The Influence of Phytosociological Cultivation and Fertilization on Polyphenolic Content of Menthae and Melissae folium and Evaluation of Antioxidant Properties through In Vitro and In Silico Methods.

Since medicinal plants are widely used in treating various diseases, phytoconstituents enrichment strategies are of high interest for plant growers. First of all, we investigated the impact of phytosociological cultivation on polyphenolic content (total flavonoids-TFL, and total polyphenols-TPC) of peppermint (Mentha piperita L.) and lemon balm (Melissa officinalis L.) leaves, using spectrophotometric methods. Secondly, the influence of chemical (NPK) and organic (BIO) fertilization on polyphenolic content and plant material quality was also assessed. Dry extracts were obtained from harvested leaves using hydroethanolic extraction solvents for further qualitative and quantitative assessment of phytoconstituents by FT-ICR MS and UHPLC-MS. Furthermore, the antioxidant activity of leaf extracts was determined in vitro using DPPH, ABTS and FRAP methods. Molecular docking simulations were employed to further evaluate the antioxidant potential of obtained extracts, predicting the interactions of identified phytochemicals with sirtuins. The concentration of polyphenols was higher in the plant material harvested from the phytosociological culture. Moreover, the use of BIO fertilizer led to the biosynthesis of a higher content of polyphenols. Higher amounts of phytochemicals, such as caffeic acid, were determined in extracts obtained from phytosociological crops. The antioxidant activity was dependent on polyphenols concentration, more potent inhibition values being observed for the extracts obtained from the phytosociological batches. Molecular docking studies and MM/PBSA calculations revealed that the obtained extracts have the potential to directly activate sirtuins 1, 5 and 6 through several polyphenolic compounds, such as rosmarinic acid, thus complementing the free radical scavenging activity with the potential stimulation of endogenous antioxidant defense mechanisms. In conclusion, growing medicinal plants in phytosociological cultures treated with biofertilizers can have a positive impact on plant material quality, concentration in active constituents and biological activity.

The Polyphenolic Profile and Antioxidant Activity of Five Vegetal Extracts with Hepatoprotective Potential.

Oxidative stress is among the major triggers for many important human functional disorders, which often lead to various metabolic or tissue diseases. The aim of the study is to obtain five standardized vegetal extracts (Cynarae extractum-CE, Rosmarini extractum-RE, Taraxaci extractum-TE, Cichorii extractum-CHE, and Agrimoniae extractum-AE) that contain active principles with an essential role in protecting liver cells against free radicals and quantify their antioxidant actions. The compounds of therapeutic interest from the analyzed extracts were identified and quantified using the UHPLC-HRMS/MS technique. Thus, the resulting identified compounds were 28 compounds in CE, 48 compounds in RE, 39 compounds in TE, 43 compounds in CHE, and 31 compounds in AE. These compounds belong to the class of flavonoids, isoflavones, phenolic acids and dicarboxylic acids, depsides, diterpenes, triterpenes, sesquiterpenes, proanthocyanidins, or coumarin derivatives. From the major polyphenolic compounds quantified in all the extracts analyzed by UHPLC-HRMS/MS, considerable amounts have been found for chlorogenic acid (619.8 µg/g extract for TE-2032.4 µg/g extract for AE), rutoside (105.1 µg/g extract for RE-1724.7 µg/g extract for AE), kaempferol (243 µg/g extract for CHE-2028.4 µg/g extract for CE), and for naringenin (383 µg/g extract for CHE-1375.8 µg/g extract for AE). The quantitative chemical analysis showed the highest content of total phenolic acids for AE (24.1528 ± 1.1936 g chlorogenic acid/100 g dry extract), the highest concentration of flavones for RE (6.0847 ± 0.3025 g rutoside/100 g dry extract), and the richest extract in total polyphenols with 31.7017 ± 1.2211 g tannic acid equivalent/100 g dry extract for AE. Several methods (DPPH, ABTS, and FRAP) have been used to determine the in vitro total antioxidant activity of the extracts to evaluate their free radical scavenging ability, influenced by the identified compounds. As a result, the correlation between the content of the polyphenolic compounds and the antioxidant effect of the extracts has been demonstrated. Statistically significant differences were found when comparing the antiradical capacity within the study groups. Although all the analyzed extracts showed good IC50 values, which may explain their antihepatotoxic effects, the highest antioxidant activity was obtained for Agrimoniae extractum (IC50ABTS = 0.0147 mg/mL) and the lowest antioxidant activity was obtained for Cynarae extractum (IC50ABTS = 0.1588 mg/mL). Furthermore, the hepatoprotective potential was evaluated in silico by predicting the interactions between the determined phytochemicals and key molecular targets relevant to liver disease pathophysiology. Finally, the evaluation of the pharmacognostic and phytochemical properties of the studied extracts validates their use as adjuvants in phytotherapy, as they reduce oxidative stress and toxin accumulation and thus exert a hepatoprotective effect at the cellular level.

