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1.
Sci Rep ; 10(1): 9992, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561783

RESUMO

Y-chromosome genetic diversity in and around its domestication origin and a better understanding of indicine-specific microsatellite alleles are imperative concerns but less -targeted. We analysed Y-chromosome markers in 301 bulls representing 19 native Indian cattle (Bos indicus) and identified new alleles and haplotypes. Compared to other indicine studies, the high Y-haplotype diversity found in Indian cattle supports the hypothesis of greater genetic variability across the centre of origin decreasing along migratory routes with increasing distance. Hence, a considerable paternal genetic diversity of Indian cattle appears to have been lost in transboundary commercial indicine breeds. The Khillar and Gir are the most diversified populations where the first tends to be the well-differentiated traditional breed carrying strikingly distinct Y-lineages with typical BM861-158 bp allele, characteristics of taurine cattle, while retaining standard indicine lineages for all other markers. Geographical distribution found to be an unreliable predictor of parental variation, and Y-lineages seemed closely related to Indian breed function/utility. The comprehensive Y-chromosome information will be useful to examine the demographic expansion/spread of Bos indicus lineages from close proximity to the domestication centre across different countries worldwide and such diversity should be preserved through effective management and conservation programs.

2.
Plant Physiol ; 183(3): 1345-1363, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32354878

RESUMO

Plant pathogens secrete cell wall-degrading enzymes that degrade various components of the plant cell wall. Plants sense this cell wall damage as a mark of infection and induce immune responses. However, the plant functions that are involved in the elaboration of cell wall damage-induced immune responses remain poorly understood. Transcriptome analysis revealed that a rice (Oryza sativa) receptor-like kinase, WALL-ASSOCIATED KINASE-LIKE21 (OsWAKL21.2), is up-regulated following treatment with either Xanthomonas oryzae pv oryzae (a bacterial pathogen) or lipaseA/esterase (LipA; a cell wall-degrading enzyme of X. oryzae pv oryzae). Overexpression of OsWAKL21.2 in rice induces immune responses similar to those activated by LipA treatment. Down-regulation of OsWAKL21.2 attenuates LipA-mediated immune responses. Heterologous expression of OsWAKL21.2 in Arabidopsis (Arabidopsis thaliana) also activates plant immune responses. OsWAKL21.2 is a dual-activity kinase that has in vitro kinase and guanylate cyclase activities. Interestingly, kinase activity of OsWAKL21.2 is necessary to activate rice immune responses, whereas in Arabidopsis, OsWAKL21.2 guanylate cyclase activity activates these responses. Our study reveals a rice receptor kinase that activates immune responses in two different species via two different mechanisms.

3.
J Med Internet Res ; 22(4): e17550, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32343256

RESUMO

BACKGROUND: Machine-learning or deep-learning algorithms for clinical diagnosis are inherently dependent on the availability of large-scale clinical datasets. Lack of such datasets and inherent problems such as overfitting often necessitate the development of innovative solutions. Probabilistic modeling closely mimics the rationale behind clinical diagnosis and represents a unique solution. OBJECTIVE: The aim of this study was to develop and validate a probabilistic model for differential diagnosis in different medical domains. METHODS: Numerical values of symptom-disease associations were utilized to mathematically represent medical domain knowledge. These values served as the core engine for the probabilistic model. For the given set of symptoms, the model was utilized to produce a ranked list of differential diagnoses, which was compared to the differential diagnosis constructed by a physician in a consult. Practicing medical specialists were integral in the development and validation of this model. Clinical vignettes (patient case studies) were utilized to compare the accuracy of doctors and the model against the assumed gold standard. The accuracy analysis was carried out over the following metrics: top 3 accuracy, precision, and recall. RESULTS: The model demonstrated a statistically significant improvement (P=.002) in diagnostic accuracy (85%) as compared to the doctors' performance (67%). This advantage was retained across all three categories of clinical vignettes: 100% vs 82% (P<.001) for highly specific disease presentation, 83% vs 65% for moderately specific disease presentation (P=.005), and 72% vs 49% (P<.001) for nonspecific disease presentation. The model performed slightly better than the doctors' average in precision (62% vs 60%, P=.43) but there was no improvement with respect to recall (53% vs 56%, P=.27). However, neither difference was statistically significant. CONCLUSIONS: The present study demonstrates a drastic improvement over previously reported results that can be attributed to the development of a stable probabilistic framework utilizing symptom-disease associations to mathematically represent medical domain knowledge. The current iteration relies on static, manually curated values for calculating the degree of association. Shifting to real-world data-derived values represents the next step in model development.

