Application of a newly constructed NGS panel with 45 X-linked microhaplotypes demonstrates the unique value of X-MH for kinship testing and mixture analysis.

X-linked microhaplotypes (X-MHs) have the potential to be a valuable supplementary tool in complex kinship identification or the resolution of DNA mixtures, because they bring together the distinctive genetic pattern of X chromosomal markers and the benefits of microhaplotypes (MHs). In this study, we used the 1000 Genome database to screen and select 63 X-MHs; 18 MHs were filtered out though a batch sequencing assessment of the DNA samples collected from 112 unrelated Chinese Han individuals. The resulting 45-plex panel performed well in comprehensive assessments including repeatability, sensitivity, species specificity, resistance to PCR inhibitors or degradation, mutation rate, and accuracy in detecting DNA mixture samples. The minimum amount of DNA template that can be tested with this panel is 0.5 ng. Additionally, the alleles of the minor contributor can be accurately detected when the mixture rate is larger than 1:9 in female-male mixture or 1:19 in male-male mixture. Then, we calculated population parameters on each MH based on the allele frequency data obtained from the sequence results of the aforementioned 112 unrelated samples. Combining these parameters on each MH, it can be calculated that TDPm, TDPf, CPET, CPEDFM, CPEDFF and CNCEP3 of the 45-plex system were 1-8.99×10-13, 1-1.62×10-19, 0.9999999995, 0.9999981, 0.9955, 0.9999971 and 0.99940, respectively, indicating that the panel is capable in personal identification and parentage testing. To reveal the unique advantage of X-MHs in the analyses of complex kinship and male DNA mixture, further assessments were made. For complex kinship identification, 22 types of individual pairs with different second-degree kinship were simulated and different types of likelihood ratios (LR) were calculated for each. The results revealed that the panel can achieve accuracy of approximately 70 %∼80 % when dividing each of the three types of second-degree kinships into three or four groups. Theoretically, such sub-division cannot be done by using independent autosomal markers. For male DNA mixture analysis without suspects, the maximum likelihood ratio strategy was derived and employed in the estimation of the number of male contributors (NOMC). Simulations were conducted to verify the efficacy of the 45-plex panel in the field and to compare it with autosomal markers by assuming the 45 MHs as autosomal ones. The results showed that X-MHs can achieve higher accuracy in the estimation of NOMC than autosomal ones when the mixed males were unrelated. The results highlighted the unique value of X-linked MHs in complex kinship and male mixture analyses.

Dysregulated Wnt/ß-catenin signaling confers resistance to cuproptosis in cancer cells.

Cuproptosis is characterized by the aggregation of lipoylated enzymes of the tricarboxylic acid cycle and subsequent loss of iron-sulfur cluster proteins as a unique copper-dependent form of regulated cell death. As dysregulation of copper homeostasis can induce cuproptosis, there is emerging interest in exploiting cuproptosis for cancer therapy. However, the molecular drivers of cancer cell evasion of cuproptosis were previously undefined. Here, we found that cuproptosis activates the Wnt/ß-catenin pathway. Mechanistically, copper binds PDK1 and promotes its interaction with AKT, resulting in activation of the Wnt/ß-catenin pathway and cancer stem cell (CSC) properties. Notably, aberrant activation of Wnt/ß-catenin signaling conferred resistance of CSCs to cuproptosis. Further studies showed the ß-catenin/TCF4 transcriptional complex directly binds the ATP7B promoter, inducing its expression. ATP7B effluxes copper ions, reducing intracellular copper and inhibiting cuproptosis. Knockdown of TCF4 or pharmacological Wnt/ß-catenin blockade increased the sensitivity of CSCs to elesclomol-Cu-induced cuproptosis. These findings reveal a link between copper homeostasis regulated by the Wnt/ß-catenin pathway and cuproptosis sensitivity, and suggest a precision medicine strategy for cancer treatment through selective cuproptosis induction.

Modulation of Ras signaling pathway by exosome miRNAs in T-2 toxin-induced chondrocyte injury.

This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21-3p, along with the downregulation of hsa-miR-152-3p and hsa-miR-186-5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.

m1A inhibition fuels oncolytic virus-elicited antitumor immunity via downregulating MYC/PD-L1 signaling.