CD11c+ T-bet+ B Cells Require IL-21 and IFN-γ from Type 1 T Follicular Helper Cells and Intrinsic Bcl-6 Expression but Develop Normally in the Absence of T-bet.

CD11c+ T-bet+ B cells generated during ehrlichial infection require CD4+ T cell help and IL-21 signaling for their development, but the exact T cell subset required had not been known. In this study, we show in a mouse model of Ehrlichia muris that type 1 T follicular helper (TFH1) cells provide help to CD11c+ T-bet+ B cells via the dual secretion of IL-21 and IFN-γ in a CD40/CD40L-dependent manner. TFH1 cell help was delivered in two phases: IFN-γ signals were provided early in infection, whereas CD40/CD40L help was provided late in infection. In contrast to T-bet+ T cells, T-bet+ B cells did not develop in the absence of B cell-intrinsic Bcl-6 but were generated in the absence of T-bet. T-bet-deficient memory B cells were largely indistinguishable from their wild-type counterparts, although they no longer underwent switching to IgG2c. These data suggest that a primary function of T-bet in B cells during ehrlichial infection is to promote appropriate class switching, not lineage specification. Thus, CD11c+ memory B cells develop normally without T-bet but require Bcl-6 and specialized help from dual cytokine-producing TFH1 cells.

TNF-α Contributes to Lymphoid Tissue Disorganization and Germinal Center B Cell Suppression during Intracellular Bacterial Infection.

Bacterial, parasitic, and viral infections are well-known causes of lymphoid tissue disorganization, although the factors, both host and/or pathogen derived, that mediate these changes are largely unknown. Ehrlichia muris infection in mice causes a loss of germinal center (GC) B cells that is accompanied by the generation of extrafollicular T-bet+ CD11c+ plasmablasts and IgM memory B cells. We addressed a possible role for TNF-α in this process because this cytokine has been shown to regulate GC development. Ablation of TNF-α during infection resulted in an 8-fold expansion of GL7+ CD38lo CD95+ GC B cells, and a 2.5- and 5-fold expansion of CD138+ plasmablasts and T-bet+ memory cells, respectively. These changes were accompanied by a reduction in splenomegaly, more organized T and B cell zones, and an improved response to Ag challenge. CXCL13, the ligand for CXCR5, was detected at 6-fold higher levels following infection but was much reduced following TNF-α ablation, suggesting that CXCL13 dysregulation also contributes to loss of lymphoid tissue organization. T follicular helper cells, which also underwent expansion in infected TNF-α--deficient mice, may also have contributed to the expansion of T-bet+ B cells, as the latter are known to require T cell help. Our findings contrast with previously described roles for TNF-α in GCs and reveal how host-pathogen interactions can induce profound changes in cytokine and chemokine production that can alter lymphoid tissue organization, GC B cell development, and extrafollicular T-bet+ B cell generation.

Paclitaxel controlled delivery using a pH-responsive functional-AuNP/block-copolymer vesicular nanocarrier composite system.

