Serendipitous discovery of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds as potent HIV-1 replication inhibitors serendipitously during the process of evaluation of triazolothienopyrimidine (TTPM) compounds. Herein, we report synthesis and biological evaluation of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds using a cell-based full replication assay to identify thienopyrimidines 6 and 30, which could be further utilized as viable lead compounds.

Next-generation Sequencing of MicroRNA in Acquired Middle Ear Cholesteatoma.

OBJECTIVES/HYPOTHESIS: The pathophysiology of cholesteatoma is not precisely understood, and research on the associated microRNAs (miRNAs) is also deficient. We demonstrated the expression of miRNA in normal skin and middle ear cholesteatoma by next-generation sequencing (NGS) technology. The profiles of miRNA and relevant molecular interaction pathways were investigated. STUDY DESIGN: Case-control experimental study. METHODS: Middle ear cholesteatoma and post-auricular skin tissue specimens were collected from 13 adult patients. Total RNA was extracted, and miRNA expression profiles were analyzed by NGS technology. Functional gene classification to predict target genes and relevant biological pathways was performed using DIANA-microT-CDS and the Kyoto Encyclopedia Gene and Genome database (KEGG) pathways. RESULTS: The expression of 2588 miRNAs from middle ear cholesteatoma and skin tissue samples was analyzed. The expression of 76 upregulated and 128 downregulated miRNAs was identified in the cholesteatoma samples compared to normal skin (FC ≥2 and p < 0.05). Ninety-nine differentially expressed miRNAs (FC ≥4 and p < 0.05) were used to explore the biological pathways involved in the etiopathogenesis of cholesteatoma. The most predicted pathway in cholesteatoma in the upregulated miRNA group was the ErbB signaling pathway and it was extracellular matrix (ECM)-receptor interaction in the downregulated miRNA group. CONCLUSIONS: This was the first study investigating small miRNAs in human acquired cholesteatoma using NGS technique. We were able to identify new miRNAs and pathways related to cholesteatoma. The results of this study are expected to be helpful in revealing new pathophysiologies of cholesteatoma. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

The therapeutic effect of hyperbaric oxygen therapy in patients with severe to profound idiopathic sudden sensorineural hearing loss.

The optimal treatment for sudden sensorineural hearing loss (SSNHL) is unclear. Hyperbaric oxygen therapy (HBOT) has been suggested as a viable option for treatment of SSNHL as it improves vascular dysfunction. In this study, we evaluated the therapeutic effects of HBOT by retrospectively reviewing the records of 2206 patients with SSNHL. 54 who had received HBOT were selected for the HBOT groups, while 59 age-matched controls who had not were selected for the control groups. The HBOT and control groups were divided into subgroups according to intratympanic steroid (ITS) use. Groups A-D had received oral steroids + HBOT, oral steroids only, oral steroids + ITS + HBOT, and oral steroids + ITS, respectively. Of the 113 SSNHL patients, 21 had diabetes mellitus (DM) (2, 0, 9, and 10 patients in Groups A-D, respectively). There was no notable difference in hearing improvement between patients receiving HBOT and those in the control group. However, among diabetic patients, those who underwent HBOT demonstrated a significant improvement in hearing when compared to the control group. The combination of HBOT and steroids could potentially be beneficial for treating severe to profound SSNHL patients with DM.

Consecutive Dual-Session Transcranial Direct Current Stimulation in Chronic Subjective Severe to Catastrophic Tinnitus with Normal Hearing.

Transcranial direct current stimulation (tDCS) is emerging as a promising non-invasive intervention for tinnitus by aiming to modulate abnormal brain activity. This study investigated the efficacy of dual-session tDCS for the relief of perception, distress, and loudness in patients with severe chronic subjective tinnitus and assessed the duration of tinnitus suppression effects compared to single-session and control groups over a 2-month follow-up. In a prospective, randomized, single-blind, placebo-controlled trial, 30 participants with severe chronic subjective tinnitus underwent bifrontal tDCS. The control group (n = 9), single-session group (n = 10), and dual-session group (n = 11) received 2 mA stimulation for 20 min per session, twice a week for one month. The treatment response was monitored weekly using the Visual Analogue Scale (VAS), with additional assessments using the Tinnitus Handicap Inventory (THI) and Beck Depression Inventory (BDI) at the fourth and eighth weeks. The single- and dual-session groups showed statistically significant improvements in VAS, THI, and BDI scores compared to the control group. THI and BDI scores showed a significant difference between the single- and dual-session groups. The dual-session group demonstrated a more sustained tinnitus suppression effect than the single-session group. tDCS has been validated as an effective intervention for the suppression of tinnitus, with the dual-session protocol showing longer-term benefits. These findings support the potential of tDCS as a treatment for tinnitus, particularly in dual-session applications.

