Production of fructooligosaccharides by Bacillus subtilis natto CCT7712 and their antiproliferative potential.

AIMS: Fructooligosaccharides (FOSs) known for their health properties and ß-(2→6)-levan-type FOSs have shown prebiotic and immunomodulatory activities that overcome those of commercial ß-(2→1)-FOSs, but costs do not favour their use. Moreover, FOSs can reach the bloodstream through the diet, and little is known about their direct effect on cells. The aim of this work was to produce high-content FOSs by Bacillus subtilis natto CCT7712 in a bioreactor using commercial sucrose and to evaluate their antiproliferative effects in OVCAR-3 cells. METHODS AND RESULTS: FOS production reached 173·60 g l-1 , 0·2 vvm aeration and uncontrolled pH. Levan-type FOSs, composed of ß-(2 â†’ 6) links and mainly GF3 (6-nystose), were identified using RMN spectroscopy, FT-IR and ESI-MS. FOSs decreased the viability and proliferation of OVCAR-3 cells, and the effects were associated with an increased pro-inflammatory response by the induction of IL-8 and TNF-α, and the repression of ER-ß genes. The metabolic profiles showed disruption of cellular homeostasis that can be associated with a decrease in proliferation. CONCLUSIONS: The high production of levan-type FOSs from B. subtilis natto CCT7712 in a bioreactor was achieved, and they showed antiproliferative potential in OVCAR-3 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: FOS could be a good target for future therapeutic studies and commercial use.

Influence of the incorporation of marine spongin into a Biosilicate®: an in vitro study.

The combination of different biomaterials can be a promising intervention for the composites manufacture, mainly by adding functional and structural characteristics of each material and guarantee the advantages of the use of these composites. In this context, the aim of this study was to develop and evaluated the influence of the incorporation of marine spongin (SPG) into Biosilicate® (BS) in different proportions be used during bone repair. For this purpose, it was to develop and investigate different BS/SPG formulations for physico-chemical and morphological characteristics by pH, loss mass, Fourier transform infrared spectrometer (FTIR) and scanning electron microscope (SEM) analysis. Additionally, the influence of these composites on cell viability, proliferation, and alkaline phosphatase (ALP) activity were investigated. The results revealed that the pH values of all BS groups (with or without SPG) increased over time. A significant mass loss was observed in all composites, mainly with higher SPG percentages. Additionaly, SEM micrographies demonstrated fibers of SPG into BS and material degradation over time. Moreover, FTIR spectral analysis revealed characteristic peaks of PMMA, BS, and SPG in BS/SPG composites. BS/SPG groups demonstrated a positive effect for fibroblast proliferation after 3 and 7 days of culture. Additionally, BS and BS/SPG formulations (at 10% and 20% of SPG) presented similar values of osteoblasts viability and proliferation after 7 days of culture. Furthermore, ALP activity demonstrated no significant difference between BS and BS/SPG scaffolds, at any composition. Based on the present in vitro results, it can be concluded that the incorporation of SPG into BS was possible and produced an improvement in the physical-chemical characteristics and in the biological performance of the graft especially the formulation with 80/20 and 90/10. Future research should focus on in vivo evaluations of this novel composite.

Aerobic exercise training and low-level laser therapy modulate inflammatory response and degenerative process in an experimental model of knee osteoarthritis in rats.

OBJECTIVE: The aim of this study was to evaluate the effects of an aerobic exercise training and low-level laser therapy (LLLT) (associated or not) on degenerative modifications and inflammatory mediators on the articular cartilage using an experimental model of knee OA. MATERIAL AND METHODS: Fifty male Wistar rats were randomly divided into five groups: control group (CG); knee OA control group (OAC); OA plus exercise training group (OAT); OA plus LLLT group (OAL); OA plus exercise training associated with LLLT group (OATL). The exercise training (treadmill; 16 m/min; 50 min/day) and the laser irradiation (two points-medial and lateral side of the left joint; 24 sessions) started 4 weeks after the surgery, 3 days/week for 8 weeks. RESULTS: The results showed that all treated groups showed (irradiated or not) a better pattern of tissue organization, with less fibrillation and irregularities along the articular surface and chondrocytes organization, a lower degenerative process measured by OARSI score and higher thickness values. Additionally, all treated group showed a reduced expression in IL-1ß, caspase-3 and MMP-13 compared to OAC. Moreover, a lower caspase-3 expression was observed in OATL compared to OAL and OAT. CONCLUSION: These results suggest that exercise training and LLLT were effective in preventing cartilage degeneration and modulating inflammatory process induced by knee OA.