4.
Theranostics ; 10(8): 3397-3412, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32206098

RESUMO

Rationale: Some studies have shown that the local activation of immunogenic cell death (ICD) by upregulating calreticulin (CRT) expression in solid tumors can improve antitumor effects. Although a promising approach, a key current challenge in ICD tumor therapy is the absence of a clinically translatable method for reproducibly inducing the CRT expression. Herein, we report a novel calreticulin-nanoparticle (CRT-NP) that enhances ICD and synergizes with focused ultrasound (FUS) to achieve local and systemic antitumor effects. Methods: Full-length clone DNA of calreticulin was encapsulated in NPs made from DOTAP and cholesterol. Three CRT-NP intratumoral injections of 20 µg each were given 2 days apart, and FUS heating (42-45°C, ~15min) was applied sequentially 24h after each injection to induce ICD. To investigate ICD specific immune effect, the splenocytes of mice vaccinated with CRT-NP (± FUS) treated B16F10 cells were evaluated ex-vivo for TRP-2 antigen specific immunity. Additionally, the long-term protection was evaluated by re-challenging with the melanoma cells in the flank regions of tumor bearing mice. Results: CRT-NP plus FUS (CFUS) upregulated CRT expression, expanded the population of melanoma TRP-2 specific functional CD4+ and CD8+ T cells and tumor-suppressing M1 phenotype, and increased PD-1 and PD-L1 marker expression in the T cells. Therapeutically, CFUS suppressed B16 melanoma growth by >85% vs. that seen in untreated controls, and >~50% vs. CRT-NP or FUS alone, and prevented tumor growth in distal untreated sites. Conclusions: CRT-NP amplifies the FUS and ICD therapeutic outcomes against melanoma, suggesting that the proposed combinatorial methodology may be clinically translatable.

5.
Pharmacol Ther ; 207: 107456, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31863820

RESUMO

Chemotherapy, surgery, and radiation are accepted as the preferred treatment modalities against cancer, but in recent years the use of immunotherapeutic approaches has gained prominence as the fourth treatment modality in cancer patients. In this approach, a patient's innate and adaptive immune systems are activated to achieve clearance of occult cancerous cells. In this review, we discuss the preclinical and clinical immunotherapeutic (e.g., immunoadjuvants (in-situ vaccines, oncolytic viruses, CXC antagonists, device activated agents), organic and inorganic nanoparticles, and checkpoint blockade) that are under investigation for cancer therapy and diagnostics. Additionally, the innovations in imaging of immune cells for tracking therapeutic responses and limitations (e.g., toxicity, inefficient immunomodulation, etc.) are described. Existing data suggest that if immune therapy is optimized, it can be a real and potentially paradigm-shifting cancer treatment frontier.

6.
BMC Plant Biol ; 19(1): 530, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783788

RESUMO

BACKGROUND: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. RESULTS: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. CONCLUSION: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/imunologia , Oryza/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Xanthomonas/fisiologia , Arabidopsis/imunologia , Parede Celular/imunologia , Oryza/imunologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Proteínas de Plantas/imunologia , Ubiquitina-Proteína Ligases/imunologia , Xanthomonas/enzimologia
7.
Int J Hyperthermia ; 36(sup1): 64-73, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31795832

