[Unsupervised clustering analysis based on multidimensional features reveals distinct clinical characteristics and associated factors of different phenotypes in patients with chronic rhinosinusitis with nasal polyp].

Objective: To utilize routinely available clinical parameters to uncover the clinical features of different clusters in patients with chronic rhinosinusitis with nasal polyp (CRSwNP) through unsupervised clustering analysis. Methods: The clinical data from 155 CRSwNP patients undergoing nasal endoscopic surgery at Renmin Hospital of Wuhan University from 2021 to 2023 were prospectively collected, including 112 males and 43 females, aged from 7 to 87 years. Unsupervised clustering analysis was conducted using various clinical parameters, including age, gender, smoking and drinking history, local eosinophil (EOS) and neutrophil (NEU) counts, comorbid allergic rhinitis (AR), comorbid asthma, recurrence status, serum-specific IgE, total IgE, cytokine levels, peripheral blood EOS count and percentage, Lund-Mackay CT score, the ratio of CT scores for the maxillary sinus and ethmoid sinus (E/M ratio), visual analogue scale (VAS) score, Lund-Kennedy endoscopic score, and other common clinical indicators to elucidate the clinical characteristics of each cluster. Statistical analysis was conducted using GraphPad Prism 9.5 software. Results: Hierarchical clustering analysis identified four main clusters (Cluster A1-A4), showcasing distinct characteristics such as mild nasal polyps with higher peripheral blood cytokines levels, nasal polyps accompanied by allergies and asthma, a subtype of nasal polyps with high recurrence rates dominated by neutrophils, and nasal polyps with high eosinophil levels. Further subset clustering revealed two clusters of mild polyps (Cluster B1-B2) featuring high cytokine expression and comorbid AR; and two clusters of severe polyps (Cluster B3-B4) presented with severe symptoms, high Lund-Mackay CT score, and high Lund-Kennedy endoscopic score. Variations between Cluster B3 and B4 included symptom complexity, the degree of eosinophil infiltration, and the probability of comorbid asthma. Further clustering analysis for eosinophilic nasal polyps revealed a cluster characterized by highly neutrophilic infiltration and recurrent nasal polyps. The comprehensive analysis of multi-index correlations demonstrated valuable insights into the relationships between common clinical parameters of nasal polyps, providing valuable information for a deeper understanding of the pathogenesis of CRSwNP. Conclusion: The clustering analysis in this study categorizes CRSwNP patients into different clusters based on clinical features and disease outcomes, providing a new perspective for more precise clinical treatment strategies.

Archetypal clustering reveals physiological mechanisms linking milk yield and fertility in dairy cattle.

Fertility in dairy cattle has declined as an unintended consequence of single-trait selection for high milk yield. The unfavorable genetic correlation between milk yield and fertility is now well documented; however, the underlying physiological mechanisms are still uncertain. To understand the relationship between these traits, we developed a method that clusters variants with similar patterns of effects and, after the integration of gene expression data, identifies the genes through which they are likely to act. Biological processes that are enriched in the genes of each cluster were then identified. We identified several clusters with unique patterns of effects. One of the clusters included variants associated with increased milk yield and decreased fertility, where the "archetypal" variant (i.e., the one with the largest effect) was associated with the GC gene, whereas others were associated with TRIM32, LRRK2, and U6-associated snRNA. These genes have been linked to transcription and alternative splicing, suggesting that these processes are likely contributors to the unfavorable relationship between the 2 traits. Another cluster, with archetypal variant near DGAT1 and including variants associated with CDH2, BTRC, SFRP2, ZFHX3, and SLITRK5, appeared to affect milk yield but have little effect on fertility. These genes have been linked to insulin, adipose tissue, and energy metabolism. A third cluster with archetypal variant near ZNF613 and including variants associated with ROBO1, EFNA5, PALLD, GPC6, and PTPRT were associated with fertility but not milk yield. These genes have been linked to GnRH neuronal migration, embryonic development, or ovarian function. The use of archetypal clustering to group variants with similar patterns of effects may assist in identifying the biological processes underlying correlated traits. The method is hypothesis generating and requires experimental confirmation. However, we have uncovered several novel mechanisms potentially affecting milk production and fertility such as GnRH neuronal migration. We anticipate our method to be a starting point for experimental research into novel pathways, which have been previously unexplored within the context of dairy production.