N1-methyladenosine (m1A) RNA methylation is critical for regulating mRNA translation; however, its role in the development, progression, and immunotherapy response of head and neck squamous cell carcinoma (HNSCC) remains largely unknown. Using Tgfbr1 and Pten conditional knockout (2cKO) mice, we found the neoplastic transformation of oral mucosa was accompanied by increased m1A modification levels. Analysis of m1A-associated genes identified TRMT61A as a key m1A writer linked to cancer progression and poor prognosis. Mechanistically, TRMT61A-mediated tRNA-m1A modification promotes MYC protein synthesis, upregulating programmed death-ligand 1 (PD-L1) expression. Moreover, m1A modification levels were also elevated in tumors treated with oncolytic herpes simplex virus (oHSV), contributing to reactive PD-L1 upregulation. Therapeutic m1A inhibition sustained oHSV-induced antitumor immunity and reduced tumor growth, representing a promising strategy to alleviate resistance. These findings indicate that m1A inhibition can prevent immune escape after oHSV therapy by reducing PD-L1 expression, providing a mutually reinforcing combination immunotherapy approach.

Enhancing Thermoelectric Performance of n-Type Bi2Te2.7Se0.3 through Incorporation of Amorphous Si3N4 Nanoparticles.

Bi2Te3-based thermoelectric (TE) materials are the state-of-the-art compounds for commercial applications near room temperature. Nevertheless, the application of the n-type Bi2Te2.7Se0.3 (BTS) is restricted by the comparatively low figure of merit (ZT) and intrinsic embrittlement. Here, we show that through dispersion of amorphous Si3N4 (a-Si3N4) nanoparticles both 14% increase in power factor (at 300 K) and 48% decrease in lattice thermal conductivity are simultaneously realized. The increased power factor comes from enhanced thermopower and reduced electrical resistivity while the reduced lattice thermal conductivity originates mainly from scattering of middle- and low-frequency phonons at the incorporated a-Si3N4 nanoparticles. As a result, a large ZTmax = 1.19 (at 373 K) and an average ZTave ∼ 1.12 (300-473 K) with better mechanical properties are achieved for the BTS/0.25 wt % Si3N4 sample. Present results demonstrate that the incorporation of a-Si3N4 is a promising way to improve TE performance.

EEPD1 is identified as a predictor of prognosis and immune microenvironment through pan-cancer analysis and related to progression of colorectal cancer.

Background: EEPD1 is vital in homologous recombination, while its role in cancer remains unclear. Methods: We performed multiple pan-cancer analyses of EEPD1 with bioinformatics methods, such as gene expression, gene alterations, Prognosis and enrichment analysis, tumor microenvironment, immune cell infiltration, TMB, MSI, immunotherapy, co-expression of genes, and drug resistance. Finally, RT-qPCR, EdU, and transwell assays helped investigate the impact of EEPD1 on CRC cells. Results: EEPD1 was dysregulated and correlated with bad prognosis in several cancers. GSVA and GSEA revealed that EEPD1 was primarily associated with the "WNT_BETA_CATENIN_SIGNALING," "ribonucleoprotein complex biogenesis," "Ribosome," and "rRNA processing." The infiltration of CD8+ T cells, MAIT cells, iTreg cells, NK cells, Tc cells, Tex cells, Tfh cells, and Th1 cells were negatively correlated with EEPD1 expression. Additionally, EEPD1 is significantly associated with TMB and MSI in COAD, while enhanced CRC cell proliferation and migration. Conclusions: EEPD1 was dysregulated in human cancers and correlated with various cancer patient prognoses. The dysregulated EEPD1 expression can affect tumor-infiltrating immune cells and immunotherapy response. Therefore, EEPD1 could act as an oncogene associated with immune cell infiltration in CRC.

Identification of Novel FZD4 Mutations in Familial Exudative Vitreoretinopathy and Investigating the Pathogenic Mechanisms of FZD4 Mutations.

