Accelerating the design of pili-enabled living materials using an integrative technological workflow.

Bacteria can be programmed to create engineered living materials (ELMs) with self-healing and evolvable functionalities. However, further development of ELMs is greatly hampered by the lack of engineerable nonpathogenic chassis and corresponding programmable endogenous biopolymers. Here, we describe a technological workflow for facilitating ELMs design by rationally integrating bioinformatics, structural biology and synthetic biology technologies. We first develop bioinformatics software, termed Bacteria Biopolymer Sniffer (BBSniffer), that allows fast mining of biopolymers and biopolymer-producing bacteria of interest. As a proof-of-principle study, using existing pathogenic pilus as input, we identify the covalently linked pili (CLP) biosynthetic gene cluster in the industrial workhorse Corynebacterium glutamicum. Genetic manipulation and structural characterization reveal the molecular mechanism of the CLP assembly, ultimately enabling a type of programmable pili for ELM design. Finally, engineering of the CLP-enabled living materials transforms cellulosic biomass into lycopene by coupling the extracellular and intracellular bioconversion ability.

Transcriptome and metabolome analysis reveals mechanism of light intensity modulating iridoid biosynthesis in Gentiana macrophylla Pall.

Light intensity is a key factor affecting the synthesis of secondary metabolites in plants. However, the response mechanisms of metabolites and genes in Gentiana macrophylla under different light intensities have not been determined. In the present study, G. macrophylla seedlings were treated with LED light intensities of 15 µmol/m2/s (low light, LL), 90 µmol/m2/s (medium light, ML), and 200 µmol/m2/s (high light, HL), and leaves were collected on the 5th day for further investigation. A total of 2162 metabolites were detected, in which, the most abundant metabolites were identified as flavonoids, carbohydrates, terpenoids and amino acids. A total of 3313 and 613 differentially expressed genes (DEGs) were identified in the LL and HL groups compared with the ML group, respectively, mainly enriched in KEGG pathways such as carotenoid biosynthesis, carbon metabolism, glycolysis/gluconeogenesis, amino acids biosynthesis, plant MAPK pathway and plant hormone signaling. Besides, the transcription factors of GmMYB5 and GmbHLH20 were determined to be significantly correlated with loganic acid biosynthesis; the expression of photosystem-related enzyme genes was altered under different light intensities, regulating the expression of enzyme genes involved in the carotenoid, chlorophyll, glycolysis and amino acids pathway, then affecting their metabolic biosynthesis. As a result, low light inhibited photosynthesis, delayed glycolysis, thus, increased certain amino acids and decreased loganic acid production, while high light got an opposite trend. Our research contributed significantly to understand the molecular mechanism of light intensity in controlling metabolic accumulation in G. macrophylla.

Genomic characterization of WRKY transcription factors related to secoiridoid biosynthesis in Gentiana macrophylla.

Gentiana macrophylla is one of Chinese herbal medicines in which 4 kinds of iridoids or secoiridoids, such as loganic acid, sweroside, swertiamarin, and gentiopicroside, are identified as the dominant medicinal secondary metabolites. WRKY, as a large family of transcription factors (TFs), plays an important role in the synthesis of secondary metabolites in plants. Therefore, WRKY genes involved in the biosynthesis of secoiridoids in G. macrophylla were systematically studied. First, a comprehensive genome-wide analysis was performed, and 42 GmWRKY genes were identified, which were unevenly distributed in 12 chromosomes. Accordingly, gene structure, collinearity, sequence alignment, phylogenetic, conserved motif and promoter analyses were performed, and the GmWRKY proteins were divided into three subfamilies based on phylogenetic and multiple sequence alignment analyses. Moreover, the enzyme-encoding genes of the secoiridoid biosynthesis pathway and their promoters were then analysed, and the contents of the four secoiridoids were determined in different tissues. Accordingly, correlation analysis was performed using Pearson's correlation coefficient to construct WRKY gene-enzyme-encoding genes and WRKY gene-metabolite networks. Meanwhile, G. macrophylla seedlings were treated with methyl jasmonate (MeJA) to detect the dynamic change trend of GmWRKYs, biosynthetic genes, and medicinal ingredient accumulation. Thus, a total of 12 GmWRKYs were identified to be involved in the biosynthesis of secoiridoids, of which 8 (GmWRKY1, 6, 12, 17, 33, 34, 38 and 39) were found to regulate the synthesis of gentiopicroside, and 4 (GmWRKY7, 14, 26 and 41) were found to regulate the synthesis of loganic acid. Taken together, this study systematically identified WRKY transcription factors related to the biosynthesis of secoiridoids in G. macrophylla, which could be used as a cue for further investigation of WRKY gene functions in secondary metabolite accumulation.

