Long-term remission and survival in patients with relapsed or refractory multiple myeloma after treatment with LCAR-B38M CAR T cells: 5-year follow-up of the LEGEND-2 trial.

BACKGROUND: The autologous anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy LCAR-B38M has been approved for the treatment of relapsed and refractory multiple myeloma in many countries across the world under the name ciltacabtagene autoleucel. LEGEND-2 was the first-in-human trial of LCAR-B38M and yielded deep and durable therapeutic responses. Here, we reported the outcomes in LEGEND-2 after a minimal 5-year follow-up. METHODS: Participants received an average dose of 0.5 × 106 cells/kg LCAR-B38M in split or single unfractionated infusions after cyclophosphamide-based lymphodepletion therapy. Investigator-assessed response, survival, safety and pharmacokinetics were evaluated. RESULTS: Seventy-four participants enrolled and had a median follow-up of 65.4 months. The 5-year progression-free survival (PFS) and overall survival (OS) rates were 21.0% and 49.1%, with progressive flattening of the survival curves over time. Patients with complete response (CR) had longer PFS and OS, with 5-year rates of 28.4% and 65.7%, respectively. Twelve patients (16.2%) remained relapse-free irrespective of baseline high-risk cytogenetic abnormality and all had normal humoral immunity reconstituted. An ongoing CR closely correlated with several prognostic baseline indices including favorable performance status, immunoglobulin G subtype, and absence of extramedullary disease, as well as a combination cyclophosphamide and fludarabine preconditioning strategy. Sixty-two (83.8%) suffered progressive disease (PD) and/or death; however, 61.1% of PD patients could well respond to subsequent therapies, among which, the proteasome inhibitor-based regimens benefited the most. Concerning the safety, hematologic and hepatic function recovery were not significantly different between non-PD and PD/Death groups. A low rate of second primary malignancy (5.4%) and no severe virus infection were observed. The patients who tested positive for COVID-19 merely presented self-limiting symptoms. In addition, a sustainable CAR T population of one case with persistent remission was delineated, which was enriched with indolently proliferative and lowly cytotoxic CD4/CD8 double-negative functional T lymphocytes. CONCLUSIONS: These data, representing the longest follow-up of BCMA-redirected CAR T-cell therapy to date, demonstrate long-term remission and survival with LCAR-B38M for advanced myeloma. TRIAL REGISTRATION: LEGEND-2 was registered under the trial numbers NCT03090659, ChiCTRONH-17012285.

Kikuchi-Fujimoto disease evolves into lupus encephalopathy characterized by venous sinus thrombosis: a case report.

Kikuchi-Fujimoto disease (KFD) is a benign, self-limiting illness that can progress to systemic lupus erythematosus (SLE) in approximately 30% of cases. Neurological injuries can occur in both diseases, albeit with distinct presentations. Venous sinus thrombosis is a serious cerebrovascular complication in patients with neuropsychiatric SLE but is rarely observed in patients with KFD. The involvement of various antibodies, particularly antiphospholipid antibodies, can cause vascular endothelial cell injury, resulting in focal cerebral ischemia and intracranial vascular embolism in SLE. However, there are cases in which thrombotic pathology occurs without antiphospholipid antibody positivity, attributed to vascular lesions. In this report, we present a case of KFD and lupus encephalopathy featuring cerebral venous sinus thrombosis, despite the patient being negative for antiphospholipid antibody. We also conducted a comparative analysis of C3 and C4 levels in cerebrospinal fluid (CSF) and peripheral blood, along with the protein ratio in CSF and serum, to elucidate the pathological changes and characteristics of lupus encephalopathy.

A novel mouse model of calcific aortic valve stenosis.

