Agent Invivo Research Articles

Studying the Efficacy of Local Hemostatic Agents In Vivo

Introduction: In modern clinical practice, there is no common and effective hemostatic agent for solid organs of the abdomen. Local agents such as hemostatic sponges, membranes, powders seem promising. The main advantages of hemostatic powders include relatively high efficacy, portability, versatility, as well as simplicity and low cost of manufacturing.Objective: To study the hemostatic efficacy of hemostatic powders’ samples based on the sodium salt of carboxymethylcellulose (Na-CMC) in an acute in vivo experiment.Materials and methods: The study was based at Kursk State Medical University. Fifty male Wistar rats (weighing 220-239 g) were selected as test animals. They were evenly divided into 5 groups: ARISTA AH, Cutanplast Powder, and Na-CMC hemostatic powders with 3 different dynamic viscosities: low (50-200 cP), medium (400-800 cP) and high (1500-3000 cP). Under general inhalation anesthesia, median laparotomy was performed in Wistar rats, the middle lobe of the liver was removed into the wound, and then marginal tangential resection was performed. We applied hemostatic powders of each group to the resulting wound, and then estimated the bleeding time and the volume of blood loss. We used the Mann-Whitney test (P ≤ .05) to assess the statistical significance of differences between the study groups.Results: We recorded the minimum bleeding time and the smallest volume of blood loss for the samples based on the Na-CMC and found no statistically significant differences between the viscosities groups.Conclusions: With Na-CMC hemostatic powder the bleeding time and the volume of blood loss were reduced by 3.6 and 2.2 times, respectively, comparing to the control group. These results were considered statistically significant (P ≤ .05).

Temperature-Responsive Bottlebrush Polymers Deliver a Stress-Regulating Agent In Vivo for Prolonged Plant Heat Stress Mitigation.

Anticipated increases in the frequency and intensity of extreme temperatures will damage crops. Methods that efficiently deliver stress-regulating agents to crops can mitigate these effects. Here, we describe high aspect ratio polymer bottlebrushes for temperature-controlled agent delivery in plants. The foliar-applied bottlebrush polymers had near complete uptake into the leaf and resided in both the apoplastic regions of the leaf mesophyll and in cells surrounding the vasculature. Elevated temperature enhanced the in vivo release of spermidine (a stress-regulating agent) from the bottlebrushes, promoting tomato plant (Solanum lycopersicum) photosynthesis under heat and light stress. The bottlebrushes continued to provide protection against heat stress for at least 15 days after foliar application, whereas free spermidine did not. About 30% of the ∼80 nm short and ∼300 nm long bottlebrushes entered the phloem and moved to other plant organs, enabling heat-activated release of plant protection agents in phloem. These results indicate the ability of the polymer bottlebrushes to release encapsulated stress relief agents when triggered by heat to provide long-term protection to plants and the potential to manage plant phloem pathogens. Overall, this temperature-responsive delivery platform provides a new tool for protecting plants against climate-induced damage and yield loss.

Effects of CBD (Cannabidiol) on the physiology of Nile tilapia (Oreochromisn niloticus) as a chronic stress mitigating agent In-vivo.

This study evaluates the effects of Cannabidiol (CBD) on the physiology of stressed and non-stressed Nile tilapia, reared in a recirculating aquaculture system. Tilapia were fed with and without CBD (0.001% of feed weight) and with and without hydrocortisone stress hormone (0.01% of body weight) every day for four weeks. This experiment compared the plasma cortisol, blood glucose and protein levels, liver and spleen somatic indices (HSI and SSI, respectively), and lysozyme activity of the fish. Stress group (S) had a significantly higher value than the control group (C) in two of the parameters, glucose and lysozyme activity, this is an indication of stress. CBD had a stress reducing effect under stressed conditions in lysozyme activity. Although not significant, the stress reducing effect of CBD on stress biomarkers such as glucose and HSI also seemed promising. Further investigation into the matter may not just be useful in stress mediation in aquatic organisms but may also have implications in human medicine as well.

