Breast tumours maintain a reservoir of subclonal diversity during expansion.

Our knowledge of copy number evolution during the expansion of primary breast tumours is limited1,2. Here, to investigate this process, we developed a single-cell, single-molecule DNA-sequencing method and performed copy number analysis of 16,178 single cells from 8 human triple-negative breast cancers and 4 cell lines. The results show that breast tumours and cell lines comprise a large milieu of subclones (7-22) that are organized into a few (3-5) major superclones. Evolutionary analysis suggests that after clonal TP53 mutations, multiple loss-of-heterozygosity events and genome doubling, there was a period of transient genomic instability followed by ongoing copy number evolution during the primary tumour expansion. By subcloning single daughter cells in culture, we show that tumour cells rediversify their genomes and do not retain isogenic properties. These data show that triple-negative breast cancers continue to evolve chromosome aberrations and maintain a reservoir of subclonal diversity during primary tumour growth.

The Black Lives Matter movement mitigates bias against racial minority actors.

Watching movies is among the most popular entertainment and cultural activities. How do viewers react when a movie sequel increases racial minority actors in the main cast ("minority increase")? On the one hand, such sequels may receive better evaluations if viewers appreciate racially inclusive casting for its novel elements (the value-in-diversity perspective) and moral appeal (the fairness perspective on diversity). On the other hand, discrimination research suggests that if viewers harbor biases against racial minorities, sequels with minority increase may receive worse evaluations. To examine these competing possibilities, we analyze a unique panel dataset of movie series released from 1998 to 2021 and conduct text analysis of 312,457 reviews of these movies. Consistent with discrimination research, we find that movies with minority increase receive lower ratings and more toxic reviews. Importantly, these effects weaken after the advent of the Black Lives Matter (BLM) movement, especially when the movement's intensity is high. These results are reliable across various robustness checks (e.g., propensity score matching, random implementation test). We conceptually replicate the bias mitigation effect of BLM in a preregistered experiment: Heightening the salience of BLM increases White individuals' acceptance of racial minority increase in a movie sequel. This research demonstrates the power of social movements in fostering diversity, equality, and inclusion.

HDGF stimulates liver tumorigenesis by enhancing reactive oxygen species generation in mitochondria.

Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor prognosis in various types of cancer. However, the interplay between HDGF and ROS generation has not been elucidated in hepatocellular carcinoma. Here, we first analyzed the profile of HDGF expression and ROS production in newly generated orthotopic hepatomas by ultrasound-guided implantation. In situ superoxide detection showed that HDGF-overexpressing hepatomas had significantly elevated ROS levels compared with adjacent nontumor tissues. Consistently, liver tissues from HDGF-deficient mice exhibited lower ROS fluorescence than those from age- and sex-matched WT mice. ROS-detecting fluorescent dyes and flow cytometry revealed that recombinant HDGF (rHDGF) stimulated the production of superoxide anion, hydrogen peroxide, and mitochondrial ROS generation in cultured hepatoma cells in a dose-dependent manner. In contrast, the inactive Ser103Ala rHDGF mutant failed to promote ROS generation or oncogenic behaviors. Seahorse metabolic flux assays revealed that rHDGF dose dependently upregulated bioenergetics through enhanced basal and total oxygen consumption rate, extracellular acidification rate, and oxidative phosphorylation in hepatoma cells. Moreover, antioxidants of N-acetyl cysteine and MitoQ treatment significantly inhibited HDGF-mediated cell proliferation and invasive capacity. Genetic silencing of superoxide dismutase 2 augmented the HDGF-induced ROS generation and oncogenic behaviors of hepatoma cells. Finally, genetic knockdown nucleolin (NCL) and antibody neutralization of surface NCL, the HDGF receptor, abolished the HDGF-induced increase in ROS and mitochondrial energetics. In conclusion, this study has demonstrated for the first time that the HDGF/NCL signaling axis induces ROS generation by elevating ROS generation in mitochondria, thereby stimulating liver carcinogenesis.

