[Clinical Significance of Genetic and Molecular Changes in Primary Myeloid Sarcoma].

OBJECTIVE: To investigate the clinical significance of genetic and molecular changes in primary myeloid sarcoma (MS). METHODS: Fourteen patients with primary MS were selected in Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, The First People's Hospital of Lianyungang from September 2010 to December 2021. AML1-ETO fusion, PML-RARα fusion and CBFß breakage were detected by fluorescence in situ hybridization (FISH), and the mutations of NPM1, CEBPA, FLT3, RUNX1, ASXL1, KIT and TP53 genes were detected by new generation sequencing (NGS). RESULTS: Among 14 patients, the MS occurred in bone, breast, epididymis, lung, chest wall, cervix, small intestine, ovary, lymph nodes and central nervous system. The tumor cells expressed MPO (13 cases), CD34 (7 cases), CD43 (8 cases), CD68 (7 cases), CD99 (8 cases) and CD117 (6 cases). Cytogenetic abnormalities were observed in 4 cases, including 3 cases of AML1-ETO fusion and 1 case of CBFß breakage, while no PML-RARα fusion was detected. There were no significant differences in overall survival (OS) and leukemia-free survival (LFS) between patients with and without AML1-ETO fusion/CBFß breakage (both P >0.05). Among the 14 patients, the number of NPM1, CEBPA, FLT3-ITD, RUNX1, ASXL1, KIT and TP53 gene mutations was 5, 3, 5, 3, 2, 2, 1, respectively, of which 7 cases had at least one mutation in FLT3-ITD, RUNX1, ASXL1 and TP53 gene. The OS and LFS of patients with FLT3-ITD, RUNX1, ASXL1 or TP53 mutation were shorter than those without mutations (both P <0.01). CONCLUSION: The genetic and molecular abnormalities of primary MS can be detected by FISH and NGS techniques. FLT3-ITD, RUNX1, ASXL1 or TP53 mutation indicates a worse prognosis, but further clinical studies are needed to confirm it.

A Grading System of Microvascular Invasion for Patients with Hepatocellular Carcinoma Undergoing Liver Resection with Curative Intent: A Multicenter Study.

Background: Microvascular invasion (MVI) is closely correlated with poor clinical outcomes in patients with hepatocellular carcinoma (HCC). A grading system of MVI is needed to assist in the management of HCC patient. Methods: Multicenter data of HCC patients who underwent liver resection with curative intent was analyzed. This grading system was established by detected number and distance from tumor boundary of MVI. Survival outcomes were compared among patients in each group. This system was verified by time-receiver operating characteristic curve, time-area under the curve, calibration curve, and decision curve analyses. Cox regression analysis was performed to study the associated factors of prognosis. Logistic analysis was used to study the predictive factors of MVI. Results: All patients were classified into 4 groups: M0: no MVI; M1: 1~5 proximal MVIs (≤1 cm from tumor boundary); M2a: >5 proximal MVIs (≤1 cm from tumor boundary); M2b: ≥1 distal MVIs (>1 cm from tumor boundary). The recurrence-free survival (RFS), overall survival (OS), and early RFS rates among all the individual groups were significantly different. Based on the number of proximal MVI (0~5 vs >5), patients in the M2b group were further divided into two subgroups which also showed different prognosis. Multiple methods showed this grading system to be significantly better than the MVI two-tiered system in prognostic evaluation. Four multivariate models for RFS, OS, early RFS, late RFS, and a predictive model of MVI were then established and were shown to satisfactorily evaluate prognosis and have a great discriminatory power, respectively. Conclusion: This MVI grading system could precisely evaluate prognosis of HCC patients after liver resection with curative intent and it could be employed in routine pathological reports. The severity of MVI from both adjacent and distant from tumor boundary should be stated. A hypothesis about two occurrence modes of distal MVI was proposed.

Multiomics analyses of two Leonurus species illuminate leonurine biosynthesis and its evolution.