A SNAI2/CTCF Interaction is Required for NOTCH1 Expression in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle with characteristics of cells blocked in differentiation. NOTCH1 is an oncogene that promotes self-renewal and blocks differentiation in the fusion negative-RMS sub-type. However, how NOTCH1 expression is transcriptionally maintained in tumors is unknown. Analyses of SNAI2 and CTCF chromatin binding and HiC analyses revealed a conserved SNAI2/CTCF overlapping peak downstream of the NOTCH1 locus marking a sub-topologically associating domain (TAD) boundary. Deletion of the SNAI2-CTCF peak showed that it is essential for NOTCH1 expression and viability of FN-RMS cells. Reintroducing constitutively activated NOTCH1-ΔE in cells with the SNAI2-CTCF peak deleted restored cell-viability. Ablation of SNAI2 using CRISPR/Cas9 reagents resulted in the loss of majority of RD and SMS-CTR FN-RMS cells. However, the few surviving clones that repopulate cultures have recovered NOTCH1. Cells that re-establish NOTCH1 expression after SNAI2 ablation are unable to differentiate robustly as SNAI2 shRNA knockdown cells; yet, SNAI2-ablated cells continued to be exquisitely sensitive to ionizing radiation. Thus, we have uncovered a novel mechanism by which SNAI2 and CTCF maintenance of a sub-TAD boundary promotes rather than represses NOTCH1 expression. Further, we demonstrate that SNAI2 suppression of apoptosis post-radiation is independent of SNAI2/NOTCH1 effects on self-renewal and differentiation.

[Short-term efficacy and safety observation of standardized mite allergen extract rush subcutaneous immunotherapy for allergic rhinitis: a prospective study].

Objective: To conduct a comparative analysis of the efficacy, safety, and cytokine changes associated with three distinct dose escalation regimens of allergen-specific immunotherapy (AIT), and to provide valuable insights into the implementation of safer and more effective accelerated immunotherapy in clinical practice. Methods: A prospective study of subcutaneous immunotherapy (SCIT) was conducted at Renmin Hospital of Wuhan University, involving patients with allergic rhinitis visited from 2019 to 2022. Participants were allocated to one of three treatment groups based on their preferences: conventional immunotherapy (CIT, 23 cases), cluster immunotherapy (CLIT, 25 cases), or rush immunotherapy (RIT, 18 cases). The RIT group received a single subcutaneous injection of 150 mg of omalizumab one week before commencing treatment. Subjective evaluation indices, including the Combined Symptom and Medication Score (CSMS), Visual Analogue Scale (VAS), and single symptom score, were recorded alongside objective evaluation indices (e.g., sIgE, tIgE, Th1/2 and Th17 cytokines) and adverse reactions. Assessments were conducted at baseline, and at 1, 7, and 15 weeks after treatment. SPSS 22.0 software was used for data processing and analysis. Results: The study included a total of 66 patients, comprising 37 males and 29 females, who completed the treatment regimen. In all three groups, CSMS and VAS scores showed significant reductions at 1, 7, and 15 weeks post-treatment (all P<0.05). Notably, the RIT group demonstrated a significantly lower VAS score (4.33±0.94) compared to the CIT (9.48±1.37) and CLIT (9.44±1.33) groups at 1 week post-treatment (P<0.05). Additionally, the RIT group (0.62±0.23) exhibited a lower CSMS score than the CIT (1.54±0.21) and CLIT (1.06±0.22) groups at 15 weeks post-treatment (P<0.05). Furthermore, at the point of reaching the maintenance dose, the RIT group (0.61±0.20) demonstrated superior improvement in nasal itching symptoms compared to the CIT (1.78±0.38) and CLIT groups (1.56±0.32), with P<0.05. The incidence of local adverse reactions in the RIT group (36/11.76%) was lower than that in the CIT (69/20.00%) and CLIT groups (62/16.53%), with P<0.05. Notably, none of the three groups reported grade 3/4 systemic adverse reactions, and there was no statistically significant difference in systemic adverse reactions among the three groups. Following treatment, IL-4, IL-5, IL-6, IL-17, sIgE, sIgE/tIgE, and Eos% exhibited varying degrees of decrease in all three groups, whereas IL-10, TNF, and IFN-γ did not show significant changes. Conclusions: All three distinct dose escalation regimens of SCIT demonstrated substantial clinical efficacy. Of note, the approach of combining a single injection of omalizumab with RIT significantly improved early-stage efficacy and exhibited the advantages of safety, effectiveness, and convenience, establishing it as a reliable immunotherapy method.

