Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Clonal evolution from B-cell acute lymphoblastic leukemia with BCR::ABL1 multilineage involvement to acute myeloid leukemia after multiple anti-CD19 chimeric antigen receptor T-cell therapy.

Not available.

Adipocyte pyroptosis occurs in omental tumor microenvironment and is associated with chemoresistance of ovarian cancer.

BACKGROUND: Ovarian carcinoma (OC) is a fatal malignancy, with most patients experiencing recurrence and resistance to chemotherapy. In contrast to hematogenous metastasizing tumors, ovarian cancer cells disseminate within the peritoneal cavity, especially the omentum. Previously, we reported omental crown-like structure (CLS) number is associated with poor prognosis of advanced-stage OC. CLS that have pathologic features of a dead or dying adipocyte was surrounded by several macrophages is well known a histologic hallmark for inflammatory adipose tissue. In this study, we attempted to clarify the interaction between metastatic ovarian cancer cells and omental CLS, and to formulate a therapeutic strategy for advanced-stage ovarian cancer. METHODS: A three-cell (including OC cells, adipocytes and macrophages) coculture model was established to mimic the omental tumor microenvironment (TME) of ovarian cancer. Caspase-1 activity, ATP and free fatty acids (FFA) levels were detected by commercial kits. An adipocyte organoid model was established to assess macrophages migration and infiltration. In vitro and in vivo experiments were performed for functional assays and therapeutic effect evaluations. Clinical OC tissue samples were collected for immunochemistry stain and statistics analysis. RESULTS: In three-cell coculture model, OC cells-derived IL-6 and IL-8 could induce the occurrence of pyroptosis in omental adipocytes. The pyroptotic adipocytes release ATP to increase macrophage infiltration, release FFA into TME, uptake by OC cells to increase chemoresistance. From OC tumor samples study, we demonstrated patients with high gasdermin D (GSDMD) expression in omental adipocytes is highly correlated with chemoresistance and poor outcome in advanced-stage OC. In animal model, by pyroptosis inhibitor, DSF, effectively retarded tumor growth and prolonged mice survival. CONCLUSIONS: Omental adipocyte pyroptosis may contribute the chemoresistance in advanced stage OC. Omental adipocytes could release FFA and ATP through the GSDMD-mediate pyroptosis to induce chemoresistance and macrophages infiltration resulting the poor prognosis in advanced-stage OC. Inhibition of adipocyte pyroptosis may be a potential therapeutic modality in advanced-stage OC with omentum metastasis.

Maternal tobacco exposure during pregnancy and atopic dermatitis in offspring: A systematic review and meta-analysis.

The main purpose of this review was to examine the evidence of the relationship between active smoking or passive smoking during pregnancy and atopic dermatitis in offspring. The protocol was written following the PRISMA Checklist and was registered in the PROSPERO database (registration number CRD42022381136). We implemented a comprehensive search in PubMed, Embase and Web of Science databases to identify all potentially related articles from inception through 1 December 2022. We assessed cohort studies and case-control studies using the Newcastle-Ottawa Scale (NOS), and the Joanna Briggs Institute (JBI) critical appraisal tool to assess the quality of cross-sectional studies. Heterogeneity was investigated by using Cochrane Q tests and I2 statistics. In addition, according to the research design, population source and population size, the reasons for the heterogeneity were analysed. A total of 15 observational studies were included in this analysis. Our meta-analysis suggests that atopic dermatitis in offspring is not associated with active smoking during pregnancy (pooled OR, 0.96 [95% CI 0.86-1.07]); however, it is related to passive smoking (OR, 1.52 [95% CI 1.36-1.70]). Passive smoking during pregnancy is associated with an increased risk of eczema development in offspring. More research is needed to explore the risk of active smoking and eczema development in offspring, especially the association between measurements of pregnancy cotinine levels in maternal body fluids and AD in offspring.

The impact of PM2.5 on lung function and chronic respiratory diseases: insights from genetic evidence.