Effects of Leukocyte-rich platelet-rich plasma and Leukocyte-poor platelet-rich plasma on cartilage in a rabbit osteoarthritis model.

Osteoarthritis is a prevalent chronic disease. One of its primary pathological processes involves the degeneration of articular cartilage. Platelet-rich plasma (PRP) contains cytokines and growth factors that can stimulate the repair and regeneration of articular cartilage tissues. PRP may also slow the progression of osteoarthritis. The purpose of this experiment is to compare the efficacy of Leukocyte poor (LP) - PRP and Leukocyte rich (LR) - PRP in treating rabbit osteoarthritis and to investigate their mechanisms of action. Analyzing the impact of leukocytes on PRP therapeutic effectiveness will provide a valuable clinical reference for the choice of which PRP is better for the treatment of osteoarthritis. A rabbit osteoarthritis model was established by injecting papain into the knee joint cavity, and LP-PRP and LR-PRP were prepared through different centrifugation methods for injection into the knee joint cavity. Eight weeks after injection, rabbit knee cartilage specimens were observed for gross changes, HE staining, senna O-solid green staining, and immunohistochemistry of type II collagen and were quantitatively compared using Pelletier's score, Mankin's pathology score, and ImageJ image processing software. Injection of papain into the knee joint cavity successfully established a rabbit model of osteoarthritis. All three evaluation indexes differed significantly from those of the blank group (P<0.05). LP-PRP and LR-PRP exhibited therapeutic effects when compared with the model group. The two PRP groups had similar gross tissue appearance and pathology (P>0.05). The LR-PRP group had higher collagen type-II expression (P < 0.05) than the LP-PRP group. Both LP-PRP and LR-PRP proved therapeutic for the rabbit papain osteoarthritis model. The difference in leukocyte content between the two groups did not yield different cartilage morphology or other factors by 8 weeks posttreatment. LR-PRP displayed the ability to release more factors relevant to the metabolism of type II collagen than LP-PRP, enabling the preservation of into cartilage collagen content of type II collagen and delaying osteoarthritis progression.

A modified open surgical revision technique for malfunctioning peritoneal dialysis catheter.

Backgrounds: The malfunction of peritoneal dialysis (PD) catheter is still an intractable problem. A modified open surgical revision technique with suturing fixation and without catheter removal for malfunctioning catheter was developed to evaluated the efficacy and safety between simultaneous catheter replacement technique.Methods: A total of 167 PD patients with malfunctioning catheter were retrospectively reviewed. For the salvage of PD catheters, patients underwent modified open surgical revision (group A) or simultaneous catheter replacement (group B). The baseline characteristics before operation, perioperative condition, complications and outcomes were compared between both groups.Results: Patients of group A showed significantly shorter operative time (67.4 ± 22.1 versus 82.8 ± 21.1 min, p = 0.009), less postoperative pain score within 24 h (median 0.0 versus 2.0, p < 0.001), quicker start of PD (1.06 ± 0.31 versus 1.89 ± 0.89 days, p < 0.001), shorter length of stay (9.89 ± 5.11 versus 12.55 ± 7.37 days, p = 0.020) than group B. In terms of complications, the incidence of recurred catheter malfunction in group A was significantly lower than those in group B (1/114 versus 12/53, p < 0.001). There were no significant differences in mechanical complications (bloody effluent, dialysate leakage, and hernia) and early peritonitis between the groups. The group A patients had a favorable catheter survival rate compared with group B (log-rank, p = 0.004).Conclusions: Our modified open surgical revision technique is a safe, simple and fast method, and offers a better outcome with minimal risk of recurrence of catheter malfunction without additional cost and equipment. This technique is worthy of clinical application.

