Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

CABP4 mutation in mice shows alteration in protein expression level and neuron discharge frequency.

Background: The calcium-binding protein 4 (CABP4) gene is a newly identified epilepsy-related gene that might be associated with a rare type of genetic focal epilepsy; that is, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In vitro, mutant CABP4 causes an increased inward flow voltage of calcium ions and a significant increase in the electrical signal discharge in hippocampus neurons; however, the role of CABP4 in epilepsy has not yet been specifically described, and there is not yet a CABP4 mutant animal model recapitulating the epilepsy phenotype. Methods: We introduced a human CABP4 missense mutation into the C57BL/6J mouse genome and generated a knock-in strain carrying a glycine-to-aspartic acid mutation in the gene. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to evaluate the CABP4 expression level. Slice patch-clamp recording was carried out on pyramidal cells of prefrontal cortex layers II and III. Results: The CABP4G155D/+ mutant mice were viable and born at an expected Mendelian ratio. Surprisingly, the heterozygous (HE) mice did not display either an abnormal appearance or an overt seizure phenotype, and there was no statistically significant difference between the HE and wild-type (WT) mice in terms of overall messenger RNA (mRNA) and protein expression. However, the HE mutant mice showed an imbalance in the amount of protein expressed in the brain regions. Additionally, the patch-clamp recordings from the HE mouse layer II/III cortical pyramidal cells revealed an increase in the frequency of micro-excitatory post-synaptic currents (mEPSCs) but no change in the amplitude was observed. Conclusions: The findings of this study suggest that the CABP4 p.G155D mutation might be one of the mechanisms underlying seizure onset.

Intestinal metabolite xylulose inhibits colorectal cancer by inducing apoptosis through the MAPK signalling pathway.

BACKGROUND: The intestinal metabolites are involved in the initiation, progression and metastasis of colorectal cancer (CRC). They are a potential source of agents for cancer therapy. Our previous study identified altered faecal metabolites between CRC patients and healthy volunteers. However, no specific metabolite was clearly illustrated for CRC therapy. RESULTS: We found that the level of xylulose was lower in the stools of CRC patients than in those of healthy volunteers. Xylulose inhibited cell growth without affecting the cell cycle by inducing apoptosis in CRC cells, which was evidenced by increased expression of the proapoptotic proteins C-PARP and C-Caspase3 and decreased expression of the antiapoptotic protein BCL-2 in CRC cells. Mechanistically, xylulose reduced the activity of the MAPK signalling pathway, represented by reduced phosphorylation of JNK, ERK, and P38. Furthermore, an ALI model was used to show the tumour killing ability of xylulose on human CRC spheres, as well as human colorectal adenoma (AD) spheres. CONCLUSION: Xylulose inhibits CRC growth by inducing apoptosis through attenuation of the MAPK signalling pathway. These results suggest that xylulose may serve as an effective agent for CRC therapy.

Norepinephrine may promote the progression of Fusobacterium nucleatum related colorectal cancer via quorum sensing signalling.

Fusobacterium nucleatum (F. nucleatum) is closely correlated with tumorigenesis in colorectal cancer (CRC). We aimed to investigate the effects of host norepinephrine on the carcinogenicity of F. nucleatum in CRC and reveal the underlying mechanism. The results revealed that both norepinephrine and bacterial quorum sensing (QS) molecule auto-inducer-2 (AI-2) were positively associated with the progression of F. nucleatum related CRC (p < 0.01). In vitro studies, norepinephrine induced upregulation of QS-associated genes and promoted the virulence and proliferation of F. nucleatum. Moreover, chronic stress significantly increased the colon tumour burden of ApcMin/+ mice infected with F. nucleatum (p < 0.01), which was decreased by a catecholamine inhibitor (p < 0.001). Our findings suggest that stress-induced norepinephrine may promote the progression of F. nucleatum related CRC via bacterial QS signalling. These preliminary data provide a novel strategy for the management of pathogenic bacteria by targeting host hormones-bacterial QS inter-kingdom signalling.

Bioactive Compounds from Vicia sativa L. and Vicia monantha Retz. with Unveiling Antiviral Potentials in Newly Green Synthesized CdO Nanoparticles.