Neurotrophin-mimicking peptides at the biointerface with gold respond to copper ion stimuli.

The peptide fragments NGF1-14 and BDNF1-12, encompassing the N-terminal domains, respectively, of the proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were used in this study for the fabrication of a hybrid gold/peptide biointerface. These peptides mimic the Trk receptor activation of the respective whole protein - with a crucial role played by copper ions - and exhibit, in bulk solution, a pH-dependent capability to complex copper. We demonstrate here the maintenance of peptide-specific responses at different pH values as well as the copper binding also for the adlayers formed upon physisorption at the gold surface. The physicochemical properties, including viscoelastic behavior of the adlayer and competitive vs. synergic interactions in sequential adsorption processes, were addressed both experimentally, by quartz crystal microbalance with dissipation monitoring (QCM-D) and circular dichroism (CD), and theoretically, by molecular dynamics (MD) calculations. Proof-of work biological assays with the neuroblastoma SY-SH5H cell line demonstrated that the developed hybrid Au/peptide nanoplatforms are very promising for implementation in pH- and metal-responsive systems for application in nanomedicine.

Infection with Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae in cancer patients.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) is an emergent pathogen in healthcare-associated infections (HAIs). The aim of this study was to describe HAIs due to KPC-Kp, as well as identify mortality risk factors in cancer patients. In patients diagnosed with HAIs due to KPC-Kp between January 2009 and July 2013, we evaluated only the first infection episode of each patient, analyzing mortality separately for patients treated for ≥48 h with at least one antimicrobial agent proven to display in vitro activity against KPC-Kp. We evaluated variables related to the malignancy, the severity and characteristics of the HAI, and the antimicrobial therapy. We identified 83 HAIs due to KPC-Kp. The 30-day mortality was 57.8 % for all infections and 72.7 % for bacteremic infections. Of the 83 patients, 60 patients received ≥48 h of appropriate treatment and 44 (53 %) developed bacteremia. Ten patients (12 %) were neutropenic at HAI diagnosis and 33 (39.8 %) had infection at the tumor site. The most common HAI was urinary tract infection, seen in 26 patients (31.3 %), followed by primary bloodstream infection, seen in 24 patients (28.9 %). Forty-four patients (73.3 %) received combination antimicrobial therapy, most often including polymyxin (68.3 %). Risk factors for 30-day mortality are high sequential organ failure assessment (SOFA) score, need for intensive care stay at diagnosis of infection, and acute kidney injury; the removal of invasive devices related to infection and treatment with effective antibiotics for KPC-Kp are protective factors. In cancer patients, high mortality is associated with HAI due to KPC-Kp and mortality risk factors are more often related to acute infection than to the underlying disease.

Effect of a new bioactive fibrous glassy scaffold on bone repair.

