A CNN-CBAM-BIGRU model for protein function prediction.

Understanding a protein's function based solely on its amino acid sequence is a crucial but intricate task in bioinformatics. Traditionally, this challenge has proven difficult. However, recent years have witnessed the rise of deep learning as a powerful tool, achieving significant success in protein function prediction. Their strength lies in their ability to automatically learn informative features from protein sequences, which can then be used to predict the protein's function. This study builds upon these advancements by proposing a novel model: CNN-CBAM+BiGRU. It incorporates a Convolutional Block Attention Module (CBAM) alongside BiGRUs. CBAM acts as a spotlight, guiding the CNN to focus on the most informative parts of the protein data, leading to more accurate feature extraction. BiGRUs, a type of Recurrent Neural Network (RNN), excel at capturing long-range dependencies within the protein sequence, which are essential for accurate function prediction. The proposed model integrates the strengths of both CNN-CBAM and BiGRU. This study's findings, validated through experimentation, showcase the effectiveness of this combined approach. For the human dataset, the suggested method outperforms the CNN-BIGRU+ATT model by +1.0 % for cellular components, +1.1 % for molecular functions, and +0.5 % for biological processes. For the yeast dataset, the suggested method outperforms the CNN-BIGRU+ATT model by +2.4 % for the cellular component, +1.2 % for molecular functions, and +0.6 % for biological processes.

A novel hybrid CNN and BiGRU-Attention based deep learning model for protein function prediction.

Proteins are the building blocks of all living things. Protein function must be ascertained if the molecular mechanism of life is to be understood. While CNN is good at capturing short-term relationships, GRU and LSTM can capture long-term dependencies. A hybrid approach that combines the complementary benefits of these deep-learning models motivates our work. Protein Language models, which use attention networks to gather meaningful data and build representations for proteins, have seen tremendous success in recent years processing the protein sequences. In this paper, we propose a hybrid CNN + BiGRU - Attention based model with protein language model embedding that effectively combines the output of CNN with the output of BiGRU-Attention for predicting protein functions. We evaluated the performance of our proposed hybrid model on human and yeast datasets. The proposed hybrid model improves the Fmax value over the state-of-the-art model SDN2GO for the cellular component prediction task by 1.9 %, for the molecular function prediction task by 3.8 % and for the biological process prediction task by 0.6 % for human dataset and for yeast dataset the cellular component prediction task by 2.4 %, for the molecular function prediction task by 5.2 % and for the biological process prediction task by 1.2 %.

Duplication of the Lacrimal Sac With Three Canaliculi.

Complex congenital lacrimal drainage anomalies are known to be associated with several syndromes and present unique surgical challenges. Duplication of human body structures is uncommon and has been reported in the uterus (uterine didelphys), ureter (duplex ureter), duodenum, transverse colon, and nose. Lacrimal drainage anomalies have been reported in proboscis lateralis. To the best of the authors' knowledge, there are no prior reports on duplication of the lacrimal sac. The present case reports a complex congenital nasolacrimal duct obstruction that was associated with duplication of the lacrimal sac and the presence of 3 canaliculi.

Treating methicillin-resistant Staphylococcus aureus (MRSA) bone infection with focused ultrasound combined thermally sensitive liposomes.

OBJECTIVE: Chronic bone infection caused by Staphylococcus aureus biofilms in children and adults is characterized by reduced antibiotic sensitivity. In this study, we assessed 'heat-targeted, on-demand' antibiotic delivery for S. aureus killing by combining ciprofloxacin (CIP)-laden low-temperature sensitive liposomes (LTSLs) with local high-intensity focused ultrasound (HIFU) induced bone heating in a rat model of bone infection. METHODS: CIP-LTSLs were prepared using the thin-film hydration and extrusion method. Bone infection was established by surgically implanting an orthopedic K-wire colonized with methicillin-resistant S. aureus (MRSA) strain into rat's femurs. For bone heating, ultrasound-guided HIFU exposures were performed to achieve a local temperature of 40-42 °C (∼15 min) concurrently with intravenous injection of CIP-LTSLs or CIP. CIP biodistribution was determined spectrophotometrically and therapeutic efficacy was determined by bacteriological, histological and scanning electron microscopy (SEM) analyses. RESULTS: CIP-LTSLs in the range of 183.5 nm ± 1.91 showed an encapsulation efficiency of >70% at 37 °C and a complete release at ∼42 °C. The metal implantation method yielded medullary osteomyelitis characterized by suppurative changes (bacterial and pus pockets) by day 10 in bones and adjoining muscle tissues. HIFU heating significantly improved CIP delivery from LTSLs in bones, resulting in a significant reduction in MRSA load compared to HIFU and CIP alone groups. These were also verified by histology and SEM, wherein a distinct reduction in S. aureus population in the infected metal wires and tissues from the combinatorial therapy was noted. CONCLUSION: HIFU improved CIP delivery to bones, achieving clearance of hard-to-treat MRSA biofilms.

