Enhanced breakage of the aggregates of nanoscale zero-valent iron via ball milling.

Aggregates of nanoscale zero-valent iron (nZVI) are commonly encountered for nZVI in aqueous solution, particularly during large-scale nZVI applications where nZVI is often in a highly concentrated slurry, and such aggregates lower nZVI mobility during its in-situ remediation applications. Herein, we report that the ball milling is an effective tool to break the nZVI aggregates and thereby improve the nZVI mobility. Results show that the milling (in just five minutes) can break the aggregates of a few tens of microns to less than one micron, which is one-tenth of the size that is acquired via the breakage using the mechanical mixing and ultrasonication. The milling breakage can also improve the efficacy of the chemical conditioning method that is commonly used for the nanoparticle stabilization and dispersion. The milling breakage is further optimized via a study of the milling operational factors including milling time, bead velocity, bead diameter, and chamber porosity, and an empirical equation is proposed combining the bead collision number during the milling. Mechanistic study shows that the high efficacy of the milling to break the aggregates can be explained by the small eddy created by the high shear rate produced by the close contact of the milling beads and may also relate to the direct mechanical pulverization effect. This study provides a high efficacy physical method to break the nanoparticle aggregates. The method can be used to improve the nZVI mobility performance by milling the nZVI slurry before its injection for in-situ remediation, and the milling may also replace the mechanical mixing during the nZVI stabilization via surface modification.

Effective of phage cocktail against Klebsiella pneumoniae infection of murine mammary glands.

Phage therapy has potential to combat antibiotic-resistant bacteria causing bovine mastitis. Our objective was to use 3 Klebsiella lytic phages to create a phage cocktail, and to compare bactericidal activity of this phage cocktail versus an individual phage, both in vitro and in vivo. Based on transmission electron microscopy, phage CM_Kpn_HB154724 belonged to Podoviridae and on double agar plates, it formed translucent plaques on the bacterial lawn of Klebsiella pneumoniae KPHB154724. In one-step growth curves, this phage had a latent period of 40 min, an outbreak period of 40 min, a burst size of 1.2 × 107 PFU/mL, and an optimal multiplicity of infection (MOI) of 1. Furthermore, it was inactivated under extreme conditions (pH ≤ 3.0 or ≥ 12.0 and temperatures of 60 or 70 °C). It had a host range of 90% and had 146 predicted genes (Illumine NovaSeq). Based on histopathology and expression of inflammatory factors interleukin-1ß, tumor necrosis factor-α, interleukin-6, and prostaglandin, phage cocktail therapy had better efficiency than an individual phage in K. pneumoniae-infected murine mammary glands. In conclusion, we used 3 Klebsiella lytic phages to create a phage cocktail and confirmed its effectiveness against K. pneumoniae both in vitro (bacterial lawn) and in vivo (infected murine mammary glands).

Streptococcus uberis induced expressions of pro-inflammatory IL-6, TNF-α, and IFN-γ in bovine mammary epithelial cells associated with inhibited autophagy and autophagy flux formation.

Autophagy is a highly conserved cellular defensive mechanism that can eliminate bacterial pathogens such as Streptococcus uberis, that causes mastitis in cows. However, S. uberis induced autophagy is still unclear. In this study, we tested if certain inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, critical in mastitis due to S. uberis infection, regulate autophagy activation in bovine mammary epithelial cells (bMECs). Using Western blot and laser scanning confocal microscope in bMECs challenged by S. uberis, showed that the expression of IL-6, TNF-α, IFN-γ oscillated with the expressions of autophagic Atg5, ULK1, PTEN, P62, and LC3ӀӀ/LC3Ӏ. S. uberis infection induced autophagosomes and LC3 puncta in bMECs with upregulation of Atg5, ULK1, PTEN, LC3ӀӀ/LC3Ӏ, and downregulation of P62. The levels of IL-6, TNF-α, and IFN-γ increased during autophagy flux formation to decrease during autophagy induction. Autophagy inhibition increased the expression of IL-6, TNF-α, and IFN-γ and increased S. uberis burden. This study indicates autophagy is induced during S. uberis infection and IL-6, TNF-α, and IFN-γ contribute to autophagy and autophagy flux formation.

Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures.

Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1ß and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1ß, and IL-1ß proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.

Using Immune-Related lncRNA Signature for Prognosis and Response to Immunotherapy in Cutaneous Melanoma.

