of the MCF approach using CardinalSim software. Trainees from Neurosurgery and Otolaryngology-Head and Neck Surgery at two Canadian academic centers performed MCF dissections on this VR platform. Randomization was used to assess the effect of enhanced VR interaction. Likert scales were used to assess the face and content validities. Performance metrics and pre- and postsimulation test scores were evaluated. Construct validity was evaluated by examining the effect of the training level on simulation performance. RESULTS: Twenty trainees were enrolled. Face and content validities were achieved in all domains. Construct validity, however, was not demonstrated. Postsimulation test scores were significantly higher than presimulation test scores ( P < .001 ). Trainees demonstrated statistically significant improvement in the time to complete dissections ( P < .001 ), internal auditory canal skeletonization ( P < .001 ), completeness of the anterior petrosectomy ( P < .001 ), and reduced number of injuries to critical structures ( P = .001 ). CONCLUSION: This MCF VR simulation created using CardinalSim demonstrated face and content validities. Construct validity was not established because no trainee included in the study had previous MCF approach experience, which further emphasizes the importance of simulation. When used as a formative educational adjunct in both Neurosurgery and Otolaryngology-Head and Neck Surgery, this simulation has the potential to enhance understanding of the complex anatomic relationships of critical neurovascular structures.

The Effect of Timing Between Traumatic Flexor Tendon Injury of the Hand and Surgical Intervention on Repair Failure Rates.

Flexor tendon injuries of the hand, especially in zone II, pose a challenge for hand surgeons because of the region's intricate pulley mechanism and local avascularity, and post-surgical complications such as repair failure are not uncommon. One proposed predictor of outcomes following flexor tendon repair has been timing of surgery from initial injury. However, the effect of the timing of flexor tendon repair on failure rates remains controversial and understudied. The purpose of this study was to compare the failure rates of zone II flexor tendon repairs in patients at various time intervals from onset of injury. A retrospective chart review was conducted using data from hand surgery specialists at our level 1 trauma center from January 1, 2010, through May 31, 2020. This retrospective review included 407 zone II flexor tendon repairs. The primary outcome was failure of repair. Among 407 flexor tendon repairs, there were 12 reported repair failures. The failure rate was 2.9%. In the non-failure group, the mean number of days between the date of injury and the date of surgery was 7±13 days. For the failure group, this value was 14±17 days. Repairs occurring within 14 days had a failure rate of 2.3%, while repairs occurring beyond 14 days had a failure rate of 7.7%. This study demonstrates that there is a benefit to repairing the tendon within a 14-day window, as evidenced by a lower failure rate. More research is required to determine if other complications and overall health of the hand are also improved when a repair is performed in a more expedient manner. [Orthopedics. 2024;47(2):113-117.].

A Virtual Inner Ear Model Selects Ramped Pulse Shapes for Vestibular Afferent Stimulation.

Bilateral vestibular deficiency (BVD) results in chronic dizziness, blurry vision when moving the head, and postural instability. Vestibular prostheses (VPs) show promise as a treatment, but the VP-restored vestibulo-ocular reflex (VOR) gain in human trials falls short of expectations. We hypothesize that the slope of the rising ramp in stimulation pulses plays an important role in the recruitment of vestibular afferent units. To test this hypothesis, we utilized customized programming to generate ramped pulses with different slopes, testing their efficacy in inducing electrically evoked compound action potentials (eCAPs) and current spread via bench tests and simulations in a virtual inner model created in this study. The results confirmed that the slope of the ramping pulses influenced the recruitment of vestibular afferent units. Subsequently, an optimized stimulation pulse train was identified using model simulations, exhibiting improved modulation of vestibular afferent activity. This optimized slope not only reduced the excitation spread within the semicircular canals (SCCs) but also expanded the neural dynamic range. While the model simulations exhibited promising results, in vitro and in vivo experiments are warranted to validate the findings of this study in future investigations.

How standardised are antibiotic regimens in otologic surgery?

BACKGROUND: Within otologic surgery, a paucity of well-controlled studies assessing the use of systemic antibiotic to reduce surgical site infections exists. Moreover, discrepancies in wound classification of procedures challenge consensus in antimicrobial prescribing patterns. We sought to compare surgeons from two different health systems to examine how surgeons' prescribing habits compared to practice guidelines for numerous otologic procedures. METHODS: An online questionnaire was distributed to 33 Canadian and 32 Austrian surgeons who regularly perform otologic surgery. Current systemic antibiotic prescribing habits for cochlear implantation, cholesteatoma surgery, stapes surgery, and tympanoplasty ± ossiculoplasty were collected. RESULTS: Eighteen of 33 (54.5%) Canadian surgeons provided responses, while 18 of 32 (56.3%) of Austrian surgeons answered. Clear consistency with clinical practice guidelines exists for pre-operative antibiotics use in cochlear implant surgery and infected cholesteatoma surgery. However, for stapes surgery and tympanoplasty ± ossiculoplasty, consensus is lacking for both pre- and post-operative antibiotic prescribing habits. Notable differences between the two countries include post-operative antibiotics for cochlear implant surgery (Austria: 36.4%, Canada: 71.4%) and uninfected cholesteatoma surgery (Austria: 33.3%, Canada: 77.8%). Across all procedures, both induction and post-operative antibiotic administration was not significantly associated with surgeon seniority when stratified by five-year increments. CONCLUSION: The lack of consensus among each country's otologic surgeons underscores the uncertainty in wound classification and thus, adherence to clinical practice guidelines.