RESUMO

The success of melanoma immunotherapy is dependent on the presence of activated and functional T-cells in tumors. The objective of this study was to investigate the impact of local-focused ultrasound (FUS) heating (∼42-45 °C) and in-situ anti-CD-40 agonistic antibody in enhancing T-cell function for melanoma immunotherapy. We compared the following groups of mice with bilateral flank B16 F10 melanoma: (1) Control, (2) FUS, (3) CD-40, and (4) CD-40 + FUS (FUS40). FUS heating was applied for ∼15 min in right flank tumor, and intratumoral injections of CD-40 were performed sequentially within 4 h. A total of 3 FUS and 4 anti-CD-40 treatments were administered unilaterally 3 days apart. Mice were sacrificed 30 days post-inoculation, and the treated tumor and spleen tissues were profiled for T-cell function and macrophage polarization. Compared to all other groups, histology and flow cytometry showed that FUS40 increased the population of tumor-specific CD-4+ and CD-8+ T cells rich in Granzyme B+, interleukin-2 (IL-2) and IFN-γ production and poor in PD-1 expression. In addition, FUS40 promoted the infiltration of tumor-suppressing M1 phenotype macrophages in the treated mice. The resultant immune-enhancing effects of FUS40 suppressed B16 melanoma growth at the treated site by 2-3-folds compared to control, FUS, and CD-40, and also achieved significant abscopal effects in untreated tumors relative to CD40 alone. Additionally, the local FUS40 prevented adverse liver toxicities in the treated mice. Our study suggests that combined FUS and CD-40 can enhance T-cell and macrophage functions to aid effective melanoma immunotherapy.


Assuntos
Antígenos CD40/antagonistas & inibidores , Calefação/métodos , Imunofenotipagem/métodos , Imunoterapia/métodos , Melanoma/diagnóstico por imagem , Melanoma/terapia , Vacinação/métodos , Animais , Modelos Animais de Doenças , Camundongos
8.
Macromol Biosci ; 19(10): e1900183, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31507074

RESUMO

Reactive oxygen species (ROS) forming enzymes are of significant interest as anticancer agents due to their potent cytotoxicity. A key challenge in their clinical translation is attaining site-specific delivery and minimizing biodistribution to healthy tissues. Here, complexes composed of the ROS enzyme glucose oxidase (GOX), poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG), and anti-prostate specific membrane antigen (anti-PSMA) monoclonal antibody are synthesized for localized delivery and uptake in prostate cancer cells. Formation of anti-PSMA-PLL-g-PEG/GOX results in nanoscale complexes ≈30 nm in diameter with a ζ-potential of 6 mV. The anti-PSMA-PLL-g-PEG/GOX complexes show significant cytotoxicity (≈60% reduction in cell viability) against PSMA-expressing LNCaP cells compared to unmodified GOX. Importantly, cytotoxicity in LNCaP cells occurrs concurrently with anti-PSMA-PLL-g-PEG/GOX uptake and increases in intracellular generation of ROS. These results demonstrate that cytotoxicity of ROS inducing enzymes can be enhanced by intracellular delivery compared to equivalent concentrations of free enzyme, providing a novel means for cancer therapy.

9.
Sci Rep ; 9(1): 7293, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086267

RESUMO

The aim of this study is to investigate the feasibility of identifying and applying quantitative imaging features computed from ultrasound images of athymic nude mice to predict tumor response to treatment at an early stage. A computer-aided detection (CAD) scheme with a graphic user interface was developed to conduct tumor segmentation and image feature analysis. A dataset involving ultrasound images of 23 athymic nude mice bearing C26 mouse adenocarcinomas was assembled. These mice were divided into 7 treatment groups utilizing a combination of thermal and nanoparticle-controlled drug delivery. Longitudinal ultrasound images of mice were taken prior and post-treatment in day 3 and day 6. After tumor segmentation, CAD scheme computed image features and created four feature pools including features computed from (1) prior treatment images only and (2) difference between prior and post-treatment images of day 3 and day 6, respectively. To predict tumor treatment efficacy, data analysis was performed to identify top image features and an optimal feature fusion method, which have a higher correlation to tumor size increase ratio (TSIR) determined at Day 10. Using image features computed from day 3, the highest Pearson Correlation coefficients between the top two features selected from two feature pools versus TSIR were 0.373 and 0.552, respectively. Using an equally weighted fusion method of two features computed from prior and post-treatment images, the correlation coefficient increased to 0.679. Meanwhile, using image features computed from day 6, the highest correlation coefficient was 0.680. Study demonstrated the feasibility of extracting quantitative image features from the ultrasound images taken at an early treatment stage to predict tumor response to therapies.