BACKGROUND: Cutaneous melanoma is a highly malignant skin tumor, and most patients have a poor prognosis. In recent years, immunotherapy has assumed an important role in the treatment of advanced cutaneous melanoma, but only a small percentage of patients benefit from immunotherapy. A growing number of studies have demonstrated that the prognosis of patients with cutaneous melanoma is closely related to long non-coding RNA and the tumor immune microenvironment. METHODS: We downloaded RNA expression data and immune-related gene lists of cutaneous melanoma patients separately from The Cancer Genome Atlas database and ImmPort website and identified immune-related lncRNAs by co-expression analysis. The prognostic model was constructed by applying least absolute shrinkage and selection operator regression, and all patients were classified into high- and low-risk groups according to the risk score of the model. We evaluated the differences between the two groups in terms of survival outcomes, immune infiltration, pathway enrichment, chemotherapeutic drug sensitivity and immune checkpoint gene expression to verify the impact of lncRNA signature on clinical prognosis and immunotherapy efficacy. RESULTS: By correlation analysis and LASSO regression analysis, we constructed an immune-related lncRNA prognostic model based on five lncRNA: HLA-DQB1-AS1, MIR205HG, RP11-643G5.6, USP30-AS1 and RP11-415F23.4. Based on this model, we plotted Kaplan-Meier survival curves and time-dependent ROC curves and analyzed its ability as an independent prognostic factor for cutaneous melanoma in combination with clinicopathological features. The results showed that these lncRNA signature was an independent prognostic factor of cutaneous melanoma with favorable prognostic ability. Our results also show a higher degree of immune infiltration, higher expression of immune checkpoint-associated genes, and better outcome of immunotherapy in the low-risk group of the lncRNA signature. CONCLUSION: The 5 immune-related lncRNA signatures constructed in our study can predict the prognosis of cutaneous melanoma and contribute to the selection of immunotherapy.

Study of Contact Electrification at Liquid-Gas Interface.

It is known that the suspended liquid droplets in clouds can generate electrostatic charges, which finally results in the lightning. However, the detailed mechanism related to the contact-electrification process on the liquid-gas (L-G) interfaces is still poorly understood. Here, by introducing an acoustic levitation method for levitating a liquid droplet, we have studied the electrification mechanism at the L-G interface. The tribo-motion between water droplets and air induced by the ultrasound wave leads to the generation of positive charges on the surface of the droplets, and the charge amount of water droplets (20 µL) gradually reaches saturation within 30 s. The mixed solid particles in droplets can increase the amount of transferred charge, whereas the increase of ion concentration in the droplet can suppress the charge generation. This charge transfer phenomenon at L-G interfaces and the related analysis can be a guidance for the study in many fields, including anti-static, harvesting rainy energy, micro/nano fluidics, triboelectric power generator, surface engineering, and so on. Moreover, the surface charge generation due to L-G electrification is an inevitable effect during ultrasonic levitation, and thus, this study can also work for the applications of the ultrasonic technique.

Bacteriophages isolated from dairy farm mitigated Klebsiella pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro.

BACKGROUND: Klebsiella pneumoniae, an environmental pathogen causing mastitis in dairy cattle, is often resistant to antibiotics. K. pneumoniae was used as the host bacteria to support bacteriophage replication; 2 bacteriophages, CM8-1 and SJT-2 were isolated and considered to have therapeutic potential. In the present study, we determined the ability of these 2 bacteriophages to mitigate cytotoxicity, pathomorphological changes, inflammatory responses and apoptosis induced by K. pneumoniae (bacteriophage to K. pneumoniae MOI 1:10) in bovine mammary epithelial cells (bMECs) cultured in vitro. RESULTS: Bacteriophages reduced bacterial adhesion and invasion and cytotoxicity (lactate dehydrogenase release). Morphological changes in bMECs, including swelling, shrinkage, necrosis and hematoxylin and eosin staining of cytoplasm, were apparent 4 to 8 h after infection with K. pneumoniae, but each bacteriophage significantly suppressed damage and decreased TNF-α and IL-1ß concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1ß, IL-6, IL-8, caspase-3, caspase-9 and cyt-c in bMECs and increased apoptosis of bMECs, although these effects were mitigated by treatment with either bacteriophage for 8 h. CONCLUSIONS: Bacteriophages CM8-1 and SJT-2 mitigated K. pneumoniae-induced inflammation in bMECs cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.

Mycoplasma bovis-generated reactive oxygen species and induced apoptosis in bovine mammary epithelial cell cultures.

