Comparative analysis of asian carps parvalbumin reveals the divergence pattern of major fish allergen.

BACKGROUND: Asian carps, a popular freshwater fish globally, are valued for their flavor and serve as a crucial protein source, especially for infants. However, grass carp parvalbumin is highly allergenic, surpassing the allergenicity of fish like salmon and cod. The allergenic potential of parvalbumin in other Asian carps remains unknown, underscoring the need for allergen identification to improve the precision of fish allergy diagnosis and treatment. OBJECTIVE: To identify all parvalbumin homologs in Asian carps and investigate the role of gene divergence in allergenic homolog formation. METHODS: Three annotated genomes of Asian carp, including grass carp, black carp and bighead carp, were constructed using a hybrid assembly approach. Through sequence homology at the genomic level, all the homologs of major fish allergens were identified. Bioinformatics tools were then employed to reveal the gene structures, expression levels, and protein conformations of parvalbumin. RESULTS: Grass carp genome analysis showed nine parvalbumin homologs, with Cid_PV2 most similar to Cten i 1. Bighead and black carp genomes had ten homologs, including potentially allergenic Mpi_PV7 and Hno_PV7. Tissue-specific expression patterns revealed alternative usage of parvalbumin homologs. Gene duplication events expanded parvalbumin copies in bony fish, with two gene clusters identified in Asian carp genomes. CONCLUSION: All the homologs of Asian carps' parvalbumin were accurately identified and gene divergence contributed to the formation of allergenic homologs. Together with a comprehensive gene sequence profile of carps' parvalbumin, those could be applied to achieve a more precise clinical diagnostic test.

An HPLC-SEC-based rapid quantification method for vesicular stomatitis virus particles to facilitate process development.

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size-exclusion chromatography (SEC) separation using high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method's performance and applicability to in-process samples were characterized. The assay's repeatability variation was <1% and its intermediate precision variation was <3%. The linear range of the method spans from 7.08 × 108 to 1.72 × 1011 VP/mL, with a limit of detection (LOD) of 7.72 × 107 VP/mL and a lower limit of quantification (LLOQ) of 4.20 × 108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.

Do Hospital Characteristics Predict Racial-and-Ethnic Disparities in Patient Experience? National Results From the HCAHPS Survey.

OBJECTIVE: Assess whether hospital characteristics associated with better patient experiences overall are also associated with smaller racial-and-ethnic disparities in inpatient experience. BACKGROUND: Hospitals that are smaller, non-profit, and serve high proportions of White patients tend to be high-performing overall, but it is not known whether these hospitals also have smaller racial-and-ethnic disparities in care. RESEARCH DESIGN: We used linear mixed-effect regression models to predict a summary measure that averaged eight Hospital CAHPS (HCAHPS) measures (Nurse Communication, Doctor Communication, Staff Responsiveness, Communication about Medicines, Discharge Information, Care Coordination, Hospital Cleanliness, and Quietness) from patient race-and-ethnicity, hospital characteristics (size, ownership, racial-and-ethnic patient-mix), and interactions of race-and-ethnicity with hospital characteristics. SUBJECTS: Inpatients discharged from 4,365 hospitals in 2021 who completed an HCAHPS survey ( N =2,288,862). RESULTS: While hospitals serving larger proportions of Black and Hispanic patients scored lower on all measures, racial-and-ethnic disparities were generally smaller for Black and Hispanic patients who received care from hospitals serving higher proportions of patients in their racial-and-ethnic group. Experiences overall were better in smaller and non-profit hospitals, but racial-and-ethnic differences were slightly larger. CONCLUSIONS: Large, for-profit hospitals and hospitals serving higher proportions of Black and Hispanic patients tend to be lower performing overall but have smaller disparities in patient experience. High-performing hospitals might look at low-performing hospitals for how to provide less disparate care whereas low-performing hospitals may look to high-performing hospitals for how to improve patient experience overall.

Dissecting the effects of METTL3 on alternative splicing in prostate cancer.