Extracorporeal photopheresis reduces inflammation and joint damage in a rheumatoid arthritis murine model.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory reactions and tissue damage in the joints. Long-term drug use in clinical practice is often accompanied by adverse reactions. Extracorporeal photopheresis (ECP) is an immunomodulatory therapy with few side effects, offering a potential and safe therapeutic alternative for RA through the induction of immune tolerance. This study aimed to investigate the therapeutic effects of ECP on RA using a collagen-induced arthritis (CIA) murine model, as well as to explore its immunomodulatory effects in vivo. Additionally, particular attention was given to the significant role of monocytes during the ECP process. METHODS: A murine model of rheumatoid arthritis was established by administering two injections of bovine type II collagen to DBA/1J mice. ECP, ECP-MD (mononuclear cells were depleted during the ECP), MTX, and PBS treatment were applied to the CIA mice. During the treatment process, clinical scores and body weight changes of CIA mice were closely monitored. After six treatment sessions, micro-CT images of the hind paws from live mice were captured. Ankle joints and paws of the mice were collected and processed for histological evaluation. Spleen samples were collected to measure the Th17/Treg cells ratio, and serum samples were collected to assess cytokine and anti-type II collagen IgG levels. Monocytes and dendritic cells populations before and after ECP in vitro were detected by flow cytometry. RESULT: ECP therapy significantly attenuated the progression of CIA, alleviated the severity of clinical symptoms in CIA mice and effectively suppressed synovial hyperplasia, inflammation, and cartilage damage. There was an expansion in the percentage of CD3 + CD4 + CD25 + FoxP3 + Tregs and a decrease in CD3 + CD4 + IL17A + Th17 cells in vivo. Furthermore, ECP reduced the serum levels of pro-inflammatory cytokines IL-6 (53.47 ± 7.074 pg/mL vs 5.142 ± 1.779 pg/mL, P < 0.05) and IL-17A (3.077 ± 0.401 pg/mL vs 0.238 ± 0.082 pg/mlL, P < 0.0001) compared with PBS. Interestingly, the depletion of monocytes during the ECP process did not lead to any improvement in clinical symptoms or histological scores in CIA mice. Moreover, the imbalance in the Th17/Treg cells ratio became even more pronounced, accompanied by an augmented secretion of pro-inflammatory cytokines IL-6 and IL-17A. In vitro, compared with cells without ECP treatment, the proportion of CD11b + cells were significantly reduced (P < 0.01), the proportion of CD11c + cells were significantly elevated (P < 0.001) 24 h after ECP treatment. Additionally, the expression of MHC II (P < 0.0001), CD80 (P < 0.01), and CD86 (P < 0.001) was downregulated in CD11c + cells 24 h after ECP treatment. CONCLUSION: Our study demonstrates that ECP exhibits a therapeutic effect comparable to conventional therapy in CIA mice, and the protective mechanisms of ECP against RA involve Th17/Treg cells ratio, which result in decreased IL-6 and IL-17A. Notably, monocytes derived from CIA mice are an indispensable part to the efficacy of ECP treatment, and the proportion of monocytes decreased and the proportion of tolerogenic dendritic cells increased after ECP treatment in vitro.

Utilizing radiomics and dosiomics with AI for precision prediction of radiation dermatitis in breast cancer patients.

