Catalytic Enantioselective Primary C-H Borylation for Acyclic All-Carbon Quaternary Stereocenters.

Creating a perfect catalyst to operate enzyme-like chiral recognition has been a long-sought aim. A challenging example in this context is constructing acyclic all-carbon quaternary stereogenic centers by transition metal-catalyzed enantioselective C-H activation. We now report highly enantioselective iridium-catalyzed primary C-H borylation of α-all-carbon substituted 2,2-dimethyl amides enabled by a tailor-made chiral bidentate boryl ligand (CBL). The success of the current transformation is attributed to the CBL/iridium catalyst, which has a confined chiral pocket. This protocol provides a diverse array of acyclic all-carbon quaternary stereocenters with excellent enantiocontrol and distinct structural features. Computational study reveals that steric hindrance of CBL could regulate the type of dominant orbital interaction between the catalyst and substrate, which is crucial to conferring high chiral induction.

Prostaglandin E2 promotes Th17 differentiation induces corneal epithelial cell apoptosis and participates in the progression of dry eye.

This study is mainly based on T helper type 17 (Th17) cells analysis of the mechanism of prostaglandin E2 (PGE2) promoting the progression of dry eye (DE). Scopolamine and dry environment were used to induce mice DE model. Celecoxib was used to inhibit PGE2. Corneal epithelial cells and CD4+ T cells were used to construct a co-culture system. The osmotic pressure was increased by adding NaCl to simulate DE in vitro. AH6809 and E7046 were used to pre-culture to inhibit EP2/4 in T cells to verify the effect of exogenous PGE2 on Th17 cell differentiation and corneal epithelial cell apoptosis. The function of Th17 cells was analyzed by detecting RORγt and interleukin-17 (IL-17). PGE2 was instilled on the ocular surface to induce DE symptoms of mice. AH6809 and E7046 were used to inhibit EP2/4. The corneal epithelial cell apoptosis was observed by TUNEL. The proportion of Th17 cells in corneal tissue and draining lymph nodes (DLNs) was detected by flow cytometry. In DE mice, the concentration of PGE2 and IL-17 increased in tears, and the proportion of Th17 increased, while inhibition of PGE2 alleviated the symptoms of DE and inhibited Th17 differentiation. Hypertonic environment induces corneal epithelial cells to secrete PGE2. PGE2 promoted the expression of EP2/4 and the differentiation of Th17 cells in vitro. The hypertonic environment promoted PGE2 level and the apoptosis of corneal epithelial cells in the co-culture system. PGE2 alone did not cause corneal epithelial cell apoptosis, while PGE2 promoted apoptosis by promoting Th17. Blocking EP2/4 reduced the induction of Th17 differentiation by PGE2 and the promoted corneal epithelial cell apoptosis. Animal experiments showed that exogenous PGE2 induced DE symptoms. Blocking EP2/4 not only inhibited the proportion of Th17, but also alleviated the apoptosis of corneal epithelial cells caused by PGE2. PGE2 induces aggravation of inflammation by promoting the level of Th17 in the ocular surface, and causes corneal epithelial cell apoptosis, thereby participating in the progression of DE.

Functional hierarchy of the angular gyrus and its underlying genetic architecture.

The angular gyrus (AG), given its rich connectivity and its location where multisensory information converges, is a functionally and anatomically heterogeneous structure. Using the state-of-the-art functional gradient approach and transcription-neuroimaging association analysis, we sought to determine whether there is an overarching hierarchical organization of the AG and if so, how it is modulated by the underlying genetic architecture. Resting-state functional MRI data of 793 healthy subjects were obtained from discovery and validation datasets. Functional gradients of the AG were calculated based on the voxel-wise AG-to-cerebrum functional connectivity patterns. Combined with the Allen Human Brain Atlas, we examined the spatial correlations between the AG functional gradient and gene expression. The dominant gradient topography showed a dorsoanterior-ventroposterior hierarchical organization of the AG, which was related to its intrinsic geometry. Concurrently, AG functional subdivisions corresponding to canonical functional networks (behavioral domains) were distributed along the dominant gradient in a hierarchical manner, that is, from the default mode network (abstract cognition) at one extreme to the visual and sensorimotor networks (perception and action) at the other extreme. Remarkably, we established a link between the AG dominant gradient and gene expression, with two gene sets strongly contributing to this link but diverging on their functional annotation and specific expression. Our findings represent a significant conceptual advance in AG functional organization, and may introduce novel approaches and testable questions to the investigation of AG function and anatomy in health and disease.