Optimization of test day for milk yield recording and sire evaluation in Murrah buffaloes.

In the present study, random regression models (RRM) were used to estimate genetic parameters for test-day milk yield in Murrah buffaloes using Legendre polynomial function (LP), with the objective to find the best combination of "minimum test-day model," which would be essential and sufficient to evaluate the trait successfully. Data included for analysis were 10,615 first lactation monthly test-day milk yield records (5th, 35th, 65th, …, 305th) from 965 Murrah buffaloes for the period 1975-2018. Cubic to octic-order orthogonal polynomials with homogeneous residual variances were used for the estimation of genetic parameters. Random regression models with sixth-order were selected based on goodness of fit criteria like lower AIC, BIC and residual variance. Heritability estimates ranged from 0.079 (TD6) to 0.21(TD10). For both ends of lactation, the additive genetic and environmental variances were higher and ranged from 0.21 ± 0.12 (TD6) to 0.85 ± 0.35 kg2 (TD1) and 3.74 ± 0.36 (TD11) to 1.36 ± 0.14 kg2 (TD9), respectively. Between adjacent test-day records, genetic correlation estimates ranged from 0.09 ± 0.31 (TD1 and TD2) to 0.97 ± 0.03 (TD3 and TD4; TD4 and TD5), but values gradually declined as the distance between test days increased. Negative genetic correlations were also obtained between TD1 with TD3 to TD9, TD2 with TD9 and TD10, and TD3 with TD10. On the basis of genetic correlations, models with 5 and/or 6 test-days combination were able to account for 86.1%-98.7% of variation along the lactation. Models with fourth and fifth-order LP functions were considered to account for variance with combinations of 5 and/or 6 test-day milk yields. The model with 6 test-day combinations had a higher rank correlation (0.93) with model using 11 monthly test-day milk yield records. On the basis of relative efficiency, the model with 6 monthly test day combinations with fifth-order was more efficient (maximum 99%) than the model using 11 monthly test-day milk yield records. Looking into the similar accuracy with the 11TD model, and the low resources requirement, we recommend the use of the "6 test-day combination model" for sire evaluation. These models may help in reducing the cost and time for data recording of milk yield.

Review of Robot-Assisted HIFU Therapy.

This paper provides an overview of current robot-assisted high-intensity focused ultrasound (HIFU) systems for image-guided therapies. HIFU is a minimally invasive technique that relies on the thermo-mechanical effects of focused ultrasound waves to perform clinical treatments, such as tumor ablation, mild hyperthermia adjuvant to radiation or chemotherapy, vein occlusion, and many others. HIFU is typically performed under ultrasound (USgHIFU) or magnetic resonance imaging guidance (MRgHIFU), which provide intra-operative monitoring of treatment outcomes. Robot-assisted HIFU probe manipulation provides precise HIFU focal control to avoid damage to surrounding sensitive anatomy, such as blood vessels, nerve bundles, or adjacent organs. These clinical and technical benefits have promoted the rapid adoption of robot-assisted HIFU in the past several decades. This paper aims to present the recent developments of robot-assisted HIFU by summarizing the key features and clinical applications of each system. The paper concludes with a comparison and discussion of future perspectives on robot-assisted HIFU.

Estimation of genetic parameters of growth traits for direct and maternal effects in Murrah buffalo.