The Lamiaceae family is renowned for its terpenoid-based medicinal components, but Leonurus, which has traditional medicinal uses, stands out for its alkaloid-rich composition. Leonurine, the principal active compound found in Leonurus, has demonstrated promising effects in reducing blood lipids and treating strokes. However, the biosynthetic pathway of leonurine remains largely unexplored. Here, we present the chromosome-level genome sequence assemblies of Leonurus japonicus, known for its high leonurine production, and Leonurus sibiricus, characterized by very limited leonurine production. By integrating genomics, RNA sequencing, metabolomics, and enzyme activity assay data, we constructed the leonurine biosynthesis pathway and identified the arginine decarboxylase (ADC), uridine diphosphate glucosyltransferase (UGT), and serine carboxypeptidase-like (SCPL) acyltransferase enzymes that catalyze key reactions in this pathway. Further analyses revealed that the UGT-SCPL gene cluster evolved by gene duplication in the ancestor of Leonurus and neofunctionalization of SCPL in L. japonicus, which contributed to the accumulation of leonurine specifically in L. japonicus. Collectively, our comprehensive study illuminates leonurine biosynthesis and its evolution in Leonurus.

Secular Trends in Sleep Conditions in Chinese Elderly Individuals: A National Population-Based Study.

Purpose: As one of the most rapidly aging countries in the world, the elderly population is expected to reach over 400 million in China by 2032. Many studies have suggested a positive association between sleep duration and adverse health events among elderly individuals. This study aimed to investigate the sleep conditions of Chinese elderly individuals between 2005 and 2018. Patients and methods: Data for 53,013 elderly individuals were taken from five cycles of the Chinese Longitudinal Healthy Longevity Survey (CLHLS) during 2005-2018. Sex- and age-specific means and 95% confidence intervals (95% CIs) were used to estimate sleep duration trends. Changes in sleep patterns were explored during this period. The prevalence of short and long sleep durations was assessed and age-standardized by the 2010 census. Finally, self-reported sleep quality was used to determine sleep conditions from another perspective among elderly individuals. Results: The mean sleep duration decreased from 7.87 (95% CI: 7.83-7.91) to 7.29 (95% CI: 7.25-7.33) hours between 2005 and 2018. Changes in sleep duration patterns were found during the study period. The proportion of the elderly population who slept ≤6 hours increased and that of those who slept ≥9 hours decreased noticeably over the past 13 years. The age-standardized prevalence of short sleep duration increased from 32.7% (95% CI: 32.7-32.9%) to 38.4% (95% CI: 38.3-38.5%). A significant decrease was observed in the prevalence of long sleep duration. Conclusion: Sleep conditions are gradually shifting toward a shorter sleep duration and poorer sleep quality among Chinese elderly individuals.

CD1d-Dependent Natural Killer T Cells Mediate Hypertension and Vascular Injury Through Interleukin-17A.

Background Different T-lymphocyte subsets, including CD1d-dependent natural killer T (NKT) cells, play distinct roles in hypertension, highlighting the importance of identifying key immune cells for its treatment. This study aimed to determine the unknown effects of CD1d-dependent NKT cells on hypertension and vascular injury. Methods and Results Hypertension models were induced in male CD1d knockout (CD1dko), wild-type, and adoptive bone marrow transfer mice by angiotensin II (Ang II) or deoxycorticosterone acetate salt. Blood pressure was measured by the tail-cuff system and radiotelemetry. Vascular injury was assessed by histologic studies or aortic ring assay. Inflammation was detected by flow cytometry, quantitative real-time polymerase chain reaction, or ELISA. Results showed that Ang II infusion significantly reduced CD1d expression and NKT cell numbers in the aorta of mice. CD1dko mice exhibited worsened blood pressure elevation, vascular injury, and inflammatory response induced by Ang II or deoxycorticosterone acetate salt. However, these effects were markedly reversed in wild-type mice treated with NKT cell-specific activator. Adoptive transfer of CD1dko bone marrow cells to wild-type mice also significantly worsened Ang II-induced responses. Mechanistically, CD1dko increased Ang II-induced interleukin-6 production and activated signal transducer and activator of transcription 3 and orphan nuclear receptor γ, subsequently inducing interleukin-17A production. Neutralizing interleukin-17A partially reversed Ang II-induced hypertension and vascular injury in CD1dko mice. In addition, levels of NKT cells were lower in the blood of patients with hypertension (n=57) compared with normotensive individuals (n=87). Conclusions These findings reveal a previously unknown role for CD1d-dependent NKT cells in hypertension and vascular injury, indicating that NKT cell activation could be a promising therapeutic target for hypertension.