BACKGROUND: in the current study, a comparative phytochemical analysis was carried out to explore the phenolic and flavonoid contents in the aerial parts of Vicia sativa L and Vicia monantha Retz growing in cultivated, reclaimed, and desert habitats. METHODS: High-performance liquid chromatography (HPLC) was used to detect Vicia methanolic extracts' individual phenolic and flavonoid constituents. The first-time synthesis of cadmium oxide nanoparticles (CdO NPs) using the aqueous extract of V. monantha has been developed using a green approach. Also, the cytotoxicity of V. monantha extract and CdO NPs was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for unveiling them as anti-HAV and anti-AdV. RESULTS: Our results indicated that in the case of desert habitat, the contents of total phenolics (76.37 mg/g) and total flavonoids (65.23 mg/g) of V. monantha were higher than those of V. sativa (67.35 mg/g and 47.34 mg/g, respectively) and the contents of these secondary metabolites were even increased in V. monantha collected from reclaimed land (phenolics: 119.77 mg/g, flavonoids: 88.61 mg/g). Also, V. monantha surpassed V. sativa in the contents of some individual HPLC constituents, and hence, V. monantha was used to synthesize the green CdO NPs and subsequent antiviral tests. The average size of CdO NPs was determined to be 24.28 nm, and the transmission electron microscopy (TEM) images of CdO NPs clearly showed their spherical form and varying particle sizes, with different diameters in the range of 19-29 nm. MTT assay was positive to the exposure of CdO NPs in the normal cell line, proposing that CdO NPs can reduce cell viability. V. monantha extract showed promising antiviral activity against Hepatitis A virus (HAV) and Adenovirus (AdV) with SI of 16.40 and 10.54. On the other hand, CdONPs had poor antiviral activity against HAV with an SI of 4.74 and moderate antiviral activity against AdV with an SI of 10.54. CONCLUSION: V. monantha is now considered a new, valuable natural resource for phenolics and flavonoids, especially when grown in reclaimed soil. The green CdO NPs based on V. monantha extract showed a promising antiviral effect against HAV and AdV.

Central nervous system anomalies in 41 Chinese children incontinentia pigmenti.

INTRODUCTION: Incontinentia pigmenti (IP) is a rare neuroectodermal dysplasia caused by a defect in the IKBKG gene. The pathogenesis of central nervous system injury is believed to be related to microvascular ischemia. Currently, few treatment strategies are available for the inflammatory phase. MATERIALS AND METHODS: This retrospective descriptive analysis included the clinical data of 41 children with IP collected from 2007 to 2021 in Xi'an, China, comprising clinical characteristics, imaging findings, blood cell analysis, skin histopathology, and genetic data. RESULTS: Fourteen children (34%) aged 4 days to 5 months exhibited clinical signs and symptoms, including convulsions, delayed psychomotor development following neurological damage, and revealed significant MRI abnormalities, including ischemia, hypoxia, cerebral hypoperfusion, hemorrhage, encephalomalacia, and cerebral atrophy. Eight of the 24 patients (33%) presented with retinal vascular tortuosity and telangiectasis, accompanied by neovascularization and hemorrhage. Thirty-eight children (93%) had elevated eosinophils (mean: 3.63 ± 4.46 × 109), and 28 children (68%) had significantly elevated platelets (mean: 420.16 ± 179.43 × 109). Histopathology of skin revealed microvascular extravasation and vasodilation with perivascular and intravascular eosinophilic infiltration. CONCLUSION: Brain injury in IP occurs during infancy until 5 months of age, which is also the acute dermatitis phase accompanied by eosinophilia and an increased platelet count. This study provides evidence of microvascular damage to the skin and fundus during the inflammatory phase. The mechanism of microvascular damage may be similar to that in the brain.

SGLT2i Alleviates Atherosclerosis by Inhibiting NHE1 Activation to Protect against Macrophage Senescence Induced by Angiotensin II.