10.
Artigo em Inglês | MEDLINE | ID: mdl-30998479

RESUMO

The order of amino acids in a protein sequence enables the protein to acquire a conformation suitable for performing functions, thereby motivating the need to analyse these sequences for predicting functions. Although machine learning based approaches are fast compared to methods using BLAST, FASTA, etc., they fail to perform well for long protein sequences (with more than 300 amino acids). In this paper, we introduce a novel method for construction of two separate feature sets for protein using bi-directional long short-term memory network based on the analysis of fixed 1) single-sized segments and 2) multi-sized segments. The model trained on the proposed feature set based on multi-sized segments is combined with the model trained using state-of-the-art Multi-label Linear Discriminant Analysis (MLDA) features to further improve the accuracy. Extensive evaluations using separate datasets for biological processes and molecular functions demonstrate not only improved results for long sequences, but also significantly improve the overall accuracy over state-of-the-art method. The single-sized approach produces an improvement of +3.37% for biological processes and +5.48% for molecular functions over the MLDA based classifier. The corresponding numbers for multi-sized approach are +5.38% and +8.00%. Combining the two models, the accuracy further improves to +7.41% and +9.21% respectively.

11.
BMC Genomics ; 20(1): 157, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808300

RESUMO

BACKGROUND: Sclerotinia sclerotiorum is a broad-host range necrotrophic pathogen which is the causative agent of Sclerotinia stem rot (SSR), and a major disease of soybean (Glycine max). A time course transcriptomic analysis was performed in both compatible and incompatible soybean lines to identify pathogenicity and developmental factors utilized by S. sclerotiorum to achieve pathogenic success. RESULTS: A comparison of genes expressed during early infection identified the potential importance of toxin efflux and nitrogen metabolism during the early stages of disease establishment. The later stages of infection were characterized by an apparent shift to survival structure formation. Analysis of genes highly upregulated in-planta revealed a temporal regulation of hydrolytic and detoxification enzymes, putative secreted effectors, and secondary metabolite synthesis genes. Redox regulation also appears to play a key role during the course of infection, as suggested by the high expression of genes involved in reactive oxygen species production and scavenging. Finally, distinct differences in early gene expression were noted based on the comparison of S. sclerotiorum infection of resistant and susceptible soybean lines. CONCLUSIONS: Although many potential virulence factors have been noted in the S. sclerotiorum pathosystem, this study serves to highlight soybean specific processes most likely to be critical in successful infection. Functional studies of genes identified in this work are needed to confirm their importance to disease development, and may constitute valuable targets of RNAi approaches to improve resistance to SSR.


Assuntos
Ascomicetos/genética , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/microbiologia , Soja/microbiologia , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Parede Celular , Resistência à Doença , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Ácido Oxálico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Metabolismo Secundário/genética , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
12.
Plant Biotechnol J ; 17(8): 1567-1581, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30672092

RESUMO

Sclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield-limiting disease of soybean called Sclerotinia stem rot. Resistance mechanisms against this pathogen in soybean are poorly understood, thus hindering the commercial deployment of resistant varieties. We used a multiomic approach utilizing RNA-sequencing, gas chromatography-mass spectrometry-based metabolomics and chemical genomics in yeast to decipher the molecular mechanisms governing resistance to S. sclerotiorum in soybean. Transcripts and metabolites of two soybean recombinant inbred lines, one resistant and one susceptible to S. sclerotiorum were analysed in a time course experiment. The combined results show that resistance to S. sclerotiorum in soybean is associated in part with an early accumulation of JA-Ile ((+)-7-iso-jasmonoyl-L-isoleucine), a bioactive jasmonate, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates, such as 4-hydroxybenzoate, cinnamic acid, ferulic acid and caffeic acid, were highly accumulated in the resistant line. In vitro assays show that these metabolites and total stem extracts from the resistant line clearly affect S. sclerotiorum growth and development. Using chemical genomics in yeast, we further show that this antifungal activity targets ergosterol biosynthesis in the fungus, by disrupting enzymes involved in lipid and sterol biosynthesis. Overall, our results are consistent with a model where resistance to S. sclerotiorum in soybean coincides with an early recognition of the pathogen, leading to the modulation of the redox capacity of the host and the production of antifungal metabolites.