Paclitaxel (PTX)-loaded gold nanoparticles functionalized with mercaptooctanoic acid (MOA) and folic acid (FA) (AuMOA-FA) were encapsulated within pH-sensitive poly(2-vinylpyridine)-b-poly(ethylene oxide) (P2VP-PEO) vesicles with the aim to develop a more selective injectable nano-formulation for PTX, lacking the side effects of the conventional PTX delivery system. The size of the resulting composite vesicles was lower than 200 nm, i.e. it is suitable for tumor targeting applications taking advantage of the enhanced permeability and retention (EPR) effect. The vesicles did not aggregate in the presence of high electrolyte concentrations, indicating the colloidal stability of the vesicles. The vesicles did not leak their AuMOA-FA or PTX content at physiological pH of 7.4. However, AuMOA-FA and PTX release were significantly accelerated at acidic pHs resembling tumor environment and acidic intracellular compartments. PTX release from the vesicles at acidic pH apparently follows AuMOA-FA release from the vesicles. Flow cytometry measurements and confocal laser scanning microscopy images showed that the vesicles could enter A549 cancer cells in culture and that cellular uptake increased with time. Blank vesicles did not exhibit cytotoxicity and did not induce apoptosis in A549 cancer cells. The PTX currying vesicles exhibited comparable or a little higher cytotoxicity than free PTX. Both the PTX currying vesicles and free PTX induced A549 cells apoptosis, however the vesicle-encapsulated PTX induced a higher percentage of late apoptotic cells than free PTX.

Injectable Hydrogel: Amplifying the pH Sensitivity of a Triblock Copolypeptide by Conjugating the N-Termini via Dynamic Covalent Bonding.

We explore the self-assembly behavior of aqueous solutions of an amphiphilic, pH-sensitive poly(l-alanine)-b-poly(l-glutamic acid)-b-poly(l-alanine), (A5E11A5) triblock copolypeptide, end-capped by benzaldehyde through Schiff base reaction. At elevated concentrations and under physiological pH (7.4) and ionic strength (0.15M), the bare copolypeptide aqueous solutions underwent a sol-gel transition after heating and slow cooling thermal treatment, forming opaque stiff gels due to a hierarchical self-assembly that led to the formation of ß-sheet-based twisted super fibers (Popescu et al. Soft Matter 2015, 11, 331-342). The conjugation of the N-termini with benzaldehyde (Bz) through a Schiff base reaction amplifies the copolypeptide pH-sensitivity within a narrow pH window relevant for in vivo applications. Specifically, the dynamic character of the imine bond allowed coupling/decoupling of the Bz upon switching pH. The presence of Bz conjugates to the N-termini of the copolypeptide resulted in enhanced packing of the elementary superfibers into thick and short piles, which inhibited the ability of the system for gelation. However, partial cleavage of Bz upon lowering pH to 6.5 prompted recovery of the hydrogel. The sol-gel transition triggered by pH was reversible, due to the coupling/decoupling of the benzoic-imine dynamic covalent bonding, endowing thus the gelling system with injectability. Undesirably, the gelation temperature window was significantly reduced, which however can be regulated at physiological temperatures by using a suitable mixture of the bare and the Bz-conjugated coplypeptide. This triblock copolypeptide gelator was investigated as a scaffold for the encapsulation of polymersome nanocarriers, loaded with a hydrophilic model drug, calcein. The polymersome/polypeptide complex system showed prolonged probe release in pH 6.5, which is relevant to extracellular tumor environment, rendering the system potentially useful for sustained delivery of anticancer drugs locally in the tumor.

Fibrinogen Reduction and Motor Function Improvement by Hematopoietic Growth Factor Treatment in Chronic Stroke in Aged Mice: A Treatment Frequency Study.

Stroke is a serious medical condition that causes long-term neurological disability in mainly elderly adults worldwide. Lack of therapy to improve functional recovery in the chronic phase of stroke is a major challenge for stroke research. Combining two hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF), our previous studies have demonstrated the neurovascular restorative efficacy of this treatment in the chronic phase of experimental stroke. Elevated plasma fibrinogen has been thought to serve as a predictor for ischemic stroke. Here we have determined the treatment frequency in reducing plasma fibrinogen and in restoring motor function in aged mice with chronic stroke. Our findings show that SCF + G-CSF treatment in chronic stroke decreases plasma fibrinogen and improves motor function in aged mice. No differences have been found between a 2-week treatment regimen and 7-day treatment in the plasma fibrinogen assay, while the 7-day treatment regimen displays a better recovery pattern with regard to motor function. This study provides new insight into understanding the potential contribution of SCF + G-CSF in both reducing the risk of recurrent ischemic stroke and enhancing stroke recovery.