Three-dimensional structure of a mycobacterial oligoribonuclease reveals a unique C-terminal tail that stabilizes the homodimer

Oligoribonucleases (Orns) are highly conserved DnaQ-fold 3'-5' exoribonucleases that have been found to carry out the last step of cyclic-di-GMP (c-di-GMP) degradation, that is, pGpG to GMP in several bacteria. Removal of pGpG is critical for c-di-GMP homeostasis, as excess uncleaved pGpG can have feedback inhibition on phosphodiesterases, thereby perturbing cellular signaling pathways regulated by c-di-GMP. Perturbation of c-di-GMP levels not only affects survival under hypoxic, reductive stress, or nutrient-limiting conditions but also affects pathogenicity in infection models as well as antibiotic response in mycobacteria. Here, we have determined the crystal structure of MSMEG_4724, the Orn of Mycobacterium smegmatis (Ms_orn) to 1.87Å resolution to investigate the function of its extended C-terminal tail that is unique among bacterial Orns. Ms_orn is a homodimer with the canonical RNase-H fold of exoribonucleases and conserved catalytic residues in the active site. Further examination of the substrate-binding site with a modeled pGpG emphasized the role of a phosphate cap and "3'OH cap" in constricting a 2-mer substrate in the active site. The unique C-terminal tail of Ms_orn aids dimerization by forming a handshake-like flap over the second protomer of the dimer. Our thermal and denaturant-induced unfolding experiments suggest that it helps in higher stability of Ms_orn as compared with Escherichia coli Orn or a C-terminal deletion mutant. We also show that the C-terminal tail is required for modulating response to stress agents invivo. These results will help in further evaluating the role of signaling and regulation by c-di-GMP in mycobacteria.

AGR2-Dependent Nuclear Import of RNA Polymerase II Constitutes a Specific Target of Pancreatic Ductal Adenocarcinoma in the Context of Wild-Type p53

Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2-RNAPII complex. We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation invivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents invivo. Of note, this therapeutic effect is efficient across various cancer types. Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Microspheres of Carboxymethyl Chitosan, Sodium Alginate, and Collagen as a Hemostatic Agent in Vivo

In the search for biocompatible composite microspheres to be used as a hemostatic agent, in a previous study, we designed a novel biomaterial, consisting of composite microspheres containing three natural biological ingredients, carboxymethyl chitosan, sodium alginate and collagen (CSCM). Furthermore, the chemical and physical properties, hemostatic ability, biocompatibility and cytotoxicity were investigated in vitro. In this work, the in vivo hemostatic performance, wound healing, hemocompatibility, histocompatibility, and biodegradability were evaluated by a series of experiments. The results showed that CSCM could both stop bleeding and enhance healing efficiency by accelerating the clotting and the wound closure rate, suggesting that CSCM acts as a hemostat, and enhances wound healing. In addition, the CSCM material had negligible intracutaneous stimulation reactions and no obvious hemolytic reactions. More importantly, CSCM can be degraded in vivo without significant impacts on physiology, biochemistry, and organization. Thus, CSCM may be a useful tool to stop bleeding in emergency conditions in both military and civilian settings.

Antibacterial Agents: Enzyme‐Coated Mesoporous Silica Nanoparticles as Efficient Antibacterial Agents In Vivo (Adv. Healthcare Mater. 10/2013)

On page 1351, Li-li Li and Hao Wang report lysozyme-coated mesoporous silica nanoparticles as anti-bacterial agents that exhibit efficient antibacterial activity both in vitro and in vivo with low cytotoxicity and negligible hemolytic side effect. The lysozyme corona provides multivalent interaction between nanoparticles and bacterial walls, promoting hydrolysis of peptidoglycans and increased membrane-perturbation abilities. The survival bacteria in colon are three orders of magnitude lower than that in the untreated group.