Although the role of METTL3 has been extensively studied in many cancers, its role in isoform switching in prostate cancer (PCa) has been poorly explored. To investigate its role, we applied standard RNA-sequencing and long-read direct RNA-sequencing from Oxford Nanopore to examine how METTL3 affects alternative splicing (AS) in two PCa cell lines. By dissecting genome-wide METTL3-regulated AS events, we noted that two PCa cell lines (representing two different PCa subtypes, androgen-sensitive or resistant) behave differently in exon skipping and intron retention events following METTL3 depletion, suggesting AS heterogeneity in PCa. Moreover, we revealed that METTL3-regulated AS is dependent on N6-methyladenosine (m6A) and distinct splicing factors. Analysis of the AS landscape also revealed cell type specific AS signatures for some genes (e.g., MKNK2) involved in key functions in PCa tumorigenesis. Finally, we also validated the clinical relevance of MKNK2 AS events in PCa patients and pointed to the possible regulatory mechanism related to m6A in the exon14a/b region and SRSF1. Overall, we characterize the role of METTL3 in regulating PCa-associated AS programs, expand the role of METTL3 in tumorigenesis, and suggest that MKNK2 AS events may serve as a new potential prognostic biomarker.

Heterogeneous Tfh cell populations that develop during enteric helminth infection predict the quality of type 2 protective response.

T follicular helper (Tfh) cells are an important component of germinal center (GC)-mediated humoral immunity. Yet, how a chronic type 1 versus protective type 2 helminth infection modulates Tfh-GC responses remains poorly understood. Here, we employ the helminth Trichuris muris model and demonstrate that Tfh cell phenotypes and GC are differentially regulated in acute versus chronic infection. The latter failed to induce Tfh-GC B cell responses, with Tfh cells expressing Τ-bet and interferon-γ. In contrast, interleukin-4-producing Tfh cells dominate responses to an acute, resolving infection. Heightened expression and increased chromatin accessibility of T helper (Th)1- and Th2 cell-associated genes are observed in chronic and acute induced Tfh cells, respectively. Blockade of the Th1 cell response by T-cell-intrinsic T-bet deletion promoted Tfh cell expansion during chronic infection, pointing to a correlation between a robust Tfh cell response and protective immunity to parasites. Finally, blockade of Tfh-GC interactions impaired type 2 immunity, revealing the critical protective role of GC-dependent Th2-like Tfh cell responses during acute infection. Collectively, these results provide new insights into the protective roles of Tfh-GC responses and identify distinct transcriptional and epigenetic features of Tfh cells that emerge during resolving or chronic T. muris infection.

Examining behavioural intention towards organ donation in Hong Kong.

Although many people report favourable attitudes towards organ donation, Hong Kong has one of the lowest rates of organ donation globally. The present study examined behavioural intention towards organ donation. A convenience sample of 225 Hong Kong Chinese adults (118 = female) aged 18-82 years were recruited to an online survey. Behavioural intention, attitudes, subjective norms, self-efficacy, knowledge and altruism were examined. ANOVA was conducted to examine key differences based on behavioural intention, regression then examined predictors of behavioural intention to donate before exploratory analysis examined the mediating role of subjective norms on the relationship between self-efficacy and behavioural intention. Findings revealed over one third (38%) of respondents were actively registered as organ donors. Women were significantly more likely to be registered as organ donors. Subjective norms and self-efficacy were strong predictors of behavioural intention to donate, and subjective norms significantly mediated the relationship between self-efficacy and behavioural intention to donate.

The Effect of Maternal Self-Regulated Motivation on Breastfeeding Continuation.