Researchers have investigated several therapeutic approaches to treat non-union fractures. Among these, bioactive glasses and glass ceramics have been widely used as grafts. This class of biomaterial has the ability to integrate with living bone. Nevertheless, bioglass and bioactive materials have been used mainly as powder and blocks, compromising the filling of irregular bone defects. Considering this matter, our research group has developed a new bioactive glass composition that can originate malleable fibers, which can offer a more suitable material to be used as bone graft substitutes. Thus, the aim of this study was to assess the morphological structure (via scanning electron microscope) of these fibers upon incubation in phosphate buffered saline (PBS) after 1, 7 and 14 days and, also, evaluate the in vivo tissue response to the new biomaterial using implantation in rat tibial defects. The histopathological, immunohistochemistry and biomechanical analyzes after 15, 30 and 60 days of implantation were performed to investigate the effects of the material on bone repair. The PBS incubation indicated that the fibers of the glassy scaffold degraded over time. The histological analysis revealed a progressive degradation of the material with increasing implantation time and also its substitution by granulation tissue and woven bone. Histomorphometry showed a higher amount of newly formed bone area in the control group (CG) compared to the biomaterial group (BG) 15 days post-surgery. After 30 and 60 days, CG and BG showed a similar amount of newly formed bone. The novel biomaterial enhanced the expression of RUNX-2 and RANK-L, and also improved the mechanical properties of the tibial callus at day 15 after surgery. These results indicated a promising use of the new biomaterial for bone engineering. However, further long-term studies should be carried out to provide additional information concerning the material degradation in the later stages and the bone regeneration induced by the fibrous material.

Japanese encephalitis virus RNA detected in Culex pipiens mosquitoes in Italy.

Mosquitoes collected in northern Italy were screened for flavivirus RNA. Positive amplicons were sequenced and found most similar to insect flavivirus (ISF), Usutu virus (USUV) and surprisingly also to Japanese encephalitis virus (JEV). The sequence (167 bp), obtained from one pool of Culex pipiens, was found identical to JEV strains from bats in China. Unfortunately additional sequence data or virus isolations were not obtained in this study. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed.

Midline mandibular osteotomy in an asymmetric patient.

This case report shows the possibility of the application of a mandibular osteotomy to resolve mandibular asymmetry with independent and discordant movements of both bony segments. The authors report the case of a 25-year-old woman referred for mandibular asymmetry, with a transverse excess of the right hemi mandible and vertical defect of the left one. The patient underwent a bilateral sagittal split osteotomy, midline osteotomy, and genioplasty, which corrected the mandibular asymmetry with contraction of the entire right hemi mandible. A slight left vertical increase was also obtained through the surgically created lateral open bite. In the follow-up assessment, the patient's face appeared symmetrical with normalization of the bizygomatic-bigonial relationships, and the facial shape corresponded to ideal anthropometric features. This technique resulted in resolution of mandibular asymmetry. In addition, mandibular osteotomy permits the esthetic management of the shape of the entire mandibular body in relation to the other third of the face.

Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs.

The eukaryotic porin, also called the Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L. The main VDAC isoform, called VDAC1 (Porin1), is expressed from the first gene of the cluster. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5' untranslated regions (UTRs). Here, we studied the influence of the two 5' UTRs, 1A-5' UTR and 1B-5' UTR, on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained using constructs containing the two 5' UTRs upstream of the GFP reporter. A short region of 15 nucleotides in the 1B-5' UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. Our data suggest that contacts between the 5' UTR and 18S rRNA sequences could modulate the translation of Drosophila 1B-VDAC mRNA. The evolutionary significance of this finding is discussed.

A model for the simulation of the SHARON process: pH as a key factor.

The SHARON process allows partial nitrification of wastewaters with high ammonium content and, when coupled with the Anammox process, represents a more sustainable alternative for N-removal than a conventional nitrification-denitrification. In this work, a mathematical model describing a continuously aerated SHARON reactor is presented. Special attention was given to the pH, because it affects substrates availability and inhibition phenomena, implementing an algorithm for its calculation. Since ammonium-oxidizing and nitrite-oxidizing organisms are inhibited by their own substrates, ammonia and nitrous acid respectively, Haldane kinetics was used in both nitrification steps. A preliminary evaluation of the model using historical experimental data generated in a lab-scale SHARON reactor, fed with synthetic substrate, is also presented, corroborating that the quality of the obtained effluent is highly dependent on pH.

Characterization and biological evaluation of the introduction of PLGA into biosilicate®.