An understanding of genetic principles and environmental factors affecting the growth traits is essential to implement optimal breeding and selection programs. Early growth is an indicator of future success in production and reproduction status of dairy animals. In this study, a total of 18,989 records of body weight were used to estimate genetic parameters of body weight at birth (BW), 3 months (3BW), 6 months (6BW), 9 months (9BW),12 months (12BW), 18 months (18BW), 24 months (24 BW), 30 months (3BW), and 36 months (36BW) in Murrah buffalo at ICAR-NDRI Karnal, Haryana, for the period 1974-2019. The genetic parameters were estimated using the average information restricted maximum likelihood (AIREML) procedure by excluding or including maternal effects. Six analytical models were fitted in order to optimize the model for each trait. The most appropriate univariate model was selected based on the log likelihood ratio test (LRT). Influencing factors like calf sex, period of birth, season of birth, and dam's parity were investigated. The results showed that the maternal genetic effects, in addition to direct additive effects, were important for unbiased and accurate genetic parameter estimates of growth traits in Murrah buffaloes. Total heritability estimates h2T1 for BW, 3BW, 6BW, 9BW, 12BW, 18BW, 24BW, 30BW, and 36BW were 0.25, 0.04, 0.14, 0.16, 0.10, 0.15, 0.21, 0.24, and 0.23, respectively. Maternal effect was significant for birth weight and accounted for 13% variation through maternal genetic and 5% variability through maternal permanent environmental effect. Maternal genetic effect was also important for other traits. However, it interfered with the estimates of variance ratios in live weight traits owing to large and negative covariance between direct and maternal genetic effects. Direct genetic correlations between body weight traits were positive and high ranging from 0.10 to 0.94. Results revealed that the Murrah herd has a sizable genetic variability for growth traits and hence, there is sufficient scope for selection for achieving better growth rate if selection in this direction is applied. Owing to higher positive genetic correlation of 6BW with later ages, the scope of indirect selection for optimum growth in later ages can be aimed at by selecting animals for their higher 6-month live weight.

Dual Activities of Receptor-Like Kinase OsWAKL21.2 Induce Immune Responses.

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.

Focused ultrasound ablation of a large canine oral tumor achieves efficient tumor remission: a case report.

Purpose: Oral cancers are one of the commonly diagnosed tumors worldwide in human and veterinary patients. Most oral cancers are surgically resected; however, obtaining an adequate margin of safety in patients without compromising their quality of life is often challenging. Herein, we investigated the ability of non-invasive focused ultrasound (FUS) to thermally ablate a biopsy confirmed canine oral cancer. Materials and Methods: A male canine patient with a large neurilemmoma (schwannoma) mass on the left maxilla, with evidence of thinning and loss of alveolar bone and pressure necrosis, was treated with FUS ablation instead of the traditional maxillectomy procedure. FUS ablations were performed in three sessions over three weeks. Tumor remission was determined with computed tomography and histopathological examination of the treated site. Additionally, the anti-tumor immune effects of FUS were assessed by flow cytometry analysis of blood and tumor samples. Results: Complete tumor remission was noted at the treated site. Treatment related adverse events were primarily thermal burns of the buccal mucosa, which were managed with periodic hyperbaric oxygen therapy and surgical coverage of the underlying exposed bones with gingival flaps. Enhanced proliferation of adaptive immunity cells (e.g., T-cells) was observed in tumor and blood samples. Conclusion: Our limited investigation in a canine oral cancer patient suggests that FUS may avoid the need for large-scale resection of bony tissues, thus potentially improving quality of life.

Using nanoparticles for in situ vaccination against cancer: mechanisms and immunotherapy benefits.

Immunotherapy to treat cancer is now an established clinical approach. Immunotherapy can be applied systemically, as done with checkpoint blockade antibodies, but it can also be injected directly into identified tumors, in a strategy of in situ vaccination (ISV). ISV is designed to stimulate a strong local antitumor immune response involving both innate and adaptive immune cells, and through this generate a systemic antitumor immune response against metastatic tumors. A variety of ISVs have been utilized to generate an immunostimulatory tumor microenvironment (TME). These include attenuated microorganisms, recombinant proteins, small molecules, physical disruptors of TME (alternating magnetic and focused ultrasound heating, photothermal therapy, and radiotherapy), and more recently nanoparticles (NPs). NPs are attractive and unique since they can load multiple drugs or other reagents to influence immune and cancer cell functions in the TME, affording a unique opportunity to stimulate antitumor immunity. Here, we describe the NP-ISV therapeutic mechanisms, review chemically synthesized NPs (i.e., liposomes, polymeric, chitosan-based, inorganic NPs, etc.), biologically derived NPs (virus and bacteria-based NPs), and energy-activated NP-ISVs in the context of their use as local ISV. Data suggests that NP-ISVs can enhance outcomes of immunotherapeutic regimens including those utilizing tumor hyperthermia and checkpoint blockade therapies.