Rational design of functional amyloid fibrillar assemblies.

Amyloid fibrillar assemblies, originally identified as pathological entities in neurodegenerative diseases, have been widely adopted by various proteins to fulfill diverse biological functions in living organisms. Due to their unique features, such as hierarchical assembly, exceptional mechanical properties, environmental stability, and self-healing properties, amyloid fibrillar assemblies have been employed as functional materials in numerous applications. Recently, with the rapid advancement in synthetic biology and structural biology tools, new trends in the functional design of amyloid fibrillar assemblies have begun to emerge. In this review, we provide a comprehensive overview of the design principles for functional amyloid fibrillar assemblies from an engineering perspective, as well as through the lens of structural insights. Initially, we introduce the fundamental structural configurations of amyloid assemblies and highlight the functions of representative examples. We then focus on the underlying design principles of two prevalent strategies for the design of functional amyloid fibrillar assemblies: (1) introducing new functions via protein modular design and/or hybridization, with typical applications encompassing catalysis, virus disinfection, biomimetic mineralization, bio-imaging, and biotherapy; and (2) dynamically regulating living amyloid fibrillar assemblies using synthetic gene circuits, with typical applications in pattern formation, leakage repair, and pressure sensing. Next, we summarize how breakthroughs in characterization techniques have contributed to unveiling the structural polymorphism of amyloid fibrils at the atomic level, and further clarifying the highly diverse regulation mechanisms of amyloid fibrillar assembly and disassembly fine-tuned by various factors. The structural knowledge may significantly aid in the structure-guided design of amyloid fibrillar assemblies with diverse bio-activities and adjustable regulatory properties. Finally, we envision that a new trend in functional amyloid design may emerge by integrating structural tunability, synthetic biology and artificial intelligence.

The efficacy of rituximab plus belimumab or telitacicept in refractory lupus nephritis.

OBJECTIVE: Lupus nephritis is a severe and common complication of systemic lupus erythematosus (SLE). The pathogenesis of lupus nephritis is characterized by B-cell activation and autoantibody formation. Rituximab and belimumab, as well as telitacicept, target B cells through different mechanisms, potentially exerting a synergistic effect in the treatment of lupus nephritis. This study aims to investigate the efficacy and safety of treatment with rituximab followed by belimumab or telitacicept in the management of refractory lupus nephritis. METHODS: We conducted a single-center, open-label, retrospective study, including 25 patients with refractory lupus nephritis. All patients received combination therapy with rituximab in individualized dosages to achieve peripheral B-cell depletion, and then followed by belimumab or telitacicept. The follow-up period was at least 12 months, and the primary end point was renal remission rate at the last follow-up. RESULTS: During a median follow-up of 19 (13, 29) months, 20 of 25 (80%) patients achieved objective remission (OR), including 19 (76%) patients achieved complete renal response (CRR). After rituximab (712 ± 416mg in average), 18 patients received belimumab and seven patients received telitacicept. In the rituximab plus telitacicept group, all patients achieved CRR; while in the rituximab plus belimumab group, 12 (66.7%) patients achieved CRR and 13 (72.2%) patients achieved OR. The mean SLEDAI-2K score decreased from 15 ± 6 to 6 ± 6, representing an average reduction of 60%. At the last follow-up, 18/25 (72%) had prednisone ≤ 5 mg/d or even discontinued prednisone use. Adverse effects were mainly immunoglobulin deficiency, respiratory tract infection, urinary tract infections, and rash. No death occurred. CONCLUSIONS: Rituximab followed by belimumab or telitacicept may be effective in inducing remission in refractory lupus nephritis, with tolerable adverse effects.

The risk factors and predictive model for cardiac valve calcification in patients on maintenance peritoneal dialysis: a single-center retrospective study.