The aims of this study were to characterize different BS/PLGA composites for their physicochemical and morphological characteristics and evaluate the in vitro and in vivo biological performance. The physicochemical and morphological modifications were analyzed by pH, mass loss, XRD, setting time, and SEM. For in vitro analysis, the osteoblast and fibroblast viability was evaluated. For in vivo evaluations, histopathology and immunohistochemistry were performed in a tibial defect in rats. After incubation, all composites presented lower values in pH and mass loss over time. Moreover, XRD and SEM analysis confirmed that the composites degraded over time. Additionally, pore formation was observed by SEM analysis after incubation mainly in BS/PLGA groups. BS/PLGA showed significantly increased in osteoblast viability 24 h. Moreover, BS/PLGA composites demonstrated an increase in fibroblast viability in all periods analyzed when compared to BS. In the in vivo study, after 2 and 6 weeks of implantation of biomaterials, histopathological findings revealed that the BS/PLGA composites degrades over time, mainly at periphery. Moreover, can be observed the presence of granulation tissue, bone formation, Runx-2, and RANKL immunoexpression in all groups. In conclusion, BS/PLGA composites present appropriate physicochemical characteristics, stimulate the cellular viability, and enhance the bone repair in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1063-1074, 2017.

Characterization and biocompatibility of a fibrous glassy scaffold.

Bioactive glasses (BGs) are known for their ability to bond to living bone and cartilage. In general, they are readily available in powder and monolithic forms, which are not ideal for the optimal filling of bone defects with irregular shapes. In this context, the development of BG-based scaffolds containing flexible fibres is a relevant approach to improve the performance of BGs. This study is aimed at characterizing a new, highly porous, fibrous glassy scaffold and evaluating its in vitro and in vivo biocompatibility. The developed scaffolds were characterized in terms of porosity, mineralization and morphological features. Additionally, fibroblast and osteoblast cells were seeded in contact with extracts of the scaffolds to assess cell proliferation and genotoxicity after 24, 72 and 144 h. Finally, scaffolds were placed subcutaneously in rats for 15, 30 and 60 days. The scaffolds presented interconnected porous structures, and the precursor bioglass could mineralize a hydroxyapatite (HCA) layer in simulated body fluid (SBF) after only 12 h. The biomaterial elicited increased fibroblast and osteoblast cell proliferation, and no DNA damage was observed. The in vivo experiment showed degradation of the biomaterial over time, with soft tissue ingrowth into the degraded area and the presence of multinucleated giant cells around the implant. At day 60, the scaffolds were almost completely degraded and an organized granulation tissue filled the area. The results highlight the potential of this fibrous, glassy material for bone regeneration, due to its bioactive properties, non-cytotoxicity and biocompatibility. Future investigations should focus on translating these findings to orthotopic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Biosilicate/PLGA osteogenic effects modulated by laser therapy: In vitro and in vivo studies.

The main purpose of the present work was to evaluate if low laser level therapy (LLLT) can improve the effects of Biosilicate®/PLGA (BS/PLGA) composites on cell viability and bone consolidation using a tibial defects of rats. The composites were characterized by scanning electron microscope (SEM) and reflection Fourier transform infrared spectrometer (FTIR). For the in vitro study, fibroblast and osteoblast cells were seeded in the extract of the composites irradiated or not with LLLT (Ga-Al-As, 808nm, 10J/cm2) to assess cell viability after 24, 48 and 72h. For the in vivo study, 80 Wistar rats with tibial bone defects were distributed into 4 groups (BS; BS+LLLT; BS/PLGA and BS/PLGA+LLLT) and euthanized after 2 and 6weeks. Laser irradiation Ga-Al-As (808nm, 30J/cm2) in the rats was performed 3 times a week. The SEM and FTIR results revealed that PLGA were successfully inserted into BS and the microparticles degraded over time. The in vitro findings demonstrated higher fibroblast viability in both BS/PLGA groups after 24h and higher osteoblast viability in BS/PLGA+LLLT in all periods. As a conclusion, animals treated with BS/PLGA+LLLT demonstrated an improved material degradation and an increased amount of granulation tissue and newly formed bone.