PURPOSE: This study explores integrating clinical features with radiomic and dosiomic characteristics into AI models to enhance the prediction accuracy of radiation dermatitis (RD) in breast cancer patients undergoing volumetric modulated arc therapy (VMAT). MATERIALS AND METHODS: This study involved a retrospective analysis of 120 breast cancer patients treated with VMAT at Kaohsiung Veterans General Hospital from 2018 to 2023. Patient data included CT images, radiation doses, Dose-Volume Histogram (DVH) data, and clinical information. Using a Treatment Planning System (TPS), we segmented CT images into Regions of Interest (ROIs) to extract radiomic and dosiomic features, focusing on intensity, shape, texture, and dose distribution characteristics. Features significantly associated with the development of RD were identified using ANOVA and LASSO regression (p-value < 0.05). These features were then employed to train and evaluate Logistic Regression (LR) and Random Forest (RF) models, using tenfold cross-validation to ensure robust assessment of model efficacy. RESULTS: In this study, 102 out of 120 VMAT-treated breast cancer patients were included in the detailed analysis. Thirty-two percent of these patients developed Grade 2+ RD. Age and BMI were identified as significant clinical predictors. Through feature selection, we narrowed down the vast pool of radiomic and dosiomic data to 689 features, distributed across 10 feature subsets for model construction. In the LR model, the J subset, comprising DVH, Radiomics, and Dosiomics features, demonstrated the highest predictive performance with an AUC of 0.82. The RF model showed that subset I, which includes clinical, radiomic, and dosiomic features, achieved the best predictive accuracy with an AUC of 0.83. These results emphasize that integrating radiomic and dosiomic features significantly enhances the prediction of Grade 2+ RD. CONCLUSION: Integrating clinical, radiomic, and dosiomic characteristics into AI models significantly improves the prediction of Grade 2+ RD risk in breast cancer patients post-VMAT. The RF model analysis demonstrates that a comprehensive feature set maximizes predictive efficacy, marking a promising step towards utilizing AI in radiation therapy risk assessment and enhancing patient care outcomes.

Exposure-response modelling of osimertinib in patients with non-small cell lung cancer.

AIMS: Osimertinib is a third-generation, irreversible, central nervous system-active, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with efficacy in EGFR-mutated non-small cell lung cancer (NSCLC). We assessed the relationship between plasma osimertinib levels and its efficacy and safety events. METHODS: Comprehensive pharmacokinetics exposure-response (E-R) modelling was performed utilizing steady state area under the curve (AUCss) data from first-line, ≥second-line and adjuvant studies from the osimertinib clinical development programme (20-240 mg once-daily dosing; N = 1689 patients). Analyses were conducted for survival using a proportional hazard model; for interstitial lung disease (ILD) and left ventricular ejection fraction (LVEF) events using a penalized logistic regression model and graphical analysis of potential confounding factors; and for rash and diarrhoea events using descriptive analysis. RESULTS: E-R modelling analyses indicated no clear trend of increasing efficacy with increasing osimertinib AUCss; efficacy in all exposure quartiles was significantly better than the control arm (comparator EGFR-TKI, chemotherapy or placebo) irrespective of treatment line. Model-based analysis suggested a potential relationship between increased osimertinib exposure and increased probability of ILD events, predominantly in Japanese patients. Additionally, there were increased probabilities of rash or diarrhoea with increasing osimertinib exposure. The probability of LVEF events showed overlapping confidence intervals for osimertinib ≤80 mg and control. CONCLUSIONS: E-R modelling in patients with EGFR-mutated NSCLC demonstrated that increased osimertinib exposure was unlikely to increase efficacy but may increase occurrence of certain adverse events. Hence, long-term treatment with doses ≥80 mg was not expected to provide additional benefit.

Transfusion outcomes and clinical safety of ABO-nonidentical platelets transfusion: A systematic review and meta-analysis.