Purpose: The purpose of this study is to report five novel FZD4 mutations identified in familial exudative vitreoretinopathy (FEVR) and to analyze and summarize the pathogenic mechanisms of 34 of 96 reported missense mutations in FZD4. Methods: Five probands diagnosed with FEVR and their family members were enrolled in the study. Ocular examinations and targeted gene panel sequencing were conducted on all participants. Plasmids, each carrying 29 previously reported FZD4 missense mutations and five novel mutations, were constructed based on the selection of mutations from each domain of FZD4. These plasmids were used to investigate the effects of mutations on protein expression levels, Norrin/ß-catenin activation capacity, membrane localization, norrin binding ability, and DVL2 recruitment ability in HEK293T, HEK293STF, and HeLa cells. Results: All five novel mutations (S91F, V103E, C145S, E160K, C377F) responsible for FEVR were found to compromise Norrin/ß-catenin activation of FZD4 protein. After reviewing a total of 34 reported missense mutations, we categorized all mutations based on their functional changes: signal peptide mutations, cysteine mutations affecting disulfide bonds, extracellular domain mutations influencing norrin binding, transmembrane domain (TM) 1 and TM7 mutations impacting membrane localization, and intracellular domain mutations affecting DVL2 recruitment. Conclusions: We expanded the spectrum of FZD4 mutations relevant to FEVR and experimentally demonstrated that missense mutations in FZD4 can be classified into five categories based on different functional changes.

Characterization of a novel heterozygous frameshift variant in NDP gene that causes familial exudative vitreoretinopathy in female patients.

Familial exudative vitreoretinopathy (FEVR) is a severe inherited disease characterized by defective retinal vascular development. With genetic and clinical heterogeneity, FEVR can be inherited in different patterns and characterized by phenotypes ranging from moderate visual defects to complete vision loss. This study was conducted to unravel the genetic and functional etiology of a 4-month-old female FEVR patient. Targeted gene panel and Sanger sequencing were utilized for genetic evaluation. Luciferase assays, western blot, quantitive real-time PCR, and immunocytochemistry were performed to verify the functional defects in the identified candidate variant. Here, we report a 4-month-old girl with bilateral retinal folds and peripheral avascularization, and identified a novel frameshift heterozygous variant c.37dup (p.Leu13ProfsTer13) in NDP. In vitro experiments revealed that the Leu13ProfsTer13 variant led to a prominent decrease in protein levels instead of mRNA levels, resulting in compromised Norrin/ß-catenin signaling activity. Human androgen receptor assay further revealed that a slight skewing of X chromosome inactivation could partially cause FEVR. Thus, the pathogenic mechanism by which heterozygous frameshift or nonsense variants in female carriers cause FEVR might largely result from a loss-of-function variant in one X chromosome allele and a slightly skewed X-inactivation. Further recruitment of more FEVR-affected females carrying NDP variants and genotype-phenotype correlation analysis can ultimately offer valuable information for the prognosis prediction of FEVR.

Boosting Thermoelectric Performance of PbBi2 Te4 via Reduced Carrier Scattering and Intensified Phonon Scattering.

Materials with low intrinsic lattice thermal conductivity are crucial in the pursuit of high-performance thermoelectric (TE) materials. Here, the TE properties of PbBi2 Te4-x Sex (0 ≤ x ≤ 0.6) samples are systematically investigated for the first time. Doping with Se in PbBi2 Te4 can simultaneously reduce carrier concentration and increase carrier mobility. The Seebeck coefficient is significantly increased by doping with Se, based on the density functional theory calculation, it is shown to be due to the increased bandgap and electronic density of states. In addition, the lattice strain is enhanced due to the difference in the size of Se and Te atoms, and the multidimensional defects formed by Se doping, such as vacancies, dislocations, and grain boundaries, enhance the phonon scattering and reduce the lattice thermal conductivity by about 37%. Finally, by using Se doping to reduce carrier concentration and thermal conductivity, a large ZTmax = 0.56 (at 574K) is achieved for PbBi2 Te3.5 Se0.5 , which is around 64% larger than those of the PbBi2 Te4 pristine sample. This work not only demonstrates that PbBi2 Te4 is a potential medium temperature thermoelectric material, but also provides a reference for enhancing thermoelectric properties through defect and energy band engineering.

The association between DNA methylation and human height and a prospective model of DNA methylation-based height prediction.