BACKGROUND: PM2.5 has been associated with various adverse health effects, particularly affecting lung function and chronic respiratory diseases. However, the genetic causality relationship between PM2.5 exposure and lung function as well as chronic respiratory diseases remains poorly understood. METHOD: We conducted a two-sample Mendelian randomization analysis to investigate the causal impact of PM2.5 on lung function and chronic respiratory diseases. Instrumental variables were carefully selected, with significance thresholds (P < 5 × 10- 8), and linkage disequilibrium with an r2 value below 0.001. Additionally, SNPs with an F-statistic exceeding 10 were included to mitigate potential bias stemming from weak instrumental variables. The primary analytical approach employed the Inverse Variance Weighted method, supplemented by the Weighted Median, MR-Egger, Simple Model, and Weighted Model. Furthermore, pleiotropy and heterogeneity were evaluated through the MR-Egger intercept test and Cochrane's Q test, with a sensitivity analysis conducted using the leave-one-out method. RESULTS: Eight SNPs significantly associated with PM2.5 exposure were identified as Instrumental variables. Mendelian randomization analysis revealed a significant causal association between PM2.5 exposure and lung function (FEV), with an OR of 0.7284 (95% CI: 0.5799-0.9150). Similarly, PM2.5 exposure demonstrated a substantial causal effect on asthma, with an OR of 1.5280 (95% CI: 1.0470-2.2299). However, no causal association was observed between PM2.5 exposure and chronic obstructive pulmonary disease, with an OR of 1.5176 (95% CI: 0.8294-2.7768). CONCLUSION: These findings emphasize the necessity for continued research efforts in environmental health to develop effective strategies for the prevention and management of chronic respiratory diseases.

Blood biomarkers to distinguish complicated and uncomplicated appendicitis in pediatric patients.

BACKGROUND: /Purpose: Acute appendicitis (AA) stands as the most prevalent cause of acute abdominal pain among children. The potential for morbidity escalates significantly when uncomplicated appendicitis (UA) progresses to complicated appendicitis (CA), which can encompass gangrenous, necrotic, or perforated appendicitis. Consequently, establishing an early and accurate diagnosis of AA, and effectively differentiating CA from UA, becomes paramount. This study explores the diagnostic utility of various blood biomarkers for distinguishing CA from UA in pediatric patients. METHODS: We conducted a retrospective review of medical records pertaining to pediatric patients who underwent surgery for AA. Patients were categorized as either having UA or CA based on histopathological examination of the appendix. The data collected and analyzed included demographic information, white blood cell (WBC) count, neutrophil proportion, lymphocyte proportion, neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR), and C-reactive protein (CRP) levels upon admission. RESULTS: Among the 192 pediatric patients who underwent surgery for AA, 150 were diagnosed with UA, while 42 were diagnosed with CA. The CA group exhibited significantly higher neutrophil proportions, NLRs, PLRs, and CRP levels, alongside lower lymphocyte proportions (all p < 0.01) compared to the UA group. Receiver operating characteristic (ROC) curve analysis disclosed that CRP exhibited the highest specificity, sensitivity, and positive and negative predictive values for predicting CA. CONCLUSION: CRP emerges as a valuable biomarker for differentiating complicated appendicitis from uncomplicated appendicitis.

Size-selective adhesion of calcium oxalate monohydrate crystals to lipid membranes.

The retention of calcium oxalate monohydrate (COM) crystals on cell membranes is pivotal in kidney stone formation. However, the mechanisms underlying COM attachment to neutral lipid membranes remain unclear. In this study, we demonstrate that COM exhibits size-selective adhesion to fluid lipid membranes composed of lipids with distinct sizes. Specifically, the (100) facet of COM induces the formation of new domains and establishes strong adhesion in the 18:1 (Δ9-Cis) PC (DOPC) membrane, while the (010) facet induces domains with strong adhesion in the 16:0-14:0 PC membrane. This selectivity is linked to the compatibility of the area per lipid in DOPC with the unit cell area of the (100) facet and the area per lipid in 16:0-14:0 PC with the (010) facet. Our Raman spectroscopic analyses reveal that the lipid acyl chains within these induced domains exhibit a higher degree of ordering compared to the typical fluid state of the membrane. This ordered structural alignment, combined with the lateral size-matching effect, suggests the potential formation of molecular arrays within the lipid bilayer that are in harmony with the lattice dimension of COM. To elucidate the strong adhesion between calcium oxalate and the phospholipid head group in the absence of a direct molecular structural correspondence, we propose that crystal water associated with COM can form hydrogen bonds with the phospholipid head group. Using structure visualization software, we demonstrate the feasibility of such hydrogen bonding networks. The formation of this network could serve to stabilize and enhance the attachment of COM to the lipid membrane. This mediation by water molecules offers a plausible explanation for the pronounced affinity at the interface.

Bioremediation of trichloroethylene-contaminated groundwater using green carbon-releasing substrate with pH control capability.