Assuntos
Ascomicetos/patogenicidade , Resistência à Doença/genética , Ergosterol/biossíntese , Doenças das Plantas/genética , Soja/genética , Soja/microbiologia , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/microbiologia , Regulação para Cima
13.
Sci Rep ; 8(1): 13062, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30166607

RESUMO

Using attenuated Salmonella that efficiently homes in solid tumors, here we developed thermobots that actively transported membrane attached low-temperature sensitive liposome (LTSL) inside colon cancer cells for triggered doxorubicin release and simultaneous polarized macrophages to M1 phenotype with high intensity focused ultrasound (HIFU) heating (40-42 °C). Biocompatibility studies showed that the synthesized thermobots were highly efficient in LTSL loading without impacting its viability. Thermobots demonstrated efficient intracellular trafficking, high nuclear localization of doxorubicin, and induced pro-inflammatory cytokine expression in colon cancer cells in vitro. Combination of thermobots and HIFU heating (~30 min) in murine colon tumors significantly enhanced polarization of macrophages to M1 phenotype and therapeutic efficacy in vivo compared to control. Our data suggest that the thermobots and focused ultrasound treatments have the potential to improve colon cancer therapy.


Assuntos
Neoplasias do Colo/terapia , Imunoterapia , Salmonella/metabolismo , Temperatura , Ultrassom , Animais , Neoplasias do Colo/sangue , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Citocinas/sangue , Feminino , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Células RAW 264.7 , Linfócitos T Auxiliares-Indutores/imunologia
14.
Sci Rep ; 8(1): 11390, 2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-30061558

RESUMO

The temporal and spatial patterns of nanoparticle that ferry both imaging and therapeutic agent in solid tumors is significantly influenced by target tissue movement, low spatial resolution, and inability to accurately define regions of interest (ROI) at certain tissue depths. These combine to limit and define nanoparticle untreated regions in tumors. Utilizing graph and matrix theories, the objective of this project was to develop a novel spectral Fiedler field (SFF) based-computational technology for nanoparticle mapping in tumors. The novelty of SFF lies in the utilization of the changes in the tumor topology from baseline for contrast variation assessment. Data suggest that SFF can enhance the spatiotemporal contrast compared to conventional method by 2-3 folds in tumors. Additionally, the SFF contrast is readily translatable for assessment of tumor drug distribution. Thus, our SFF computational platform has the potential for integration into devices that allow contrast and drug delivery applications.


Assuntos
Algoritmos , Neoplasias do Colo/diagnóstico por imagem , Meios de Contraste/química , Diagnóstico por Imagem , Nanopartículas/química , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Lipossomos , Camundongos , Temperatura , Ultrassonografia
15.
Int J Hyperthermia ; 34(2): 189-200, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29498309

RESUMO

Implants are commonly used as a replacement for damaged tissue. Many implants, such as pacemakers, chronic electrode implants, bone screws, and prosthetic joints, are made of or contain metal. Infections are one of the difficult to treat complications associated with metal implants due to the formation of biofilm, a thick aggregate of extracellular polymeric substances (EPS) produced by the bacteria. In this study, we treated a metal prosthesis infection model using a combination of ciprofloxacin-loaded temperature-sensitive liposomes (TSL) and alternating magnetic fields (AMF). AMF heating is used to disrupt the biofilm and release the ciprofloxacin-loaded TSL. The three main objectives of this study were to (1) investigate low- and high-temperature-sensitive liposomes (LTSLs and HTSLs) containing the antimicrobial agent ciprofloxacin for temperature-mediated antibiotic release, (2) characterise in vitro ciprofloxacin release and stability and (3) study the efficacy of combining liposomal ciprofloxacin with AMF against Pseudomonas aeruginosa biofilms grown on metal washers. The release of ciprofloxacin from LTSL and HTSL was assessed in physiological buffers. Results demonstrated a lower transition temperature for both LTSL and HTSL formulations when incubated in serum as compared with PBS, with a more pronounced impact on the HTSLs. Upon combining AMF with temperature-sensitive liposomal ciprofloxacin, a 3 log reduction in CFU of Pseudomonas aeruginosa in biofilm was observed. Our initial studies suggest that AMF exposure on metal implants can trigger release of antibiotic from temperature sensitive liposomes for a potent bactericidal effect on biofilm.