BACKGROUND: ABO-nonidentical platelets transfusion has been frequently employed to address clinical platelets insufficiencies. The significance of ABO compatibility for platelets transfusion is not clearly defined. This study is aimed to explore the transfusion outcomes and clinical safety of ABO-nonidentical platelets transfusion. STUDY DESIGN AND METHODS: A systematic articles search was performed for eligible studies published up to November 30, 2023 through the PubMed, Embase, Cochrane library, Chinese National Knowledge Infrastructure database, Wanfang database and SinMed. Meta-analysis Of Observational Studies in Epidemiology study guidelines for observational studies and Newcastle Ottawa bias scale were implemented to assess studies. Meta-analysis was performed using Manager 5.3. This study is registered with PROSPERO, number CRD42023417824. RESULTS: A total of 11 retrospective cohort studies and 7 prospective cohort studies with a sample size of 104,359 platelets transfusions were included. There was significant difference in transfusion effectiveness between the ABO-identical and ABO-nonidentical platelets transfusions (RR 1.20, 95 % CI 1.11-1.38, P < 0.00001, I2 = 21 %), also the ABO-identical platelets transfusions showed more platelets increment than ABO-nonidentical ones, but it was not statistically significant (MD 0.34, 95 % CI - 0.01 to 0.70, P = 0.06, I2 = 0 %). Allergy and fever occurred more in ABO-nonidentical platelets transfusions in terms of adverse reactions (RR 0.63, 95 % CI 0.41-0.96, P = 0.03, I2 = 0 %; RR 0.59, 95 % CI 0.37-0.94, P = 0.03, I2 = 31 %). When it comes to the mortality, the ABO-identical platelets transfusions did not statistically improve survival in patients who received multiple platelets transfusions (RR 0.77, 95 % CI 0.72-0.83, P = 0.17, I2 = 38 %) and who only received less than 3 transfusions (RR 0.74, 95 % CI 0.52-1.06, P = 0.10, I2 = 47 %) compared with the ABO-nonidentical platelets transfusions. CONCLUSION: In comparison to ABO-identical platelets transfusions, nonidentical platelets transfusions exhibited lower transfusion efficacy. However, the clinical safety between these two groups was similar, which indicated that ABO-nonidentical transfusions are acceptable, albeit inferior to ABO-identical ones.

Leukocyte cell-derived chemotaxin 2 regulates epithelial-mesenchymal transition and cancer stemness in hepatocellular carcinoma.

Leukocyte cell-derived chemotaxin 2 (LECT2) acts as a tumor suppressor in hepatocellular carcinoma (HCC). However, the antineoplastic mechanism of LECT2, especially its influence on hepatic cancer stem cells (CSCs), remains largely unknown. In The Cancer Genome Atlas cohort, LECT2 mRNA expression was shown to be associated with stage, grade, recurrence, and overall survival in human HCC patients, and LECT2 expression was downregulated in hepatoma tissues compared with the adjacent nontumoral liver. Here, we show by immunofluorescence and immunoblot analyses that LECT2 was expressed at lower levels in tumors and in poorly differentiated HCC cell lines. Using functional assays, we also found LECT2 was capable of suppressing oncogenic behaviors such as cell proliferation, anchorage-independent growth, migration, invasiveness, and epithelial-mesenchymal transition in hepatoma cells. Moreover, we show exogenous LECT2 treatment inhibited CSC functions such as tumor sphere formation and drug efflux. Simultaneously, hepatic CSC marker expression was also downregulated, including expression of CD133 and CD44. This was supported by infection with adenovirus encoding LECT2 (Ad-LECT2) in HCC cells. Furthermore, in animal experiments, Ad-LECT2 gene therapy showed potent efficacy in treating HCC. We demonstrate LECT2 overexpression significantly promoted cell apoptosis and reduced neovascularization/CSC expansion in rat hepatoma tissues. Mechanistically, we showed using immunoblot and immunofluorescence analyses that LECT2 inhibited ß-catenin signaling via the suppression of the hepatocyte growth factor/c-MET axis to diminish CSC properties in HCC cells. In summary, we reveal novel functions of LECT2 in the suppression of hepatic CSCs, suggesting a potential alternative strategy for HCC therapy.

Fluorescent Quinolinium Derivative as Novel Mitochondria Probe and Function Modulator by Targeting Mitochondrial RNA.