BACKGROUND: Cardiovascular calcification includes cardiac valve calcification (CVC) and vascular calcification. We aimed to analyze risk factors for CVC, and construct a predictive model in maintenance peritoneal dialysis (MPD) patients. METHODS: We retrospectively analyzed MPD patients who began peritoneal dialysis between January 2014 and September 2021. Patients were randomly assigned to the derivation cohort and validation cohort in a 7:3 ratio. The patients in the derivation cohort were divided into the CVC group and non-CVC group. Logistic regression was used to analyze risk factors, then the rms package in R language was used to construct a nomogram model to predict CVC. RESULTS: 1,035 MPD patients were included, with the age of 50.0 ± 14.2 years and 632 males (61.1%). Their median follow-up time was 25 (12, 46) months. The new-onset CVC occurred in 128 patients (12.4%). In the derivation cohort, multivariate logistic regression indicated old age, female, high systolic blood pressure (SBP), high calcium-phosphorus product (Ca × P), high Charlson comorbidity index (CCI) and long dialysis time were independent risk factors for CVC (p < 0.05). We constructed a nomogram model for predicting CVC in the derivation cohort, with a C index of 0.845 (95% CI 0.803-0.886). This model was validated with a C index of 0.845 (95%CI 0.781-0.909) in the validation cohort. CONCLUSION: We constructed a nomogram model for CVC in MPD patients, using independent risk factors including age, sex, SBP, Ca × P, CCI and dialysis time. This model achieved high efficiency in CVC prediction.

The survival nomograms for anti-neutrophil cytoplasmic antibody-associated glomerulonephritis patients with a follow-up of more than one year.

OBJECTIVES: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis with kidney injury, manifested as ANCA-associated glomerulonephritis (AAGN), often portends a poor prognosis of renal function and life survival in long term. METHODS: A cohort of 339 AAGN patients were enrolled retrospectively. These patients survived and were followed up for at least 12 months after diagnosis in our centre. Multivariate Cox regression analysis and nomogram models were performed to determine the risk factors associated with renal survival and patient survival. RESULTS: The median follow-up time of all 339 patients was 65.2 (IQR 45.1, 91.3) months and the median age was 61(IQR 53, 69) years. In order to analyse the impact of the factors on renal survival, we divided the patients into 2 groups: non-dialysis group (204 patients without dialysis at the final visit) and dialysis group (135 patients with maintaining dialysis). The patients in dialysis group had lower haemoglobin level, lower eGFR level, lower platelets count, more daily urine protein, and higher Birmingham Vasculitis Activity Score (BVAS) at admission than those in non-dialysis group. Multivariate Cox regression revealed that low haemoglobin (HR=0.977, 95%CI 0.965-0.990, p<0.001), low eGFR (HR=0.957, 95%CI 0.941-0.973, p<0.001) and high proteinuria (HR=1.139, 95%CI 1.055-1.230, p=0.001) at admission were independent risk factors for developing maintaining dialysis. A nomogram was established based on the results of multivariate Cox analysis and the internal bootstrap resampling approach showed the C-index of the nomogram was 0.83. Then we divided all patients into death group (n=99) and survival group (n=240). The patients in death group had older age, more hypertension, more chronic lung disease, lower platelets count, lower serum albumin, higher BVAS and lower eGFR at admission than those in survival group. Multivariate Cox regression revealed that the status of maintaining dialysis (HR 3.51, 95% CI 1.91-6.47, p<0.001) and old age (HR 1.07, 95% CI 1.04-1.09, p<0.001) were independent risk factors for all-cause mortality. Again, a nomogram was established and the C-index was 0.74. CONCLUSIONS: We analysed the independent risk factors for maintaining dialysis and all-cause mortality in AAGN patients with a follow-up of more than 12 months. The two proposed nomograms were of predictive value.

Systemic sclerosis complicated with renal thrombotic microangiopathy: a case report and literature review.