BACKGROUND: Calcific aortic valve stenosis (CAVS) is one of the most challenging heart diseases in clinical with rapidly increasing prevalence. However, study of the mechanism and treatment of CAVS is hampered by the lack of suitable, robust and efficient models that develop hemodynamically significant stenosis and typical calcium deposition. Here, we aim to establish a mouse model to mimic the development and features of CAVS. METHODS: The model was established via aortic valve wire injury (AVWI) combined with vitamin D subcutaneous injected in wild type C57/BL6 mice. Serial transthoracic echocardiography was applied to evaluate aortic jet peak velocity and mean gradient. Histopathological specimens were collected and examined in respect of valve thickening, calcium deposition, collagen accumulation, osteogenic differentiation and inflammation. RESULTS: Serial transthoracic echocardiography revealed that aortic jet peak velocity and mean gradient increased from 7 days post model establishment in a time dependent manner and tended to be stable at 28 days. Compared with the sham group, simple AVWI or the vitamin D group, the hybrid model group showed typical pathological features of CAVS, including hemodynamic alterations, increased aortic valve thickening, calcium deposition, collagen accumulation at 28 days. In addition, osteogenic differentiation, fibrosis and inflammation, which play critical roles in the development of CAVS, were observed in the hybrid model. CONCLUSIONS: We established a novel mouse model of CAVS that could be induced efficiently, robustly and economically, and without genetic intervention. It provides a fast track to explore the underlying mechanisms of CAVS and to identify more effective pharmacological targets.

Development and Validation of an ADA-Tolerant Assay for Quantification of an Exatecan-Based ADC in Monkey Plasma.

BACKGROUND: The development of an anti-drug antibody (ADA)-tolerant pharmacokinetic (PK) assay is important when the drug exposure is irrelevant to toxicity in the presence of ADA. We aimed to develop and validate an ADA-tolerant assay for an exatecan-based antibody-drug conjugate (ADC) in monkey plasma. RESULTS: The assay tolerated 5.00 µg/mL of ADA at 12 µg/mL of ADC. Its accuracy and precision results satisfied the acceptance criteria. Furthermore, the assay was free from hook and matrix effects and exhibited good dilutional linearity. Additionally, the ADC in plasma samples was stable under different storage conditions. METHOD: An ADA-tolerant ADC assay was configured with an anti-payload antibody for capture, and a drug-target protein combined with a horseradish peroxidase (HRP)-labeled antibody against a drug-target-protein tag for detection. Samples were firstly acidified to dissociate drug and ADA complexes, and to convert the carboxylate form to the lactone form of exatecan molecules; then, the ADAs in the samples were removed with a naked antibody-coated microplate. The treated samples were further incubated with coated anti-payload antibody and captured ADC molecules were quantified by the detection reagent. The developed assay was optimized and validated against regulatory guidelines. CONCLUSIONS: The assay met both methodological and sample-related ADA tolerance requirements, and was applicable to a nonclinical study in cynomolgus monkeys.

Separable Microneedle for Integrated Hyperglycemia Sensing and Photothermal Responsive Metformin Release.

Microdevices that offer hyperglycemia monitoring and controllable drug delivery are urgently needed for daily diabetes management. Herein, a theranostic separable double-layer microneedle (DLMN) patch consisting of a swellable GelMA supporting base layer for glycemia sensing and a phase-change material (PCM) arrowhead layer for hyperglycemia regulation has been fabricated. The Cu-TCPP(Fe)/glucose oxidase composite and 3,3',5,5'-tetramethylbenzidine coembedded in the supporting base layer permit a visible color shift at the base surface in the presence of glucose via a cascade reaction, allowing for the in situ detection of glucose in interstitial fluid. The PCM arrowhead layer is encapsulated with water monodispersity melanin nanoparticles from Sepia officinalis and metformin that is imparted with a near-infrared ray photothermal response feature, which is beneficial to the controllable release of metformin for suppression of hyperglycemia. By applying the DLMN patch to the streptozotocin-induced type 2 diabetic Sprague-Dawley rat model, the results demonstrated that it can effectively extract dermal interstitial fluid, read out glucose levels, and regulate hyperglycemia. This DLMN-integrated portable colorimetric sensor and self-regulated glucose level hold great promise for daily diabetes management.