OBJECTIVE: Women frequently report breastfeeding problems in the early postpartum period. Women who have self-endorsed beliefs that breastfeeding benefits their babies and themselves are more likely to continue breastfeeding despite breastfeeding barriers. Maternal self-endorsed beliefs is a key component of maternal self-regulated motivation. The present study examined the association between maternal self-regulated motivation, breastfeeding duration and exclusivity in Chinese women. METHODS: This was a prospective cohort study, of which we recruited participants in postnatal maternity units of publicly funded hospitals in Hong Kong. Postpartum women were asked to fill in the validated breastfeeding self-regulation questionnaire (BSRQ) before hospital discharge and their breastfeeding status was assessed by telephone follow-ups at 6 and 12 weeks postpartum. Multiple logistic regression was used to study the relationship between breastfeeding self-regulated motivation and the duration of breastfeeding at follow-up. RESULTS: At 6 and 12 weeks postpartum, women who breastfed exclusively scored significantly higher in self-regulated motivation than those who formula-fed. The self-regulated motivation was associated with higher odds of exclusive breastfeeding at 6 weeks and any breastfeeding at 12 weeks postpartum. CONCLUSIONS FOR PRACTICE: The study found that self-regulated motivation was positively related to breastfeeding duration. Maternal self-regulated motivation toward breastfeeding could be enhanced by the availability of social support and breastfeeding-friendly facilities, resulting in longer breastfeeding duration.

c-Rel Is Required for IL-33-Dependent Activation of ILC2s.

Group 2 innate lymphoid cells (ILC2s) are emerging as important cellular regulators of homeostatic and disease-associated immune processes. The cytokine interleukin-33 (IL-33) promotes ILC2-dependent inflammation and immunity, with IL-33 having been shown to activate NF-κB in a wide variety of cell types. However, it is currently unclear which NF-κB members play an important role in IL-33-dependent ILC2 biology. Here, we identify the NF-κB family member c-Rel as a critical component of the IL-33-dependent activation of ILC2s. Although c-Rel is dispensable for ILC2 development, it is critical for ILC2 function in the lung, with c-Rel-deficient (c-Rel-/- ) mice present a significantly reduced response to papain- and IL-33-induced lung inflammation. We also show that the absence of c-Rel reduces the IL-33-dependent expansion of ILC2 precursors and lower levels of IL-5 and IL-13 cytokine production by mature ILC2s in the lung. Together, these results identify the IL-33-c-Rel axis as a central control point of ILC2 activation and function.

Rates of Disenrollment From Medicare Advantage Plans Are Higher for Racial/Ethnic Minority Beneficiaries.

BACKGROUND: Each year, about 10% of Medicare Advantage (MA) enrollees voluntarily switch to another MA contract, while another 2% voluntarily switch from MA to fee-for-service Medicare. Voluntary disenrollment from MA plans is related to beneficiaries' negative experiences with their plan, disrupts the continuity of care, and conflicts with goals to reduce Medicare costs. Little is known about racial/ethnic disparities in voluntary disenrollment from MA plans. OBJECTIVE: The objective of this study was to investigate differences in rates of voluntary disenrollment from MA plans by race/ethnicity. SUBJECTS: A total of 116,770,319 beneficiaries enrolled in 736 MA plans in 2015. METHODS: Differences in rates of disenrollment across racial/ethnic groups [Asian or Pacific Islander (API), Black, Hispanic, and White] were summarized using 4 types of logistic regression models: adjusted and unadjusted models estimating overall differences and adjusted and unadjusted models estimating within-plan differences. Unadjusted overall models included only racial/ethnic group probabilities as predictors. Adjusted overall models added age, sex, dual eligibility, disability, and state of residence as control variables. Between-plan differences were estimated by subtracting within-plan differences from overall differences. RESULTS: Adjusted rates of disenrollment were significantly (P<0.001) higher for Hispanic (+1.2 percentage points), Black (+1.2 percentage points), and API beneficiaries (+2.4 percentage points) than for Whites. Within states, all 3 racial/ethnic minority groups tended to be concentrated in higher disenrollment plans. Within plans, API beneficiaries voluntarily disenrolled considerably more often than otherwise similar White beneficiaries. CONCLUSION: These findings suggest the need to address cost, information, and other factors that may create barriers to racial/ethnic minority beneficiaries' enrollment in plans with lower overall disenrollment rates.