Assuntos
Antibacterianos/uso terapêutico , Ciprofloxacino/uso terapêutico , Lipossomos/metabolismo , Antibacterianos/farmacologia , Biofilmes , Ciprofloxacino/farmacologia , Humanos , Campos Magnéticos , Microscopia Eletrônica de Varredura
16.
Ultrasound Med Biol ; 44(4): 909-914, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29395679

RESUMO

Chronic wounds typically require long-duration treatment with a combination of antibiotics administered systemically. This incurs adverse side effects and can require aversive surgical treatments and limb amputations. To improve non-invasive antimicrobial therapy, the objective of this study was to investigate antimicrobial chemotherapy combined with high-intensity focused ultrasound (HIFU) heating (HT). A Staphylococcus aureus abscess (80 ± 30 mm3) was generated in the mouse flank region. Once the average temperature (~42 °C-46 °C) in the abscess was reached with HIFU-HT, a broad-spectrum antimicrobial (ciprofloxacin, 10 mg/kg) and perfusion marker (Evans blue dye, 40 mg/kg wt) were administered intravenously via the tail vein. Four hours later, mean abscess perfusion and colony-forming units (CFUs) per gram of abscess were determined. HIFU-HT increased abscess perfusion by ~2.5-fold (4 ± 0.6 µg/mL Evans blue) compared with control (1.5 ± 0.7 µg/mL), and improved antimicrobial efficacy to decrease percentage average survival of S. aureus by ~20% (46 ± 7 CFUs/g of abscess) versus that seen with ciprofloxacin alone (61 ± 4 CFU/g). Our in vivo data suggest that HIFU-HT can improve antimicrobial treatment responses against deep-seated bacteria in abscess wounds via enhanced perfusion.


Assuntos
Abscesso/tratamento farmacológico , Antibacterianos/uso terapêutico , Ciprofloxacino/uso terapêutico , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus , Animais , Terapia Combinada/métodos , Modelos Animais de Doenças , Temperatura Alta , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento
17.
Int J Hyperthermia ; 34(2): 201-208, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29278945

RESUMO

Musculoskeletal infections caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in children and adults can lead to adverse outcomes including a need for extensive surgical debridement and limb amputation. To enable targeted antimicrobial release in infected tissues, the objective of this study was to design and investigate novel elastin-like polypeptide (ELP)-based thermally sensitive liposomes in vitro. ELP biopolymers can change their phase behaviour at higher temperatures. We hypothesised that ELP-TSL will improve therapeutic efficacy by releasing antimicrobial payloads locally at higher temperatures (≥39 °C). ELP-TSL library were formulated by varying cholesterol and phospholipid composition by the thin film and extrusion method. A broad-spectrum antimicrobial (Ciprofloxacin or Cipro) was encapsulated inside the liposomes by the ammonium sulphate gradient method. Cipro release from ELP-TSLs was assessed in physiological buffers containing ∼25% serum by fluorescence spectroscopy, and efficacy against Staphylococcus aureus and Pseudomonas aeruginosa was assessed by disc diffusion and planktonic assay. Active loading of Cipro achieved an encapsulation efficiency of 40-70% in the ELP-TSL depending upon composition. ELP-TSL Cipro release was near complete at ≥39 °C; however, the release rates could be delayed by cholesterol. Triggered release of Cipro from ELP-TSL at ∼42 °C induced significant killing of S. aureus and P. aeruginosa compared to 37 °C. Our in vitro data suggest that ELP-TSL may potentially improve bacterial wound therapy in patients.