CD1d-dependent natural killer T-cells inactivation aggravates sepsis-induced myocardial injury via T lymphocytes infiltration and IL-6 production in mice.

Myocardial edema mediated by endothelial dysfunction plays an important role in sepsis-induced cardiomyopathy (SIC); however, its mechanism is unclear. The current study aimed to provide evidence on the cardioprotection of CD1d-dependent natural killer T (NKT) cells and clarify the possible mechanism in a mouse model of sepsis. Wild-type (WT) and CD1d-dependent NKT-cells inactivation (CD1dko) mice were subjected to sepsis induced by intraperitoneal injection of lipopolysaccharide (LPS). The NKT-cells number and CD1d expression were both increased in the hearts and blood of WT mice after LPS treatment. Compared with WT mice, CD1dko mice exhibited remarkably accelerated LPS-induced mortality, cardiac dysfunction, myocardial injury, endothelial apoptosis, microvascular damage, microvascular permeability and cardiac edema. Mechanistically, CD1d deficiency further increased LPS-induced accumulation of T lymphocytes in the myocardium and upregulation of IL-6 protein levels. Administration of an IL-6 neutralizing antibody to CD1dko mice improved cardiac dysfunction, myocardial injury and edema induced by LPS. Our study identified that CD1d-dependent NKT-cells inactivation exacerbated SIC via T lymphocytes infiltration and IL-6 production. Hence, activation of CD1d-dependent NKT cells may be a potential candidate strategy for SIC treatment.

Screening epitope peptides based on a phage-displayed random peptide and peptide microarrays to contribute to improving the diagnostic efficiency of systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is one of the most common autoimmune diseases in China. At present, there are hundreds of autoantibodies in SLE patients; however, only a dozen of the autoantibodies can be routinely detected, and the available diagnostic antibodies are not sufficient for diagnosis or differential diagnosis of SLE patients with atypical clinical manifestations or other autoimmune diseases. Therefore, it is necessary to find new diagnostic markers to improve the diagnostic effect of SLE. METHODS: The displayed random peptide library and peptide microarray were combined to identify SLE-related epitope peptides. A case-control design was used. The IgG antibodies in the sera from SLE patients, healthy controls, and other autoimmune disease controls underwent a reaction with the phage-display random peptide library, respectively. Selected epitope peptides were used to construct a peptide chip. A total of 644 serum samples (including 296 SLE patients, 168 disease controls, and 180 healthy controls) were used for further screening and verification. Peptides with an area under the curve (AUC) > 0.650 were further verified by ELISA. Finally, 500 serum samples (including 200 SLE patients, 150 disease controls, and 150 healthy controls) were used to verify and evaluate the diagnostic and differential diagnostic efficacy of the selected peptides. RESULTS: After the previous screening, five epitope peptides (SLE_P19, SLE_P20, SLE_P27, SLE_P28, and SLE_P29) may have potential as SLE diagnostic markers. Additionally, SLE_P27 was superior to the other four peptides in the diagnosis and differential diagnosis of SLE and rheumatoid arthritis (RA). The AUC of SLE_P27 was 0.938, the sensitivity was 76.00%, the specificity was 92.70%, the positive likelihood ratio was 10.411, the negative likelihood ratio was 0.259, and the accuracy was 84.40%. The diagnostic efficacy of SLE can be increased by combining the five selected peptides with the anti-double stranded DNA antibody (anti-dsDNA) and anti-Smith antibodies (anti-Sm). CONCLUSIONS: In this study, we identified five peptides that may serve as potential biomarkers for SLE diagnosis using the strategy of combining the displayed random peptide library with the peptide microarray. The combination of selected peptides and existing autoantibodies can significantly improve the diagnostic efficiency. These specific peptides are expected to be new diagnostic markers for SLE.