Forecasting Tourist Arrivals for Hainan Island in China with Decomposed Broad Learning before the COVID-19 Pandemic.

This study proposes a decomposed broad learning model to improve the forecasting accuracy for tourism arrivals on Hainan Island in China. With decomposed broad learning, we predicted monthly tourist arrivals from 12 countries to Hainan Island. We compared the actual tourist arrivals to Hainan from the US with the predicted tourist arrivals using three models (FEWT-BL: fuzzy entropy empirical wavelet transform-based broad learning; BL: broad Learning; BPNN: back propagation neural network). The results indicated that US foreigners had the most arrivals in 12 countries, and FEWT-BL had the best performance in forecasting tourism arrivals. In conclusion, we establish a unique model for accurate tourism forecasting that can facilitate decision-making in tourism management, especially at turning points in time.

[Research Progress and New Immunotherapy Strategies of Tumor Microenvironment Metabolism].

The tumor microenvironment (TME), the environment of tumorigenesis and tumor progression, incorporates multiple types of cells and non-cellular components. TME plays an important role in tumorigenesis and tumor progression. Due to the abnormal proliferation of tumors, the TME has a unique chemophysiology environment and complex metabolic patterns, which subsequently affects the role of immune cells. Understanding the metabolic patterns of TME can help us develop immunotherapy regimens that target TME. Microbial metabolism and lipid metabolism, the key metabolic processes of TME, have emerged as important foci of research. The metabolites released by the microbiome and the reprogramming of cellular lipid metabolism affect the subsistence of tumor and immune cells. In this review, we summarized the composition and metabolic characteristics of TME and discussed the latest research progress in microbial metabolism and lipid metabolism in TME. We also provided an update on relevant metabolic regulatory targets and immunotherapy strategies, stressing that identifying highly effective therapeutic targets, in spite of the apparent difficulty, is what future research should be focused on.

Genome-wide colocalization of RNA-DNA interactions and fusion RNA pairs.

Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, we compared 16,410 fusion transcripts detected in cancer (from a published cohort of 9,966 tumor samples of 33 cancer types) with genome-wide RNA-DNA interactions mapped in two normal, noncancerous cell types [using iMARGI, an enhanced version of the mapping of RNA-genome interactions (MARGI) assay]. Among the top 10 most significant RNA-DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA-DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA-DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA-DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts.

An Organic Laser Based on Thermally Activated Delayed Fluorescence with Aggregation-Induced Emission and Local Excited State Characteristics.

The spatial separation between the highest occupied and the lowest unoccupied molecular orbitals (HOMO and LUMO) in thermally activated delayed fluorescent (TADF) molecules leads to charge transfer (CT) states, which degrade the oscillator strength of emission transition and sacrifices high solid-state photoluminescence quantum yield (PLQY), together limiting its application in organic solid-state lasers (OSSLs). Here, we demonstrated organic microwire lasers from TADF emitters that combine aggregation induced emission (AIE) and local excited (LE) state characteristics. The unique AIE and LE feature lead to a PLQY approaching 50 % and a high optical gain of 870 cm-1 for TADF microwires. The regenerated singlet excitons by reverse intersystem crossing (RISC) process are conducive to population inversion. As a result, we demonstrated microwire lasers around 465 nm with a low threshold of 3.74 µJ cm-2 . Therefore, our work provides insight to design TADF materials for OSSLs.

Large-scale functional network connectivity mediate the associations of gut microbiota with sleep quality and executive functions.