Neutrophil activation and clonal CAR-T re-expansion underpinning cytokine release syndrome during ciltacabtagene autoleucel therapy in multiple myeloma.

Cytokine release syndrome (CRS) is the most common complication of chimeric antigen receptor redirected T cells (CAR-T) therapy. CAR-T toxicity management has been greatly improved, but CRS remains a prime safety concern. Here we follow serum cytokine levels and circulating immune cell transcriptomes longitudinally in 26 relapsed/refractory multiple myeloma patients receiving the CAR-T product, ciltacabtagene autoleucel, to understand the immunological kinetics of CRS. We find that although T lymphocytes and monocytes/macrophages are the major overall cytokine source in manifest CRS, neutrophil activation peaks earlier, before the onset of severe symptoms. Intracellularly, signaling activation dominated by JAK/STAT pathway occurred prior to cytokine cascade and displayed regular kinetic changes. CRS severity is accurately described and potentially predicted by temporal cytokine secretion signatures. Notably, CAR-T re-expansion is found in three patients, including a fatal case characterized by somatic TET2-mutation, clonal expanded cytotoxic CAR-T, broadened cytokine profiles and irreversible hepatic toxicity. Together, our findings show that a latent phase with distinct immunological changes precedes manifest CRS, providing an optimal window and potential targets for CRS therapeutic intervention and that CAR-T re-expansion warrants close clinical attention and laboratory investigation to mitigate the lethal risk.

Comparative study of acetalated-dextran microparticle fabrication methods for a clinically translatable subunit-based influenza vaccine.

The most common influenza vaccines are inactivated viruses produced in chicken eggs, which is a time-consuming production method with variable efficacy due to mismatches of the vaccine strains to the dominant circulating strains. Subunit-based vaccines provide faster production times in comparison to the traditional egg-produced vaccines but often require the use of an adjuvant to elicit a highly protective immune response. However, the current FDA approved adjuvant for influenza vaccines (MF59) elicits a primarily helper T-cell type 2 (Th2)-biased humoral immune response. Adjuvants that can stimulate a Th1 cellular response are correlated to have more robust protection against influenza. The cyclic dinucleotide cGAMP has been shown to provide a potent Th1 response but requires the use of a delivery vehicle to best initiate its signalling pathway in the cytosol. Herein, acetalated dextran (Ace-DEX) was used as the polymer to fabricate microparticles (MPs) via double-emulsion, electrospray, and spray drying methods to encapsulate cGAMP. This study compared each fabrication method's ability to encapsulate and retain the hydrophilic adjuvant cGAMP. We compared their therapeutic efficacy to Addavax, an MF59-like adjuvant, and cGAMP Ace-DEX MPs provided a stronger Th1 response in vaccinated BALB/c mice. Furthermore, we compared Ace-DEX MPs to spray dried MPs composed from a commonly used polymer for drug delivery, poly(lactic-co-glycolic acid) (PLGA). We observed that all Ace-DEX MPs elicited similar humoral and cellular responses to the PLGA MPs. Overall, the results shown here indicate Ace-DEX can perform similarly to PLGA as a polymer for drug delivery and that spray drying can provide an efficient way to produce MPs to encapsulate cGAMP and stimulate the immune system.

A novel automated multi-cycle magnetic solid-phase extraction coupled to LC-MS/MS to study the disorders of six functional B vitamins in patients with gastroenterology and hyperhomocysteinemia.