Successful treatment of patients with refractory idiopathic membranous nephropathy with low-dose Rituximab: A single-center experience.

BACKGROUND: The recognition of idiopathic membranous nephropathy (IMN) as an autoimmune disease has paved the way for the use of B-cell-depleting agents, such as Rituximab (RTX), which is now a first-line drug for treating IMN with proven safety and efficacy. Nevertheless, the usage of RTX for the treatment of refractory IMN remains controversial and challenging. AIM: To evaluate the efficacy and safety of a new low-dose RTX regimen for the treatment of patients with refractory IMN. METHODS: A retrospective study was performed on refractory IMN patients that accepted a low-dose RTX regimen (RTX, 200 mg, once a month for five months) in the Xiyuan Hospital of Chinese Academy of Chinese Medical Sciences' Department of Nephrology from October 2019 to December 2021. To assess the clinical and immune remission data, we performed a 24 h urinary protein quantification (UTP) test and measured the serum albumin (ALB) and serum creatinine (SCr) levels, phospholipase A2 receptor (PLA2R) antibody titer, and CD19+ B-cell count every three months. RESULTS: A total of nine refractory IMN patients were analyzed. During follow-up conducted twelve months later, the results from the 24 h UTP decreased from baseline [8.14 ± 6.05 g/d to 1.24 ± 1.34 g/d (P < 0.05)] and the ALB levels increased from baseline [28.06 ± 8.42 g/L to 40.93 ± 5.85 g/L (P < 0.01)]. Notably, after administering RTX for six months, the SCr decreased from 78.13 ± 16.49 µmol/L to 109.67 ± 40.87 µmol/L (P < 0.05). All of the nine patients were positive for serum anti-PLA2R at the beginning, and four patients had normal anti-PLA2R titer levels at six months. The level of CD19+ B-cells decreased to 0 at three months, and CD19+ B-cell count remained at 0 up until six months of follow-up. CONCLUSION: Our low-dose RTX regimen appears to be a promising treatment strategy for refractory IMN.

Spontaneous ventilation anesthesia combined with uniportal and tubeless thoracoscopic sympathectomy in selected patients with primary palmar hyperhidrosis.

BACKGROUND: To assess the feasibility and safety of tubeless video-assisted thoracoscopic sympathectomy (VATS) with a single 5 mm port under nonintubated, intravenous anesthesia with spontaneous ventilation in selected patients with primary palmar hyperhidrosis (PPH). METHODS: Adults (aged between 18 and 60 years) with moderate or severe PPH symptoms were enrolled. Demographic information and clinical data were obtained from 172 consecutive patients undergoing thoracoscopic surgery for PPH from March 2014 to December 2020. The primary outcomes were the rate of complications, including death, and the intraoperative conversion rate to 3-port VATS. The secondary outcomes were the conversion rate to intubated anesthesia during the operation and the surgical duration and pain score of postoperative day 0. RESULTS: In total, 172 patients were included with 88 males and 84 females. The median age was was 25 years (IQR:21-30 years). No mortalities or major morbidities occurred in any patient. The overall median surgical duration was 53 min (IQR:37-72 min). The median length of postoperative hospital stay was one day (IQR:one-one day). The median pain score of POD0 was 2 (IQR:2-2). Intraoperative conversion to 3-port VATS followed by drainage tube insertion occurred in one (0.6%) patient due to extensive pleural adhesions. No patients required conversion to intubated anesthesia during surgery. No postoperative mechanical ventilation was noted in any patient. CONCLUSIONS: For selected patients with PPH, tubeless VATS with a single 5 mm port using spontaneous ventilation anesthesia can be considered a feasible and safe operation. The surgical wound is extremely small and the operation time is shorter than the conventional technique. Trial registration This study was in conformity with the Declaration of Helsinki, and was approved by the National Ethics Committee of the University of the Hong Kong-Shenzhen Hospital (Approval number: [2020]70). We registered the study in the Chinese Clinical Trial Registry (Registration number: ChiCTR2100049063) in 2021.Informed consent was collected from all the participants of this study. URL for this clinical trial registration is: https://www.chictr.org.cn/index.aspx .