Integrative analysis reveals the recurrent genetic etiologies in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is increasingly recognized as a chronic, progressive and fatal lung disease with an unknown etiology. Current studies focus on revealing the genetic factors in the risk of IPF, making the integrative analysis of genetic variations and transcriptomic alterations of substantial value. AIM: This study aimed to improve the understanding of the molecular basis of IPF through an integrative analysis of whole-exome sequencing (WES), bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) data. METHODS: WES is a powerful tool for studying the genetic basis of IPF, allowing for the identification of genetic variants that may be associated with the development of the disease. RNA-seq data provide a comprehensive view of the transcriptional changes in IPF patients, while scRNA-seq data offer a more granule view of cell-type-specific alterations. RESULTS: In this study, we identified a comprehensive mutational landscape of recurrent genomic and transcriptomic variations, including single-nucleotide polymorphisms, CNVs and differentially expressed genes, in IPF populations, which may play a significant role in the development and progression of IPF. CONCLUSIONS: Our study provided valuable insights into the genetic and transcriptomic variations associated with IPF, revealing changes in gene expression that may contribute to disease development and progression. These findings highlight the importance of an integrative approach to understanding the molecular mechanisms underlying IPF and may pave the way for identifying potential therapeutic targets.

[Study on the recurrence pattern of rectal cancer patients undergoing radical surgery after neoadjuvant chemoradiotherapy].

Objective: To analyze the recurrence pattern of rectal cancer patients with radical surgery after neoadjuvant chemoradiotherapy. Methods: The clinicopathological characteristics and follow-up information of rectal cancer patients with radical surgery after neoadjuvant chemoradiotherapy in the Cancer Hospital of the Chinese Academy of Medical Sciences from June 2004 to December 2017 were retrospectively collected. The recurrence pattern including the time and site was investigated. Results: The age of 537 patients was (55.5±11.7) years, of whom 361 were male (67.2%). The median follow-up time [M(Q1,Q3)] was 77.9 (64.5, 95.6) months. Moreover, 30.7% (165/537) of patients had distant metastasis or local recurrence; 26.8% (144/537) of patients had distant metastasis; 5.6% (30/537) of patients had local recurrence; 1.7% (9/537) of patients had both distant metastasis and local recurrence. In all the recurrent patients, 23.6% (39/165) were in the first year after surgery, followed by 27.3% (45/165) in the second year, 17.0% (28/165) in the third year, and 15.8% (26/165) after five years. According to the risk curve drawn by the life table, the highest metastasis risk of patients occurred in the second year after surgery, and the metastasis risk peak occurred again after more than five years. The lung was the most common metastatic organ, accounting for 47.9% (69/144), followed by the liver (18.8%, 27/144). The ratios of the recurrent patients in each ypTNM stage were 9.5% (8/84), 12.0% (12/100), 26.5% (41/155), 52.5% (104/198), respectively. The proportion of recurrent patients in tumor regression grade (TRG) 1-2 and TRG 3-5 patients were 19.2% (38/198) and 37.5% (127/339), respectively. Conclusions: The recurrence pattern of patients undergoing radical surgery after neoadjuvant chemoradiotherapy is mainly distant metastasis. The lung is the primary metastatic organ. The risk of distant metastasis and local recurrence is high in the first three years after surgery, and there is still high risk of recurrence after five years. For patients with ypTNM stage 2, 3 and TRG3-5, the postoperative adjuvant chemotherapy and long-term follow-up should be strengthened.

[A long-term follow-up study on recurrence and benefit of standardized postoperative chemotherapy of rectal cancer patients with complete pathological response after neoadjuvant chemoradiotherapy].