Network neuroscience has broadly conceptualized the functions of the brain as complex communication within and between large-scale neural networks. Nevertheless, whether and how the gut microbiota influence functional network connectivity that in turn impact human behaviors has yet to be determined. We collected fecal samples from 157 healthy young adults and used 16S sequencing to assess gut microbial diversity and enterotypes. Large-scale inter- and intranetwork functional connectivity was measured using a combination of resting-state functional MRI data and independent component analysis. Sleep quality and core executive functions were also evaluated. Then, we tested for potential associations between gut microbiota, functional network connectivity and behaviors. We found significant associations of gut microbial diversity with internetwork functional connectivity between the executive control, default mode and sensorimotor systems, and intranetwork connectivity of the executive control system. Moreover, some internetwork functional connectivity mediated the relations of microbial diversity with sleep quality, working memory, and attention. In addition, there was a significant effect of enterotypes on intranetwork connectivity of the executive control system, which could mediate the link between enterotypes and executive function. Our findings not only may expand existing biological knowledge of the gut microbiota-brain-behavior relationships from the perspective of large-scale functional network organization, but also may ultimately inform a translational conceptualization of how to improve sleep quality and executive functions through the regulation of gut microbiota.

Germline genetic patterns underlying familial rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren's syndrome highlight T cell-initiated autoimmunity.

OBJECTIVES: Familial aggregation of primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and co-aggregation of these autoimmune diseases (ADs) (also called familial autoimmunity) is well recognised. However, the genetic predisposition variants that explain this clustering remains poorly defined. METHODS: We used whole-exome sequencing on 31 families (9 pSS, 11 SLE, 6 RA and 5 mixed autoimmunity), followed by heterozygous filtering and cosegregation analysis of a family-focused approach to document rare variants predicted to be pathogenic by in silico analysis. Potential importance in immune-related processes, gene ontology, pathway enrichment and overlap analyses were performed to prioritise gene sets. RESULTS: A range from 1 to 50 rare possible pathogenic variants, including 39 variants in immune-related genes across SLE, RA and pSS families, were identified. Among this gene set, regulation of T cell activation (p=4.06×10-7) and T cell receptor (TCR) signalling pathway (p=1.73×10-6) were particularly concentrated, including PTPRC (CD45), LCK, LAT-SLP76 complex genes (THEMIS, LAT, ITK, TEC, TESPA1, PLCL1), DGKD, PRKD1, PAK2 and NFAT5, shared across 14 SLE, RA and pSS families. TCR-interactive genes P2RX7, LAG3, PTPN3 and LAX1 were also detected. Overlap analysis demonstrated that the antiviral immunity gene DUS2 variant cosegregated with SLE, RA and pSS phenotypes in an extended family, that variants in the TCR-pathway genes CD45, LCK and PRKD1 occurred independently in three mixed autoimmunity families, and that variants in CD36 and VWA8 occurred in both RA-pSS and SLE-pSS families. CONCLUSIONS: Our preliminary results define common genetic characteristics linked to familial pSS, SLE and RA and highlight rare genetic variations in TCR signalling pathway genes which might provide innovative molecular targets for therapeutic interventions for those three ADs.

Suppression of the innate cancer-killing activity in human granulocytes by stress reaction as a possible mechanism for affecting cancer development.

Psychological stress may be linked to cancer incidence; however, more direct evidence is required to support this viewpoint. In this study, we investigated the effects of stress on immunosurveillance against cancer cells using a previously established examination stress model. We showed that the cancer killing activity (CKA) of granulocytes (also known as polymorphic nuclear cells, PMNs) is sharply reduced during examination stress stimulation in some donors who are psychologically sensitive to examination stress, with the concentration of plasma stress hormones (cortisone, epinephrine, and norepinephrine) increasing accordingly. The effects of stress hormones on immune cell CKA were also investigated under two in vitro co-incubation conditions, with all three hormones found to exert inhibitory effects on the CKA of PMNs and mononuclear cells. We showed that stress triggered the release of stress hormones which had profound inhibitory effects on the innate anticancer functions of PMNs. These results provide a possible explanation for the relationship between psychological stress and cancer incidence.

Delphinidin induced protective autophagy via mTOR pathway suppression and AMPK pathway activation in HER-2 positive breast cancer cells.

