The Development of Aspergillus flavus and Biosynthesis of Aflatoxin B1 are Regulated by the Golgi-Localized Mn2+ Transporter Pmr1.

Microorganisms rely on diverse ion transport and trace elements to sustain growth, development, and secondary metabolism. Manganese (Mn2+) is essential for various biological processes and plays a crucial role in the metabolism of human cells, plants, and yeast. In Aspergillus flavus, we confirmed that Pmr1 localized in cis- and medial-Golgi compartments was critical in facilitating Mn2+ transport, fungal growth, development, secondary metabolism, and glycosylation. In comparison to the wild type, the Δpmr1 mutant displayed heightened sensitivity to environmental stress, accompanied by inhibited synthesis of aflatoxin B1, kojic acid, and a substantial reduction in pathogenicity toward peanuts and maize. Interestingly, the addition of exogenous Mn2+ effectively rectified the developmental and secondary metabolic defects in the Δpmr1 mutant. However, Mn2+ supplement failed to restore the growth and development of the Δpmr1Δgdt1 double mutant, which indicated that the Gdt1 compensated for the functional deficiency of pmr1. In addition, our results showed that pmr1 knockout leads to an upregulation of O-glycosyl-N-acetylglucose (O-GlcNAc) and O-GlcNAc transferase (OGT), while Mn2+ supplementation can restore the glycosylation in A. flavus. Collectively, this study indicates that the pmr1 regulates Mn2+ via Golgi and maintains growth and metabolism functions of A. flavus through regulation of the glycosylation.

De novo transcriptome sequencing of Impatiens uliginosa and the analysis of candidate genes related to spur development.

BACKGROUND: Spur, a structure capable of producing and storing nectar, not only plays a vital role in the pollination process but also promotes the rapid diversification of some plant lineages, which is considered a key innovation in plants. Spur is the focus of many studies, such as evolution and ecological hypothesis, but the current understanding of spur development is limited. High-throughput sequencing of Impatiens uliginosa was carried out to study the molecular mechanism of its spur development, which is believed to provide some insights into the spur development of Impatiens. RESULTS: Transcriptomic sequencing and analysis were performed on spurs and limbs of I. uliginosa at three developmental stages. A total of 47.83 Gb of clean data were obtained, and 49,716 unigene genes were assembled. After comparison with NR, Swiss-Prot, Pfam, COG, GO and KEGG databases, a total of 27,686 genes were annotated successfully. Through comparative analysis, 19,356 differentially expressed genes were found and enriched into 208 GO terms and 146 KEGG pathways, among which plant hormone signal transduction was the most significantly enriched pathway. One thousand thirty-two transcription factors were identified, which belonged to 33 TF families such as MYB, bHLH and TCP. Twenty candidate genes that may be involved in spur development were screened and verified by qPCR, such as SBP, IAA and ABP. CONCLUSIONS: Transcriptome data of different developmental stages of spurs were obtained, and a series of candidate genes related to spur development were identified. The importance of genes related to cell cycle, cell division, cell elongation and hormones in spur development was clarified. This study provided valuable information and resources for understanding the molecular mechanism of spur development in Impatiens.

SARS-CoV-2 nucleocapsid protein triggers hyperinflammation via protein-protein interaction-mediated intracellular Cl- accumulation in respiratory epithelium.

SARS-CoV-2, the culprit pathogen of COVID-19, elicits prominent immune responses and cytokine storms. Intracellular Cl- is a crucial regulator of host defense, whereas the role of Cl- signaling pathway in modulating pulmonary inflammation associated with SARS-CoV-2 infection remains unclear. By using human respiratory epithelial cell lines, primary cultured human airway epithelial cells, and murine models of viral structural protein stimulation and SARS-CoV-2 direct challenge, we demonstrated that SARS-CoV-2 nucleocapsid (N) protein could interact with Smad3, which downregulated cystic fibrosis transmembrane conductance regulator (CFTR) expression via microRNA-145. The intracellular Cl- concentration ([Cl-]i) was raised, resulting in phosphorylation of serum glucocorticoid regulated kinase 1 (SGK1) and robust inflammatory responses. Inhibition or knockout of SGK1 abrogated the N protein-elicited airway inflammation. Moreover, N protein promoted a sustained elevation of [Cl-]i by depleting intracellular cAMP via upregulation of phosphodiesterase 4 (PDE4). Rolipram, a selective PDE4 inhibitor, countered airway inflammation by reducing [Cl-]i. Our findings suggested that Cl- acted as the crucial pathological second messenger mediating the inflammatory responses after SARS-CoV-2 infection. Targeting the Cl- signaling pathway might be a novel therapeutic strategy for COVID-19.