Mitochondria have a crucial role in regulating energy metabolism and their dysfunction has been linked to tumorigenesis. Cancer diagnosis and intervention have a great interest in the development of new agents that target biomolecules within mitochondria. However, monitoring and modulating mitochondria RNA (mtRNA), an essential component in mitochondria, in cells is challenging due to limited functional research and the absence of targeting agents. In this study, we designed and synthesized a fluorescent quinolinium derivative, QUCO-1, which actively lit up with mtRNA in both normal and cancer cells in vitro. Additionally, we evaluated the function of QUCO-1 as an mtRNA ligand and found that it effectively induced severe mitochondrial dysfunction and OXPHOS inhibition in RKO colorectal cancer cells. Treatment with QUCO-1 resulted in apoptosis, cell cycle blockage at the G2/M phase, and the effective inhibition of cell proliferation. Our findings suggest that QUCO-1 has great potential as a promising probe and therapeutic agent for mtRNA, with the potential for treating colorectal cancer.

R-praziquantel integrated population pharmacokinetics in preschool- and school-aged African children infected with Schistosoma mansoni and S. haematobium and Lao adults infected with Opisthorchis viverrini.

Racemic praziquantel (PZQ) is the standard treatment for schistosomiasis and liver fluke infections (opisthorchiasis and clonorchiasis). The development of an optimal pediatric formulation and dose selection would benefit from a population pharmacokinetic (popPK) model. A popPK model was developed for R-PZQ, the active enantiomer of PZQ, in 664 subjects, 493 African children (2-15 years) infected with Schistosoma mansoni and S. haematobium, and 171 Lao adults (15-78 years) infected with Opisthorchis viverrini. Racemate tablets were administered as single doses of 20, 40 and 60 mg/kg in children and 30, 40 and 50 mg/kg in 129 adults, and as 3 × 25 mg/kg apart in 42 adults. Samples collected by the dried-blood-spot technique were assayed by LC-MS/MS. A two-compartment disposition model, with allometric scaling and dual first-order and transit absorption, was developed using Phoenix™ software. Inversely parallel functions of age described the apparent oral bioavailability (BA) and clearance maturation in children and ageing in adults. BA decreased slightly in children with dose increase, and by 35% in adults with multiple dosing. Crushing tablets for preschool-aged children increased the first-order absorption rate by 64%. The mean transit absorption time was 70% higher in children. A popPK model for R-PZQ integrated African children over 2 years of age with schistosomiasis and Lao adults with opisthorchiasis, and should be useful to support dose optimization in children. In vitro hepatic and intestinal metabolism data would help refining and validating the model in younger children as well as in target ethnic pediatric and adult groups.

Improvement After Celecoxib Treatment in Patients with Thalamic Hemorrhage - A Case Report.

PURPOSE: Perihematomal edema of intracerebral hemorrhage (ICH) is caused by a hematoma-induced inflammatory reaction, which usually contributes to delayed deterioration of neurological function and poor outcomes. Celecoxib is a commonly used nonsteroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2. High-dose celecoxib (400 mg twice daily) for 14 days has been shown to reduce perihematomal edema and hematoma enlargement in patients with ICH, but without improvement in long-term functional outcome, which may be confounded by the heterogeneity of hematoma location. Low-dose celecoxib may be an effective management for symptoms caused by perihematomal edema in patients with ICH, particularly those involving the thalamus. CASE REPORT: We reported two patients with acute thalamic ICH; a common symptom between the two was delayed onset of drowsiness caused by perihematomal edema involving the thalamus. Their consciousness improved after low-dose celecoxib (200 mg once daily) administration for 3 and 2 days in case A and B, respectively. Furthermore, other symptoms that concomitantly improved included poor appetite caused by perihematomal edema involving the left hypothalamus in case A, and limb weakness caused by perihematomal edema of the internal capsule in case B. CONCLUSION: These cases revealed that low-dose celecoxib may be an effective management for symptoms caused by perihematomal edema in patients with ICH, particularly those involving the thalamus.

Polymyxin Triple Combinations against Polymyxin-Resistant, Multidrug-Resistant, KPC-Producing Klebsiella pneumoniae.