In this research, a sustainable substrate, termed green and long-lasting substrate (GLS), featuring a blend of emulsified substrate (ES) and modified rice husk ash (m-RHA) was devised. The primary objective was to facilitate the bioremediation of groundwater contaminated with trichloroethylene (TCE) using innovative GLS for slow carbon release and pH control. The GLS was concocted by homogenizing a mixture of soybean oil, surfactants (Simple Green™ and soya lecithin), and m-RHA, ensuring a gradual release of carbon sources. The hydrothermal synthesis was applied for the production of m-RHA production. The analyses demonstrate that m-RHA were uniform sphere-shape granules with diameters in micro-scale ranges. Results from the microcosm study show that approximately 83% of TCE could be removed (initial TCE concentration = 7.6 mg/L) with GLS supplement after 60 days of operation. Compared to other substrates without RHA addition, higher TCE removal efficiency was obtained, and higher Dehalococcoides sp. (DHC) population and hydA gene (hydrogen-producing gene) copy number were also detected in microcosms with GLS addition. Higher hydrogen concentrations enhanced the DHC growth, which corresponded to the increased DHC populations. The addition of the GLS could provide alkalinity at the initial stage to neutralize the acidified groundwater caused by the produced organic acids after substrate biodegradation, which was advantageous to DHC growth and TCE dechlorination. The addition of m-RHA reached an increased TCE removal efficiency, which was due to the fact that the m-RHA had the zeolite-like structure with a higher surface area and lower granular diameter, and thus, it resulted in a more effective initial adsorption effect. Therefore, a significant amount of TCE could be adsorbed onto the surface of m-RHA, which caused a rapid TCE removal through adsorption. The carbon substrates released from m-RHA could then enhance the subsequent dechlorination. The developed GLS is an environmentally-friendly and green substrate.

CsMYB67 participates in the flavonoid biosynthesis of summer tea leaves.

Flavonoids are important compounds in tea leaves imparting bitter and astringent taste, which also play key roles in tea plants responding to environmental stress. Our previous study showed that the expression level of CsMYB67 was positively correlated with the accumulation of flavonoids in tea leaves as exposed to sunlight. Here, we newly reported the function of CsMYB67 in regulating flavonoid biosynthesis in tea leaves. CsMYB67 was localized in the nucleus and responded to temperature. The results of transient expression assays showed the co-transformation of CsMYB67 and CsTTG1 promoted the transcription of CsANS promoter in the tobacco system. CsTTG1 was bound to the promoter of CsANS based on the results of yeast one-hybrid (Y1H) and transient expression assays, while CsMYB67 enhanced the transcription of CsANS through protein interaction with CsTTG1 according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Thus, CsMYB67-CsTTG1 module enhanced the anthocyanin biosynthesis through up-regulating the transcription of CsANS. Besides, CsMYB67 also enhanced the transcription of CsFLS and CsUFGT through forming transcription factor complexes. The function of CsMYB67 on flavonoid biosynthesis in tea leaves was validated by gene suppression assay. As CsMYB67 was suppressed, the transcriptional level of CsFLS was greatly reduced, leading to a significant increase in the contents of total catechins and total anthocyanidins. Hence, CsMYB67 plays an important role in regulating the downstream pathway of flavonoid biosynthesis in summer tea leaves.

Evaluating the efficacy of balloon pulmonary angioplasty using quantitative analysis of SPECT pulmonary ventilation/perfusion scintigraphy in patients with chronic thromboembolic pulmonary hypertension.

Background: Single-photon emission computed tomography (SPECT) ventilation perfusion imaging is the main imaging method for the diagnosis of pulmonary embolism, and its application in the diagnosis and efficacy evaluation of chronic thromboembolic pulmonary hypertension (CTEPH) has been paid more and more attention. In recent years, with the development of computer software technology, ventilation/perfusion (V/Q) imaging quantitative analysis technology has become more and more mature. The objective of this study was to investigate the utility of quantitative analysis of pulmonary V/Q scintigraphy in evaluating the efficacy of balloon pulmonary angioplasty (BPA) in patients with CTEPH. Methods: In this retrospective analysis, we collected data of patients diagnosed with CTEPH who underwent BPA at the China-Japan Friendship Hospital from April 2018 to September 2020. The sample consisted of 23 males and 28 females, with an average age of 55.1±12.7 years. All patients underwent V/Q scintigraphy within one week before surgery, and we reviewed the pulmonary angiography within 1-3 months following the last BPA procedure. We repeated V/Q scintigraphy within 1 week before or after the pulmonary angiography, at the time of collecting clinical and hemodynamic parameters of these patients. We divided the patients into two groups based on the presence of residual pulmonary hypertension post-surgery and compared the pre- and post-operative quantitative pulmonary perfusion defect percentage scores (PPDs%) using the t-test. Results: In all, 102 V/Q scintigraphy scans were performed in 51 patients. The quantitative PPDs% were positively correlated with the hemodynamic indexes mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance (PVR), and mean right ventricular pressure (RVP) (r=0.605, 0.391, and 0.464, respectively, all P<0.001) and negatively correlated with the 6-minute walking distance (6MWD) (r=-0.254, P=0.010). The average preoperative quantitative PPDs% were (49.0±15.6)% which significantly decreased to (33.5±13.9)% after surgery (t=11.249, P<0.001). The preoperative quantitative PPDs% were (54.7±15.7)% and (44.0±13.8)% in the residual pulmonary hypertension group and the non-residual pulmonary hypertension group, respectively (t=2.599, P=0.012). The postoperative quantitative PPDs% were (41.5±12.5)% and (26.3±11.0)%, in the residual pulmonary hypertension group and the non-residual pulmonary hypertension group, respectively (t=4.647, P<0.001). Conclusions: In this study, we found that quantitative analysis of SPECT pulmonary V/Q scintigraphy adequately reflected the pulmonary artery pressure and clinical status in patients with CTEPH. Our results demonstrate its definite utility in predicting residual pulmonary hypertension and in evaluating the postoperative efficacy of BPA in patients with CTEPH.

