Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

Dihydroartemisinin is an inhibitor of trained immunity through Akt/mTOR/HIF1α signaling pathway.

Trained immunity is mechanistically defined as the metabolically and epigenetically mediated long-term functional adaptation of the innate immune system, characterized by a heightened response to a secondary stimulation. Given appropriate activation, trained immunity represents an attractive anti-infective therapeutic target. Nevertheless, excessive immune response and subsequent inflammatory cascades may contribute to pathological tissue damage, indicating that the negative impacts of trained immunity appear to be significant. In this study, we show that innate immune responses such as the production of extracellular traps, pro-inflammatory cytokines, and autophagy-related proteins were markedly augmented in trained BMDMs. Furthermore, heat-killed C. albicans priming promotes the activation of the AIM2 inflammasome, and AIM2-/- mice exhibit impaired memory response induced by heat-killed C. albicans. Therefore, we establish that the AIM2 inflammasome is involved in trained immunity and emerges as a promising therapeutic target for potentially deleterious effects. Dihydroartemisinin can inhibit the memory response induced by heat-killed C. albicans through modulation of mTOR signaling and the AIM2 inflammasome. The findings suggest that dihydroartemisinin can reduce the induction of trained immunity by heat-killed C. albicans in C57BL/6 mice. Dihydroartemisinin is one such therapeutic intervention that has the potential to treat of diseases characterized by excessive trained immunity.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

IFI204 protects host defense against Staphylococcus aureus-induced pneumonia by promoting extracellular traps formation.

Interferon-inducible protein 204 (IFI204) is an intracellular DNA receptor that can recognize DNA viruses and intracellular bacteria. Extracellular traps (ETs) have been recognized as an indispensable antimicrobial barrier that play an indispensable role in bacterial, fungal, parasitic, and viral infections. However, how ETs form and the mechanisms by which IFI204 function in Staphylococcus aureus pneumonia are still unclear. Moreover, by in vitro experiments, we proved that IFI204 deficiency decreases the formation of ETs induced by Staphylococcus aureus in a NOX-independent manner. More importantly, Deoxyribonuclease I (DNase I) treatment significantly inhibited the formation of ETs. IFI204 contributed to ETs formation by promoting citrullination of histone H3 and the expression of PAD4 (peptidylarginine deiminase 4). Altogether, these findings highlight the potential importance of IFI204 for host defense against S. aureus USA300-TCH1516 infection.

Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.

Akkermansia muciniphila Suppresses High-Fat Diet-Induced Obesity and Related Metabolic Disorders in Beagles.

Obesity is one of the prevalent chronic diseases in human and companion animals usually associated with several metabolic disorders. The gut commensal bacterium Akkermansia muciniphila (A. muciniphila) is known for its therapeutic effects on metabolic disorders and inflammations. Here, we isolated the A. muciniphila AKK2 strain from the feces of interferon-inducible protein 204-/- (IFI204-/-) mice and further evaluated its anti-obesity effects on high-fat diet (HFD)-fed C57BL/6J mice and beagles. The results showed that it effectively controlled weight gain. Microbiome analysis using 16S rRNA gene sequencing revealed that HFD alters gut microbiota composition and A. muciniphila AKK2 increases the Firmicutes/Bacteroidetes (F/B) ratio in beagles. Furthermore, we prepared microcapsules containing A. muciniphila AKK2, and tolerance tests showed the encapsulation maintained high viability and stability in an aerobic environment and simulated the secretion of gastrointestinal fluids. Overall, this study widens the spectrum of A. muciniphila applications to prevent obesity.

Peripheral immunological features of COVID-19 patients in Taizhou, China: A retrospective study.

BACKGROUND: Abnormal peripheral immunological features are associated with the progression of coronavirus disease 2019 (COVID-19). METHODS: Clinical and laboratory data were retrieved in a cohort of 146 laboratory-confirmed COVID-19 patients. Potential risk factors for the development of severe COVID-19 were evaluated. RESULTS: On admission, lymphocytes, CD3+, CD4+ and CD8+ T cells, eosinophils, and albumin and pre-albumin were dramatically lower, whereas neutrophils, and interleukin (IL)-10, C-reactive protein (CRP), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were significantly higher in severe cases. By the second week after discharge, all variables improved to normal levels. Covariate logistic regression results showed that the CD8+ cell count and CRP level were independent risk factors for severe COVID-19. CONCLUSION: Lower peripheral immune cell subsets in patients with severe disease recovered to normal levels as early as the second week after discharge. CD8+ T cell counts and CRP levels on admission are independent predictive factors for severe COVID-19.