BACKGROUND: We have previously demonstrated the anticancer effect of anthocyanins. In this study, we explored the biological activities of delphinidin, the most common of the anthocyanidin monomers, that were related to autophagy in HER-2 positive breast cancer MDA-MB-453 and BT474 cells. METHODS: The effects of various doses of delphinidin on the proliferation and apoptosis of MDA-MB-453 and BT474 cells were analysed. Autophagy was identified as a critical factor that influenced chemotherapy, and the autophagic mechanism in delphinidin-treated cells was investigated. The autophagy inhibitors, 3-MA and BA1, were used to analyse the effects of autophagy inhibition. RESULTS: Delphinidin inhibited proliferation, promoted apoptosis, and induced autophagy in MDA-MB-453 and BT474 cells in a dose-dependent manner. The inhibition of autophagy enhanced the delphinidin-induced apoptosis and antiproliferative effect in both HER-2 positive breast cancer cells. In addition, delphinidin induced autophagy via suppression of the mTOR signalling pathway and activation of the AMPK signalling pathway in HER-2 positive breast cancer cells. CONCLUSIONS: Collectively, the results showed that delphinidin induced apoptosis and autophagy in HER-2 positive breast cancer cells and that autophagy was induced via the mTOR and AMPK signalling pathways. The suppression of autophagy promoted the anticancer effects of delphinidin.

[Delphinidin induces autophagy in HER-2+ breast cancer cells via inhibition of AKT/mTOR pathway].

OBJECTIVE: To explore the effect of delphinidin on breast cancer and the underlying mechanisms.
 Methods: Human epidermal growth factor receptor-2 (HER-2) positive breast cancer cells MDA-MB-453 were treated by delphinidin. Proliferation of MDA-MB-453 cells was detected by CCK-8 after 48 h. TdT-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to explore apoptotic status for MDA-MB-453 cells. Fluorescence dot assay, immunofluorescence, and Western blot were used to identify autophagy in breast cancer cells.
 Results: Delphinidin suppressed proliferation of MDA-MB-453 cells. Delphinidin increased the number of TUNEL positive cells. Delphinidin downregulated the expression of caspase-3 and caspase-9, while upregulated the expression of cleaved caspase-3 and cleaved caspase-9 in a dose-dependent manner. Delphinidin enhanced the number of GFP-LC3 punctate dots, LC3 immunofluorescence dots and the expression of LC3-II and ATG5. Delphinidin inhibited the expression of proteins in mTOR signaling pathway, including AKT, mTOR, eIF4E and p70s6k.
 Conclusion: Delphinidin induced apoptosis and autophagy by inhibition of AKT/mTOR pathway in HER positive breast cancer cells.

[Geinsten inhibits the proliferation of VCaP castration-resistant prostate cancer cells].

OBJECTIVE: To explore the inhibitory effect of genistein (GEN) on the proliferation of VCaP castration-resistant prostate cancer (CRPC) cells. METHODS: VCaP CRPC cells were treated with GEN at the concentrations of 0, 12.5, 25, 50, 100, and 200 µmol/L for 24, 48, and 72 hours followed by determination of their proliferation by CCK-8 assay and their cycle by flow cytometry. The expression of Ki-67 in the cells was detected by immunocytochemistry and the levels of PSA, Cyclin D1, PCNA, and P53 determined by Western blot. RESULTS: After 72 hours of treatment with GEN at 12.5, 25, 50, 100, and 200 µmol/L, the inhibition rates of the VCaP cells were (25.38±0.02)%, (31.14±0.29)%, (45.27±0.03)%, (52.19±0.05)%, and (68.21±0.19)%, respectively, all significantly higher than in the 0 µmol/L group (ï¼»10.08±0.02ï¼½%)(P<0.05). GEN caused the arrest of the VCaP cells in the G2/M phase (P<0.05) and inhibited the expression of Ki-67. The expressions of PSA, Cyclin D1, and PCNA were gradually down-regulated while that of P53 up-regulated with the increased concentration of GEN (P<0.05). CONCLUSIONS: GEN inhibits the proliferation of VCaP CRPC cells by arresting the cell cycle with related protein expression changes.

[Development and assessment of a dry eye questionnaire applicable to the Chinese population].