Mycoplasma bovis is an important cause of bovine mastitis in China and worldwide. We hypothesized that M. bovis damages bovine mammary epithelial cells (bMEC), with the degree of damage varying among field isolates. Our objective was to evaluate 2 novel sequence type (ST) field strains of M. bovis (ST172 and ST173) for their ability to induce oxidative stress, cytotoxicity, pathomorphological changes, and apoptosis in bMEC, as a model for pathogenesis of M. bovis-induced bovine mastitis. Cytotoxicity (as indicated by release of lactate dehydrogenase, LDH) from bMEC depended on multiplicity of infection (MOI), with a high MOI (1:1,000) being required to induce cytotoxicity. Morphological changes in bMEC, including shrinkage, loss of cell integrity, and heavy staining (hematoxylin and eosin) of cytoplasm were apparent 24 h after infection with ST172 or ST173 M. bovis, with more severe changes being induced by the latter strain. Adhesion and invasion assays both had curvilinear patterns, peaking 12 h after infection with MOI of 1:1,000. Both production of reactive oxygen species (ROS) and proportion of apoptotic cells increased with time after infection. Increased Bax/Bcl-2 ratios and activation of caspase-3 implied involvement of mitochondria-dependent pathways of apoptosis. Furthermore, intracellular ROS generation, apoptosis, and cleaved caspase-3 were mitigated by N-acetyl-l-cysteine, a ROS scavenger. Both interleukin (IL)-1ß and IL-6 were significantly upregulated by ST172 and ST173 M. bovis, with little change in expression of tumor necrosis factor-α. One ST173 M. bovis isolate had the greatest cytotoxicity of all of our field isolates, with the highest LDH release, adhesion, invasion, ROS production, and apoptosis. In conclusion, our hypothesis was supported: M. bovis damaged bMEC by generating ROS and initiating a mitochondria-dependent pathway of apoptosis, with the degree of damage varying among field isolates. This study provided new knowledge regarding pathogenesis of M. bovis-induced bovine mastitis.

Klebsiella pneumoniae isolated from bovine mastitis is cytopathogenic for bovine mammary epithelial cells.

Klebsiella pneumoniae, a common cause of clinical mastitis (CM) in dairy cows, can cause severe clinical symptoms. However, its pathogenicity in the bovine mammary gland is not well understood. Our objectives were to establish an in vitro infection model of K. pneumoniae on bovine mammary epithelial cells (bMEC) to assess (1) cytopathogenicity (adhesive and invasive ability, damage and apoptosis, pro-inflammatory effects) of K. pneumoniae on bMEC and (2) the role of hypermucoviscous (HMV) phenotype on cytopathogenicity. Two K. pneumoniae isolates from CM cows, 1 HMV and 1 non-HMV, were used to infect bMEC. Adhesion and invasion ability, release of lactate dehydrogenase (LDH), ultrastructural morphology, apoptosis, transcriptional expression of pro-inflammatory genes and production of pro-inflammatory cytokines were characterized at various intervals. Both K. pneumoniae isolates rapidly adhered to and invaded bMEC within 1 h post infection (pi), causing ultrastructural damage (swelling of mitochondria and vesicle formation on cell surface) after 3 h pi and apoptotic death after 9 h pi. In addition, K. pneumoniae promoted transcriptional expression of pro-inflammatory genes IL-6, IL-8, IL-1ß, and tumor necrosis factor (TNF)-α and production of IL-8, IL-1ß, and TNF-α cytokines. Compared with non-HMV K. pneumoniae, the HMV isolate had lower adhesive and invasive abilities but caused more serious cellular damage. In conclusion, K. pneumoniae was cytopathogenic on bMEC and induced a pro-inflammatory response; however, the HMV phenotype did not have a key role in pathogenicity. Therefore, more attention should be paid to milk loss, and targeted prevention and treatment strategies should be implemented in Klebsiella mastitis episodes.

Exenatide Reduces Graft Injury in a Rat Transplantation Model Using Kidneys Donated after Cardiac Death.