BACKGROUND: Sodium-dependent glucose transporter (SGLT2) inhibitors (SGLT2i) have been found to have anti-atherosclerotic effects in clinical treatment. OBJECTIVES: The aim of this study was to explore whether angiotensin II (Ang II) induces changes in the expression of Na+/H+ exchanger of cytoplasmic membrane channel proteins (NHE1) and SGLT2 in macrophages and whether dapagliflozin (DAPA), an SGLT2i, protects against Ang II induced macrophage senescence by inhibiting NHE1 activation to alleviate Atherosclerosis (AS). METHODS: After intervention with DAPA plus gavage or feeding them a high-fat diet, the mice's aortas were dissected, and oil red O staining was performed. Cell proliferation and toxicity detection, western blot, immunofluorescence, and ß-galactosidase staining methods were adopted to detect cell activity, expressions of senescence-related genes, and number of senescent cells after different concentrations of Ang II or DAPA or plasmid NHE1 were treated with RAW264.7 cells. RESULTS: (1) The formation of AS plaques in ApoE -/- mice showed a downward trend under DAPA. (2) After the intervention of Ang II, the cell activity of RAW264.7 decreased, and the expression of senescent cells and related genes increased. (3) Under the Ang II condition, the expression of SGLT2 and NHE1 increased, and SGLT2, NHE1, and senescence-related genes decreased with the addition of DAPA. (4) The expression of NHE1, senescent cells and related genes decreased in RAW264.7 cells after DAPA treatment with plasmid NHE1 intervention. CONCLUSION: SGLT2i alleviates atherosclerosis by inhibiting NHE1 activation to protect against macrophage senescence induced by Ang II.

The Norepinephrine-QseC Axis Aggravates F. nucleatum-associated Colitis Through Interkingdom Signaling.

AIMS: Inflammatory bowel disease (IBD) is associated with F. nucleatum, and chronic stress can increase the risk of aggravation. However, whether norepinephrine (NE) can enhance the pathogenicity of F. nucleatum to aggravate dextran sulfate sodium salt (DSS)-induced colitis is unclear. METHODS: Transcriptome sequencing was used to identify differentially expressed genes in bacteria treated with NE. Affinity testing and molecular docking were applied to calculate and predict the binding of NE and Quorum sensing  regulators C (QseC). The pathogenicity of Fusobacterium nucleatum treated with NE and QseC inhibitors was examined in vitro and further verified using the IBD mouse model induced by DSS. RESULTS: Norepinephrine could bind to QseC directly to upregulate the quorum sensing pathway of F. nucleatum and enhance its virulence gene expression (FadA, FomA, Fap2) and invasiveness in vitro. Meanwhile, it promoted the invasion of F. nucleatum into the intestine and increased the expression of host inflammatory cytokines (IL-6, IL-1ß) to aggravate colonic inflammation in IBD mice. The QseC inhibitor LED209 inhibited the effect of NE on F. nucleatum and partially restored short-chain fatty acid (SCFA)-producing bacteria (Prevotellaceae, Lactobacillaceae) to attenuate colonic inflammation in IBD mice. CONCLUSIONS: Generally, the NE-QseC axis enhanced the pathogenicity of F. nucleatum through interkingdom signaling to aggravate colonic inflammation in IBD mice. We see that QseC may be a potential target for microbiota management of IBD under chronic pressure.

Norepinephrine could bind to QseC directly to enhance the pathogenicity of F. nucleatum to aggravate colonic inflammation. The QseC inhibitor inhibited the effect of NE on F. nucleatum and partially restored short-chain fatty acid­producing bacteria to attenuate colonic inflammation.

Improving image quality consistency and diagnostic accuracy in lower extremity CT angiography using a split-bolus contrast injection protocol.

OBJECTIVES: To evaluate the clinical value of using a split-bolus contrast injection protocol in improving image quality consistency and diagnostic accuracy in lower extremity CT angiography (CTA). METHODS: Fifty (mean age, 66 ± 12 years) and 39 (mean age, 66 ± 11 years) patients underwent CTA in the lower extremity arteries using split-bolus and fixed-bolus injection schemes, respectively. The objective and subjective image quality of the 2 groups were compared and the diagnostic efficacy for the degree of vessel stenosis was compared using digital subtraction angiography as the gold standard. A P < .05 was considered statistically significant. RESULTS: In comparison with the fixed-bolus scheme, the split-bolus scheme greatly improved the consistency of image quality of the low extremities by significantly increasing the arterial enhancement (337.87 ± 64.67HU vs. 254.74 ± 71.58HU, P < .001), signal-to-noise ratio (22.58 ± 11.64 vs. 7.14 ± 1.98, P < .001), and contrast-to-noise ratio (37.21 ± 10.46 vs. 31.10 ± 15.40, P = .041) in the infrapopliteal segment. The subjective image quality was better (P < .001) and the diagnostic accuracy was higher in the split-bolus group than in the fixed-bolus group (96.00% vs. 91.67%, P < .05, for diagnosing >50% stenosis, and 97.00% vs. 89.10%, P < .05, for diagnosing occlusion) for the infrapopliteal segment arteries. CONCLUSIONS: Compared with the fixed-bolus injection scheme, the split-bolus injection scheme improves the image quality consistency and diagnostic accuracy especially for the infrapopliteal segment arteries in lower extremity CTA. ADVANCES IN KNOWLEDGE: (1) The split-bolus injection scheme of CTA of the lower extremity arteries improves the overall image quality, uniformity of contrast enhancement. (2) Compared with the fixed-bolus injection scheme, the split-bolus injection scheme especially improves the infrapopliteal segment arteries image quality and diagnostic efficacy.