OBJECTIVE: To develop and assess a new dry eye questionnaire applicable to the Chinese population. METHODS: Based on literature review and clinical practice, a dry eye questionnaire was developed and optimized to apply to Chinese dry eye patients in the language expression and culture background. Participants (78 patients with dry eye and 82 controls) completed the dry eye questionnaire and the ocular surface disease index (OSDI) questionnaire, and ophthalmic examinations were performed, including slit lamp examination, tear breakup time, fluorescein staining, Schirmer I test and meibomian gland assessment. The original questionnaire was optimized with factor analysis according to the answers from respondents and clinical evaluations. The Cronbach α and intraclass correlation coefficient (ICC) were used to evaluate the internal consistency reliability and test-retest reliability. Factor analysis was used to assess the construct validity, concurrent validity was obtained by Spearman correlation analysis, and discriminant validity was obtained by ANOVA and Wilcoxon rank sum test. Receiver operator characteristics curves were generated to identify the sensitivity and specificity of each questionnaire for diagnosis of dry eye. RESULTS: The questionnaire was optimized to 12 items by factor analysis. The response rate from respondents to the dry eye questionnaire and the OSDI was 100% and 91.25%, respectively. The Cronbachαof the dry eye questionnaire and the OSDI was 0.794 and 0.925, respectively, whilst the ICC of both questionnaires was 0.99, indicating good to excellent reliability. The factor analysis suggested that these two questionnaires had good construct validity. The Spearman correlation analysis indicated that the dry eye questionnaire score correlated positively with the OSDI score (r = 0.812, P < 0.01) and had a greater correlation relationship with the clinical evaluations compared with the OSDI score (r for each was 0.613 and 0.605, P < 0.01). The discriminant validity analysis suggested that there was significant difference in the dry eye questionnaire score between the dry eye group and non-dry eye group (P < 0.01). When the dry eye questionnaire score of 7 was used as the diagnostic threshold, the sensitivity and specificity were 83.33% and 70.73%, respectively, and the area under roc curve was 0.814, which was higher than 0.772 of the OSDI (P < 0.01). CONCLUSION: The dry eye questionnaire we developed is applicable to the Chinese population with Chinese culture characteristics, high reliability, validity, specificity, and sensitivity, and holds a better diagnostic value than the OSDI for Chinese patients with dry eye.

Atypical neurological manifestations in anti-IgLON5 disease: a case report.

Anti-IgLON5 disease is a recently discovered autoimmune encephalopathy with sleep disorder as a hallmark in the majority of reported cases. Additional neurological manifestations include bulbar dysfunction, gait problems, movement disorders, oculomotor abnormalities, and hyperexcitability of the nervous system. At present, an increasing number of publications have dealt with the course and possible treatment options for anti-IgLON5 disease, and its clinical spectrum has expanded wider and more heterogeneous. Here, we report a case of a 66-year-old female with cognitive impairment accompanied by slow reaction, impaired memory, and decreased orientation. A positive cerebral MRI change and serum and cerebrospinal fluid (CSF) antibodies against IgLON5 were found during the diagnostic course. Subsequently the patient received immunotherapy and was generally in good health with no new symptoms during follow-up. Early testing for IgLON5 antibodies should be considered in patients with atypical neurological symptoms such as cognitive impairment, slow reaction, or decreased orientation. In clinical practice, immunotherapy should be considered in all cases of anti-IgLON5 encephalopathies.

Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

BACKGROUND AND PURPOSE: Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis. EXPERIMENTAL APPROACH: A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. KEY RESULTS: Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. CONCLUSION AND IMPLICATIONS: According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

Organoids in ovarian cancer: a platform for disease modeling, precision medicine, and drug assessment.