Correction: Transcriptional Effects of E3 Ligase Atrogin-1/MAFbx on Apoptosis, Hypertrophy and Inflammation in Neonatal Rat Cardiomyocytes.

[This corrects the article DOI: 10.1371/journal.pone.0053831.].

Transplantation of rat frozen-thawed testicular tissues: Does fragment size matter?

Cryopreservation of testicular tissue from pre-pubertal boys before gonadotoxic treatment is an important step in fertility preservation. Yet, this approach remains experimental, and there is still few study measuring the effect of tissue size on the graft after cryopreservation and transplantation. The objective of this study is to detect the effect of varying tissue sizes on the efficacy of rat testicular tissue cryopreservation and transplantation. Varying sizes of rat testicular tissues were frozen-thawed and autografted. At the 30th day after grafting, the grafts were collected for histology assessment and immunohistochemistry assay for MAGE-A4 (germ cell marker) and CD34 (blood vessel marker). The transplant recovery, seminiferous tubule integrity, tubular diameter, spermatogonia number, and microsvessel density in testicular fragments sizing in 3 mm in length, 3 mm wide, and 3 mm in thickness were significantly lower than other groups. Whereas, the absorption rate of graft sizing in 1 mm in length, 1 mm in wide, and 1 mm in thickness was significantly higher than other groups. Testicular fragment sizing in 2-3 mm in length, 2-3 mm in wide, and 2 mm in thickness (8 mm3-18 mm3) is suitable for rat testicular tissue cryopreservation and transplantation.

Erratum: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy: Erratum.

[This corrects the article DOI: 10.7150/thno.27706.].

Targeting against the activity of the NLRP3 inflammasome is a potential therapy for rat testicular tissue cryopreservation and transplantation.

The objective of the present experiment was to explore the role of NLRP3 inflammasome in the testicular tissue freezing, thawing and grafting; furthermore, the potential effect of a NLRP3 inhibitor on the function of testis transplant was explored. Tissues from male Wistar rats in pre-pubertal age were cryopreserved, thawed and auto-transplanted into the scrotum treated or not treated with the MCC950 (a NLRP3 inhibitor). After grafting, cryopreserved tissue was removed and analysed. Quantitative morphometric, immunohistochemical techniques and Western blotting were used to evaluate the survival of spermatogonia and the activation of the NLRP3 inflammasome after freezing/thawing/grafting. Moreover, serum IL-1ß level was assessed with ELISA kits. The testicular transplants exhibited upregulated expression of the NLRP3 pathway meditors (NLRP3, IL-1ß). In NLRP3 inhibition group, the rate of recovered grafts, the percentage of intact tubules and spermatogonial number were significantly higher than that in cryopreserved graft group. Moreover, serum concentration of IL-1ß in NLRP3 inhibition group was significantly lower than that in cryopreserved graft group. Testicular tissue cryopreservation and transplantation exhibited upregulated expression of NLRP3 pathway and NLRP3 inflammasome blockade improves testicular graft function. These finding suggest that NLRP3 inflammasome is a therapeutic target for testicular tissue cryopreservation and transplantation.

Testicular damage during cryopreservation and transplantation.