B vitamins are essential for human life and their disorders can cause a variety of diseases. Solid-phase extraction (SPE) coupled to LC-MS/MS is a preferred technique for determining multiple B vitamins, however, their complexity in real biological matrices makes it hard to achieve satisfactory recovery and accuracy when simultaneous detection. In this study, a novel automated multi-cycle magnetic SPE (MSPE) coupled to the LC-MS/MS method was established using a mixed-mode anion exchange magnetic adsorbent for the simultaneous extraction of six functional B vitamins, including methylmalonic acid, riboflavin, pantothenic acid, 4-pyridoxic acid, folic acid, and 5-methyltetrahydrofolate. After three consecutive MSPE cycles, the recoveries of all analytes were between 51.5% and 89.6%. The method exhibited excellent sensitivity and linearity, with a dynamic range of 200-fold (R > 0.99 for all analytes), exceptional accuracy (ranging between 95.4% and 105.6%) and precision (with RSDs ≤ 6.2%) without significant matrix effects or interferences. Compared to manual SPE method, the automated multi-cycle MSPE method has better feasibility and greater vitamin coverage. It shows a high correlation with the manual method for the detection of 5-methyltetrahydrofolate and folate (R > 0.99). A study of patients from the gastroenterology department showed that those undergoing surgery and those with malignancies may be at risk of folate deficiency. In addition, patients with hyperhomocystinemia had higher levels of methylmalonic acid and lower levels of 5-methyltetrahydrofolate, which correlated with homocysteine levels (R = 0.404 and -0.311, respectively) and showed dose-response relationships. This method is highly automated and cost-effective, with minimal systematic error, making it suitable for the analysis of clinical samples.

The TLR2/TLR6 ligand FSL-1 mitigates radiation-induced hematopoietic injury in mice and nonhuman primates.

Thrombocytopenia, hemorrhage, anemia, and infection are life-threatening issues following accidental or intentional radiation exposure. Since few therapeutics are available, safe and efficacious small molecules to mitigate radiation-induced injury need to be developed. Our previous study showed the synthetic TLR2/TLR6 ligand fibroblast stimulating lipopeptide (FSL-1) prolonged survival and provided MyD88-dependent mitigation of hematopoietic acute radiation syndrome (H-ARS) in mice. Although mice and humans differ in TLR number, expression, and function, nonhuman primate (NHP) TLRs are like those of humans; therefore, studying both animal models is critical for drug development. The objectives of this study were to determine the efficacy of FSL-1 on hematopoietic recovery in small and large animal models subjected to sublethal total body irradiation and investigate its mechanism of action. In mice, we demonstrate a lack of adverse effects, an easy route of delivery (subcutaneous) and efficacy in promoting hematopoietic progenitor cell proliferation by FSL-1. NHP given radiation, followed a day later with a single subcutaneous administration of FSL-1, displayed no adversity but showed elevated hematopoietic cells. Our analyses revealed that FSL-1 promoted red blood cell development and induced soluble effectors following radiation exposure. Cytologic analysis of bone marrow aspirates revealed a striking enhancement of mononuclear progenitor cells in FSL-1-treated NHP. Combining the efficacy of FSL-1 in promoting hematopoietic cell recovery with the lack of adverse effects induced by a single administration supports the application of FSL-1 as a viable countermeasure against H-ARS.

Antigen-induced chimeric antigen receptor multimerization amplifies on-tumor cytotoxicity.

Ligand-induced receptor dimerization or oligomerization is a widespread mechanism for ensuring communication specificity, safeguarding receptor activation, and facilitating amplification of signal transduction across the cellular membrane. However, cell-surface antigen-induced multimerization (dubbed AIM herein) has not yet been consciously leveraged in chimeric antigen receptor (CAR) engineering for enriching T cell-based therapies. We co-developed ciltacabtagene autoleucel (cilta-cel), whose CAR incorporates two B-cell maturation antigen (BCMA)-targeted nanobodies in tandem, for treating multiple myeloma. Here we elucidated a structural and functional model in which BCMA-induced cilta-cel CAR multimerization amplifies myeloma-targeted T cell-mediated cytotoxicity. Crystallographic analysis of BCMA-nanobody complexes revealed atomic details of antigen-antibody hetero-multimerization whilst analytical ultracentrifugation and small-angle X-ray scattering characterized interdependent BCMA apposition and CAR juxtaposition in solution. BCMA-induced nanobody CAR multimerization enhanced cytotoxicity, alongside elevated immune synapse formation and cytotoxicity-mediating cytokine release, towards myeloma-derived cells. Our results provide a framework for contemplating the AIM approach in designing next-generation CARs.