As a vital anthropometric characteristic, human height information not only helps to understand overall developmental status and genetic risk factors, but is also important for forensic DNA phenotyping. We utilized linear regression analysis to test the association between each CpG probe and the height phenotype. Next, we designed a methylation sequencing panel targeting 959 CpGs and subsequent height inference models were constructed for the Chinese population. A total of 11,730 height-associated sites were identified. By employing KPCA and deep neural networks, a prediction model was developed, of which the cross-validation RMSE, MAE and R2 were 5.62 cm, 4.45 cm and 0.64, respectively. Genetic factors could explain 39.4% of the methylation level variance of sites used in the height inference models. Collectively, we demonstrated an association between height and DNA methylation status through an EWAS analysis. Targeted methylation sequencing of only 959 CpGs combined with deep learning techniques could provide a model to estimate human height with higher accuracy than SNP-based prediction models.

Investigating the Impact of Dimer Interface Mutations on Norrin's Secretion and Norrin/ß-Catenin Pathway Activation.

Purpose: This study aimed to investigate the impact of 21 NDP mutations located at the dimer interface, focusing on their potential effects on protein assembly, secretion efficiency, and activation of the Norrin/ß-catenin signaling pathway. Methods: The expression level, secretion efficiency, and protein assembly of mutations were analyzed using Western blot. The Norrin/ß-catenin signaling pathway activation ability after overexpression of mutants or supernatant incubation of mutant proteins was tested in HEK293STF cells. The mutant norrin and wild-type (WT) FZD4 were overexpressed in HeLa cells to observe their co-localization. Immunofluorescence staining was conducted in HeLa cells to analyze the subcellular localization of Norrin and the Retention Using Selective Hook (RUSH) assay was used to dynamically observe the secretion process of WT and mutant Norrin. Results: Four mutants (A63S, E66K, H68P, and L103Q) exhibited no significant differences from WT in all evaluations. The other 17 mutants presented abnormalities, including inadequate protein assembly, reduced secretion, inability to bind to FZD4 on the cell membrane, and decreased capacity to activate Norrin/ß-catenin signaling pathway. The RUSH assay revealed the delay in endoplasmic reticulum (ER) exit and impairment of Golgi transport. Conclusions: Mutations at the Norrin dimer interface may lead to abnormal protein assembly, inability to bind to FZD4, and decreased secretion, thus contributing to compromised Norrin/ß-catenin signaling. Our results shed light on the pathogenic mechanisms behind a significant proportion of NDP gene mutations in familial exudative vitreoretinopathy (FEVR) or Norrie disease.

Comprehensive characterization of small noncoding RNA profiles in hypoxia-induced pulmonary hypertension (HPH) rat tissues.

Hypoxia-induced pulmonary hypertension (HPH) is a fatal cardiovascular disease characterized by an elevation in pulmonary artery pressure, resulting in right ventricular dysfunction and eventual heart failure. Exploring the pathogenesis of HPH is crucial, and small noncoding RNAs (sncRNAs) are gaining recognition as potential regulators of cellular responses to hypoxia. In this study, we conducted a comprehensive analysis of sncRNA profiles in eight tissues of male HPH rats using high-throughput sequencing. Our study unveiled several sncRNAs, with the brain, kidney, and spleen exhibiting the highest abundance of microRNA (miRNA), tRNA-derived small RNA (tDR), and small nucleolar RNA (snoRNA), respectively. Moreover, we identified numerous tissue-specific and hypoxia-responsive sncRNAs, particularly miRNAs and tDRs. Interestingly, we observed arm switching in miRNAs under hypoxic conditions and a significant increase in the abundance of 5' tRNA-halves among the total tDRs during hypoxia. Overall, our study provides a comprehensive characterization of the sncRNA profiles in HPH rats.

An approach to unified formulae for likelihood ratio calculation in pairwise kinship analysis.