Resistance to polymyxin antibiotics is increasing. Without new antibiotic classes, combination therapy is often required. We systematically investigated bacterial killing with polymyxin-based combinations against multidrug-resistant (including polymyxin-resistant), carbapenemase-producing Klebsiella pneumoniae Monotherapies and double- and triple-combination therapies were compared to identify the most efficacious treatment using static time-kill studies (24 h, six isolates), an in vitro pharmacokinetic/pharmacodynamic model (IVM; 48 h, two isolates), and the mouse thigh infection model (24 h, six isolates). In static time-kill studies, all monotherapies (polymyxin B, rifampin, amikacin, meropenem, or minocycline) were ineffective. Initial bacterial killing was enhanced with various polymyxin B-containing double combinations; however, substantial regrowth occurred in most cases by 24 h. Most polymyxin B-containing triple combinations provided greater and more sustained killing than double combinations. Standard dosage regimens of polymyxin B (2.5 mg/kg of body weight/day), rifampin (600 mg every 12 h), and amikacin (7.5 mg/kg every 12 h) were simulated in the IVM. Against isolate ATH 16, no viable bacteria were detected across 5 to 25 h with triple therapy, with regrowth to ∼2-log10 CFU/ml occurring at 48 h. Against isolate BD 32, rapid initial killing of ∼3.5-log10 CFU/ml at 5 h was followed by a slow decline to ∼2-log10 CFU/ml at 48 h. In infected mice, polymyxin B monotherapy (60 mg/kg/day) generally was ineffective. With triple therapy (polymyxin B at 60 mg/kg/day, rifampin at 120 mg/kg/day, and amikacin at 300 mg/kg/day), at 24 h there was an ∼1.7-log10 CFU/thigh reduction compared to the starting inoculum for all six isolates. Our results demonstrate that the polymyxin B-rifampin-amikacin combination significantly enhanced in vitro and in vivo bacterial killing, providing important information for the optimization of polymyxin-based combinations in patients.

Lipid A profiling and metabolomics analysis of paired polymyxin-susceptible and -resistant MDR Klebsiella pneumoniae clinical isolates from the same patients before and after colistin treatment.

BACKGROUND: The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern. OBJECTIVES: To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates. METHODS: Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted. RESULTS: The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P < 0.05]. Similarly, nucleotides, amino acids and key metabolites in glycerophospholipid metabolism, namely sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine, were significantly reduced across all polymyxin-resistant isolates (FC > 1.5, P < 0.05) compared with polymyxin-susceptible isolates. However, higher glycerophospholipid levels were evident in polymyxin-resistant ATH 8 and ATH 16 (FC > 1.5, P < 0.05) compared with their corresponding susceptible isolates. CONCLUSIONS: To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae.

Metabolomics Study of the Synergistic Killing of Polymyxin B in Combination with Amikacin against Polymyxin-Susceptible and -Resistant Pseudomonas aeruginosa.

In the present study, we employed untargeted metabolomics to investigate the synergistic killing mechanism of polymyxin B in combination with an aminoglycoside, amikacin, against a polymyxin-susceptible isolate of Pseudomonas aeruginosa, FADDI-PA111 (MIC = 2 mg/liter for both polymyxin B and amikacin), and a polymyxin-resistant Liverpool epidemic strain (LES), LESB58 (the corresponding MIC for both polymyxin B and amikacin is 16 mg/liter). The metabolites were extracted 15 min, 1 h, and 4 h following treatment with polymyxin B alone (2 mg/liter for FADDI-PA111; 4 mg/liter for LESB58), amikacin alone (2 mg/liter), and both in combination and analyzed using liquid chromatography-mass spectrometry (LC-MS). At 15 min and 1 h, polymyxin B alone induced significant perturbations in glycerophospholipid and fatty acid metabolism pathways in FADDI-PA111 and, to a lesser extent, in LESB58. Amikacin alone at 1 and 4 h induced significant perturbations in peptide and amino acid metabolism, which is in line with the mode of action of aminoglycosides. Pathway analysis of FADDI-PA111 revealed that the synergistic effect of the combination was largely due to the inhibition of cell envelope biogenesis, which was driven initially by polymyxin B via suppression of key metabolites involved in lipopolysaccharide, peptidoglycan, and membrane lipids (15 min and 1 h) and later by amikacin (4 h). Overall, these novel findings demonstrate that the disruption of cell envelope biogenesis and central carbohydrate metabolism, decreased levels of amino sugars, and a downregulated nucleotide pool are the metabolic pathways associated with the synergistic killing of the polymyxin-amikacin combination against P. aeruginosa This mechanistic study might help optimize synergistic polymyxin B combinations in the clinical setting.