BACKGROUND: Systemic sclerosis (SSc) may overlap with other connective tissue diseases, which is named overlap syndrome. Scleroderma renal crisis (SRC) is a rare but severe complication of SSc. SSc related thrombotic microangiopathy (SSc-TMA) is an infrequent pathology type of SRC, while SSc-TMA accompanied by overlap syndrome is very rare. CASE PRESENTATION: This study reported a case of acute kidney injury (AKI) accompanied with overlap syndrome of SSc, systemic lupus erythematosus (SLE) and polymyositis (PM). The renal pathology supported the diagnosis of SSc-TMA but not SLE or PM-related renal injury, characterized by renal arteriolar thrombosis, endothelial cells edema, little cast in tubules and mild immune complex deposition. The primary TMA related factors (ADAMTS13 and complement H factor) were normal. Thus, this case was diagnosed as secondary TMA associated with SSc. The patient was treated with renin angiotensin system inhibitors, sildenafil, supportive plasma exchange/dialysis, and rituximab combined with glucocorticoids. After 2 months of peritoneal dialysis treatment, her renal function recovered and dialysis was stopped. CONCLUSION: This study presented a case of SSc-TMA with overlap syndrome. Rituximab can be used as a treatment option in patients with high SRC risk or already manifesting SRC.

Multiple diagnostic methods for mucormycosis: A retrospective case series.

BACKGROUND: Mucormycosis is a rare invasive fungal disease with high mortality. Early diagnosis and targeted drugs are crucial to improving clinical outcomes. METHODS: We searched the electronic hospital database of the First Affiliated Hospital of Zhejiang University School of Medicine for adult patients with mucormycosis between 2000 and 2021. Demographic, clinical, treatment, and outcome data were collected and compared with the data in the relevant literature. RESULTS: Eleven cases of mucormycosis-four of multisite infection, one of skin infection, five of lung infection, and one of gastrointestinal infection-were found and analyzed. The patients were diagnosed mainly based on pathological and histological findings, and three patients had metagenomic next-generation sequencing findings. Delayed diagnosis (i.e., diagnosis >7 days after patient admission or >30 days after onset of symptoms) results in poor prognosis compared with early diagnosis. CONCLUSIONS: Improving awareness and shortening diagnosis time may improve the prognosis of mucormycosis. If mucormycosis is suspected, appropriate samples should be collected as soon as possible and submitted for biopsy, culture, or mNGS to confirm the diagnosis.

Sexual dysfunction associated with chronic retention of foreign bodies in the low urinary tract.

We sought to evaluate the association between sexual dysfunction and chronic retention of foreign bodies in the lower urinary tract (LUT) for long-term periods (≧4 weeks) in patients seen at three medical centres between January 2015 and September 2020, followed by assessing the impact of long-term retention of a foreign body in the LUT on sexual function. Thirty-eight patients were studied in the long-term group, among whom the aetiology of the foreign bodies included sexual desire with masturbation (n = 22, 58%), sexual inquisitiveness (n = 10, 26%), dysuria (n = 3, 8%) and seeking to relieve itching (n = 3, 8%). There were various types of foreign bodies, including a string of magnetic beads (n = 13), a thermometer (n = 5), plastic electric wire (n = 5) and others (n = 15). All cases presented with sexual dysfunction and LUT symptoms. Three months after foreign body removal, sexual dysfunction symptoms were significantly improved in 22 male cases and seven female cases. We found that chronic retention of foreign bodies in the LUT causes sexual dysfunction in both men and women. The psychological effects of fear may prevent these patients from seeking medical help. Thus, education on sexual medicine and timely removal of foreign bodies is necessary to avert sexual dysfunction and urinary tract infection.

Down-regulated LINC00115 inhibits prostate cancer cell proliferation and invasion via targeting miR-212-5p/FZD5/Wnt/ß-catenin axis.