Characteristics of CXE family of Salvia miltiorrhiza and identification of interactions between SmGID1s and SmDELLAs.

Carboxylesterase (CXE) is a class of hydrolases that contain an α/ß folding domain, which plays critical roles in plant growth, development, and stress responses. Based on the genomic and transcriptomic data of Salvia miltiorrhiza, the SmCXE family was systematically analyzed using bioinformatics. The results revealed 34 SmCXE family members in S. miltiorrhiza, and the SmCXE family could be divided into five groups (Group I, Group II, Group III, Group IV, and Group V). Cis-regulatory elements indicated that the SmCXE promoter region contained tissue-specific and development-related, hormone-related, stress-related, and photoresponsive elements. Transcriptome analysis revealed that the expression levels of SmCXE2 were highest in roots and flowers (SmCXE8 was highest in stems and SmCXE19 was highest in leaves). Further, two GA receptors SmCXE1 (SmGID1A) and SmCXE2 (SmGID1B) were isolated from the SmCXE family, which are homologous to other plants. SmGID1A and SmGID1B have conserved HGGSF motifs and active amino acid sites (Ser-Asp-Val/IIe), which are required to maintain their GA-binding activities. SmGID1A and SmGID1B were significantly responsive to gibberellic acid (GA3) and methyl jasmonate (MeJA) treatment. A subcellular assay revealed that SmCXE1 and SmCXE2 resided within the nucleus. SmGID1B can interact with SmDELLAs regardless of whether GA3 exists, whereas SmGID1A can only interact with SmDELLAs in the presence of GA3. A Further assay showed that the GRAS domain mediated the interactions between SmGID1s and SmDELLAs. This study lays a foundation for further elucidating the role of SmCXE in the growth and development of S. miltiorrhiza.

Imaging and detecting intercellular tensile forces in spheroids and embryoid bodies using lipid-modified DNA probes.

Cells continuously experience and respond to different physical forces that are used to regulate their physiology and functions. Our ability to measure these mechanical cues is essential for understanding the bases of various mechanosensing and mechanotransduction processes. While multiple strategies have been developed to study mechanical forces within two-dimensional (2D) cell culture monolayers, the force measurement at cell-cell junctions in real three-dimensional (3D) cell models is still pretty rare. Considering that in real biological systems, cells are exposed to forces from 3D directions, measuring these molecular forces in their native environment is thus highly critical for the better understanding of different development and disease processes. We have recently developed a type of DNA-based molecular probe for measuring intercellular tensile forces in 2D cell models. Herein, we will report the further development and first-time usage of these molecular tension probes to visualize and detect mechanical forces within 3D spheroids and embryoid bodies (EBs). These probes can spontaneously anchor onto live cell membranes via the attached lipid moieties. By varying the concentrations of these DNA probes and their incubation time, we have first characterized the kinetics and efficiency of probe penetration and loading onto tumor spheroids and stem cell EBs of different sizes. After optimization, we have further imaged and measured E-cadherin-mediated forces in these 3D spheroids and EBs for the first time. Our results indicated that these DNA-based molecular tension probes can be used to study the spatiotemporal distributions of target mechanotransduction processes. These powerful imaging tools may be potentially applied to fill the gap between ongoing research of biomechanics in 2D systems and that in real 3D cell complexes.

The impact of electroacupuncture on anxiety-like behavior and gut microbiome in a mouse model of chronic restraint stress.