Early Risk Factors for the Duration of Severe Acute Respiratory Syndrome Coronavirus 2 Viral Positivity in Patients With Coronavirus Disease 2019.

BACKGROUND: Pneumonia coronavirus disease 2019 (COVID-19) has became a pandemic. However, information on early risk factors for the duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral positivity is not yet available. METHODS: In this prospective study, a cohort of 137 patients with confirmed SARS-CoV-2 were enrolled. Clinical information and laboratory data were retrieved from electronic medical records. Viral positivity duration was calculated by the interval from the day of confirmed SARS-CoV-2 positive results to the day SARS-CoV-2 testing showed negative results in these 137 patients with COVID-19. Early risk factors for the duration of SARS-CoV-2 viral positivity were evaluated. RESULTS: The median SARS-CoV-2 viral positivity duration is 12 days (range, 4 to ~45) for this cohort. Cox regression results showed a significantly shorter viral positivity duration was related to younger age (hazard ratio [HR], .658; P = .017); disease not being severe (HR, .653; P = .076); higher lymphocyte (HR, 1.464; P = .033), eosinophil (HR, 1.514; P = .020), and CD8+ T-cell (HR, 1.745; P = .033) counts; and lower IL-6 (HR, .664; P = .036) and IL-10 (HR, .631; P = .021). Multivariate analysis with covariable-adjusted results showed that the CD8+ T-cell count (HR, 2.376; P= .114) was a predominant risk factor for the duration of SARS-CoV-2 viral positivity. CONCLUSIONS: Our findings show early laboratory parameters such as CD8+ T-cell count to be risk factors for the duration of SARS-CoV-2 viral positivity, which has significance in the control and prevention of the disease.

Complex mode-locking oscillations and Stern-Brocot derivation tree in a CSTR reaction with impulsive perturbations.

A complex mode-locking (entrainment) topology underlying the continuous stirred tank reactor reaction model subjected to impulsive perturbations is identified. Employing high-resolution stability diagrams, we exhibit the global structure of mode-locking oscillations and describe how they are interconnected and how their complexity unfolds with control parameters varying. The scenarios shown in the bi-parametric planes revealed that the skeleton of Arnold's tongues is organized according to the symmetric Stern-Brocot sum tree. Moreover, the mode-locking organization is controlled by an invariant torus (a pair of frequencies) initiated from Hopf bifurcations. Interestingly, the mode-locking order is unfolded in an elusive way, that is, in perfect agreement with the reciprocal of the Stern-Brocot sum tree. The findings reported here contribute to providing a description and classification of mode-locking oscillations for the impulsive system.

Significance of plasma MACC1 levels on the prognostic stratification in patients with colorectal cancer.

The clinical significance of metastasis-associated in colon cancer-1 (MACC1) has been investigated but the relevance of peripheral MACC1 levels was rather limited. Herein, our data revealed that plasma MACC1 levels in 117 colorectal cancer patients (CRC) were dramatically higher than that in normal controls (P < 0.001), and with a strong discrimination power between the two groups (AUC = 0.960, P < 0.001). Moreover, MACC1 is an independent prognostic factor for CRC patients. When clinical parameters stratified by MACC1low and MACC1high , MACC1 levels exhibited further significant predictive value. Summary, plasma MACC1 levels could be a useful prognostic and diagnostic biomarker, and could improve the prognostic value of traditional prognosticators for colorectal cancer patients.

[Advances in indole alkaloids from traditional Chinese medicine of Uncariae Ramulus Cum Uncis documented in Chinese Pharmacopoeia].

Uncaria genus( Rubiaceae) contains 34 species all over the world,of which 11 species and one variant are present in China. Five species,namely U. rhynchophylla,U. macrophylla,U. hirsuta,U. sinensis and U. sessilifructus,are documented in Chinese Pharmacopoeia as the raw materials of Uncariae Ramulus Cum Uncis. Indole alkaloids are the characteristic constituents of Uncaria plants,in addition to triterpenes,lignans and flavones. This paper reviews the progress of indole alkaloids and their distribution within the five Uncaria species documented in Chinese Pharmacopoeia for better understanding the active constituents of Uncariae Ramulus Cum Uncis.

Dereplication-guided isolation of a new indole alkaloid triglycoside from the hooks of Uncaria rhynchophylla by LC with ion trap time-of-flight MS.