Epinodosin suppresses the proliferation, invasion, and migration of esophageal squamous cell carcinoma by mediating miRNA-143-3p/Bcl-2 axis.

Epinodosin has shown antibacterial and antitumor biological characteristics in the documents. We found that Epinodosin has an effective inhibitory effect on esophageal squamous cell carcinoma (ESCC). However, the potential roles and mechanisms of Epinodosin in ESCC remain unclear. We performed many experiments to clarify the effect and mechanism of Epinodosin on ESCC. In this study, cell viability, invasion, migration, and apoptosis were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,-diphenytetrazoliumromide (MTT), Transwell, and flow cytometry. The differentially expressed miRNAs were screened through RNA transcriptome sequencing. The expression levels of miRNA-143-3p and some proteins were measured by real-time polymerase chain reaction (PCR) and Western blot. The anticancer effects of Epinodosin in vivo were determined by a nude mouse model. Epinodosin suppressed cell proliferation/invasion/migration and induced ESCC cell apoptosis. Epinodosin remarkably affected the protein expression of mitogen-activated protein kinase (MAPK) signaling pathway. The animal experiments demonstrated that Epinodosin could attenuate the growth of ESCC tumors in nude mice. The expression of p53, Bim, and Bax was upregulated, while that of Bcl-2 was downregulated in tumor tissues. In conclusion, Epinodosin suppresses cell viability/invasion/migration, while induces ESCC cell apoptosis by mediating miRNA-143-3p and Bcl-2, and can markedly attenuate the growth of ESCC tumors in nude mice.

Human umbilical cord mesenchymal stem cell­derived exosomes improve ovarian function in natural aging by inhibiting apoptosis.

Prolonging the reproductive lifespan is beneficial for preserving the physical and psychological health of women. The transplantation of mesenchymal stem cell (MSC)­derived exosomes (MSC­Exos) has been reported to be a promising regenerative therapeutic strategy for restoring the function of aging ovaries. The present study thus evaluated the therapeutic efficacy of exosomes derived from human umbilical cord­MSCs (hUCMSC­Exos) in a mouse model of natural ovarian aging (NOA), and further investigated the role of exosomal microRNAs (miRNAs/miRs) in the mechanisms of this creative therapy. Specifically, following the administration of hUCMSC­Exos in mice with NOA, ovarian function was found to improve, as indicated by the restoration of follicle numbers and hormone levels. These exosomes were found to exhibit the ability to inhibit PTEN expression and suppress apoptosis both in vivo and in vitro. Subsequently, miRNA sequencing of the exosomes was performed, following which bioinformatics analysis was used to identify the highly expressed miRNAs that are capable of targeting PTEN expression. Through high­throughput sequencing and molecular analyses, miR­21­5p was found to be the highest in ranking in terms of expression, suggesting that hUCMSC­Exos can preserve ovarian function by suppressing PTEN expression to inhibit apoptosis by delivering miR­21­5p. On the whole, the results of the present study suggest that the application of exosomes can be used to restore ovarian function in mice with NOA. These positive findings also suggest that the transplantation of exosomes derived from MSCs holds promise as an agent against ovarian aging.

Enhanced therapeutic effects of mesenchymal stem cell-derived extracellular vesicles within chitosan hydrogel in the treatment of diabetic foot ulcers.

Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (hUCMSCs) have emerged as promising candidates for cell-free therapy in various diseases, including chronic cutaneous wounds. However, the lack of standardized protocols for EVs' preparation and identification poses a significant challenge to their clinical application. Thus, the objective was to develop a safe and efficient method for the large-scale production of hUCMSC-derived EVs while establishing a comprehensive identification protocol encompassing morphology, particle size distribution, protein expression, and purity. This study observed that most of the EVs acquired through the protocol exhibited either a cup-shaped or round-shaped structure, with a median diameter of ~73.25 nm. The proportions of EVs positive for CD9, CD63, and CD81 were 37.5%, 38.6%, and 19.8%, respectively. To enhance their therapeutic potential in wound treatment, EVs were incorporated into chitosan hydrogel, forming chitosan hydrogel-EVs (CS-EVs). Furthermore, it was demonstrated that CS-EVs exhibited continuous release of EVs into the surrounding environment and, importantly, that the released EVs were internalized by human umbilical vein endothelial cells (HUVECs), resulting in significant enhancement of cell migration and angiogenesis. Additionally, in a rat model of diabetic foot ulcers, CS-EVs demonstrated a robust therapeutic effect in promoting wound healing. Following a 15-day treatment period, the group treated with CS-EVs demonstrated an impressive 93.3% wound closure ability, accompanied by a high degree of re-epithelialization. In contrast, the control group exhibited only a 71.5% reduction in wound size. In summary, this study offers solutions for the purification, characterization, and application of EVs in clinical wound treatment. These results not only offer fresh perspectives on the involvement of hUCMSC-derived EVs in wound healing but also introduce a non-invasive approach for applying EVs that holds practical significance in skin repair.

Smilagenin induces expression and epigenetic remodeling of BDNF in alzheimer's disease.

BACKGROUND: Smilagenin (SMI) is a lipid-soluble steroidal sapogenin, extracted from traditional Chinses medicinal herbs Radix Asparagi, which is extracted from the dry root of Asparagus cochinchinensis (Lour.) Merr. We previously found that SMI significantly increased brain-derived neurotrophic factor (BDNF) expression in Aß-intoxicated SH-SY5Y cells. METHODS: In this study, we performed behavioral tests to analyze cognitive function of WT and APP/PS1 mice treated with or without SMI, and found that SMI could significantly improve the learning and memory ability of APP/PS1 mice. Moreover, immunofluorescence and ELISA results showed that SMI pretreatment could effectively reduce the deposition of ß-amyloid plaques in the cortex and hippocampus of APP/PS1 mice (26 mg/kg/day for 60 days) and inhibit the secretion of Aß1-42 in N2a/APPswe cells (10 µM concentration for 24 hours). RESULTS: Mechanistically, SMI enhanced BDNF mRNA expression, elevated the global level of H3AC and H4AC, and increased the expression of P300 in AD models. Furthermore, chromatin immunoprecipitation results showed that SMI could increase the levels of H3AC and H4AC at the promoter of BDNF promoter Ⅱ and Ⅳ, indicating that SMI epigenetically regulates BDNF expression through HAT enhancement. To further verify the critical role of P300 by which SMI upregulated histone acetylation in BDNF, AD mice were treated with SMI and C646 simultaneously. Behavioral experiments showed that the improvement effects of SMI on cognitive impairment were abolished after P300 inhibition in APP/PS1 mice. CONCLUSIONS: Our research for the first time demonstrated that SMI showed neuroprotective effects by increasing the expression of P300 protein, thus upregulating histone acetylation levels in the promoter region of BDNF and promoting its transcription. Our findings provide an important theoretical basis for the treatment of Alzheimer's disease with SMI extracted from Asparagus cochinchinensis (Lour.) Merr.

Impact of Infection Patterns on the Outcomes of Patients with Hematological Malignancies in Southwest China: A 10-Year Retrospective Case-Control Study.