Incentivizing Excellent Care to At-Risk Groups with a Health Equity Summary Score.

BACKGROUND: Social risk factors (SRFs) such as minority race-and-ethnicity or low income are associated with quality-of-care, health, and healthcare outcomes. Organizations might prioritize improving care for easier-to-treat groups over those with SRFs, but measuring, reporting, and further incentivizing quality-of-care for SRF groups may improve their care. OBJECTIVE: To develop, as a proof-of-concept, a Health Equity Summary Score (HESS): a succinct, easy-to-understand score that could be used to promote high-quality care to those with SRFs in Medicare Advantage (MA) health plans, which provide care for almost twenty million older and disabled Americans and collect extensive quality measure and SRF data. DESIGN: We estimated, standardized, and combined performance scores for two sets of quality measures for enrollees in 2013-2016 MA health plans, considering both current levels of care, within-plan improvement, and nationally benchmarked improvement for those with SRFs (specifically, racial-and-ethnic minority status and dual-eligibility for Medicare and Medicaid). PARTICIPANTS: All MA plans with publicly reported quality scores and 500 or more 2016 enrollees. MAIN MEASURES: Publicly reported clinical quality and patient experience measures. KEY RESULTS: Almost 90% of plans measured for MA Star Ratings received a HESS; plans serving few patients with SRFs were excluded. The summary score was moderately positively correlated with publicly reported overall Star Ratings (r = 0.66-0.67). High-scoring plans typically had sizable enrollment of both racial-and-ethnic minorities (38-42%) and dually eligible beneficiaries (29-38%). CONCLUSIONS: We demonstrated the feasibility of developing and estimating a HESS that is intended to promote and incentivize excellent care for racial-and-ethnic minorities and dually eligible MA enrollees. The HESS measures SRF-specific performance and does not simply duplicate overall plan Star Ratings. It also identifies plans that provide excellent care to large numbers of those with SRFs. Our methodology could be extended to other SRFs, quality measures, and settings.

Robot-assisted real-time sentinel lymph node mapping in oral cavity cancer: preliminary experience.

This study aims to assess the feasibility of using indocyanine green and robotic near infra-red fluorescent imaging (Firefly®) for sentinel lymph node biopsy in cN0 oral cavity cancer. Ten patients with early squamous cell carcinoma of the tongue (n = 8) and buccal mucosa (n = 2) were included. Peritumoral injection of 10 mg indocyanine green and real-time mapping of sentinel lymph nodes in the neck was performed using Firefly® via a retro-auricular trans-hairline incision. Sentinel lymph node was detected in all patients at 1.2 sentinel lymph node per person. Majority were situated in level II (91.7%). Mean time to detection of sentinel lymph node was 171.0 (68.0-312.0)s. Mean signal-to-background ratio was 5.62 (3.51-7.91). Frozen section of one sentinel lymph node was positive for malignancy, paraffin section of which confirmed the presence of metastatic disease. Modified radical neck dissection was performed for that particular patient, paraffin section of which did not show any tumor deposit. Frozen section and paraffin section of all other sentinel lymph nodes (n = 11) and neck dissection specimens yielded no malignancy. All resection margins were clear. Three patients completed adjuvant radiotherapy for pT2N0 (n = 2) and pT2N1 (n = 1) carcinoma of the tongue. Mean follow-up was 12.0 (4.0-18.0) months. All patients were alive at last follow-up with no disease recurrence. There were no adverse outcomes associated with the use of indocyanine green and robot-assisted neck dissection. Indocyanine green and Firefly® for sentinel lymph node biopsy in cN0 oral cavity cancer is feasible with no adverse effects.

The Methyltransferase DOT1L Controls Activation and Lineage Integrity in CD4+ T Cells during Infection and Inflammation.

CD4+ T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T-cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and are resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th2 cell lineage commitment and stability and suggest that inhibition of DOT1L may provide a therapeutic strategy to limit type 2 immune responses.