Microarray analysis of Drosophila dicer-2 mutants reveals potential regulation of mitochondrial metabolism by endogenous siRNAs.

RNA interference is a eukaryotic regulatory mechanism by which small non-coding RNAs typically mediate specific silencing of their cognate genes. In Drosophila, the RNase III enzyme Dicer-2 (Dcr-2) is essential for biogenesis of endogenous small interfering RNAs (endo-siRNAs), which have been implicated in regulation of endogenous protein-coding genes. Although much is known about microRNA-based regulatory networks, the biological functions of endo-siRNAs in animals remain poorly understood. We performed gene expression profiling on Drosophila dcr-2 null mutant pupae to investigate transcriptional effects caused by a severe defect in endo-siRNA production, and found 306 up-regulated and 357 down-regulated genes with at least a twofold change in expression compared with the wild type. Most of these up-regulated and down-regulated genes were associated with energy metabolism and development, respectively. Importantly, mRNA sequences of 39% of the up-regulated genes were perfectly complementary to the sequences of previously reported endo-siRNAs, suggesting they may be direct targets of endo-siRNAs. We confirmed up-regulation of five selected genes matching endo-siRNAs and concomitant down-regulation of the corresponding endo-siRNAs in dcr-2 mutant pupae. Most of the potential endo-siRNA target genes were associated with energy metabolism, including the citric acid cycle and oxidative phosphorylation in mitochondria, implying that these are major metabolic processes directly affected by endo-siRNAs in Drosophila. Consistent with this finding, dcr-2 null mutant pupae had lower ATP content compared with controls, indicating that mitochondrial energy production is impaired in these mutants. Our data support a potential role for the endo-siRNA pathway in energy homeostasis through regulation of mitochondrial metabolism.

Synthesis and biological evaluation of triazolothienopyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of triazolothienopyrimidine (TTPM) compounds as potent HIV-1 replication inhibitors during a high-throughput screening campaign that evaluated more than 200,000 compounds using a cell-based full replication assay. Herein, we report the optimization of the antiviral activity in a cell-based assay system leading to the discovery of aryl-substituted TTPM derivatives (38, 44, and 45), which exhibited significant inhibition of HIV-1 replication with acceptable safety margins. These novel and potent TTPMs could serve as leads for further development.

Craniofacial Phenotype in Obstructive Sleep Apnea and Its Impact on Positive Airway Pressure (PAP) Adherence.

Positive airway pressure (PAP) is an important treatment tool for patients with moderate and severe obstructive sleep apnea (OSA), and adherence to PAP significantly affects treatment outcomes. Disease severity, adverse effects, and psychosocial factors are known to predict medication adherence. Cephalometric parameters have been reported to positively correlate with upper airway collapse. However, research on the correlation between these cephalometric parameters and PAP adherence remains insufficient. This study aimed to identify this relationship. This study included 185 patients with OSA who were prescribed PAP. Polysomnography (PSG) was performed to diagnose OSA, and paranasal sinus computed tomography (PNS CT) was performed to check for comorbidities of the upper airway. In addition, cephalometric parameters such as the hyoid-posterior nasal spine (H-PNS), posterior nasal spine-mandibular plane (PNS-MP), and hyoid-mandibular plane (H-MP) were measured in the midsagittal and axial CT views. Adherence was evaluated 3-12 months after the PAP prescription. A total of 136 patients were PAP-adherent, and 49 were nonadherent. There were more males in the adherent group and a higher average height in the adherent group. The PSG results showed that the apnea-hypopnea index (AHI), respiratory disturbance index (RDI), oxygen desaturation index (ODI), arousal index (AI), rapid eye movement (REM) AHI, and supine AHI were significantly higher, and the lowest oxygen saturation was lower in the adherent group. In the analysis of covariance (ANCOVA) model adjusted for sex and height, among the cephalometric parameters, H-MP was significantly longer in the adherent group (p = 0.027), and H-PNS showed a longer tendency (p = 0.074). In the logistic regression analysis model, the odds ratio (OR) and 95% confidence intervals (95% CI) of adherence and severe OSA in the third tertile compared to the first tertile of H-MP were 2.93 (1.25-6.86) and 4.00 (1.87-8.56). In the case of H-PNS, they were 2.58 (1.14-5.81) and 4.86 (2.24-10.54), respectively. This study concluded that an inferiorly placed hyoid bone in adult patients is associated with PAP adherence and disease severity.