In situ vaccination with nanoparticles for cancer immunotherapy: understanding the immunology.

FDA approval of anti-CTLA4 in 2011 for melanoma immunotherapy was paradigm shifting and dramatically accelerated cancer immunotherapy research. The investment and effort have been exceptionally large, with a commensurate impressive pace of discovery. Historical and current research has validated the following key points: tumors are recognized by the immune system; tumors develop an immunosuppressive environment which suppresses the antitumor immune response; successful immunotherapy must overcome that tumor-mediated immunosuppression. While cancer immunotherapy research expanded, a parallel effort developing nanoparticles (NP) for cancer diagnosis and therapy also received major investment and expanded. Initially the two efforts appeared to have minimal synergy. Systemically administered nanoparticles are rapidly ingested by phagocytic leukocytes, and therefore nanotechnologists developed strategies to avoid NP ingestion by leukocytes in order to accomplish nanoparticle accumulation in tumors rather than liver and spleen. Recently, nanotechnology and cancer immunotherapy have increasingly merged since phagocytic leukocytes are the key to reversing the local tumor immunosuppression and the tendency of NP to be phagocytosed can be exploited to manipulate phagocytes for immunotherapy. This review focuses on in situ vaccination (ISV), an immunotherapy approach that can utilize direct injection of immunostimulatory reagents, including NPs, into tumors to disrupt the local immunosuppression, stimulate effective immune response against the treated tumor, and most importantly, generate a systemic antitumor immune response to eliminate metastatic tumors. While there are many specific options for using NP for ISV (reviewed further in this special issue), this review focuses on immunology concepts needed to understand and design successful NP ISV approaches.

Artificial Intelligence-Based Differential Diagnosis: Development and Validation of a Probabilistic Model to Address Lack of Large-Scale Clinical Datasets.

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.

Gene regulation of Sclerotinia sclerotiorum during infection of Glycine max: on the road to pathogenesis.

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.

Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis.

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.

Resistance against Sclerotinia sclerotiorum in soybean involves a reprogramming of the phenylpropanoid pathway and up-regulation of antifungal activity targeting ergosterol biosynthesis.

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.

In-situ vaccination using focused ultrasound heating and anti-CD-40 agonistic antibody enhances T-cell mediated local and abscopal effects in murine melanoma.

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.

Temperature-sensitive liposomal ciprofloxacin for the treatment of biofilm on infected metal implants using alternating magnetic fields.

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.

Elastin-like polypeptide incorporated thermally sensitive liposome improve antibiotic therapy against musculoskeletal bacterial pathogens.

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.

Plant-derived antifungal agent poacic acid targets ß-1,3-glucan.

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited ß-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding ß-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

Molecular and Biocompatibility Characterization of Red Blood Cell Membrane Targeted and Cell-Penetrating-Peptide-Modified Polymeric Nanoparticles.

Red blood cells (RBCs) express a variety of immunomodulatory markers that enable the body to recognize them as self. We have shown that RBC membrane glycophorin A (GPA) receptor can mediate membrane attachment of protein therapeutics. A critical knowledge gap is whether attaching drug-encapsulated nanoparticles (NPs) to GPA and modification with cell-penetrating peptide (CPP) will impact binding, oxygenation, and the induction of cellular stress. The objective of this study was to formulate copolymer-based NPs containing model fluorescent-tagged bovine serum albumin (BSA) with GPA-specific targeting ligands such as ERY1 (ENPs), single-chain variable antibody (scFv TER-119, SNPs), and low-molecular-weight protamine-based CPP (LNPs) and to determine their biocompatibility using a variety of complementary high-throughput in vitro assays. Experiments were conducted by coincubating NPs with RBCs at body temperature, and biocompatibility was evaluated by Raman spectroscopy, hemolysis, complement lysis, and oxidative stress assays. Data suggested that LNPs effectively targeted RBCs, conferring 2-fold greater uptake in RBCs compared to ENPs and SNPs. Raman spectroscopy results indicated no adverse effect of NP attachment or internalization on the oxygenation status of RBCs. Cellular stress markers such as glutathione, malondialdehyde, and catalase were within normal limits, and complement-mediated lysis due to NPs was negligible in RBCs. Under the conditions tested, our data demonstrates that molecular targeting of the RBC membrane is a feasible translational strategy for improving drug pharmacokinetics and that the proposed high-throughput assays can prescreen diverse NPs for preclinical and clinical biocompatibility.