Objective: To analyze the clinicopathological factors affecting long-term disease-free survival and the characteristics of local recurrence or distance metastasis of rectal cancer patients with complete pathological response after neoadjuvant chemoradiotherapy. Methods: The clinicopathological data and follow-up information of patients with a complete pathological response of rectal cancer after neoadjuvant chemoradiotherapy in the Cancer Hospital of Chinese Academy of Medical Sciences from June 2004 to December 2019 were retrospectively collected. The clinicopathological factors affecting the long-term disease-free survival of patients were analyzed to build a prediction model of local recurrence and distant metastasis and to evaluate the benefits of postoperative chemotherapy. Results: The age of 108 patients was(56.3±11.6) years, of which 68 were males (63.0%); The median follow-up time was 79.9 (61.8, 112.6) months. There were 12 patients (11.1%) who had a local recurrence or distant metastasis. The 5-year disease-free survival rate was 91.1% with 9 patients who experienced recurrence. Multivariate Cox proportional hazards regression analysis showed that the maximum diameter of the residual tumor or scar (HR=8.41, 95%CI: 1.08-65.22, P=0.042) and the distance from the lower edge of the tumor to the anal margin before treatment (HR=4.54, 95%CI: 1.23-16.81, P=0.023) were independent risk factors affecting the prognosis. The prognosis of patients was stratified based on relevant factors. The 5-year cumulative disease-free survival rate of those patients receiving postoperative standardized chemotherapy was 92.0%, while for patients who did not receive or complete standardized chemotherapy, the 5-year cumulative disease-free survival rate was 82.3%. Conclusions: The maximum diameter of the residual tumor or scar and the distance from the lower edge of the tumor to the anal margin before treatment were independent risk factors affecting the prognosis of patients with a complete pathological response. Patients with independent risk factors could benefit from the standardized postoperative chemotherapy.

Sensitization to Ionizing Radiation by MEK Inhibition Is Dependent on SNAI2 in Fusion-Negative Rhabdomyosarcoma.

In fusion-negative rhabdomyosarcoma (FN-RMS), a pediatric malignancy with skeletal muscle characteristics, >90% of high-risk patients have mutations that activate the RAS/MEK signaling pathway. We recently discovered that SNAI2, in addition to blocking myogenic differentiation downstream of MEK signaling in FN-RMS, represses proapoptotic BIM expression to protect RMS tumors from ionizing radiation (IR). As clinically relevant concentrations of the MEK inhibitor trametinib elicit poor responses in preclinical xenograft models, we investigated the utility of low-dose trametinib in combination with IR for the treatment of RAS-mutant FN-RMS. We hypothesized that trametinib would sensitize FN-RMS to IR through its downregulation of SNAI2 expression. While we observed little to no difference in myogenic differentiation or cell survival with trametinib treatment alone, robust differentiation and reduced survival were observed after IR. In addition, IR-induced apoptosis was significantly increased in FN-RMS cells treated concurrently with trametinib, as was increased BIM expression. SNAI2's role in these processes was established using overexpression rescue experiments, where overexpression of SNAI2 prevented IR-induced myogenic differentiation and apoptosis. Moreover, combining MEK inhibitor with IR resulted in complete tumor regression and a 2- to 4-week delay in event-free survival (EFS) in preclinical xenograft and patient-derived xenograft models. Our findings demonstrate that the combination of MEK inhibition and IR results in robust differentiation and apoptosis, due to the reduction of SNAI2, which leads to extended EFS in FN-RMS. SNAI2 thus is a potential biomarker of IR insensitivity and target for future therapies to sensitize aggressive sarcomas to IR.

SNAI2-Mediated Repression of BIM Protects Rhabdomyosarcoma from Ionizing Radiation.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

[Clinical analysis of 126 patients with coronavirus disease 2019 with pharyngeal symptoms].

Objective: To investigate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with pharyngeal symptoms. Methods: From January 10 to May 15, 2020, clinical data of 1 228 patients with COVID-19 in Renmin Hospital of Wuhan University was collected (554 males and 674 females, with the range of age from 10 to 95 years old, the average age was 55.2 years old). The patients were divided into pharyngeal symptoms group (PS, 126 cases) and non-pharyngeal symptoms group (Non-PS, 1 102 cases) according to the presence or absence of pharyngeal symptoms such as pharyngalgia, pharyngeal dryness, pharyngeal itching, and pharyngeal foreign body sensation. The clinical data in terms of age, sex, medical history, duration of symptoms, treatment time, clinical classification, pulmonary imaging findings, whole blood cell count, serum hypersensitivity C-reactive protein, C-reactive protein, procalcitonin were statistically analyzed between the two groups. Chi-square, Fisher's exact test and Mann-Whitney U test were used for statistical analysis. Results: The most common pharyngeal symptoms were pharyngalgia (59.52%, 75/126), followed by foreign body sensation (23.02%, 29/126), pharyngeal dryness (8.73%, 11/126), and itching (8.73%, 11/126). The median age of the patients in the PS group was 51.50 years old, which was less than 57.50 years old in the non-PS group, showing a significant difference (P<0.05). The female cases accounted for 65.08% (82/126), which was higher than 53.72% (592/1 102) of the non-PS group (P<0.05). The incidence of bilateral lung inflammation confirmed by CT images was 73.81% (93/126), which was significantly lower than 83.48% (920/1 102) in the non-PS group (P<0.05). No significant differences were shown in the proportion of patients with clinical types, treatment days, duration of symptoms, white blood cell count, lymphocyte count, lymphocyte percentage, eosinophil count, eosinophil percentage, hypersensitive C-reactive protein, C-reactive protein, D-dimer, procalcitonin and other indicators (P>0.05). Conclusions: The incidence of pharyngeal symptoms in patients with COVID-19 is 10.26%. Most of these symptoms occur before or at the same time as the common symptoms of the disease. Therefore, patients with such symptoms may bring a greater risk of infection to otolaryngologist. According to the current clinical classification criteria, pharyngeal symptoms have no obvious correlation with the degree of the disease; but the presence of pharyngeal symptoms may suggest a milder clinical presentation and a better prognosis.