Enzyme‐Coated Mesoporous Silica Nanoparticles as Efficient Antibacterial Agents In Vivo

Despite the fact that pathogenic infections are widely treated by antibiotics in the clinic nowadays, the increasing risk of multidrug-resistance associated with abuse of antibiotics is becoming a major concern in global public health. The increased death toll caused by pathogenic bacterial infection calls for effective antibiotic alternatives. Lysozyme-coated mesoporous silica nanoparticles (MSNs⊂Lys) are reported as antibacterial agents that exhibit efficient antibacterial activity both in vitro and in vivo with low cytotoxicity and negligible hemolytic side effect. The Lys corona provides multivalent interaction between MSNs⊂Lys and bacterial walls and consequently raises the local concentration of Lys on the surface of cell walls, which promotes hydrolysis of peptidoglycans and increases membrane-perturbation abilities. The minimal inhibition concentration (MIC) of MSNs⊂Lys is fivefold lower than that of free Lys in vitro. The antibacterial efficacy of MSNs⊂Lys is evaluated in vivo by using an intestine-infected mouse model. Experimental results indicate that the number of bacteria surviving in the colon is three orders of magnitude lower than in the untreated group. These natural antibacterial enzyme-modified nanoparticles open up a new avenue for design and synthesis of next-generation antibacterial agents as alternatives to antibiotics.

Probing the Membrane Interaction of Antimicrobial Agents In Vivo with Intact Bacteria by 2H NMR

The cell membrane is an important biological interface often targeted by bioactive molecules such as antimicrobial peptides (AMPs) which can disrupt the bacterial lipid barrier. Identification and study of AMPs is a dynamic research field motivated by the emergence of antibiotic-resistant pathogens. To develop new efficient therapies, the action mechanism of membrane-disrupting antimicrobial agents should thus be studied. 2H solid-state NMR is a useful method to monitor changes in organization and dynamics of phospholipids in bilayers. Usually carried out with model systems, the complex composition of bacterial membranes - especially the presence of lipopolysaccharides (LPS) - encourages such studies on intact organisms. We have therefore developed a protocol to 2H-label phospholipids in Escherichia coli membranes without mutating. About 75% of the phospholipids had deuterated acyl chains. We have thus investigated in vivo the interaction mechanism of antimicrobial agents, i.e., the antibiotic polymyxin B (PxB), the detergent cetyltrimethylammonium chloride (CTAC) and fullerenol nanoparticles. 2H NMR spectra and spectral moment analysis support the insertion of the PxB's lipid tail in the bacterial membrane. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent. The use of intact bacteria seems important in cases where the antibiotic action relies on an interaction with LPS. This is shown with fullerenol nanoparticles for which a different effect is seen on intact cells as compared to model DPPC/DPPG membranes, as will be detailed. This is further demonstrated with the blue pigment marennine produced by marine microalgae which would also interact with LPS. A different perturbation of the bacterial membranes by the intra- and extracellular forms of this pigment was also evidenced, thus shedding light on the action mechanism of this potential natural antibiotic.

Quercetin: Critical Evaluation as an Antileishmanial Agent In Vivo in Hamsters Using Different Vesicular Delivery Modes

Chemotherapy is still a major challenge for in vivo drug targeting to macrophages. Toxicity remains the major obstacle for the most potent drugs already known in the therapy of leishmaniasis. Thus, new drugs and new delivery systems are sought. By using different vesicular delivery modes e.g. liposomes, niosomes, microspheres and nanoparticles, attempts have been made to deliver an indigenous antileishmanial compound, quercetin, to treat experimental leishmaniasis in the hamster model so as to increase its efficacy as well as to reduce the toxicity. At equivalent quercetin concentration, the nanocapsulated quercetin was found to be the most potent in reducing the parasite burden in the spleen as well as in reducing hepatotoxcity and renaltoxicity compared to free drug or drug in other vesicular forms. An inverse relationship between the efficacy and the size of the vesicles was established. Such a drug vehicle formulation especially in the nanocapsulated form may be considered for clinical trials.