SfgA Renders Aspergillus flavus More Stable to the External Environment.

sfgA is known as a key negative transcriptional regulator gene of asexual sporulation and sterigmatocystin production in Aspergillus nidulans. However, here, we found that the homolog sfgA gene shows a broad and complex regulatory role in governing growth, conidiation, sclerotia formation, secondary metabolism, and environmental stress responses in Aspergillus flavus. When sfgA was deleted in A. flavus, the fungal growth was slowed, but the conidiation was significantly increased, and the sclerotia formation displayed different behavior at different temperatures, which increased at 30 °C but decreased at 36 °C. In addition, sfgA regulated aflatoxin biosynthesis in a complex way that was associated with the changes in cultured conditions, and the increased production of aflatoxin in the ∆sfgA mutant was associated with a decrease in sclerotia size. Furthermore, the ∆sfgA mutant exhibited sensitivity to osmotic, oxidative, and cell wall stresses but still produced dense conidia. Transcriptome data indicated that numerous development- and secondary-metabolism-related genes were expressed differently when sfgA was deleted. Additionally, we also found that sfgA functions downstream of fluG in A. flavus, which is consistent with the genetic position in FluG-mediated conidiation in A. nidulans. Collectively, sfgA plays a critical role in the development, secondary metabolism, and stress responses of A. flavus, and sfgA renders A. flavus more stable to the external environment.

Therapeutic Potential of Perillaldehyde in Ameliorating Vulvovaginal Candidiasis by Reducing Vaginal Oxidative Stress and Apoptosis.

Vulvovaginal candidiasis (VVC) is one of the most frequent diseases induced by Candida albicans (C. albicans) during pregnancy, which results in enormous pain to women and their partners in daily life. Perillaldehyde (PAE), a natural monoterpenoid, has significant anti-microbial, anti-inflammatory and anti-oxidation effects. Reactive oxygen species (ROS) are key factors for the host to resist the invasion of fungi. However, excess ROS can cause additional damage independent of the pathogen itself, and the mechanism of ROS in VVC has not been investigated. In this murine study, we revealed that C. albicans infection increased the expression of NADPH oxidase 2 (NOX2) and the content of malonaldehyde (MDA). C. albicans inhibited the activity of antioxidant enzymes in the vagina, including superoxide dismutase (SOD), Catalase (CAT), glutathione peroxidase (GSH-PX) and heme oxygenase (HO-1), which were returned to normal levels after treatment with PAE. Furthermore, PAE inhibited the activities of Keap1 and promoted Nrf2 transfer from cytoplasm to nucleus, which were mediated by excessive accumulation of ROS in the VVC mice. In this study, we also indicated that PAE inhibited the apoptosis of vagina cells via Caspase 9- Caspase 7-PARP pathway and prevented the release of IL-1ꞵ in VVC mice. In summary, this study revealed that the treatment of VVC in mice with PAE might be mediated by inhibition of ROS, and established the therapeutic potential of PAE as an antifungal agent for the treatment of VVC.

STAMP2 suppresses autophagy in prostate cancer cells by modulating the integrated stress response pathway.

Six Transmembrane Protein of Prostate 2 (STAMP2) is critical for prostate cancer (PCa) growth. We previously showed that STAMP2 regulates the expression of stress induced transcription factor ATF4, which is implicated in starvation-induced autophagy. We therefore investigated whether STAMP2 is involved in the regulation of autophagy in PCa cells. Here we show that STAMP2 suppresses autophagy in PCa cells through modulation of the integrated stress response axis. We also find that STAMP2 regulates mitochondrial respiration. These findings suggest that STAMP2 has significant metabolic effects through mitochondrial function and autophagy, both of which support PCa growth.

Sequential CD19 and BCMA-specific CAR T-cell treatment elicits sustained remission of relapsed and/or refractory myeloma.