Introduction: The likelihood ratio (LR) can be an efficient means of distinguishing various relationships in forensic fields. However, traditional list-based methods for derivation and presentation of LRs in distant or complex relationships hinder code editing and software programming. This paper proposes an approach for a unified formula for LRs, in which differences in participants' genotype combinations can be ignored for specific identification. This formula could reduce the difficulty of by-hand coding, as well as running time of large-sample-size simulation. Methods: The approach is first applied to a problem of kinship identification in which at least one of the participants is alleged to be inbred. This can be divided into two parts: i) the probability of different identical by descent (IBD) states according to the alleged kinship; and ii) the ratio of the probability that specific genotype combination can be detected assuming the alleged kinship exists between the two participants to the similar probability assuming that they are unrelated, for each state. For the probability, there are usually recognized results for common identification purposes. For the ratio, subscript letters representing IBD alleles of individual A's alleles are used to eliminate differences in genotype combinations between the two individuals and to obtain a unified formula for the ratio in each state. The unification is further simplified for identification cases in which it is alleged that both of the participants are outbred. Verification is performed to show that the results obtained with the unified and list-form formulae are equivalent. Results: A series of unified formulae are derived for different identification purposes, based on which an R package named KINSIMU has been developed and evaluated for use in large-size simulations for kinship analysis. Comparison between the package with two existing tools indicated that the unified approach presented here is more convenient and time-saving with respect to the coding process for computer applications compared with the list-based approach, despite appearing more complicated. Moreover, the method of derivation could be extended to other identification problems, such as those with different hypothesis sets or those involving multiple individuals. Conclusion: The unified approach of LR calculation can be beneficial in kinship identification field.

Deciphering a crucial dimeric interface governing Norrin dimerization and the pathogenesis of familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease that could cause blindness. It has been established that Norrin forms dimers to activate ß-catenin signaling, yet the core interface for Norrin dimerization and the precise mechanism by which Norrin dimerization contributes to the pathogenesis of FEVR remain elusive. Here, we report an NDP variant, c.265T>C (p.Phe89Leu), that interrupted ß-catenin signaling by disrupting Norrin dimerization. Structural and functional analysis revealed that the Phe-89 of one Norrin monomer interacts with Pro-98, Ser-101, Arg-121, and Ile-123 of another, forming two core symmetrical dimerization interfaces that are pivotal for the formation of a "hand-by-arm" dimer. Intriguingly, we proved that one of the two core symmetrical interfaces is sufficient for dimerization and activation of ß-catenin signaling, with a substantial contribution from the Phe-89/Pro-98 interaction. Further functional analysis revealed that the disruption of both dimeric interfaces eliminates potential binding sites for LRP5, which could be partially restored by over-expression of TSPAN12. In conclusion, our findings unveil a core dimerization interface that regulates Norrin/LRP5 interaction, highlighting the essential role of Norrin dimerization on ß-catenin signaling and providing potential therapeutic avenues for the treatment of FEVR.

Early-onset esophageal squamous cell carcinoma with achalasia: A case report.

RATIONALE: Individuals afflicted with achalasia of the cardia (AC) are more susceptible to the development of esophageal cancer (EC). However, the presence of esophageal retention obscured observation, making it difficult to detect EC early, which leads to misdiagnosis and poor prognosis in AC patients with EC. Besides, the persistence of high-risk factors may have contributed to the rapid progression of EC shortly after per-oral endoscopic myotomy (POEM). Therefore, it is imperative to alert clinicians to this extremely rare and instructive early-onset cancer. PATIENT CONCERNS: The patient was a 67-year-old male who developed dysphagia 3 years ago without obvious causes, with intermittent onset and aggravating trend, accompanied by weight loss. He usually eats high-temperature foods and pickled foods, and has a family history of esophageal squamous cell carcinoma. DIAGNOSIS AND INTERVENTIONS: The patient was initially diagnosed with AC 2 years ago and subsequently underwent POEM surgery. One year after surgery, he was found to have mid-upper EC during follow-up and underwent partial esophagectomy in time. OUTCOMES: The patient's symptoms have significantly improved with weight gain, and he is still adhering to regular follow-up and endoscopic examination. LESSONS: In rare cases, EC develops early in patients with achalasia after POEM surgery. To avoid missed diagnosis, a comprehensive examination to improve the accuracy to diagnose achalasia and identify possible early-onset cancer is very important in clinical practice. Especially for patients with AC who have a family history of EC or other high-risk factors may develop EC early after POEM surgery. Therefore, regular endoscopic follow-up after POEM surgery is essential.

Low Thermal Conductivity and High Thermoelectric Performance of Nb-Doped Quarternary Mixed Crystal Nb0.05W0.95-xMox(Se1-xSx)2.