Investigations of the prognostic value of RUNX1 mutation in acute myeloid leukemia patients: Data from a real-world study.

RUNX1 is one of the recurrent mutated genes in newly diagnosed acute myeloid leukemia (AML). Although historically recognized as a provisional distinct entity, the AML subtype with RUNX1 mutations (AML-RUNX1mut) was eliminated from the 2022 WHO classification system. To gain more insight into the characteristics of AML-RUNX1mut, we retrospectively analyzed 1065 newly diagnosed adult AML patients from the First Affiliated Hospital of Soochow University between January 2017 and December 2021. RUNX1 mutations were identified in 112 patients (10.5%). The presence of RUNX1 mutation (RUNX1mut) conferred a lower composite complete remission (CRc) rate (40.2% vs. 58.4%, P＜0.001), but no significant difference was observed in the 5-year overall survival (OS) rate (50.2% vs. 53.9%; HR=1.293; P=0.115) and event-free survival (EFS) rate (51.5% vs. 49.4%; HR=1.487, P=0.089), even within the same risk stratification. Multivariate analysis showed that RUNX1mut was not an independent prognostic factor for OS (HR=1.352, P=0.068) or EFS (HR=1.129, P=0.513). When patients were stratified according to induction regimen, RUNX1mut was an unfavorable factor for CRc both on univariate and multivariate analysis in patients receiving conventional chemotherapy, and higher risk stratification predicted worse OS. In those who received venetoclax plus hypomethylating agents, RUNX1mut was not predictive of CRc and comparable OS and EFS were seen between intermediate-risk and adverse-risk groups. The results of this study revealed that the impact of RUNX1mut is limited. Its prognostic value depended more on treatment and co-occurrent abnormalities. VEN-HMA may abrogate the prognostic impact of RUNX1, which merits a larger prospective cohort to illustrate.

C-C chemokine receptor 4 (CCR4)-positive regulatory T cells interact with tumor-associated macrophages to facilitate metastatic potential after radiation.

PURPOSE: Our previous study revealed that elevated C-C motif chemokine ligand 2 (CCL2) secretion by irradiated cancer cells recruited C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and polarized M2-type tumor-associated macrophages (TAMs), promoting lung metastasis in an established mouse model. This study investigated the impact of CCL2 and TAMs on adaptive immunity. METHODS: We assessed the influence of CCL2 and TAMs on adaptive immunity through two ectopic allograft mouse models constructed with MB49 bladder cancer cells and Lewis lung carcinoma cells. Both models exhibited delayed primary tumor growth following radiation therapy (RT), but RT promoted the development of pulmonary metastases in C57BL/6 mice. Additionally, we employed a direct coculture system to investigate the interaction between macrophages and target cells in the context of adaptive immunity. RESULTS: C-C motif chemokine receptor 4 (CCR4)-positive regulatory T cells (Tregs) were recruited to the postirradiated tumor microenvironment (TME). Utilizing a CCR4 antagonist to inhibit CCL2-CCR4 activation reversed the infiltration of CCR4 + Tregs and reduced the incidence of pulmonary metastases. In addition, a positive feedback loop between M2-type TAMs and Tregs was observed. The combined blockade of the CCL2-CCR4 and CCL2-CCR2 signaling pathways further decreased the risk of RT-promoted lung metastasis. CONCLUSION: The recruitment of CCR4 + Tregs to the postirradiated TME increases the metastatic potential of tumor cells through increased interactions with M2-type TAMs. A significant reduction in post-RT lung metastases in ectopic mouse models was achieved by disrupting the recruitment of both CCR4 + Tregs and CCR2 + myeloid cells, which are TAM precursors.