Orbital localization error of density functional theory in shear properties of vanadium and niobium.

It is believed that the density functional theory (DFT) describes most elements with s, p, and d orbitals very well, except some materials that have strongly localized and correlated valence electrons. In this work, we find that the widely employed exchange-correlation (XC) functionals, including local-density approximation (LDA), generalized gradient approximation (GGA), and meta-GGA, underestimate the shear modulus and phase stability of V and Nb greatly. The advanced hybrid functional that is usually better for correlated systems, on the other hand, completely fails in these two simple metals. This striking failure is revealed due to the orbital localization error in GGA, which is further deteriorated by hybrid functionals. This observation is corroborated by a similar failure of DFT+U and van der Waals functionals when applied to V and Nb. To remedy this problem, a semiempirical approach of DFT+J is proposed, which can delocalize electrons by facilitating the on-site exchange. Furthermore, it is observed that including density derivatives slightly improves the performance of the semilocal functionals, with meta-GGA outperforms GGA, and the latter is better than LDA. This discovery indicates the possibility and necessity to include higher-order density derivatives beyond the Laplacian level for the purpose of removing the orbital localization error (mainly from d orbitals) and delocalization error (mainly from s and p orbitals) completely in V and Nb so that a better description of their electronic structures is achieved. The same strategy can be applied to the other d electron system and f electron system.

Tricellulin Modulates Transport of Macromolecules in the Salivary Gland.

Volume and composition of saliva are crucial for oral and systemic health. How substances, particularly macromolecules, are transported across the salivary gland epithelium has not been established in detail. Tricellulin is a component of tricellular tight junctions that form a central tube to serve as an important route for macromolecule transport. Whether tricellulin is expressed in the submandibular gland (SMG) and involved in salivation has been unknown. Here, by using Western blotting and immunofluorescence, tricellulin was found to be characteristically localized at tricellular contacts of human, rat, and mouse SMGs. Knockdown of tricellulin significantly increased, whereas overexpression of tricellulin decreased, paracellular permeability for 40-kDa but not for 4-kDa fluorescein isothiocyanate-dextran, while transepithelial electrical resistance was unaffected. Conversely, claudin-4 knockdown and overexpression affected transepithelial electrical resistance but not 40-kDa fluorescein isothiocyanate-dextran transport, suggesting that tricellulin regulated transport of macromolecules but not ions, which were mainly regulated by bicellular tight junctions (bTJs). Moreover, tricellulin was dynamically redistributed from tri- to bicellular membranes in cholinergically stimulated SMG tissues and cells. Immunoglobulin-like domain-containing receptor 1 (ILDR1) recruits tricellulin to tricellular contacts. The proportion of macromolecules in the saliva was increased, whereas the amount of stimulated saliva was unchanged in Ildr1-/- mice, which displayed abnormal tricellulin distribution in SMGs. Furthermore, tricellulin interacted with bTJ proteins, such as occludin, claudin-1, claudin-3, claudin-4, and ZO-1, in rat SMG epithelial polarized cell line SMG-C6. Knockdown of tricellulin decreased occludin levels. Thus, we revealed a specific expression pattern of tricellulin in SMG epithelium. Tricellulin not only functioned as a barrier for macromolecules but also modulated the connection of bTJs to the tight junction complex. Alterations in tricellulin expression and distribution could thereby change salivary composition. Our study provided novel insights on salivary gland tight junction organization and function.