Background: This study aimed to assess the effect of infection patterns on the outcomes of patients with hematological malignancies (HM) and to identify the determinants of in-hospital mortality. Methods: A case-control study was retrospectively conducted in a tertiary teaching hospital in Chongqing, Southwest China from 2011 to 2020. Clinical characteristics, microbial findings, and outcomes of HM patients with infections were retrieved from the hospital information system. Chi-square or Fisher's exact test was adopted to test the significance of mortality rate. Kaplan-Meier survival analysis and Log rank test were applied to evaluate and compare the 30-day survival rates of those groups. Binary logistic regression, Cox proportional hazards regression, and receiver operating characteristic curves were used to investigate the determinants of in-hospital mortality. Results: Of 1,570 enrolled participants, 43.63% suffered from acute myeloid leukemia, 69.62% received chemotherapy, and 25.73% had hematopoietic stem cell transplantation (HSCT). Microbial infection was documented in 83.38% of participants. Co-infection and septic shock were reported in 32.87% and 5.67% of participants, respectively. Patients with septic shock suffered a significantly lower 30-day survival rate, while those with distinct types of pathogens or co-infections had a comparable 30-day survival rate. The all-cause in-hospital mortality was 7.01% and higher mortality rate was observed in patients with allo-HSCT (7.20%), co-infection (9.88%), and septic shock (33.71%). Cox proportional hazards regression illustrated that elderly age, septic shock, and elevated procalcitonin (PCT) were independent predictors of in-hospital mortality. A PCT cut-off value of 0.24 ng/mL predicted in-hospital mortality with a sensitivity of 77.45% and a specificity of 59.80% (95% CI = 0.684-0.779, P<0.0001). Conclusion: Distinct infectious patterns of HM inpatients were previously unreported in Southwest China. It was the severity of infection, not co-infection, source of infection, or type of causative pathogen that positively related to poor outcome. PCT guided early recognition and treatment of septic shock were advocated.

Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01.

The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.

Human antigen R regulates autophagic flux by stabilizing autophagy-associated mRNA in calcific aortic valve disease.

AIMS: The incidence of calcific aortic valve disease (CAVD) has risen over the last decade and is expected to continue rising; however, pharmacological approaches have proven ineffective. In this study, we evaluated the role and underlying mechanisms of human antigen R (HuR)-mediated post-transcriptional regulation in CAVD. METHODS AND RESULTS: We found that HuR was significantly upregulated in human calcified aortic valves and primary aortic valvular interstitial cells (VICs) following osteogenic stimulation. Subsequent functional studies revealed that HuR silencing ameliorated calcification both in vitro and in vivo. For the first time, we demonstrated that HuR directly interacted with the transcript of phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A), which mediates phosphatidylinositol signalling, facilitates autophagy, and acts as an mRNA stabilizer. HuR positively modulated PIP4K2A expression at the post-transcriptional level and consequently influenced the AKT/mTOR/ATG13 pathway to regulate autophagy and CAVD progression. CONCLUSION: Our study provides new insights into the post-transcriptional regulatory role of HuR in modulating autophagy-positive factors to regulate the pathogenesis of CAVD. Our findings highlight the potential of HuR as an innovative therapeutic target in CAVD treatment.

[Anatomical observation and CT three-dimensional reconstruction of Five-shu acupoints of Shaoyin Heart Meridian in rabbit's forelimb].