Introduction: Electroacupuncture (EA) is a beneficial physiotherapy approach for addressing neuropsychiatric disorders. Nevertheless, the impact of EA on the gut microbiome in relation to anxiety disorders remains poorly understood. Methods: To address this gap, we conducted a study using a chronic restraint stress (CRS) mouse model to investigate the anti-anxiety outcome of EA and its influence on gut microbiota. Our research involved behavioral tests and comprehensive sequencing of full-length 16S rRNA microbiomes. Results: Our findings revealed that CRS led to significant anxiety-like behaviors and an imbalance in the gut microbiota. Specifically, we identified 13 species that exhibited changes associated with anxiety-like behaviors. Furthermore, EA partially alleviated both behaviors related to anxiety and the dysbiosis induced by CRS. Discussion: In summary, this study sheds light on the alterations in gut microbiota species resulting from CRS treatment and brings new light into the connection between EA's anti-anxiety effects and the gut microbiota.

Gut microbiota-derived autoinducer-2 regulates lung inflammation through the gut-lung axis.

OBJECTIVE: This study aimed to determine whether autoinducer-2 (AI-2), a crucial bacterial metabolite and quorum sensing molecule, is involved in lung immunity through the gut-lung axis. METHODS: The level of AI-2 and the gut microbiome composition were analysed in the stools from pneumonic patients and the mouse model of acute lung injury. The effect of AI-2 on lung inflammation was further investigated in the mouse model. RESULTS: The diversity of the faecal microbiota was reduced in pneumonic patients treated with antibiotics compared with healthy volunteers. The AI-2 level in the stool was positively correlated with inflammatory molecules in the serum of pneumonic patients. Intraperitoneal injection of AI-2 reinforced lung inflammation in the acute lung injury mouse model, characterized by increased secretion of inflammatory molecules, including IL-6, IL-1ß, C-C chemokines, and CXCL chemokines, which were alleviated by the AI-2 inhibitor D-ribose. CONCLUSIONS: Our results suggested that gut microbiota-derived AI-2 could modulate lung inflammation through the gut-lung axis.

Effects of MEA Type and Curing Temperature on the Autogenous Deformation, Mechanical Properties, and Microstructure of Cement-Based Materials.

MgO expansive agent (MEA) has the potential to meet the shrinkage compensation demands for concrete in different types of structures due to its designable reactivity and expansion properties. This study investigated the impact of three types of MEAs with different reactivities as well as curing temperature on the autogenous deformation, mechanical properties, and the microstructure of cement-based materials. The results showed that MEA type R exhibits a faster and larger hydration degree and expansion in cement mortars than MEA type M or type S in early ages under 20 °C, while when the curing temperature increases to 40 °C and 60 °C, MEA type M and type S present with significant accelerations in the hydration degree, leading to accelerated expansion rates and significantly increased expansion values compared to MEA type R. Under 40 °C, 5% MEA type M and type S present with 2.2 times and 1.1 times higher expansion in mortars than 5% MEA type R, respectively, and 8% MEA type M and type S present with 7.1 times and 5.6 times higher expansion in mortars than 8% MEA type R, respectively. Under 60 °C, 5% MEA type M and type S present 4.0 times and 3.1 times higher expansion in mortars than 5% MEA type R, respectively, and 8% MEA type M and type S present 7.0 times and 6.6 times higher expansion in mortars than 8% MEA type R, respectively. However, the increase in porosity, especially for large pores with pore size greater than 50 nm as well as the microcracks induced by the 8% dosage of MEA type M, type S, and high curing temperature of 60 °C, result in a decrease in strength of about 30% for the cement mortars. The results indicate that MEA type R is more suitable for shrinkage compensation of cement-based materials with lower temperatures, while MEA type M and type S are more suitable for shrinkage compensation of cement-based materials with higher temperatures. Under high-temperature and low-constraint conditions, the dosage of MEA needs to be strictly controlled to prevent negative effects on the microstructure and strength of cement-based materials.

Targeted RNA condensation in living cells via genetically encodable triplet repeat tags.

Living systems contain various membraneless organelles that segregate proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many protein-based synthetic compartments have been engineered in vitro and in living cells. Here, we introduce a genetically encoded CAG-repeat RNA tag to reprogram cellular condensate formation and recruit various non-phase-transition RNAs for cellular modulation. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes and densities of these cellular RNA condensates. The cis- and trans-regulation functions of these CAG-repeat tags in cellular RNA localization, life time, RNA-protein interactions and gene expression have also been investigated. Considering the importance of RNA condensation in health and disease, we expect that these genetically encodable modular and self-assembled tags can be widely used for chemical biology and synthetic biology studies.

Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs.

Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as "sequential Fluorogenic RNA Imaging-Enabled Sensor" (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study five in vitro orthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in ~20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.

Targeted RNA Condensation in Living Cells via Genetically Encodable Triplet Repeat Tags.

Living systems contain various functional membraneless organelles that can segregate selective proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many synthetic compartments have been engineered in vitro and in living cells, mostly focused on protein-scaffolded systems. Herein, we introduce a nature-inspired genetically encoded RNA tag to program cellular condensate formations and recruit non-phase-transition target RNAs to achieve functional modulation. In our system, different lengths of CAG-repeat tags were tested as the self-assembled scaffold to drive multivalent condensate formation. Various selective target messenger RNAs and noncoding RNAs can be compartmentalized into these condensates. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes, and densities of these cellular RNA condensates. The regulation functions of these CAG-repeat tags on the cellular RNA localization, lifetime, RNA-protein interactions, and gene expression have also been investigated. Considering the importance of RNA condensation in both health and disease conditions, these genetically encodable modular and self-assembled tags can be potentially widely used for chemical biology and synthetic biology studies.

[The Characteristics of T Lymphocyte Reconstitution after Haploid Hematopoietic Stem Cell Transplantation in SAA and Its Relationship with aGVHD].

OBJECTIVE: To investigate the recovery characteristics of T cell subsets in patients with severe aplastic anemia (SAA) who received haploid hematopoietic stem cell transplantation(HSCT) and its relationship with acute graft-versus-host disease(aGVHD). METHODS: The clinical data of 29 SAA patients who received haploid hematopoietic stem cell transplantation in the department of hematology, Shanxi Bethune Hospital from June 2018 to January 2022 were retrospectively analyzed. The absolute counts of CD3+T, CD4+T, CD8+T lymphocytes and the ratio of CD4+T/CD8+T lymphocytes in all patients before transplantation, 14, 21, 30, 60, 90 and 120 days after transplantation were analyzed. The proportion of T lymphocytes was compared in the non-aGVHD group, the grade Ⅰ-Ⅱ aGVHD group and the grade III-IV aGVHD group. RESULTS: The counts of all T cells in 27 patients were far below the normal level at 14 and 21 days after transplantation, but there was obvious heterogeneity. There was a certain relationship between T cell immune reconstitution and conditioning regimen, age, and immunosuppressive treatment before transplantation. CD3+T cells showed a steady upward trend at 30, 60, 90, and 120 days after transplantation, and returned to the normal levels at 120 days after transplantation; faster recovery of CD4+T cells was closely related to aGVHD, which was at 30, 60, 90, 120 days after transplantation showed a slow upward trend, and which was still far below the normal level of 120 days after transplantation. CD8+T cell counts began to recover at 14 and 21 days after transplantation, and the recovery was earlier than the CD4+T cells, and its recovery speed was rapid 30 and 60 days after transptantation, which showed an upward trend and exceeded the normal levels 90 days after transplantation. Since CD8+ T cells reconstituted quickly, while the CD4+ T cells reconstitution was slowly, which made the long-term CD4+T/CD8+T cell ratio after transplantation was inverted . Compared with the non-aGVHD group, the absolute counts of CD3+T, CD4+T, and CD8+T cells in the aGVHD group were significantly higher than those in the non-aGVHD group at each time period after transplantation. In the aGVHD group, grade Ⅲ-Ⅳ aGVHD occurred more frequently in the early post-transplantation period (within 14-21 days), the grade Ⅰ-Ⅱ aGVHD group mostly occurred within 30-90 days after transplantation, and CD3+T, CD4+T, CD8+T cell counts in the grade Ⅲ-Ⅳ aGVHD group were significantly higher than those in the grade Ⅰ-Ⅱ aGVHD group; and the greater the proportion of CD4+T, the more severe the degree of aGVHD. CONCLUSION: The speed of T cell immune reconstitution after SAA haploid transplantation is different, which is related to the conditioning regimen, age, and immunosuppressive therapy before transplantation. The rapid recovery of CD4+ T cells is closely related to the occurrence of aGVHD.