CD38 triggers inflammasome-mediated pyroptotic cell death in head and neck squamous cell carcinoma.

BACKGROUND: Pyroptosis is a form of inflammatory cell death. Although it is recognized that NLRP3 (nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3) inflammasome is involved in pyroptosis activation, the mechanism by which head and neck squamous cell carcinoma (HNSCC) inhibits pyroptotic cell death remains undefined. This study aims to delineate the role of calcium regulator CD38 in NLRP3 inflammasome-dependent pyroptosis in HNSCC. METHODS: CD38 overexpressing HNSCC cell lines (SAS, CAL27, SNU899) were generated using lentiviral vectors. NLRP3 and gasdermin D (GSDMD) quantity were detected using Western blot. Caspase-1 activity changes were measured using the Caspase-Glo® 1 inflammasome assay. Cell death proportion was determined by flow cytometry analysis. Proliferation assay was performed using xCELLigence RTCA system. Mouse xenotransplantation was performed to evaluate the potential oncogenic or tumor-suppressive function of CD38. ChIP assay was conducted to verify whether transcription factor NFAT1-mediated NLRP3 expression. RESULTS: Exogenous calcium treatment can lead to a significant increase in caspase-1 activity in HNSCC. This feature was also observed in HNSCC cells with stable CD38 overexpression. CD38-overexpressing cell lines showed a significant reduction in proliferation. Further, expression of NLRP3 protein level was significantly increased in CD38-overexpressing cell lines. The N-terminal effector domain of GSDMD was remarkably increased in the CD38-overexpressing HNSCC. ChIP assay indicated that calcium-sensitive transcription factor NFAT1 was possibly involved in the transcriptional upregulation of NLRP3 observed in CD38-overexpressing HNSCC. The pre-clinical xenograft model revealed that CD38 expression had an inhibiting function on HNSCC progression. CONCLUSION: In conclusion, our results suggested that activation of pyroptosis in HNSCC is a calcium-dependent process. Reduced expression of calcium ion regulator CD38 functions could prevent inflammasome-induced pyroptosis in HNSCC. CD38 may function as a tumor suppressor in HNSCC progression.

The development of a patient-reported functional limitations index.

OBJECTIVES: To develop an easy-to-interpret, patient-reported Functional Limitations Index (FLI) that can be used to assess and monitor the full spectrum of functioning in a community-dwelling population. STUDY DESIGN: Observational design using nationally representative survey data. METHODS: We used self-rated health as a criterion for empirically assigning weights to 5 National Health Interview Survey items assessing difficulty with seeing, hearing, walking, cognition, and self-care. In addition to succinctly summarizing cumulative limitations, we addressed 2 main questions: (1) Which limitations have stronger associations with self-rated health? and (2) How does severity (from 0, no difficulty, to 3, unable to do) relate to self-rated health? We generated a respondent-level summary score based on a model predicting self-rated health from the 5 linearly scored (0-3) items and used splines to account for nonlinear severity-self-rated health associations. RESULTS: The strongest association of specific functional limitations with self-rated health involved mobility; the weakest associations involved sensory limitations. The association of severity with self-rated health was nonlinear and largest moving from no difficulty to somewhat difficult. Nationally, 5% of noninstitutionalized adults were considered most limited, 8% somewhat limited, and 87% least limited. Great mobility limitations (defined as a lot of difficulty or unable to do) most distinguished limitation groups (present in 0% of least limited, 25% of somewhat limited, and 70% of most limited). CONCLUSIONS: The FLI is an easy-to-administer, easy-to-interpret, and valid summary measure of disability that health plans and health care organizations can use for quality-of-care monitoring across a variety of settings to improve care for patients with disabilities.

NADPH oxidase 5α promotes the formation of CD271 tumor-initiating cells in oral cancer.