Assuntos
Antibacterianos/uso terapêutico , Bactérias/patogenicidade , Elastina/metabolismo , Lipossomos/metabolismo , Peptídeos/metabolismo , Antibacterianos/farmacologia , Humanos
18.
Mol Plant Pathol ; 19(3): 700-714, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28378935

RESUMO

The plant membrane-localized NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), play crucial roles in various cellular activities, including plant disease responses, and are a major source of reactive oxygen species (ROS). Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen that causes Sclerotinia stem rot (SSR) in soybean. Via a key virulence factor, oxalic acid, it induces programmed cell death (PCD) in the host plant, a process that is reliant on ROS generation. In this study, using protein sequence similarity searches, we identified 17 soybean RBOHs (GmRBOHs) and studied their contribution to SSR disease development, drought tolerance and nodulation. We clustered the soybean RBOH genes into six groups of orthologues based on phylogenetic analysis with their Arabidopsis counterparts. Transcript analysis of all 17 GmRBOHs revealed that, of the six identified groups, group VI (GmRBOH-VI) was specifically and drastically induced following S. sclerotiorum challenge. Virus-induced gene silencing (VIGS) of GmRBOH-VI using Bean pod mottle virus (BPMV) resulted in enhanced resistance to S. sclerotiorum and markedly reduced ROS levels during disease development. Coincidently, GmRBOH-VI-silenced plants were also found to be drought tolerant, but showed a reduced capacity to form nodules. Our results indicate that the pathogenic development of S. sclerotiorum in soybean requires the active participation of specific host RBOHs, to induce ROS and cell death, thus leading to the establishment of disease.


Assuntos
Ascomicetos/patogenicidade , NADPH Oxidases/metabolismo , Proteínas de Plantas/metabolismo , Soja/metabolismo , Soja/microbiologia , Secas , NADPH Oxidases/genética , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo
19.
Front Plant Sci ; 8: 1495, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28912790

RESUMO

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen of soybean that can cause significant yield losses to growers when environmental conditions are favorable for the disease. The development of resistant varieties has proven difficult. However, poor resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. The objective of this project was to develop soybean germplasm lines that have a high level of Sclerotinia stem rot resistance to be used directly as cultivars or in breeding programs as a source of improved Sclerotinia stem rot resistance. Sclerotinia stem rot-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to Heterodera glycines and Cadophora gregata in addition to favorable agronomic traits. Following greenhouse evaluations of 1,076 inbred lines derived from these crosses, 31 lines were evaluated for resistance in field tests during the 2014 field season. Subsequently, 11 Sclerotinia stem rot resistant breeding lines were moved forward for field evaluation in 2015, and seven elite breeding lines were selected and evaluated in the 2016 field season. To better understand resistance mechanisms, a marker analysis was conducted to identify quantitative trait loci linked to resistance. Thirteen markers associated with Sclerotinia stem rot resistance were identified on chromosomes 15, 16, 17, 18, and 19. Our markers confirm previously reported chromosomal regions associated with Sclerotinia stem rot resistance as well as a novel region of chromosome 16. The seven elite germplasm lines were also re-evaluated within a greenhouse setting using a cut petiole technique with multiple S. sclerotiorum isolates to test the durability of physiological resistance of the lines in a controlled environment. This work presents a novel and comprehensive classical breeding method for selecting lines with physiological resistance to Sclerotinia stem rot and a range of agronomic traits. In these studies, we identify four germplasm lines; 91-38, 51-23, SSR51-70, and 52-82B exhibiting a high level of Sclerotinia stem rot resistance combined with desirable agronomic traits, including high protein and oil contents. The germplasm identified in this study will serve as a valuable source of physiological resistance to Sclerotinia stem rot that could be improved through further breeding to generate high-yielding commercial soybean cultivars.

20.
Chem Biol Interact ; 275: 86-94, 2017 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-28756151

RESUMO

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30-33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers.


Assuntos
Butirilcolinesterase/metabolismo , Peptídeo Hidrolases/metabolismo , Polietilenoglicóis/química , Polilisina/análogos & derivados , Biocatálise , Butirilcolinesterase/química , Butirilcolinesterase/genética , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ensaios Enzimáticos , Temperatura Alta , Humanos , Cinética , Microscopia Eletrônica de Varredura , Paraoxon/química , Paraoxon/metabolismo , Tamanho da Partícula , Polilisina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
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