The low rate of durable response against relapsed and/or refractory multiple myeloma (RRMM) in recent studies indicates that chimeric antigen receptor T-cell (CART) treatment is yet to be optimized. This study aims to investigate the safety and efficacy of sequential infusion of CD19-CART and B-cell maturation antigen (BCMA)-CARTs for RRMM with a similar 3 + 3 dose escalation combined with a toxicity sentinel design. We enrolled 10 patients, among whom 7 received autologous infusion and 3 received allogeneic infusion. The median follow-up time was 20 months. The most common grade 3/4 treatment-emergent toxicities were hematological toxicities. Cytokine-release syndrome (CRS) adverse reactions were grade 1/2 in 9 out of 10 subjects. No dose-limited toxicity (DLT) was observed for BCMA-CAR-positive T cells ≤5 × 107 /kg), while two patients with dose-levels of 5-6.5 × 107 /kg experienced DLTs. The overall response rate was 90% (five partial responses and four stringent complete responses). Three out of four patients with stringent complete responses to autologous CART had progression-free survival for over 2 years. The three patients with allogeneic CART experienced disease progression within 2 months. These results evidence the sequential infusion's preliminarily tolerability and efficacy in RRMM, and present a simple and safe design applicable for the establishment of multiple CART therapy.

Perillaldehyde: A promising antifungal agent to treat oropharyngeal candidiasis.

Perillaldehyde (PAE), a natural monoterpenoid agent extracted from Perilla frutescence, PAE has been reported to present various physiological capabilities, such as anti-inflammation, anti-oxidative and anti-fungal. In this study, we show that PAE exhibits strong antifungal activity against Candida albicans (C. albicans). C. albicans, a fungal pathogen with high incidence of antifungal resistance in clinical settings, is the major cause of oropharyngeal candidiasis (OPC). OPC is characterized by inflammatory immunological responses to fungal infections. Our in vitro results show PAE inhibited several virulence attributes of C. albicans including biofilm formation, yeast-to-hyphal transition and secreted aspartic proteinases (SAPs) gene expression. Using an experimental murine model of OPC, we found that PAE inhibited NLRP3 inflammasome assembly, reduced the excessive accumulation of ROS and prevented the p65 transfer in nuclear; processes all leading to reduced inflammation burden in the host. Together, this supports use PAE as a promising new agent to improve OPC.

Protein acetylation in mitochondria plays critical functions in the pathogenesis of fatty liver disease.

BACKGROUND: Fatty liver is a high incidence of perinatal disease in dairy cows caused by negative energy balance, which seriously threatens the postpartum health and milk production. It has been reported that lysine acetylation plays an important role in substance and energy metabolism. Predictably, most metabolic processes in the liver, as a vital metabolic organ, are subjected to acetylation. Comparative acetylome study were used to quantify the hepatic tissues from the severe fatty liver group and normal group. Combined with bioinformatics analysis, this study provides new insights for the role of acetylation modification in fatty liver disease of dairy cows. RESULTS: We identified 1841 differential acetylation sites on 665 proteins. Among of them, 1072 sites on 393 proteins were quantified. Functional enrichment analysis shows that higher acetylated proteins are significantly enriched in energy metabolic pathways, while lower acetylated proteins are significantly enriched in pathways related to immune response, such as drug metabolism and cancer. Among significantly acetylated proteins, many mitochondrial proteins were identified to be interacting with multiple proteins and involving in lipid metabolism. Furthermore, this study identified potential important proteins, such as HADHA, ACAT1, and EHHADH, which may be important regulatory factors through modification of acetylation in the development of fatty liver disease in dairy cows and possible therapeutic targets for NAFLD in human beings. CONCLUSION: This study provided a comprehensive acetylome profile of fatty liver of dairy cows, and revealed important biological pathways associated with protein acetylation occurred in mitochondria, which were involved in the regulation of the pathogenesis of fatty liver disease. Furthermore, potential important proteins, such as HADHA, ACAT1, EHHADH, were predicted to be essential regulators during the pathogenesis of fatty liver disease. The work would contribute to the understanding the pathogenesis of NAFLD, and inspire in the development of new therapeutic strategies for NAFLD.

Activities of Nerol, a natural plant active ingredient, against Candida albicans in vitro and in vivo.