Low-intensity online mindfulness-based intervention for university students with anxiety during the COVID-19 pandemic-A randomized controlled trial with 3-month follow-up.

Objective: This study investigated the effectiveness of a low-intensity online mindfulness-based Intervention (iMBI) for alleviating anxiety in university students during the COVID-19 pandemic. Methods: In a randomized controlled trial involving 134 participants from a local university in Hong Kong, subjects were randomly assigned to either the intervention group (n = 67) or the inactive control group (n = 67). The intervention group participated in a low-intensity iMBI comprising 16 online modules and two half-day online mindfulness workshops over an eight-week period. Outcomes were measured via an online platform using standardized assessment scales, including the Beck Anxiety Inventory and the Chinese Five Facets Mindfulness Questionnaire, at three different time points: pre-intervention, post-intervention and at a three-month follow-up. Results: Intent-to-treat analysis using 2 (group) × 3 (time) repeated measures of covariance (ANCOVA) showed that the intervention group, compared to the control group, showed a significant reduction in anxiety symptoms with a medium effect size (Cohen's d = 0.5) and a significant improvement in mindfulness skills with a medium effect size (Cohen's d = 0.5) at post-intervention. The effects of the intervention in reducing anxiety and improving mindfulness persisted at the three-month follow-up. Conclusion: The results of this study demonstrate the effectiveness of the low-intensity iMBI in alleviating anxiety among university students.

A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

Ancestral, Delta, and Omicron (BA.1) SARS-CoV-2 strains are dependent on serine proteases for entry throughout the human respiratory tract.

BACKGROUND: The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and became the globally dominant variant by January 2022. Authentic virus and pseudovirus systems have shown Omicron spike has an increased dependence on the endosomal pathway for entry. METHODS: We investigated the entry mechanisms of Omicron, Delta, and ancestral viruses in cell models that represent different parts of the human respiratory tract, including nasal epithelial cells (hNECs), large-airway epithelial cells (LAECs), small-airway epithelial cells, and embryonic stem cell-derived type II alveolar cells. FINDINGS: Omicron had an early replication advantage in LAECs, while Delta grew to higher titers in all cells. Omicron maintained dependence on serine proteases for entry in all culture systems. While serine protease inhibition with camostat was less robust for Omicron in hNECs, endosomal entry was not enhanced. CONCLUSIONS: Our findings demonstrate that entry of Omicron BA.1 SARS-CoV-2 is dependent on serine proteases for entry throughout the respiratory tract. FUNDING: This work was supported by The Medical Research Future Fund (MRF9200007; K.S., J.M.P.) and the DHHS Victorian State Government grant (Victorian State Government; DJPR/COVID-19; K.S, J.M.P.). K.S. is supported by a National Health and Medical Research Council of Australia Investigator grant (APP1177174).

Gene mutations as a non-invasive measure of adult cochlear implant performance: Variable outcomes in patients with select TMPRSS3 mutations.

BACKGROUND: The cochlear implant (CI) has proven to be a successful treatment for patients with severe-to-profound sensorineural hearing loss, however outcome variance exists. We sought to evaluate particular mutations discovered in previously established sensory and neural partition genes and compare post-operative CI outcomes. MATERIALS AND METHODS: Utilizing a prospective cohort study design, blood samples collected from adult patients with non-syndromic hearing loss undergoing CI were tested for 54 genes of interest with high-throughput sequencing. Patients were categorized as having a pathogenic variant in the sensory partition, pathogenic variant in the neural partition, pathogenic variant in both sensory and neural partition, or with no variant identified. Speech perception performance was assessed pre- and 12 months post-operatively. Performance measures were compared to genetic mutation and variant status utilizing a Wilcoxon rank sum test, with P<0.05 considered statistically significant. RESULTS: Thirty-six cochlear implant patients underwent genetic testing and speech understanding measurements. Of the 54 genes that were interrogated, three patients (8.3%) demonstrated a pathogenic mutation in the neural partition (within TMPRSS3 genes), one patient (2.8%) demonstrated a pathogenic mutation in the sensory partition (within the POU4F3 genes). In addition, 3 patients (8.3%) had an isolated neural partition variance of unknown significance (VUS), 5 patients (13.9%) had an isolated sensory partition VUS, 1 patient (2.8%) had a variant in both neural and sensory partition, and 23 patients (63.9%) had no mutation or variant identified. There was no statistically significant difference in speech perception scores between patients with sensory or neural partition pathogenic mutations or VUS. Variable performance was found within patients with TMPRSS3 gene mutations. CONCLUSION: The impact of genetic mutations on post-operative outcomes in CI patients was heterogenous. Future research and dissemination of mutations and subsequent CI performance is warranted to elucidate exact mutations within target genes providing the best non-invasive prognostic capability.