Prostate cancer is the second most frequent malignancy in men worldwide, and its incidence is increasing. Therefore, it is urgently required to clarify the underlying mechanisms of prostate cancer. Although the long non-coding RNA LINC00115 was identified as an oncogene in several cancers, the expression and function of LINC00115 in prostate cancer have not been explored. Our results showed that LINC00115 was significantly up-regulated in prostate cancer tissues, which was significantly associated with a poor prognosis for prostate cancer patients. Functional studies showed that knockdown LINC00115 inhibited cell proliferation and invasion. In addition, LINC00115 served as a competing endogenous RNA (ceRNA) through sponging miR-212-5p to release Frizzled Family Receptor 5 (FZD5) expression. The expression of miR-212-5p was noticeably low in tumour tissues, and FZD5 expression level was down-regulated with the knockdown of LINC00115. Knockdown LINC00115 inhibited the Wnt/ß-catenin signalling pathway by inhibiting the expression of FZD5. Rescue experiments further showed that LINC00115 inhibits prostate cancer cell proliferation and invasion via targeting miR-212-5p/ FZD5/ Wnt/ß-catenin axis. The present study provided clues that LINC00115 may be a promising novel therapeutic target for prostate cancer patients.

Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda.

BACKGROUND: Infestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii) can cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while using its stylet to probe the tender shoots of tea plants (Camellia sinensis). This study identified and analyzed proteases expressed specifically in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays complemented with an integrated analysis of transcriptomic and proteomic data. RESULTS: In total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut but moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans, including Nilaparvata lugens, Laodelphax striatellus, Acyrthosiphum pisum, Halyomorpha halys and Nephotettix cincticeps. Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans formed two distinct clusters. CONCLUSIONS: Altogether, this study provides insightful information on the digestive system of E. onukii. Compared to five other hemipteran species, we observed different patterns of proteases abundant in the SG and gut of E. onukii. These results will be beneficial in understanding the interaction between tea plants and E. onukii.

Cell active and functionally-relevant small-molecule agonists of calcitonin receptor.

The natural calcitonin (CT) receptor and its peptide agonists are considered validated targets for drug discovery. A small molecule agonist, SUN-B8155, has previously been shown to efficiently activate cellular CTR. Herein, we report the synthesis of a series of compounds (S8155 1-9) derived from SUN-B8155, and investigate the structural-functional relationship, bias properties and their cellular activity profile. We discover that the N-hydroxyl group from the pyridone ring is required for G protein activity and its affinity to the CT receptor. Among the compounds studied, S8155-7 exhibits improved G protein activity while S8155-4 displays a significant ß-arrestin-2 signaling bias. Finally, we show that both S8155-4 and S8155-7 inhibit tumour cell invasion through CTR activation. These two compounds are anticipated to find extensive applications in chemical biology research as well drug development efforts targeting CT receptor.

Saponins Extracted from Tea (Camellia Sinensis) Flowers Induces Autophagy in Ovarian Cancer Cells.

Tea flower saponins (TFS) possess effective anticancer properties. The diversity and complexity of TFS increases the difficulty of their extraction and purification from tea flowers. Here, multiple methods including solvent extraction, microporous resin separation and preparative HPLC separation were used to obtain TFS with a yield of 0.34%. Furthermore, we revealed that TFS induced autophagy-as evidenced by an increase in MDC-positive cell populations and mCherry-LC3B-labeled autolysosomes and an upregulation of LC3II protein levels. 3-MA reversed the decrease in cell viability induced by TFS, showing that TFS induced autophagic cell death. TFS-induced autophagy was not dependent on the Akt/mTOR/p70S6K signaling pathway. TFS-induced autophagy in OVCAR-3 cells was accompanied by ERK pathway activation and reactive oxygen species (ROS) generation. This paper is the first report of TFS-mediated autophagy of ovarian cancer cells. These results provide new insights for future studies of the anti-cancer effects of TFS.

Standardized Saponin Extract from Baiye No.1 Tea (Camellia sinensis) Flowers Induced S Phase Cell Cycle Arrest and Apoptosis via AKT-MDM2-p53 Signaling Pathway in Ovarian Cancer Cells.