The aim of this study is to do a study of cryoinjury and ischaemic injury on testicular graft during cryopreservation and transplantation. According to time at 1, 3, 7 and 14 days after transplantation, the grafts were collected for immunohistochemistry assay for CD34 (blood vessel marker), VEGF (neoangiogenesis marker), caspase-3 (apoptosis marker) MAGE-A4 (germ cell marker). A significant increase was observed in the density of VEGF-positive blood vessels on day 3, reached a peak on day 7. On post-transplant day 3, a sharp increase occurred in the rate of spermatogonia-expressing caspase-3 until the day 7. At 14th day after transplantation, the spermatogonia number per round tubule of nonfrozen grafts was 41 ± 5.9% from that of fresh control tissues, while, in frozen-thawed grafts, the spermatogonia number per round tubule was 36.8 ± 4.6% from that of fresh control tissues. In testicular grafts, angiogenesis initiated reperfusion from day 3, and the formation of new blood vessel generally is completed about 7 days after transplantation. Angiogenesis in grafts after transplantation plays a crucial role in the restoration of function. Therefore, minimising ischaemic injury as well as improvement of cryopreservation protocols are needed to improve testicular graft after freezing, thawing and grafting.

Dynamic Changes in Plasma Urotensin II and Its Correlation With Plaque Stability.

ABSTRACT: Urotensin II (UII) is involved in the formation of atherosclerosis, but its role in the stability of atherosclerotic plaques is unknown. The purpose of this study was to observe the dynamic changes in plasma UII and analyze its relationship to the stability of atherosclerotic plaques. One hundred thirty-five consecutive patients with acute coronary syndrome (ACS) were enrolled. The plasma UII levels were measured immediately after admission and during three-month follow-up. A vulnerable plaque model was established using local transfection of a recombinant P53 adenovirus into plaques in rabbits fed with a high-cholesterol diet and subjected to balloon arterial injury. The levels of plasma UII were measured weekly. The changes in plasma UII during the formation of atherosclerotic plaques and before and after plaque transfection were observed. The morphology of the plaques and the expression, distribution, and quantitative expression of UII in the plaques also were observed. Our results showed that the levels of plasma UII in patients with ACS at admission were lower than levels observed at the three-month follow-up. UII dynamic changes and its correlation with plaque stabilities were further verified in rabbits with atherosclerotic vulnerable plaques. The UII levels in rabbits were significantly decreased immediately after the P53 gene transfection, which led to plaque instability and rupture. These results suggested that UII expression was down-regulated in ACS, which may be related to its ability to modulate mechanisms involved in plaque stability and instability.

CD74 regulates cellularity and maturation of medullary thymic epithelial cells partially by activating the canonical NF-κB signaling pathway.

Thymic epithelial cells (TECs) are indispensable for T cell development, T cell receptor (TCR) repertoire selection, and specific lineage differentiation. Medullary thymic epithelial cells (mTECs), which account for the majority of TECs in adults, are critical for thymocyte selection and self-tolerance. CD74 is a nonpolymorphic transmembrane glycoprotein of major histocompatibility complex class II (MHCII) that is expressed in TECs. However, the exact role of CD74 in regulating the development of mTEC is poorly defined. In this research, we found that loss of CD74 resulted in a significant diminution in the medulla, a selective reduction in the cell number of mature mTECs expressing CD80 molecules, which eventually led to impaired thymic CD4+ T cell development. Moreover, RNA-sequence analysis showed that CD74 deficiency obviously downregulated the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in mTECs. Our results suggest that CD74 positively controls mTEC cellularity and maturation partially by activating the canonical NF-κB signaling pathway.

Corrigendum: Thymic Epithelial Cells Contribute to Thymopoiesis and T Cell Development.

[This corrects the article DOI: 10.3389/fimmu.2019.03099.].

A Versatile Nonviral Delivery System for Multiplex Gene-Editing in the Liver.

Recent advances in CRISPR present attractive genome-editing toolsets for therapeutic strategies at the genetic level. Here, a liposome-coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene-editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome-coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss-of-function mutation in the lipid-metabolism-related genes pcsk9, apoc3, and angptl3 would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene-editing efficiency, besting the state-of-art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene-editing at each gene target despite reduced dosage of target-specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post-treatment with lipoMSN carrying both pcsk9 and angptl3-targeted RNPs, could not be reached with a single gene-editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene-editing therapeutics.