Oral tongue squamous cell carcinoma (OTSCC) has a distinctive cell sub-population known as tumor-initiating cells (TICs). CD271 is a functional TIC receptor in head and neck cancers. The molecular mechanisms governing CD271 up-regulation remains unclear. Oxidative stress is a contributing factor in TIC development. Here, we explored the potential role of NADPH oxidase 5 (NOX5) and its regulatory mechanism on the development of CD271-expressing OTSCC. Our results showed that the splice variant NOX5α is the most prevalent form expressed in head and neck cancers. NOX5α enhanced OTSCC proliferation, migration, and invasion. Overexpression of NOX5α increased the size of OTSCC xenograft significantly in vivo. The tumor-promoting functions of NOX5α were mediated through the reactive oxygen species (ROS)-generating property. NOX5α activated ERK singling and increased CD271 expression at the transcription level. Also, NOX5α reduces the sensitivity of OTSCC to cisplatin and natural killer cells. The findings indicate that NOX5α plays an important part in the development of TIC in OTSCC.

Glucocorticoid signalling drives reduced versican levels in the fetal mouse lung.

Glucocorticoid (GC) signaling via the glucocorticoid receptor (GR) is essential for lung maturation in mammals. Previous studies using global or conditional mouse model knockouts of the GR gene have established that GR-mediated signaling in the interstitial mesenchyme of the fetal lung is critical for normal lung development. Screens for downstream GC-targets in conditional mesenchymal GR deficient mouse lung (GRmesKO) identified Versican (Vcan), an important extracellular matrix component and cell proliferation regulator, as a potential GR-regulated target. We show that, of the five major VCAN isoforms, the VCAN-V1 isoform containing the GAGß domain is the predominant VCAN isoform in the fetal mouse lung distal mesenchyme at both E16.5 and E18.5, whereas the GAGα-specific VCAN-V2 isoform was only localized to the smooth muscle surrounding proximal airways. Both Vcan-V1 mRNA and protein levels were strongly overexpressed in the GRmesKO lung at E18.5. Finally, we investigated the GC regulation of the ECM protease ADAMTS 12 and showed that Adamts 12 mRNA levels were markedly reduced at E18.5 in GRmesKO fetal mouse lung and were strongly induced by both cortisol and betamethasone in cultures of primary rat fetal lung fibroblasts. ADAMTS12 protein immunoreactivity was also strongly increased in the distal lung at E18.5, after dexamethasone treatment in utero. In summary, glucocorticoid signaling via GR represses GAGß domain-containing VCAN isoforms in distal lung mesenchyme in vivo by repressing Vcan gene expression and, in part, by inducing the ECM protease ADAMTS12, thereby contributing to the control of ECM remodelling and lung cell proliferation prior to birth.

Transplantation of Retinal Ganglion Cells Derived from Male Germline Stem Cell as a Potential Treatment to Glaucoma.

Glaucoma is characterized by retinal ganglion cell (RGC) degeneration and is the second leading cause of blindness worldwide. However, current treatments such as eye drop or surgery have limitations and do not target the loss of RGC. Regenerative therapy using embryonic stem cells (ESCs) holds a promising option, but ethical concern hinders clinical applications on human subjects. In this study, we employed spermatogonial stem cells (SSCs) as an alternative source of ESCs for cell-based regenerative therapy in mouse glaucoma model. We generated functional RGCs from SSCs with a two-step protocol without applying viral transfection or chemical induction. SSCs were first dedifferentiated to embryonic stem-like cells (SSC-ESCs) that resemble ESCs in morphology, gene expression signatures, and stem cell properties. The SSC-ESCs then differentiated toward retinal lineages. We showed SSC-ESC-derived retinal cells expressed RGC-specific marker Brn3b and functioned as bona fide RGCs. To allow in vivo RGC tracing, Brn3b-EGFP reporter SSC-ESCs were generated and the derived RGCs were subsequently transplanted into the retina of glaucoma mouse models by intravitreal injection. We demonstrated that the transplanted RGCs could survive in host retina for at least 10 days after transplantation. SSC-ESC-derived RGCs can thus potentially be a novel alternative to replace the damaged RGCs in glaucomatous retina.