Ovarian cancer is considered to be one of the most serious malignant tumors in women. Natural compounds have been considered as important sources in the search for new anti-cancer agents. Saponins are characteristic components of tea (Camellia sinensis) flower and have various biological activities, including anti-tumor effects. In this study, a high purity standardized saponin extract, namely Baiye No.1 tea flower saponin (BTFS), which contained Floratheasaponin A and Floratheasaponin D, were isolated from tea (Camellia sinensis cv. Baiye 1) flowers by macroporous resin and preparative liquid chromatography. Then, the component and purity were detected by UPLC-Q-TOF/MS/MS. This high purity BTFS inhibited the proliferation of A2780/CP70 cancer cells dose-dependently, which is evidenced by the inhibition of cell viability, reduction of colony formation ability, and suppression of PCNA protein expression. Further research found BTFS induced S phase cell cycle arrest by up-regulating p21 proteins expression and down-regulating Cyclin A2, CDK2, and Cdc25A protein expression. Furthermore, BTFS caused DNA damage and activated the ATM-Chk2 signaling pathway to block cell cycle progression. Moreover, BTFS trigged both extrinsic and intrinsic apoptosis-BTFS up-regulated the expression of death receptor pathway-related proteins DR5, Fas, and FADD and increased the ratio of pro-apoptotic/anti-apoptotic proteins of the Bcl-2 family. BTFS-induced apoptosis seems to be related to the AKT-MDM2-p53 signaling pathway. In summary, our results demonstrate that BTFS has the potential to be used as a nutraceutical for the prevention and treatment of ovarian cancer.

[Study of centrifuge conditions for preparing rabbit leukocyte-poor platelet-rich plasma by single centrifugation].

Objective: To explore the best centrifuge condition for preparing rabbit leukocyte-poor platelet-rich plasma (LP-PRP) by using single centrifugation method. Methods: Sixteen healthy New Zealand rabbits, aged 3-4 months, were utilized in the investigation. A total of 15 mL anticoagulated blood was extracted from the central ear artery of each rabbit, with a repeat of the blood collection procedure after 1 and 2 months. The obtained blood specimens were individually subjected to centrifugation at a radius of 16.7 cm and speeds of 1 200, 1 300, 1 400, and 1 500 r/min (equivalent to centrifugal forces of 269× g, 315× g, 365× g, and 420× g) for durations of 2, 3, 4, and 5 minutes, resulting in a total of 16 groups. Following centrifugation, collect plasma from each group to a distance of 1.5 mL from the separation plane. The volumes, platelet enrichment coefficient, and platelet recovery rates of LP-PRP in each group, under varying centrifugation conditions, were methodically computed and subsequently compared. Results: The volume of LP-PRP obtained under all centrifugation conditions ranged from 1.8 to 7.6 mL. At a consistent centrifugal speed, an extension of centrifugation time leaded to a significant increase in the volume of LP-PRP, accompanied by a declining trend in the platelet enrichment coefficient of LP-PRP. When centrifuged for 2 minutes, the volume of LP-PRP at speeds of 1 200 and 1 300 r/min was less than 2.0 mL, while the volume of LP-PRP obtained under other conditions was more than 2.0 mL. When centrifuged for 4 and 5 minutes, the volume of LP-PRP obtained at each speed was more than 4 mL. LP-PRP with a platelet enrichment coefficient more than 2.0 could be prepared by centrifuging at 1 200 r/min for each time group and 1 300 r/min for 2 and 3 minutes, and the highest LP-PRP platelet enrichment coefficient could be obtained by centrifugation for 2 minutes at a speed of 1 200 r/min. The platelet recovery rates of LP-PRP obtained by centrifugation at 1 200 r/min for 4 and 5 minutes, as well as centrifugation at 1 400 r/min for 5 minutes, were both greater than 60%. There was no significant difference between the groups when centrifuged at 1 200 r/min for 4 and 5 minutes ( P>0.05). Conclusion: In the process of preparing rabbit LP-PRP using a single centrifugation method, collecting 15 mL of blood and centrifuging at a radius of 16.7 cm and speed of 1 200 r/min for 4 minutes can prepare LP-PRP with a volume exceeding 2.0 mL, platelet enrichment coefficient exceeding 2.0, and platelet recovery rate exceeding 60%. This centrifugal condition can achieve the optimal LP-PRP action parameters in the shortest possible time.

Five Pairs of Enantiomer as Rearrangement Products from Secoiridoids in Gentiana macrophylla Pall.