Candida albicans invasion is one of the most serious fungal infections in clinical history. In recent years, because of the widespread use of immunosuppressive drugs, chemotherapy drugs, glucocorticoids, and broad-spectrum antibiotics, serious drug resistance has been reported; therefore, a new type of antifungal drug needs to be developed. In this study, we found that Nerol (NEL) had strong antimicrobial activity and 0.77 µL/mL NEL was the minimum inhibitory concentration (MIC) effective against C. albicans. We determined the change of the growth curve of NEL for C. albicans, to identify the trend of NEL activity against C. albicans. Through the determination of the ergosterol content and glucose-induced extracellular fluid acidification of NEL on C. albicans, we found that NEL inhibits the growth of C. albicans by destroying cell membranes. This finding was also supported by the expression of SAP (secreted aspartyl proteinase) involved in cell membrane synthesis. Finally, demonstrations of phenotype investigation, colony-forming unit (CFU) counts, and PAS (periodic acid-Schiff) staining were conducted to prove that NEL had the ability to treated mouse oral C. albicans infection and vaginal C. albicans infection. This research may help us to investigate new antimicrobial agents for treating C. albicans infections. KEY POINTS: • NEL can inhibit the growth of C. albicans. • NEL destroys the cell membrane formation and permeability of C. albicans. • NEL can treat vulvovaginal candidiasis and oropharyngeal candidiasis in mice. • NEL could be used as a possible antifungal agent.

Cinnamaldehyde inhibits Candida albicans growth by causing apoptosis and its treatment on vulvovaginal candidiasis and oropharyngeal candidiasis.

The invasion of Candida albicans is one of the most common fungal infections seen in clinical practice, and serious drug resistance has been reported in recent years. Therefore, new anti-C. albicans drugs must be introduced. In this research, it was demonstrated that cinnamaldehyde (CA) shows strong antimicrobial activity, with 0.26 mg/mL CA being the minimum inhibitory concentration to manage C. albicans. Extraordinarily, we detected that CA accumulated the intracellular reactive oxygen species (ROS) and enhanced the calcium concentration in the cytoplasm and mitochondria through flow cytometry. In addition, we observed that C. albicans cells released Cytochrome c from the mitochondria to the cytoplasm, depolarized the mitochondrial membrane potential, and activated the metacaspase when exposed to 0.065, 0.13, 0.26, and 0.52 mg/mL CA. Furthermore, to confirm that CA introduces the C. albicans apoptosis, we discovered that when the phosphatidylserine was exposed, DNA damage and chromatin condensation occurred, which were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining. Finally, demonstrations of phenotype investigation, colony-forming unit (CFU) counts, and periodic acid-Schiff (PAS) staining were conducted to prove that CA possessed the ability to treat oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC). From the above, our research indicates that CA is a promising antifungal candidate when applied to C. albicans infections.

Effect of Perillaldehyde on Prophylaxis and Treatment of Vaginal Candidiasis in a Murine Model.

Vulvovaginal candidiasis is a common fungal infection afflicting women which is primarily caused by the yeast Candida albicans (C. albicans). It is imperative to introduce new drug classes to counter this threat due to the continuous emergence of drug-resistant cases in recent years. The purpose of this study was to clarify the in vivo antifungal activity of perillaldehyde (PAE) against C. albicans and to prove that PAE is a promising candidate for the control of vaginal candidiasis. An animal model of vaginitis was developed to demonstrate the therapeutic and preventive effects of PAE on vaginal candidiasis, and these were evaluated through fungal and histopathological examinations. In clarifying the mechanism of PAE, standard hematological test results indicated that white blood cells (WBC) were elevated abnormally in mice infected with C. albicans, whereas when the mice were treated with various concentrations of PAE, the number of WBC in the blood was reduced. Flow cytometry was used to detect the populations of neutrophils, macrophages and CD4 T cells in the vaginal tissue of the mice. PAE was found to reduce these immune cells, which all play a key role in the inflammatory response, and the related interleukin and pro-inflammatory cytokines, including IL-17, IL-22 and TNF-α. These were detected using ELISA. Finally, we detected the expression levels of E-cadherin in the PAE treatment mouse group and discovered that it had recovered to its normal levels, but in the infection mouse group, the E-cadherin expression was clearly suppressed by the presence of C. albicans. Our data demonstrated that PAE targets these cytokines and possesses the ability to fight the fungal infection while also reducing the levels of the inflammatory factors identified. Our results demonstrated that PAE has a significant preventative and therapeutic effect on vaginal candidiasis and is a potential candidate for the treatment of vaginal Candida infections.

Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes.

BACKGROUND: Chronic ER stress and dysfunction is a hallmark of obesity and a critical contributor to metaflammation, abnormal hormone action and altered substrate metabolism in metabolic tissues, such as liver and adipocytes. Lack of STAMP2 in lean mice induces inflammation and insulin resistance on a regular diet, and it is dysregulated in the adipose tissue of obese mice and humans. We hypothesized that the regulation of STAMP2 is disrupted by ER stress. METHODS: 3T3-L1 and MEF adipocytes were treated with ER stress inducers thapsigargin and tunicamycin, and inflammation inducer TNFα. The treatments effect on STAMP2 expression and enzymatic function was assessed. In addition, 3T3-L1 adipocytes and HEK cells were utilized for Stamp2 promoter activity investigation performed with luciferase and ChIP assays. RESULTS: ER stress significantly reduced both STAMP2 mRNA and protein expression in cultured adipocytes whereas TNFα had the opposite effect. Concomitant with loss of STAMP2 expression during ER stress, intracellular localization of STAMP2 was altered and total iron reductase activity was reduced. Stamp2 promoter analysis by reporter assays and chromatin immunoprecipitation, showed that induction of ER stress disrupts C/EBPα-mediated STAMP2 expression. CONCLUSION: These data suggest a clear link between ER stress and quantitative and functional STAMP2-deficiency.

IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling.

Activation of endoplasmic reticulum (ER) stress/the unfolded protein response (UPR) has been linked to cancer, but the molecular mechanisms are poorly understood and there is a paucity of reagents to translate this for cancer therapy. Here, we report that an IRE1α RNase-specific inhibitor, MKC8866, strongly inhibits prostate cancer (PCa) tumor growth as monotherapy in multiple preclinical models in mice and shows synergistic antitumor effects with current PCa drugs. Interestingly, global transcriptomic analysis reveal that IRE1α-XBP1s pathway activity is required for c-MYC signaling, one of the most highly activated oncogenic pathways in PCa. XBP1s is necessary for optimal c-MYC mRNA and protein expression, establishing, for the first time, a direct link between UPR and oncogene activation. In addition, an XBP1-specific gene expression signature is strongly associated with PCa prognosis. Our data establish IRE1α-XBP1s signaling as a central pathway in PCa and indicate that its targeting may offer novel treatment strategies.

Cinnamaldehyde, a Promising Natural Preservative Against Aspergillus flavus.

The problem of food spoilage due to Aspergillus flavus (A. flavus) needs to be resolved. In this study, we found that the minimum inhibitory concentration of cinnamaldehyde (CA) that inhibited A. flavus was 0.065 mg/ml and that corn can be prevented from spoiling at a concentration of 0.13 mg/cm3. In addition to inhibiting spore germination, mycelial growth, and biomass production, CA can also reduce ergosterol synthesis and can cause cytomembrane damage. Our intention was to elucidate the antifungal mechanism of CA. Flow cytometry, fluorescence microscopy, and western blot were used to reveal that different concentrations of CA can cause a series of apoptotic events in A. flavus, including elevated Ca2+ and reactive oxygen species, decrease in mitochondrial membrane potential (Δψ m ), the release of cytochrome c, the activation of metacaspase, phosphatidylserine (PS) externalization, and DNA damage. Moreover, CA significantly increased the expression levels of apoptosis-related genes (Mst3, Stm1, AMID, Yca1, DAP3, and HtrA2). In summary, our results indicate that CA is a promising antifungal agent for use in food preservation.

Correlation analysis between injury patterns of medial patellofemoral ligament and vastus medialis obliquus after acute first-time lateral patellar dislocation.