Discovery of 3,4-dihydropyrimidin-2(1H)-ones with inhibitory activity against HIV-1 replication.

3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) were selected and derivatized through a HIV-1 replication assay based on GFP reporter cells. Compounds 14, 25, 31, and 36 exhibited significant inhibition of HIV-1 replication with a good safety profile. Chiral separation of each enantiomer by fractional crystallization showed that only the S enantiomer retained anti-HIV activity. Compound (S)-40, a novel and potent DHPM analog, could serve as an advanced lead for further development and the determination of the mechanism of action.

A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability.

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.

Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

Microbiome of Acute Otitis Externa.

BACKGROUND: Ear canal skin is directly attached to bone or cartilage, and is also connected to the eardrum. Acute otitis externa is cellulitis of the ear canal skin and subdermal tissue associated with acute inflammation and variable edema. We characterized the microbiome of the normal ear canal and ear canal with otitis externa. METHODS: In total, 28 samples (14 each from the ear canal skin of patients with acute otitis externa and normal healthy controls) were collected using swabs. DNA extraction and bacterial microbiome analysis via 16S rRNA gene sequencing were performed. RESULTS: The diversity index (mean amplicon sequence variants and Shannon index) were lower in the otitis externa than control group. According to linear discriminant effect size (LEfSe) analysis, a number of taxa differed significantly between the groups. Pseudomonas at the genus level and Staphylococcus warneri at the species level were identified in the otitis externa group. CONCLUSION: Our results show the importance of the microbiome in the pathogenesis of otitis externa and provide a basis for treating acute otitis externa by targeting the microbiome.

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.

Conformational changes involved in G-protein-coupled-receptor activation.

Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the M(3) muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.

An immune-responsive Serpin regulates the melanization cascade in Drosophila.

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.

In situ visualization of gene expression using polymer-coated quantum-dot-DNA conjugates.

Imaging of specific mRNA targets in cells is of great importance in understanding gene expression and cell signaling processes. Subcellular localization of mRNA is known as a universal mechanism for cells to sequester specific mRNA for high production of required proteins. Various gene expressions in Drosophila cells are studied using quantum dots (QDs) and the fluorescence in situ hybridization (FISH) method. The excellent photostability and highly luminescent properties of QDs compared to conventional fluorophores allows reproducible obtainment of quantifiable mRNA gene expression imaging. Amine-modified oligonucleotide probes are designed and covalently attached to the carboxyl-terminated polymer-coated QDs via EDC chemistry. The resulting QD-DNA conjugates show sequence-specific hybridization with target mRNAs. Quantitative analysis of FISH on the Diptericin gene after lipopolysaccharide (LPS) treatment shows that the intensity and number of FISH signals per cell depends on the concentration of LPS and correlates well with quantitative real-time PCR results. In addition, our QD-DNA probes exhibit excellent sensitivity to detect the low-expressing Dorsal-related immunity factor gene. Importantly, multiplex FISH of Ribosomal protein 49 and Actin 5C using green and red QD-DNA conjugates allows the observation of cellular distribution of the two independent genes simultaneously. These results demonstrate that highly fluorescent and stable QD-DNA probes can be a powerful tool for direct localization and quantification of gene expression in situ.

Western blot analysis using metal-nitrilotriacetate conjugated CdSe/ZnS quantum dots.