Uncaria rhynchophylla (Gou-Teng) as the monarch herb of many formulae (Fufang), e.g. "Tian-Ma-Gou-Teng-Yin," "Ling-Jiao-Gou-Teng-Yin," and "Yi-Gan-San", is a famous traditional Chinese medicine documented in the Chinese pharmacopoeia for mental and cardiovascular diseases. In the traditional Chinese medicine system, only the hook-bearing stems are used as the crude materials for Gou-Teng, and the hooks are always considered more effective than the stems. Focusing on the mono-herb and its active constituents from combinatorial formulae is the core idea of reductionism of traditional Chinese medicine theory. Detailed liquid chromatography with mass spectrometry analysis on the hooks of U. rhynchophylla was performed to profile the chemical constituents based on tandem mass spectrometry fragmentation and UV absorption. Under the guidance of liquid chromatography with ion trap/time-of-flight mass spectrometry, one new indole alkaloid triglycoside (1), together with five known compounds 2-6 as the main constituents, were isolated from the hooks of U. rhynchophylla by various column chromatography methods. Compound 1 showed moderate activity on MT1 and MT2 melatonin receptors with agonistic rates of 79.6 and 46.3% at the concentration of 1 mM. This dereplication strategy can be equally applicable to rapidly disclose the active constituents of other Chinese herbs through targeted purification.

Chemical and biological comparison of different sections of Uncaria rhynchophylla (Gou-Teng).

Uncaria rhynchophylla (Gou-Teng in Chinese) is officially documented in Chinese pharmacopoeia as one of the authentic sources for the crude drug of Gou-Teng which has long been used for mental and cardiovascular diseases. Indole alkaloids are the characteristic constituents responsible for the desired hypotensive effect; however, the psychiatric active constituents of Gou-Teng are still unclear. According to traditional Chinese medicine theory, only the hook-bearing stems of U. rhynchophylla are used as the crude materials for Gou-Teng, while its leaves and fruits are scarcely used. The present study aimed to compare the metabolic fingerprints of different parts (hooks, stems, leaves and fruits) of U. rhynchophylla by LC-DAD-MS/MS analysis and further evaluate their psychiatric activities on HEK293 cell line in vitro. A total of 38 constituents including 26 alkaloids, six flavonoids, two triterpenoids, two chlorogenic acid analogs and two other compounds were characterized. The different parts of U. rhynchophylla can be well differentiated from their chemical profiles. Leaves displayed the most potent activity on both MT1 and MT2 receptors, with agonistic rates of 39.7% and 97.6%. For 5-HT1A and 5-HT2C receptors, hooks showed the strongest activity with agonistic rates of 92.6% and 83.1%, respectively. This investigation provided valuable information for understanding the chemical divergence between different parts of U. rhynchophylla, and their substantial bases for psychiatric purposes.

Prognostic risk stratification value of MACC1 expression in patients with gastric cancer.

BACKGROUND: The prognostic significance of metastasis-associated in colon cancer-1 (MACC1) has been explored in a variety of malignancies. However, its clinical relevance in patients with gastric cancer (GC) is limited, also remains controversial. METHOD: In this study, we retrospectively evaluated the prognostic value of lesion MACC1 expression in 347 GC patients. Lesion MACC1 expression was analyzed with immunohistochemistry and grouped as MACC1low (n = 172) and MACC1high (n = 175) cases. RESULTS: Data revealed that the degree of MACC1 expression is not related to patient sex, age and disease stage (all p > 0.05). Survival analysis showed that only post-operation advanced pT (p = 0.018), pN (p < 0.001), pM (p = 0.001) and AJCC stages (p < 0.001) are significantly associated with shorter survival, while no obvious difference was observed between MACC1low and MACC1high cases (p = 0.158). However, we found that survival for female (p = 0.032), older (p = 0.028), and early disease stage (pT stage I + II, p = 0.033) patients with MACC1high are remarkably worse than those with MACC1low. CONCLUSION: In summary, our findings revealed that, though MACC1 expression is not associated with the survival of the whole cohort, the prognostic risk stratification value of lesion MACC1 expression in subgroups of patients with gastric cancer should be noted.

Dual-Mechanism Peptide SR25 has Broad Antimicrobial Activity and Potential Application for Healing Bacteria-infected Diabetic Wounds.