Novel Polymyxin Combination with the Antiretroviral Zidovudine Exerts Synergistic Killing against NDM-Producing Multidrug-Resistant Klebsiella pneumoniae.

Polymyxins are used as a last-line therapy against multidrug-resistant (MDR) New Delhi metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae However, polymyxin resistance can emerge with monotherapy; therefore, novel strategies are urgently needed to minimize the resistance and maintain their clinical utility. This study aimed to investigate the pharmacodynamics of polymyxin B in combination with the antiretroviral drug zidovudine against K. pneumoniae Three isolates were evaluated in static time-kill studies (0 to 64 mg/liter) over 48 h. An in vitro one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model (IVM) was used to simulate humanized dosage regimens of polymyxin B (4 mg/liter as continuous infusion) and zidovudine (as bolus dose thrice daily to achieve maximum concentration of drug in broth [Cmax] of 6 mg/liter) against K. pneumoniae BM1 over 72 h. The antimicrobial synergy of the combination was further evaluated in a murine thigh infection model against K. pneumoniae 02. In the static time-kill studies, polymyxin B monotherapy produced rapid and extensive killing against all three isolates followed by extensive regrowth, whereas zidovudine produced modest killing followed by significant regrowth at 24 h. Polymyxin B in combination with zidovudine significantly enhanced the antimicrobial activity (≥4 log10 CFU/ml) and minimized bacterial regrowth. In the IVM, the combination was synergistic and the total bacterial loads were below the limit of detection for up to 72 h. In the murine thigh infection model, the bacterial burden at 24 h in the combination group was ≥3 log10 CFU/thigh lower than each monotherapy against K. pneumoniae 02. Overall, the polymyxin B-zidovudine combination demonstrates superior antimicrobial efficacy and minimized emergence of resistance to polymyxins.

Novel HDGF/HIF-1α/VEGF axis in oral cancer impacts disease prognosis.

BACKGROUND: Hepatoma-derived growth factor (HDGF) participates in angiogenesis and represents a negative prognostic factor in oral cancer. The current study was designed to elucidate the regulatory mechanism between HDGF and vascular endothelial growth factor (VEGF) and the clinical impact of oral cancer. METHODS: TCGA data and surgical samples from oral cancer patients were used for the clinicopathological parameter and survival analysis. Human oral cancer SCC4 and SAS cells were treated with recombinant HDGF protein. VEGF gene expression and protein level were analyzed by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. The signaling pathways for regulating VEGF expression were investigated. The nucleolin neutralizing antibody and HIF-1α inhibitor were applied to SCC4 cells to investigate their effects on the HDGF-stimulated VEGF pathways. RESULTS: TCGA and immunohistochemical analysis revealed a positive correlation between HDGF and VEGF expression in oral cancer tissues. Recombinant HDGF significantly increased VEGF gene and protein expression in oral cancer SCC4 cells in a dose-dependent manner. HDGF enhanced the phosphorylation levels of AKT and IkB and the protein level of HIF-1α and NF-κB. The nucleolin-neutralizing antibody abolished HDGF-stimulated HIF-1α, NF-κB and VEGF protein expression in SCC4 cells. The HIF-1α inhibitor antagonized the HDGF-induced VEGF gene expression. High VEGF expression was strongly correlated with HDGF expression, advanced disease, and poor survival. CONCLUSION: This study postulated a new pathway in which HDGF activated HIF-1α and then induced VEGF expression through binding to membrane nucleolin under normoxic conditions, leading to poor disease control. The HDGF/HIF-1α/VEGF axis is important for developing future therapeutic strategies.