Five racemates (1-5) were isolated from Gentiana macrophylla, in which 2-5 were successfully separated into four pairs of enantiomers (2a and 2b, 3a and 3b, 4a and 4b, and 5a and 5b), whereas the resolution of 1 failed due to the hemiacetal functionality at the stereogenic center. Using electronic circular dichrosim calculation, the relationship of the molecular rotation direction and the carbon R/S chirality was revealed, and each pair of enantiomer was identified as (-)-(S)-gentianmacrol B (2a) and (+)-(R)-gentianmacrol B (2b), (-)-(S)-8-methoxy-gentianol (3a) and (+)-(R)-8-methoxy-gentianol (3b), (+)-(S)-8-methyl-gentianadine (4a) and (-)-(R)-8-methyl-gentianadine (4b), and (-)-(S)-gentianol (5a) and (+)-(R)-gentianol (5b). Besides, these compounds could be divided into two series, 1-2 containing the benzene ring moiety and 3-5 containing the pyridine ring moiety. Considering that their molecular skeleton could not be generated from the classical biosynthesis pathway in plants, the plausible biosynthesis pathways of 1-5 were deduced to be transformed from secoiridoids in G. macrophylla. Due to the significant difference in the pharmacological effect for the optical factor, our research provided new diverse molecules for further optical activity studies in drug research.

Obinutuzumab in Refractory Membranous Nephropathy: A Case Series.

Rationale & Objective: Membranous nephropathy (MN), recognized as an autoimmune kidney disease, responds well to anti-CD20 monoclonal antibodies. Obinutuzumab, a type Ⅱ humanized anti-CD20 and immunoglobulin G1 Fc-optimized monoclonal antibody, when compared with rituximab, has demonstrated superior efficacy in B-cell leukemia and lymphoma, especially in rituximab-resistant cases. However, the efficacy and safety of obinutuzumab in MN remain unclear. Study Design: A case series study. Setting & Participants: A total of 18 patients were diagnosed with MN and had received obinutuzumab at our center without secondary MN, undergoing dialysis, having a history of kidney transplantation, or infections requiring treatment. Exposure: Obinutuzumab treatment. Outcomes: Primary outcomes included remission rate, time to first remission, and first relapse-free survival time during the follow-up period. Analytical Approach: Survival analysis was performed with Cox proportional hazards models, log-rank test, and Kaplan-Meier survival analysis. Results: Patients with MN (median age of 52.5 years, 83.3% males) received an average dose of 2.1 ± 0.8 g of obinutuzumab during a median follow-up period of 13.6 months. During the follow-up, 17 patients (94.4%) achieved remission, with 12 patients (66.7%) achieving partial remission, and 5 patients (27.8%) achieving complete remission. The median time to first remission and first relapse-free survival time was 2.7 (1.0-6.1) months and 9.8 (2.6-11.2) months, respectively. Of 12 patients with previous rituximab treatment, all achieved remission successfully, with 8 (66.7%) achieving partial remission and 4 (33.3%) achieving complete remission. Adverse events were mostly mild, and no severe treatment-related adverse events were observed. Limitations: Limited or missing data; risks of selection bias; or recall bias; underestimated first relapse-free survival time because of a limited follow-up period; unmonitored counts of CD19+ B-cells and other lymphocyte subsets. Conclusions: Obinutuzumab demonstrated promising efficacy and safety in inducing remission in MN, particularly in patients with an unsatisfactory response to rituximab.

Membranous nephropathy (MN), an autoimmune kidney disease, usually responds favorably to rituximab, a chimeric anti-CD20 monoclonal antibody. Nevertheless, certain patients exhibit inadequate responses to rituximab. Obinutuzumab, a novel humanized anti-CD20 monoclonal antibody, has shown enhanced efficacy in cases where rituximab fails to address B-cell leukemias and lymphomas. However, its efficacy and safety in MN treatment remain uncertain. A case series involving 18 patients treated with obinutuzumab at our center demonstrated promising results, suggesting favorable efficacy and safety in inducing and maintaining remission, particularly among patients who did not respond well to rituximab previously. These findings signify a potential alternative for MN treatment, though further research is needed to confirm them.