Besides being used in the therapy of type 2 diabetes, exenatide reduces cerebral ischemia-reperfusion (I/R) injury. We evaluated the potential effects of exenatide on inhibition of apoptosis in kidney grafts donated after cardiac death and on reduction of I/R injury after kidney transplantation (KTx) in a rat model. We used a rat syngeneic KTx model with kidney grafts obtained after cardiac death, and apoptosis was detected in the graft before KTx. Graft function, rat survival, morphologic examination, and activation of inflammatory molecules were analyzed after KTx. By the end of the cold storage, exenatide pretreatment donors had significantly reduced caspase pathway activation, terminal deoxynucleotidyl transferase dUTP nick-end labeling--positive cells, release of mitochondrial porin proteins into the cytosol, and expression of cleaved caspase-3 and poly (ADP-ribose) polymerase in kidney grafts. Exenatide pretreatment improved renal function survival rate with lower scores of acute tubular necrosis, infiltrating macrophages, and interstitial fibrosis as well as reduced messenger RNA expression of inflammatory mediators (tumor necrosis factor α, interleukin-6, interleukin-1ß, and intercellular adhesion molecule-1) after KTx. Our study showed that exenatide reduced I/R injury in kidneys donated after cardiac death in a rat transplantation model and improved recipient survival and graft function.

Extending Hypochlorite Sensing from Cells to Elesclomol-Treated Tumors in Vivo by Using a Near-Infrared Dual-Phosphorescent Nanoprobe.

Reactive oxygen species (ROS), when beyond the threshold, can exhaust the capacity of cellular antioxidants and ultimately trigger cell apoptosis in tumor biology. However, the roles of hypochlorite (ClO-) in this process are much less clear compared with those of ROS, and its detection is easily obstructed by tissue penetration and endogenous fluorophores. Herein, we first synthesized a near-infrared (NIR) ratiometric ClO- probe (Ir NP) composed of two kinds of phosphorescent iridium(III) complexes (Ir1 and Ir2) encapsulated with amphiphilic DSPE-mPEG5000. Ir NPs are dual-emissive and show obvious changes in phosphorescence intensity ratios and lifetimes of two emission bands upon exposure to ClO-. During the ClO- detection, ratiometric photoluminescence imaging is much more reliable over the intensity-based one for its self-calibration, while time-resolved photoluminescence imaging (TRPI) could distinguish the phosphorescence with long lifetime of Ir NPs from short-lived autofluorescence of tissues, resulting in the high accuracy of ClO- determination. With NIR emission, a long phosphorescence lifetime, fast response, and excellent biocompatibility, Ir NPs were applied to the detection of ClO- in vitro and in vivo by means of ratiometric phosphorescence imaging and TRPI with high signal-to noise-ratios (SNR). Importantly, we demonstrated the elevated ClO- in elesclomol-stimulated tumors in living mice for the first time, which holds great potential for the visualization of the boost of ClO- in anti-carcinogen-treated tumors and the further investigation of ROS-related oncotherapeutics.

Demethylation of the MIR145 promoter suppresses migration and invasion in breast cancer.

miR-145 has been implicated in the progression of breast cancer. Here, we report that its expression is decreased in breast cancer specimens and cell lines and that this low level of expression is associated with DNA methylation of its gene, MIR145. Methylation of MIR145 has previously been correlated with cell migration and invasion, both in vivo and in vitro. We found that demethylation of MIR145 reactivates miR-145 and contributes to the anti-cancer properties of 5-aza-2'-deoxyazacytidine (5-AzaC). Therefore, miR-145 is a potentially valuable biomarker for breast cancer.

A case of intravascular large B cell lymphoma presenting as nodular goiter.

BACKGROUND: Intravascular large B-cell lymphoma (IVLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that is rare and highly aggressive and that may progressively involve many organs. CNS (central nervous system), BM (bone marrow) and skin are the most common systems involved. To date, only 2 cases of IVLBCL involving the thyroid have been reported. CASE PRESENTATION: Here, we report a case of IVLBCL involving the thyroid and accompanied by bilateral nodular goiter. In this case, a thyroid mass was identified in a physical examination of a 68-year-old male who initially presented with dyspnea accompanied by intermittent headache for approximately 1 month. Computed tomography scans revealed that the left lobar thyroid was occupied by a large, slightly lower density mass (5.8 × 4.7 × 8.4 cm). However, the patient had no hyperthyroidism or hoarseness. Levels of thyroid hormones and anti-thyroid autoantibodies in the serum were normal preoperatively. Thyroid mass resection was performed to establish a diagnosis and to relieve symptoms. CONCLUSIONS: Pathological results of the surgical specimen revealed that large atypical lymphoma cells filled the capillaries in the lesion area. Immunohistochemical staining revealed that the large-sized tumor cells were positive for CD20, PAX-5, MUM-1 and BCL-2, and were negative for CD3, CD5, CD43, CD10, CD23, CyclinD1, CD138, CD30, ALK, CD56, MPO, S-100, TTF-1, TG (thyroglobulin) and CT (calcitonin). The Ki-67 index was estimated to be approximately 85%. The patient was subsequently diagnosed as "Classical" IVLBCL non-germinal center B-cell type. The patient declined chemotherapy and died in the fifth month after operation.