PURPOSE: To evaluate the correlation between injury patterns of the medial patellofemoral ligament (MPFL) and vastus medialis obliquus (VMO) after acute first-time lateral patellar dislocation (LPD) in adults. METHODS: Magnetic resonance imaging (MRI) was prospectively performed in 132 consecutive adults with acute first-time LPD. Images were acquired and evaluated using standardized protocols. Injury patterns of MPFL were grouped by location and severity for analysis of the prevalence of VMO injury. RESULTS: MRI demonstrated VMO injury in 63 (47.7%) patients. Twenty (38.5%) and 43 cases (56.6%) were present in partial and complete MPFL tear subgroups, respectively. Compared with partial MPFL tears, complete tears showed a higher prevalence of VMO injury (P = 0.044). The mean coronal (28.5 mm) and mean sagittal VMO elevations (20.7 mm) were higher in the complete MPFL tear subgroup than in the partial tear subgroup (19.8 mm, P = 0.005; 11.9 mm, P < 0.001). No correlations were identified between the prevalence of VMO injury and location subgroups of MPFL injury (n.s.). Mean VMO elevations were higher in isolated femoral-side (FEM) and combined MPFL tear (COM) subgroups (mean coronal VMO elevation of 29 mm and mean sagittal VMO elevation of 20.8 mm in the FEM subgroup; mean coronal VMO elevation of 29.6 mm and mean sagittal VMO elevation of 23.1 mm in the COM subgroup) than in the isolated patellar-side MPFL tear (PAT) subgroup (P = 0.022, P < 0.001) (mean coronal VMO elevation of 20.7 mm and mean sagittal VMO elevation of 10.6 mm). CONCLUSIONS: Complete MPFL tear predisposes to VMO injury and has a higher elevation of torn VMO after acute first-time LPD in adults. Isolated femoral-side and combined MPFL tears predispose to higher elevation of torn VMO. LEVEL OF EVIDENCE: IV.

STAMP2 is required for human adipose-derived stem cell differentiation and adipocyte-facilitated prostate cancer growth in vivo.

Six Transmembrane Protein of Prostate 2 (STAMP2) has been implicated in both prostate cancer (PCa) and metabolic disease. STAMP2 has unique anti-inflammatory and pro-metabolic properties in mouse adipose tissue, but there is limited information on its role in human metabolic tissues. Using human adipose-derived stem cells (ASCs), we report that STAMP2 expression is dramatically upregulated during adipogenesis. shRNA-mediated STAMP2 knockdown in ASCs significantly suppresses adipogenesis and interferes with optimal expression of adipogenic genes and adipocyte metabolic function. Furthermore, ASC-derived adipocyte-mediated stimulation of prostate tumor growth in nude mice is significantly reduced upon STAMP2 knockdown in ASC adipocytes. These results suggest that STAMP2 is crucial for normal ASC conversion into adipocytes and their metabolic function, as well as their ability to facilitate PCa growth in vivo.

Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

AIM: To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. MAIN METHODS: Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. KEY FINDINGS: Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. SIGNIFICANCE: This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation.

In vivo and in vitro assessment of the biocompatibility and degradation of high-purity Mg anastomotic staples.

Titanium (Ti) staples are not biodegradable, and anastomotic complications related to Ti staples are reported frequently. In the present study, the biocompatibility and degradation behavior of high-purity magnesium (HP Mg) staples with the small intestine were investigated. HP Mg staples did not affect the relative growth rate, cell cycle and apoptosis of primary rectal mucosal epithelial cells (IEC-6) in vitro. At one, two and three days after immersion in intestinal juice, the weight of the 30 rinsed HP Mg staples reduced by 7.5 ± 1.6, 10.6 ± 2.2 and 13.5 ± 2.1 mg, respectively, and those in the Hanks' solution reduced by 3.9 ± 0.8, 6.1 ± 1.2 and 7.1 ± 2.4 mg. Extracts of HP Mg staples were bio-safe for IEC-6, and the corrosion rate of HP staples was faster in the small intestinal juice than in the Hanks' solution. In the in vivo experiments, the small intestine of the minipigs was anastomosed by HP Mg and Ti staples. HP Mg staples neither affected important bio-chemical parameters nor induced serious inflammation or necrosis in the anastomosis tissues. The residual weight of a HP Mg staples (0.81 ± 0.13 mg) was 89.7% of the original weight (9 ± 0.09 mg) one month after surgery. The in vivo corrosion rate for one HP Mg staple was determined to be∼0.007 ± 0.001 mm·month-1. The preliminary results of the biocompatibility and degradation of high-purity Mg anastomotic staples are promising, and further studies will be initiated to study in more detail.