To co-opt the remarkable optical properties and benefits of quantum dots and broadly used metal-NTA:histidine tag interactions, we generated metal-NTA conjugated quantum dots and applied them to Western blot analysis. In our hands, this application dramatically reduced the time and effort required for Western blot analysis, whereas the sensitivity was comparable to that of the conventionally available anti-histidine tag antibody. Our quantum dots were stable up to 6 months without precipitation. Interestingly, under our conditions, cobalt-NTA showed better detection ability than did nickel-NTA. Our new method may be able to facilitate and simplify the routinely used protein detection procedure.

M1-M3 muscarinic acetylcholine receptor-deficient mice: novel phenotypes.

The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.

Cloning and characterization of an eukaryotic initiation factor-2alpha kinase from the silkworm, Bombyx mori.

Eukaryotic initiation factor 2alpha (eIF-2alpha) kinases are involved in the translational regulations that occur in response to various types of environmental stress, and play an important role in the cellular defense system operating under unfavorable conditions. The identification of additional eIF-2alpha kinases and the elucidation of their functions are necessary to understand how different eIF-2alpha kinases can specifically respond to distinct stimuli. Here, we report a novel eIF-2alpha kinase, termed BeK, from the silkworm, Bombyx mori. This gene encodes 579 amino acids and contains all 11 catalytic domains of protein-serine/threonine kinases. Most notably, it contains an "Ile-Gln-Met-Xaa-Xaa-Cys" motif, which is highly conserved from yeast to mammalian eIF-2alpha kinases. BeK does not show any significant homology in the NH(2) terminal regulatory domain, suggesting a distinct regulatory mechanism of this novel eIF-2alpha kinase. BeK is ubiquitously expressed in the various tissues throughout the final larval stage. Importantly, BeK is activated in Drosophila Schneider cells following heat shock and osmotic stress, and activated-BeK has been shown to phosphorylate an eIF-2alpha subunit at the Ser(50) site. However, other forms of stress, such as immune stress, endoplasmic reticulum stress and oxidative stress, cannot significantly elicit BeK activity. Interestingly, the baculovirus gene product, PK2, can inhibit BeK enzymatic activity, suggesting that BeK may be an endogenous target for a viral gene product. Taken together, these data indicate that BeK is a novel eIF-2alpha kinase involved in the stress response in B. mori.

Long-term stable, low-temperature remote silicate phosphor thick films printed on a glass substrate.

A critical step in providing better phosphor solution for white light emitting diode (LED) is to utilize inexpensive silicate phosphors with strong thermal stability. Here, we demonstrate yellow silicate phosphor-embedded glass thick films with a high luminous efficacy of ∼32 lm/W at 200 mA as a nonconventional remote-phosphor approach. The simple screen-printing process of a paste consisting of (Ba,Sr,Ca)2SiO4:Eu²âº phosphor and a low softening point glass creates a planar remote structure on a regular soda lime silicate glass with controllable film thickness and location (top vs bottom) of the phosphor layer. The glass matrix provides promising densification and adhesion with the substrate at the optimal low temperature of 410 °C, with the long-term stability in luminous efficacy over 500 h of operation. The proposed phosphor structure has important implications to overcome current limitations as phosphors.

High-throughput in vivo genotoxicity testing: an automated readout system for the somatic mutation and recombination test (SMART).

Genotoxicity testing is an important component of toxicity assessment. As illustrated by the European registration, evaluation, authorization, and restriction of chemicals (REACH) directive, it concerns all the chemicals used in industry. The commonly used in vivo mammalian tests appear to be ill adapted to tackle the large compound sets involved, due to throughput, cost, and ethical issues. The somatic mutation and recombination test (SMART) represents a more scalable alternative, since it uses Drosophila, which develops faster and requires less infrastructure. Despite these advantages, the manual scoring of the hairs on Drosophila wings required for the SMART limits its usage. To overcome this limitation, we have developed an automated SMART readout. It consists of automated imaging, followed by an image analysis pipeline that measures individual wing genotoxicity scores. Finally, we have developed a wing score-based dose-dependency approach that can provide genotoxicity profiles. We have validated our method using 6 compounds, obtaining profiles almost identical to those obtained from manual measures, even for low-genotoxicity compounds such as urethane. The automated SMART, with its faster and more reliable readout, fulfills the need for a high-throughput in vivo test. The flexible imaging strategy we describe and the analysis tools we provide should facilitate the optimization and dissemination of our methods.