[Expression of amphiregulin in different types of nasal polyps and its correlation with tissue remodeling].

Objective: To explore the expression of amphiregulin (AREG) in nasal polyps patients with different degrees of eosinophil infiltration, and to analyze the correlation between AREG and tissue remodeling. Methods: Forty-eight patients underwent endoscopic sinus surgery in the Department of Otorhinolaryngology Head and Neck Surgery, Remin Hospital, Wuhan University from July 2017 to August 2018 were recruited, including 40 males and 8 females, aged from 16 to 60 years old. The subjects were divided into three groups: control group (n=14), eosinophilic chronic sinusitis with nasal polyps (ECRSwNP) group (n=19) and noneosinophilic chronic rhinosinusitis with nasal polyps (non-ECRSwNP) group (n=15). The relative expression of AREG in nasal mucosa was detected by Western blot assay and immunohistochemical staining. Tissue remodeling was detected by HE staining, AB-PAS staining and Masson staining. Kruskal-Wallis test was used for comparison among multiple groups, and Spearman correlation analysis was conducted between the expression level of AREG and the related indexes of tissue remodeling. Results: The expression of AREG in ECRSwNP group was significantly higher than that in non-ECRSwNP group and control group (median protein expression of Western blot was 1.592 vs 0.617 vs0.582, all P<0.05). The degree of epithelial injury and goblet cell metaplasia in ECRSwNP group was significantly higher than that in control group (all P<0.05), the percentage of collagen fibrosis area in ECRSwNP group was significantly lower than that in control group (P=0.01). In chronic rhinosinusitis with nasal polyps (CRSwNP) patients, the area of mucous glands was negatively correlated with the expression of AREG (r=-0.616, P<0.05), and the percentage of collagen fibrosis area was negatively correlated with the expression of AREG (r=-0.738, P<0.05). Conclusion: The expression of AREG is higher in ECRSwNP patients, which is related to the process of tissue remodeling.

Proteomics analysis of suckling mouse brain infected with attenuated rabies virus strain SRV9.

Rabies virus is an enveloped negative-stranded RNA virus belonging to the family Rhabdoviridae. It can be successfully controlled by vaccination however, there are still tens of thousands of deaths each year caused by rabies virus due to its mutations and complexity. A better understanding of the interaction between the rabies virus and the host might help solve this problem. Therefore, in this study, we used two-dimensional electrophoresis to investigate the protein expression of rabies virus-infected mice. This can help us to understand the impact of rabies virus on host protein expression during infection. For our experiment, two-dimensional electrophoresis was used to analyze the differential proteomics of the brain of 10- and 20-day-old suckling mice infected with attenuated rabies virus strain SRV9. The results showed that the expression levels of 10 protein spots had been up- or down-regulated at least 2-fold. Using MALDI-TOF-MS, we identified 8 differentially expressed proteins. We have identified proteins, namely hnRNP L, DPYSL3, NECAPs, and transaldolase that might be closely related to the susceptibility of SRV9 in suckling mice. Keywords: rabies virus; attenuated strain; suckling mouse; two-dimensional electrophoresis; proteomics.

Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms.

The nematode Caenorhabditis elegans is a suitable model organism in drug screening. Traditionally worms are grown on agar plates, posing many challenges for long-term culture and phenotyping of animals under identical conditions. Microfluidics allows for 'personalized' phenotyping, as microfluidic chips permit collecting individual responses over worms' full life. Here, we present a multiplexed, high-throughput, high-resolution microfluidic approach to culture C. elegans from embryo to the adult stage at single animal resolution. We allocated single embryos to growth chambers, for observing the main embryonic and post-embryonic development stages and phenotypes, while exposing worms to up to 8 different well-controlled chemical conditions. Our approach allowed eliminating bacteria aggregation and biofilm formation-related clogging issues, which enabled us performing up to 80 hours of automated single worm culture studies. Our microfluidic platform is linked with an automated phenotyping code that registers organism-associated phenotypes at high-throughput. We validated our platform with a dose-response study of the anthelmintic drug tetramisole by studying its influence through the life cycle of the nematodes. In parallel, we could observe development effects and variations in single embryo and worm viability due to the bleaching procedure that is standardly used for harvesting the embryos from a worm culture agar plate.