Stimuli responsive fibrous hydrogels from hierarchical self-assembly of a triblock copolypeptide.

In this work, the self-assembly behavior and pH responsiveness of a triblock copolypeptide in aqueous media are demonstrated. The copolypeptide was composed of a central pH responsive poly(l-glutamic acid) (PGA), flanked by two hydrophobic poly(l-alanine) blocks (PAla) (PAla5-PGA11-PAla5). This system showed a pH-responsive transition from short tapes to spherical aggregates by increasing the pH, as a result of deprotonation of the PGA block and a conformational change from α-helix to random coil. Increasing the ionic strength to physiological conditions (0.15 M) has triggered fibrillar self-assembly through intermolecular hydrogen bonding of PAla end-blocks that form ß-sheet nanostructures, in conjunction with charge screening of the central random coil PGA segments. At elevated concentrations a thermo-responsive free supporting hydrogel was obtained, consisting of rigid ß-sheet based twisted superfibers, resulting from hierarchical self-assembly of the copolypeptide. Yet, morphological transformation of this nanostructure was observed upon switching the pH from physiological conditions to pH 4. An unexpected morphology constituted of α-helix-based giant nanobelts was observed as a consequence of the secondary peptide transitions.

Technological update of the video-endoscopic approach of the diagnosis and staging of tumors in children.

The pediatric patient is often difficult to diagnose, especially since the surgical pathology is tumoral. Establishing the histopathological diagnosis of a tumor, staging of a disease and certifying the existence of rare pediatric affections are three of the motives for which the specialists frequently appeal to an exploratory laparoscopy, accompanied by biopsy procedures. The paper presents the laparoscopic biopsy experience of the team from the Department of Pediatric Surgery, "Maria Sklodowska Curie" Emergency Clinical Hospital for Children, Bucharest, Romania. From 2000 to 2013, 95 such procedures were performed (7.76% of a total of 1224 laparoscopic interventions). In many cases, the laparoscopy had an exclusive diagnostic purpose, of whose success has been primarily responsible the targeted biopsy. Current issues are discussed, centered on the most advanced technologies used in minimally invasive approach of pediatric malignancies, referring to the effect of minimizing the possible complications that can arise from this type of intervention. The authors concluded that laparoscopic technique is the method of choice in comparison to other ways of biopsy (classical surgery, ultrasound guidance, tomography, etc.), and it is characterized by a high diagnostic value.

Validation of spectrophotometric method for Se(IV) determination: analytical applications.

As selenium is an important part of the antioxidant enzymes and also because there are several studies suggesting a possible link between cancer and selenium deficiency, this paper presents a spectrophotometric method for the assay of Se(IV), using N,N-diethyl-p-phenylenediamine monohydrochloride as reagent. The proposed method is based on the reaction between the selenium and potassium iodide in low acidic medium, when iodine is released. This last product will further oxidise the new reagent. The final obtained product is strongly coloured in red and has an absorption maximum at 552 nm and molar extinction coefficient (ε) of 6.1 × 10(4) L mol(-1) cm(-1). The optimum working conditions were established, and the developed method was validated, being characterised by a good linearity (in the range of 0.5-3.0 µg/mL), a limit of detection (0.0573 µg/mL) and a limit of quantification (0.1737 µg/mL). At the same time, the repeatability, the precision of the method and the accuracy were established. The proposed and validated method was applied with good results for the determination of Se(IV) in spring and bottled water from Iasi and also in pharmaceutical and cosmetic products.

Controlled Delivery of Functionalized Gold Nanoparticles by pH-Sensitive Polymersomes.