Design, synthesis, and preliminary pharmacological evaluation of N-acyl-3-aminoglutarimides as broad-spectrum chemokine inhibitors in vitro and anti-inflammatory agents in vivo.

A series of N-substituted 3-aminoglutarimides have been synthesized and tested for inhibitory activity against a range of chemokines in vitro and for suppression of lipopolysaccharide-induced inflammation in vivo. The results show that they represent the first class of small molecules with broad-spectrum chemokine inhibitory effects. Among the compounds studied, 10 (NR58,4) was the most potent, being active at doses between 5 and 15 nM in vitro and at 0.3 mg kg(-1) in vivo.

Endogenous Nitric Oxide Acts as a Natural Antithrombotic Agent In Vivo by Inhibiting Platelet Aggregation in the Pulmonary Vasculature

Nitric oxide (NO) is a powerful vasodilator and an inhibitor of platelet aggregation in vitro. While the ability of NO to modulate vascular tone in vivo has been proven, only a few studies have assessed its platelet inhibitory activity in vivo. We have employed two complementary animal models of pulmonary platelet thromboembolism to assess the antithrombotic activity of endogenous NO in vivo. The inhibition of nitric oxide synthase (NOS) by L-NAME significantly potentiated while the administration of the NOS substrate L-arginine significantly reduced the accumulation of 111In-labelled platelets in the pulmonary vasculature of rabbits induced by intravenous collagen plus epinephrine. L-NAME or L-arginine did not, however, modify 111In-labelled erythrocyte distribution in lungs and phenylephrine had no effect on platelet accumulation following collagen + adrenaline, suggesting that the effects of L-NAME were not due to vasoconstriction but rather to a direct modification of platelet function. In mice, L-NAME significantly reduced the dose of collagen + adrenaline required to induce thromboembolic mortality, increased the fall in circulating platelets and increased the % of pulmonary vessels occluded by platelet thrombi. The effects of L-NAME were reversed by L-arginine but not by a dose of nicardipine exerting maximal vasodilatation. Phenylephrine did not potentiate collagen + adrenaline-induced mortality. In the pulmonary vasculature in vivo, endogenous NO inhibits collagen + adrenaline-induced aggregation and enhances platelet disaggregation. This natural modulator function of NO is exerted via a direct effect on platelets and not as a result of haemodynamic changes.

Kinetic Analysis of Malignant Fibrous Histiocytoma Cells Treated with Anticancer Agents In Vivo.

To clarify the in vivo effects and the changes in kinetic parameters of soft tissue sarcoma caused by anticancer agents, we performed double-labeling using bromo- (BrdU) and iododeoxyuridine (IUdR). A malignant fibrous histiocytoma cell line was transplanted into nude mice, and treated with 3 anticancer agents: cisplatin, doxorubicin and vincristine. The following parameters were determined: BrdU labeling index (LI), duration of S-phase (Ts), total cell cycle time (Tc), Ki-67 labeling index (KLI), PCNA labeling index (PCNA-LI), and actual tumor volume doubling time (Tv). In addition, the apoptotic cell ratio (apoptotic index; Al) was calculated in the serial specimens using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL).Vincristine inhibited the tumor growth the most effectively (Tv: -5.8 days). Doxorubicin suppressed the tumor growth better (Tv: 12.7 days) than cisplatin (Tv: 9.1 days). Tc values showed significant differences among the drug groups (ANOVA analysis; p 0.7, p<0.05) on Day 1. Al peaked on Day 10 and decreased afterward in every drug group.Anticancer agents effectively suppress tumor growth not only by necrosis and apoptosis, but also by prolonging Tc. Tc values are possibly useful for planning chemotherapy for sarcoma patients.

CHOLIC ACID AS A DEMYELINATING AGENT IN VIVO.

CHOLIC ACID AS A DEMYELINATING AGENT IN VIVO.