The rise of antibiotic resistance poses a significant public health crisis, particularly due to limited antimicrobial options for the treatment of infections with Gram-negative pathogens. Here, an antimicrobial peptide (AMP) SR25 is characterized, which effectively kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism without detectable resistance. Meanwhile, an SR25-functionalized hydrogel is developed for the efficient treatment of infected diabetic wounds. SR25 is obtained through genome mining from an uncultured bovine enteric actinomycete named Nonomuraea Jilinensis sp. nov. Investigations reveal that SR25 has two independent cellular targets, disrupting bacterial membrane integrity and restraining the activity of succinate:quinone oxidoreductase (SQR). In a diabetic mice wound infection model, the SR25-incorporated hydrogel exhibits high efficacy against mixed infections of Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA), accelerating wound healing. Overall, these findings demonstrate the therapeutic potential of SR25 and highlight the value of mining drugs with multiple mechanisms from uncultured animal commensals for combating challenging bacterial pathogens.

Biobanking of fresh-frozen human colon tissues: impact of tissue ex-vivo ischemia times and storage periods on RNA quality.

BACKGROUND: Biobanking plays an important role in translational cancer research. The impact of tissue ex-vivo ischemia time and storage period on RNA integrity is not well documented. METHODS: Fresh-frozen colon tissues were collected in Taizhou Hospital of Zhejiang Province in China since 2004. Fifty-one colon cancer tissues with tumor cell content higher than 70 % and matched normal tissues during four storage periods (less than 15 months, 16-20 months, 21-25 months, and 26-40 months) were chosen to detect RNA quality. Fresh colon cancer tissues from 5 patients were cut into pieces and kept at room temperature or on ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was determined by microcapillary electrophoresis by the RNA integrity number (RIN) algorithm. RESULTS: Sixty-seven percent of normal colon tissues and 94 % of colon cancer specimens yielded RNA with a RIN of ≥7. Matched colon cancer and normal tissues showed significant difference in RNA quality. RNA remained stable in colon cancer tissues kept at room temperature and on ice for up to 4 h, and long-term storage of banked colon specimens did not negatively influence RNA quality (RNA with RIN of ≥7 banked less than 15 months, 83 %; 16-20 months, 78 %; 21-25 months, 77 %; 26-40 months, 90 %). CONCLUSIONS: Frozen colon tissues yield high-quality RNA in approximately 80 % of specimens. Ex-vivo ischemia times and storage periods did not adversely affect RNA quality. This study showed that standard operation protocols and the maintenance of high-quality tissue repositories were the keys to translational medicine research.

[Effect of jianpi liqi recipe on mRNA expressions of TP, DPD, and CYP3A4 in mice with human gastric cancer transplanted tumor].

OBJECTIVE: To study the effect of Jianpi Liqi Recipe (JLR) on 5-fluorouracil (5-FU) relevant metabolic enzymes and CYP3A4 (the same enzyme of many chemotherapeutics) of mice with human gastric cancer transplanted tumor. METHODS: Totally 80 mice were randomly divided into the model group, the chemotherapy group, the JLR group, and the combination group (using chemotherapy combined JLR), 20 in each group. The human gastric cancer transplanted tumor mouse model was duplicated by hypodermic inoculating MKN-8 tumor cell suspension from the left armpit. Physiological saline or JLR was given to those in the model group or the JLR group at 0.25 mL each time, twice daily by gastrogavage from the 2nd day after transplantation. Mice in the chemotherapy group were given 0.25 mL physiological saline, twice daily by gastrogavage 2 days after transplantation, for 5 days in succession, and then they were peritoneal injected with 5-FU at the daily dose of 20 mg/kg, once daily for 5 days in succession from the 7th day of transplantation. Those in the combination were given 0.25 mL JLR, twice daily by gastrogavage, for 5 days in succession, and then they were peritoneal injected with 5-FU at the daily dose of 20 mg/kg, once daily for 5 days in succession from the 7th day of transplantation. The mRNA expressions of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and CYP3A4 were detected using RT-PCR. RESULTS: Compared with the model group and the chemotherapy group, mRNA expressions of TP and CYP3A4 obviously increased, mRNA expression of DPD obviously decreased in the JLR group and the combination group (P < 0.01). There was no statistical difference in mRNA expressions of TP, DPD, and CYP3A4 between the JLR group and the combination group (P > 0.05). CONCLUSION: JLR could promote the activation of 5-FU, suppress the decomposition and inactivation of 5-FU in the tumor tissue of mice, and improve the chemotherapeutic efficacy through up-regulating mRNA expressions of TP and CYP3A4, and suppressing the mRNA expression of DPD.