Exendin-4 ameliorates renal ischemia-reperfusion injury in the rat.

BACKGROUND: Glucagon-like peptide-1 receptor (GLP-1R) activation exerts protective effects against reactive oxygen species by inducing the oxidative defense gene heme oxygenase-1 (HO-1), and provides protection in mice against transient focal cerebral ischemia and ischemia-reperfusion injury in the rat heart. GLP-1R is also expressed in the kidney, but it is unknown whether GLP-1R activation is able to protect against ischemia-reperfusion injury in the rat kidney. MATERIALS AND METHODS: We used a rat model of renal ischemia-reperfusion injury. The rats were pretreated with the GLP-1R agonist, exendin-4 before reperfusion. We used real-time polymerase chain reaction to evaluate expression of the oxidative defense gene HO-1 and Western blot analysis for HO-1 and GLP-1R. Renal function was assessed at baseline and 24 and 72 h after reperfusion. The kidneys were processed for histologic and morphometric analysis, caspase-3, and ED1 immunohistochemistry at 72 h. The degree of apoptosis of the renal tubular cells was determined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling assays. RESULTS: Exendin-4 pretreatment resulted in GLP-1R activation and upregulation of HO-1. Preconditional activation of GLP-1R significantly improved the serum creatinine levels compared with vehicle (P < 0.05). Furthermore, tissue injury, caspase-3 and ED1 expression, and apoptosis were less severe, as quantified by application of a standardized histologic scoring system in a blinded manner. CONCLUSIONS: These results have demonstrated that preconditional activation of the GLP-1R with exendin-4 in the kidney significantly protected against ischemia-reperfusion injury in rats by increasing HO-1 expression.

Evaluation of 2 celecoxib derivatives: analgesic effect and selectivity to cyclooxygenase-2/1.

AIM: To evaluate the analgesic effects of 2 celecoxib derivatives and their inhibitory effects on cyclooxygenase (COX). METHODS: Four antinociceptive assays were used: the acetic acid-induced writhing test, hot plate test, hot tail-flick test and formalin test. Three doses were used in the analgesic assays and ED50 values were calculated. For the selectivity assay, macrophages were incubated with test compounds at various concentrations and then stimulated with calcimycin or lipopolysaccharide (LPS). The amounts of 6-keto-prostaglandin F1alpha(6-keto-PGF1alpha) and prostaglandin E2 (PGE2) in the supernatant were examined by radioimmunoassay (RIA). The selectivity of the test compounds was expressed as the IC(50,COX-1)/IC(50,COX-2) value. RESULTS: Celecoxib and its 2 derivatives had a significant analgesic effect. The ED50 values of celecoxib, PC-406 and PC-407 were 94.2, 67.9, and 63.3 mg/kg, respectively, for the acetic acid-induced writhing test; 104.7, 89.1, and 30.0 mg/kg, respectively, for the hot tail-flick response test; 60.7, 56.7, and 86.2 mg/kg, respectively, for the hot plate response test; 67.1, 55.8, and 68.8 mg/kg, respectively, for the formalin-induced response. That is, the ED50 of PC-406 was the lowest for the formalin and hot plate tests, which focus on changes above the spinal cord level; however, the ED50 of PC-407 was lowest for the tail-flick and writhing tests, which focus on changes at the spinal cord level. Celecoxib and PC-407 inhibited COX-1 with IC50 values of 39.8 and 27.5 nmol/L, respectively. PC-406 inhibited COX-1 with an IC50 value of more than 1000 nmol/L. The IC50 values for the effect of celecoxib, PC-406 and PC-407 on COX-2 were 4.8, 8.9, and 1.9 nmol/L respectively. The IC(50, COX-1)/IC(50,COX-2) ratios for celecoxib and PC-407 were 8.3 and 14.4, respec-tively. For PC-406, the ratio was greater than 112.2. CONCLUSION: Derivatives of celecoxib via substitution with an isopropyl or naphthyl group at the 5 position in the pyrazole ring still have analgesic effects and the ability to selectively inhibit COX-2. Substitution with a naphthyl group may have more effect on the peripheral pain pathway, whereas substitution with an isopropyl group may have more effect on the central pain pathway. This phenomenon occurs partly because substitution with an isopropyl group is more beneficial for COX-2 selectivity than is substitution with a naphthyl group.