The present study reports on the development of composite gold nanoparticles (AuNPs)/polymersome formulations, based on pH-responsive biocompatible polymer vesicles integrating prefunctionalized AuNPs, doped with a hydrophobic model probe for improved multimodal drug delivery. The polymer vesicles were prepared from an amphiphilic pentablock terpolymer poly(ε-caprolactone)-b-poly(ethylene oxide)-b-poly(2-vinylpyridine)-b-poly(ethylene oxide)-b-poly(ε-caprolactone) (PCL-PEO-P2VP-PEO-PCL), consisting of a pH-sensitive and biodegradable P2VP/PCL membrane, surrounded by neutral hydrophilic PEO looping chains. Additionally, partial quaternization of the P2VP block has been performed to introduce cationic moieties. Water-dispersible AuNPs carrying a hydrophobic molecule were encapsulated in the hydrophilic aqueous lumen of the vesicles, and the release was monitored at pH conditions simulating physiological and tumor environments. The complex delivery of the cargos from these vesicles showed improved and controlled kinetics relative to the individual nanocarriers, which could be further tuned by pH and chemical modification of the membrane forming block.

Laparoscopic exploration in pediatric surgery emergencies.

The laparoscopic approach of pediatric surgery emergencies represents a specific preoccupation in hospitals everywhere in the world. Nowadays, when confronted with this pathology, pediatric surgeons are able to apply certain well-defined therapeutic protocols, depending on the technical equipment at their disposal and their laparoscopic expertise and training. We hereby present some of the surgical pediatric emergencies that have been subjected to minimally invasive celioscopic techniques, in the Department of Pediatric Surgery "Maria Sklodowska Curie" Hospital, from August 1999 to July 2007. Out of 83 exploratory laparoscopies, 12 were performed for emergency pathology, other than acute appendicitis (in its various forms, including peritonitis) or acute cholecystitis. However, during the above-mentioned period, the number of therapeutic laparoscopies for emergencies has grown significantly (239 from a total of 663 laparoscopies), reflecting to a large extent the activity of a clinic with an emergency surgery profile. The authors conclude that exploratory laparoscopies in pediatric surgery emergencies are suited for surgical teams with a solid experience in celioscopy and a certain professional maturity, necessary to correctly appreciate the surgical and anesthetic risks involved by each individual case. It is not recommended that inexperienced laparoscopic surgeons embark on the "adventure" of this minimally invasive approach for this type of pathology. Only when the training and learning process is fully and correctly completed, specialists are offered the advantage of continuing a celioscopic exploration by performing a minimally invasive therapeutic procedure, even for a pediatric emergency case.

Exploratory laparoscopy--diagnosis method in pediatric surgery pathology.

The purpose of this article is to establish the important place of laparoscopy in the diagnosis management of pediatric surgery pathology. In addition, it is intended to become a pleading, concerning the true and realistic benefit, for the pediatric patient, of minimally invasive surgery. In our country, the method was taken over from general surgery and had a few years of latency in imposing itself. We consider that each pediatric surgeon is required to have a mandatory period of training in this topic. This is the reason why we appeal to the young generations of pediatric surgeons but also to the surgeons with many years of experience in classical surgical techniques, who are able to perfect the diagnostic and treatment methods in many medical specialties. The clinical research took place in a retro and prospective manner from August 1999 to July 2007 in the Department of Pediatric Surgery "Maria Sklodowska Curie" Hospital and studied 663 laparoscopic surgeries. 83 of them were exploratory laparoscopies (39 boys and 44 girls) and the other 580 were therapeutic procedures with accurate pre-operatory diagnostics. The surgical activity of our department concerning exploratory laparoscopy was also appreciated due to the decisional and therapeutic algorithms that were conceived. The motivations for exploratory laparoscopic procedures are multiple and arise from many causes, from right lower quadrant pain syndrome and abdominal tumors to congenital malformations and non-palpable testis. The processed data revealed the utility of laparoscopy, concerning most of the pediatric surgical pathologies and in the same time allowing them to continue the exploratory method of diagnosis with a minimally invasive therapy. The discrepancy between the exploratory benefit (sometimes minor) and the surgical trauma of an open exploratory surgery, explains the utility of laparoscopy that took over most of open surgery indications.