OBJECTIVE: To investigate the location and anatomical structure of "Shaochong"(HT9), "Shaofu"(HT8), "Shenmen"(HT7), "Lingdao"(HT4) and "Shaohai"(HT3) in the rabbit's forelimb. METHODS: Sixteen rabbits (half male and half female) were used in the present study. By referring to the national standards on the location of acupoints in the human body and the literature about the location of acupoints in the rabbit, and by using the method of comparative anatomy, the location and needling operation of the Five-shu acupoints of Shaoyin Heart Meridian on the rabbit's forelimb were defined, and these acupoints were needled and CT three-dimensional reconstruction were conducted. Then, the rabbits were killed, and intravascular perfusion was performed, followed by inserting acupuncture needles into these five acupoints for observing the anatomical relationship between the inserted acupuncture needle and the structure of surrounding tissues. RESULTS: HT9 is located at the medial side of the little finger of forelimb, about 1 mm beside the nail root, and is adjacent to the superficial flexor tendon of the finger, the dorsal branches of the proper palmar digital artery and vein, and the endings of dorsal branch of palmar digital proper nerve of the ulnar nerve on the fifth finger side. HT8 is located at the palm side of the forelimb, horizontally parallel to the proximal end of the 5th metacarpophalangeal joint and between the 4th and 5th metacarpal bones, and is adjacent to the lumbricalis, the 4th and 5th interossei, and common palmar digital artery and vein and the palmar digital proper nerve of the ulnar nerve. HT7 is located at the medial margin of the extensor carpal tendon on the ulnar side, between the distal end of the ulna and the ulnar carpal bone, and is adjacent to the tendons of flexor carpi ulnaris and extensor carpi ulnaris, ulnar artery, ulnar vein and ulnar nerve. HT4 is located at the medial border of the ulnar flexor tendon, about 1.5 cun superior to HT7, and is adjacent to extensor carpi ulnaris, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, ulnar artery, vein and ulnar nerve. HT3 is located at the depression, medial to the condyle of humerus when the elbow is bent at 90°, its neighbor structure is composed of pronator teres, biceps brachii, brachial artery and vein, radial collateral artery, radial collateral vein, medial antebrachial cutaneous nerve and median nerve. CONCLUSION: In the rabbit, there is a close relationship between HT9, HT8, HT7, HT4 and HT3 regions and brachial vascular and its branches, cephalic vein and its branches, medial antebrachial cutaneous nerve, median nerve and ulnar nerve, which is the morphological basis of the Five-shu acupoints of Shaoyin Heart Meridian for treating some related clinical disorders.

Orthometric multicolor encoded hybridization chain reaction amplifiers for multiplexed microRNA profiling in living cells.

Multiplexed microRNA (miRNA) profiling of more than four types in living cells is challenging due to fluorescent spectral overlap, representing a significant limitation in studying the complex interactions related to the occurrence and development of diseases. Herein, we report a multiplexed fluorescent imaging strategy based on an orthometric multicolor encoded hybridization chain reaction amplifier named multi-HCR. The targeting miRNA can trigger this multi-HCR strategy due to the specific sequence recognition, and then its self-assembly to amplify the programmability signals. We take the four-colored chain amplifiers, showing that the multi-HCR can form 15 combinations simultaneously. In a living process of hypoxia-induced apoptosis and autophagy under complicated mitochondria and endoplasmic reticulum stress, the multi-HCR demonstrates excellent performance in detecting eight different miRNA changes. The multi-HCR provides a robust strategy for simultaneously profiling multiplexed miRNA biomarkers in studying complicated cellular processes.

Smartphone-Based Photothermal Lateral Flow Immunoassay Using Rhenium Diselenide Nanosheet.

Developing a rapid antibody-based detection method is of great importance for preventing and controlling the spread of coronavirus disease 2019 (COVID-19). Among the antibody-based methods for point-of-care (POC) detection, lateral flow immunoassay (LFIA) is the most widely used. However, LFIA still has the disadvantage of low sensitivity. In this work, an ReSe2 nanosheet with a thickness of 10-20 nm was prepared by liquid exfoliation and applied as the label in a photothermal LFIA due to its high photothermal conversion efficiency and high photothermal stability. An integrated detection device was introduced for rapid, on-site, and highly sensitive assay of the human antisevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein IgG antibodies. The device mainly included a rhenium diselenide (ReSe2) nanosheet-based photothermal LFIA, a portable laser, and a smartphone with a portable thermal imager, which was used to record and analyze the thermal signal of the LFIA test zone. The human anti-SARS-COV-2 S protein IgG antibodies in buffer solution can be detected in a portable box within 10 min, with a thermal signal detection limit of 0.86 ng mL-1, which was 108-fold lower than that of the colorimetric signal. The integrated device can detect values as low as 2.76 ng mL-1 of the human anti-SARS-COV-2 S protein IgG antibodies in 50% serum. The integrated device showed great potential for rapid and home self-testing diagnosis of COVID-19.