Ovarian cancer (OC) is a major cause of gynecological cancer mortality, necessitating enhanced research. Organoids, cellular clusters grown in 3D model, have emerged as a disruptive paradigm, transcending the limitations inherent to conventional models by faithfully recapitulating key morphological, histological, and genetic attributes. This review undertakes a comprehensive exploration of the potential in organoids derived from murine, healthy population, and patient origins, encompassing a spectrum that spans foundational principles to pioneering applications. Organoids serve as preclinical models, allowing us to predict how patients will respond to treatments and guiding the development of personalized therapies. In the context of evaluating new drugs, organoids act as versatile platforms, enabling thorough testing of innovative combinations and novel agents. Remarkably, organoids mimic the dynamic nature of OC progression, from its initial formation to the spread to other parts of the body, shedding light on intricate details that hold significant importance. By functioning at an individualized level, organoids uncover the complex mechanisms behind drug resistance, revealing strategic opportunities for effective treatments.

Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells.

RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL (Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13 effectors that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineer Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13 effectors, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.

A case report of a family with developmental arrest of human prokaryotic stage zygote.

To study the genetic variation leading to the arrest phenotype of pronuclear (PN) zygotes. We recruited a family characterized by recurrent PN arrest during in vitro fertilization (IVF) and intracytoplasmic sperm injection cycles (ICSI) and performed whole-exome sequencing for 2 individuals. The transcriptome profiles of PN-arrest zygotes were assessed by single-cell RNA sequencing analysis. The variants were then validated by PCR amplification and Sanger sequencing in the affected individuals and other family members. A family characterized by recurrent PN arrest during IVF and ICSI cycles were enrolled after giving written informed consent. Peripheral blood samples were taken for DNA extraction. Three PN-arrest zygotes from patient III-3 were used for single-cell RNA-seq as described. This phenotype was reproduced after multiple cycles of egg retrieval and after trying different fertilization methods and multiple ovulation regimens. The mutant genes of whole exon sequencing were screened and verified. The missense variant c. C1630T (p.R544W) in RGS12 was responsible for a phenotype characterized by paternal transmission. RGS12 controls Ca2+ oscillation, which is required for oocyte activation after fertilization. Single-cell transcriptome profiling of PN-arrest zygotes revealed defective established translation, RNA processing and cell cycle, which explained the failure of complete oocyte activation. Furthermore, we identified proximal genes involved in Ca2+ oscillation-cytostatic factor-anaphase-promoting complex (Ca2+ oscillation-CSF-APC) signaling, including upregulated CaMKII, ORAI1, CDC20, and CDH1 and downregulated EMI1 and BUB3. The findings indicate abnormal spontaneous Ca2+ oscillations leading to oocytes with prolonged low CSF level and high APC level, which resulted in defective nuclear envelope breakdown and DNA replication. We have identified an RGS12 variant as the potential cause of female infertility characterized by arrest at the PN stage during multiple IVF and ICSI.

Comprehensive analysis of ATP6V1s family member, ATP6V1C2, with prognostic and drug development values in colorectal cancer.

Member of the V-type ATPase family have attracted vast attention in tumor progression. Nevertheless, the specific member of V-ATPase, ATP6V1C2, its regulatory function in colorectal cancer (CRC) progression was poorly understood. In this study, comprehensive analyses demonstrated the role of ATP6V1C2 in CRC progression and drug screening based on ATP6V1C2 was carried out. As a result, among the ATPV1s family, ATP6V1C2 was significantly highly expressed in CRC. Immuno-infiltration analysis suggests that, the interaction between CRC cells and immune cells resulting in reduced immune and estimate scores. GSEA analysis found that, ATP6V1C2 negatively correlates with immune cells，especially CD8T cells. Next, Ecotyper database queries indicated that ATP6V1C2 was negatively correlates with characteristic gene expression in CD8T cells. Then, COX regression analysis and survival curves made it clear that ATP6V1C2 is positively correlates with clinicopathological progression leading to poor CRC prognosis. CellMiner explore told us LOR-253 and Sonidegib may be effective in CRC cancer treatment. Molecular Docking between ATP6V1C2 and 9 first-line and 9 natural drugs showed that ATP6V1C2 was recognized by the best geometrical and energetic matching pattern of 2 First-line and 4 natural drugs. RT-PCR and immunoblotting confirmed that ATP6V1C2 was significantly overexpressed in CRC. Four natural drugs screened by molecular docking were effective in cell proliferation inhibition by CCK8 test. In summary, ATP6V1C2 may be a new therapeutic target for CRC. The illustration is shown in Figure 9.