Motilimonas pumila sp. nov., isolated from the gut of sea cucumber Apostichopus japonicus.

A facultatively anaerobic and Gram-negative bacterium, designated strain PLHSC7-2T, was isolated from the gut of sea cucumber Apostichopusjaponicus that had been collected from the coastal area of Yantai, China. The cells were rod-shaped and motile by means of polar flagella. The novel isolate grew optimally at 28-30 °C, in the presence of 2.0-3.0 % (w/v) NaCl and at pH 7.0-7.5. The sole respiratory quinone was Q-8 and the major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C17 : 0. The predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain PLHSC7-2T was phylogenetically affiliated with the genus Motilimonas, and exhibited sequence similarity of 96.2 % toMotilimonas eburnea YH6T. The DNA G+C content was 45.5 mol%. On the basis of phenotypic , phylogenetic and genetic distinctiveness, strain PLHSC7-2T (=MCCC 1K03522T=KCTC 62589T) was classified as a novel species within the genus Motilimonas, for which the name Motilimonas pumila sp. nov. is proposed.

Differential impact of age on survival in head and neck cancer according to classic Cox regression and decision tree analysis.

OBJECTIVES: To assess the impact of age on the survival of patients with head and neck squamous cell carcinoma (HNSCC) using different statistical methods. DESIGN: A retrospective population-based study. SETTING: Surveillance, Epidemiology, and End Results database. SUBJECTS AND METHODS: A total of 28 639 patients with newly diagnosed HNSCC were enrolled between 1 January 2007 and 31 December 2013. The effect of age on 5-year disease-specific survival was calculated using a Kaplan-Meier method and compared using log-rank tests. A Cox proportional hazards model was used for a multivariate analysis. A classification and regression tree (CART) analysis that partitioned patients with significantly different Kaplan-Meier curves was introduced to identify the important cancer-related parameters influencing survival. RESULTS: Uni- and multivariate analyses indicated that patients who were older than 60 years had poorer 5-year disease-specific survival regardless of tumour subsite and tumor-node-metastasis (TNM) stage. However, the CART analysis determined that age played only a minor role in survival after comparing with other prognosticators. The relative importance of age using the Gini index was as follows: 3.21% for oral cancer, 8.32% for oropharyngeal cancer, 2.56% for hypopharyngeal cancer and 16.51% for laryngeal cancer. CONCLUSIONS: Different to traditional statistical methods, the CART analysis which was used to identify homogeneous populations revealed that the impact of age varied for different patient groups according to the presence or absence of other prognosticators. This important information could help to guide our clinical decisions and future researches.

A Proof-of-Concept Study of the Efficacy of Systemically Administered Polymyxins in Mouse Burn Wound Infection Caused by Multidrug-Resistant Gram-Negative Pathogens.

The efficacy of subcutaneously administered polymyxins against burn wound infections caused by Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae was examined in a murine infection model. Subcutaneously administered colistin and polymyxin B (30 mg/kg thrice daily) achieved a ≥2-log10 reduction in the bacterial load for P. aeruginosa and A. baumannii infections, whereas wound infections by K. pneumoniae were less responsive (<1-log10 reduction). This study highlights the potential therapeutic benefits of parenteral polymyxins for treating burn wound infections.

Alterations of Metabolic and Lipid Profiles in Polymyxin-Resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa presents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance in P. aeruginosa has been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistant P. aeruginosa strains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-type P. aeruginosa strain K ([PAK] polymyxin B MIC, 1 mg/liter) and its paired pmrB mutant strains, PAKpmrB6 and PAKpmrB12 (polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6 and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) in speE (encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6 compared to that in PAKpmrB12 Our results indicate that spermidine may play an important role in high-level polymyxin resistance in P. aeruginosa Interestingly, both pmrB mutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12 mutant exhibited much lower levels of phospholipids than the PAKpmrB6 mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance in P. aeruginosa and highlights its impacts on bacterial metabolism.