Transition-metal dichalcogenide WSe2 has attracted increasing interest due to its large thermopower (S), low-cost, and environment-friendly constituents. However, its thermoelectric figure of merit, ZT, of WSe2 is limited due to its large lattice thermal conductivity (κL) and low electrical conductivity. In view of WSe2 and MoS2 having the same crystal structure, here we designed and prepared Nb-doped quarternary mixed crystal (MC) Nb0.05W0.95-xMox(Se1-xSx)2 (0 ≤ x ≤ 0.095). The results indicate that the κL of the MC can reach as low as 0.12 W m K-1 at 850 K, being 93% smaller than that of WSe2. Our analysis reveals that its low κL originates chiefly from intense scattering of both high-frequency phonons from point defects (mainly alloying elements) and mid/low-frequency phonons from MoS2 inclusions residual within MC. In addition, the alloying of WSe2 with MoS2 causes a 5-fold increase in cation vacancies (VW‴'), leading to a large increase in hole concentration and electrical conductivity, which gives rise to a ∼7.5 times increase in power factor (reaching 4.2 µ W cm-1 K-2 at 850 K). As a result, a record high ZTmax = 0.63 is achieved at 850 K for the MC sample with x = 0.076, which is 20 times larger than that of WSe2, demonstrating that MC Nb0.05W0.95-xMox(Se1-xSx)2 is a promising thermoelectric material.

Comparative Analysis of Differentially Expressed Genes in Chondrocytes from Rats Exposed to Low Selenium and T-2 Toxin.

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin-eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD.

Critical view on oligo(dT)-based RNA-seq: bias arising, modeling, and mitigating.

The precise biological interpretation of oligo(dT)-based RNA sequencing (RNA-seq) datasets, particularly in single-cell RNA-seq (scRNA-seq), is invaluable for understanding complex biological systems. However, the presence of biases can lead to misleading results in downstream analysis. This study has now identified two additional biases that are not accounted for in established bias models: poly(A)-tail length bias and fixed-position GC-content bias. These biases have a significant negative impact on the overall quality of oligo(dT)-based RNA-seq data. To address these biases, we have developed a universal bias-mitigating method based on the lower-affinity binding of short and nonanchored oligo(dT) primers to poly(A) tails. This method significantly reduces poly(A) length bias and completely eliminates fixed-position GC bias. Furthermore, the use of short oligo(dT) with impartial binding behavior toward the diverse poly(A) tails renders RNA-seq with more reliable measurements. The findings of this study are particularly beneficial for scRNA-seq datasets, where accurate benchmarking is critical.

Frameshift variants in the C-terminal of CTNNB1 cause familial exudative vitreoretinopathy by AXIN1-mediated ubiquitin-proteasome degradation condensation.

The ß-catenin has two intrinsically disordered regions in both C- and N-terminal domains that trigger the formation of phase-separated condensates. Variants in its C-terminus are associated with familial exudative vitreoretinopathy (FEVR), yet the pathogenesis and the role of these variants in inducing abnormal condensates, are unclear. In this study, we identified a novel heterozygous frameshift variant, c.2104-2105insCC (p.Gln703ProfsTer33), in CTNNB1 from a FEVR-affected family. This variant encodes an unstable truncated protein that was unable to activate Wnt signal transduction, which could be rescued by the inhibition of proteasome or phosphorylation. Further functional experiments revealed the propensity of the Gln703ProfsTer33 variant to form cytoplasmic condensates, exhibiting a lower turnover rate after fluorescent bleaching due to enhanced interaction with AXIN1. LiCl, which specifically blocks GSK3ß-mediated phosphorylation, restored signal transduction, cell proliferation, and junctional integrity in primary human retinal microvascular endothelial cells over-expressed with Gln703ProfsTer33. Finally, experiments on two reported FEVR-associated mutations in the C-terminal domain of ß-catenin exhibited several functional defects similar to the Gln703ProfsTer33. Together, our findings unravel that the C-terminal region of ß-catenin is pivotal for the regulation of AXIN1/ß-catenin interaction, acting as a switch to mediate nucleic and cytosolic condensates formation that is implicated in the pathogenesis of FEVR.

Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.