Copper binding to naturally occurring, lactam form of angiogenin differs from that to recombinant protein, affecting their activity.

Angiogenin is a member of the ribonuclease family and a normal constituent of human plasma. It is one of the most potent angiogenic factors known and is overexpressed in different types of cancers. Copper is also an essential cofactor in angiogenesis and, during this process, it is mobilized from inside to outside of the cell. To date, contrasting results have been reported about copper(ii) influencing angiogenin activity. However, in these studies, the recombinant form of the protein was used. Unlike recombinant angiogenin, that contains an extra methionine with a free terminal amino group, the naturally occurring protein present in human plasma starts with a glutamine residue that spontaneously cyclizes to pyroglutamate, a lactam derivative. Herein, we report spectroscopic evidence indicating that copper(ii) experiences different coordination environments in the two protein isoforms, and affects their RNase and angiogenic activity differently. These results show how relatively small differences between recombinant and wild type proteins can result in markedly different behaviours.

Bloodstream infection caused by extensively drug-resistant Acinetobacter baumannii in cancer patients: high mortality associated with delayed treatment rather than with the degree of neutropenia.

This study aimed to describe severe infections with extensively drug-resistant Acinetobacter baumannii-calcoaceticus complex (XDR-ABC), as well as to investigate risk factors for mortality, in cancer patients. It was a retrospective study including all patients diagnosed with XDR-ABC bacteraemia during hospitalization in the intensive care unit of a cancer hospital between July 2009 and July 2013. Surveillance cultures were collected weekly during the study period, and clonality was analysed using pulsed field gel electrophoresis (PFGE). We analysed underlying diseases, oncology therapy, neutrophil counts, infection site and management of infection, in terms of their correlation with 30-day mortality. During the study period, 92 patients with XDR-ABC bacteraemia were identified, of whom 35 (38.0%) were patients with haematological malignancy. We identified XDR-ABC strains with four different profile patterns, 91.3% of patients harbouring the predominant PFGE type. Of the 92 patients with XDR-ABC bacteraemia, 66 (71.7%) had central line-associated bloodstream infections; infection occurred during neutropenia in 22 (23.9%); and 58 (63.0%) died before receiving the appropriate therapy. All patients were treated with polymyxin, which was used in combination therapy in 30 of them (32.4%). The 30-day mortality rate was 83.7%. Multivariate analysis revealed that septic shock at diagnosis of XDR-ABC infection was a risk factor for 30-day mortality; protective factors were receiving appropriate therapy and invasive device removal within the first 48 h. Among cancer patients, ineffective management of such infection increases the risk of death, more so than do features such as neutropenia and infection at the tumour site.

[Surgical treatment of abdominal trauma in pediatric age]. / Traumi chirurgici addominali in età pediatrica.

AIM OF THE STUDY: to value the appropriateness and the efficacy of non-operative treatment in children with blunt abdominal trauma. PATIENTS AND METHODS: in this research 14 children with abdominal trauma, secondary mostly to road accidents, were studied; 9 of these had single organ injury while 5 had multiple organ injuries. Spleen has been the most injured organ (9 children), followed by liver (5 cases) and kidney (2 cases). Five children were admitted at emergency department in hypovolemic conditions, promptly corrected by resuscitative measures. All patients underwent abdominal ultrasound and/or C.T. scans in order to detect any intraperitoneal free fluid or organic injuries. Six children (43%) were followed by non-operative treatment, while other eight underwent surgery. RESULTS: all children, those treated conservatively as well as those operated, were cured, without any important complication. DISCUSSION AND CONCLUSIONS: nowadays, with the great help of ultrasound and C.T. scans, is possible to treat 40-50% of children affected by abdominal trauma with non-operative measures, with return to normal functions by the injured organs. The surgical approach is today accomplished only in presence of severe injuries or haemodynamic instability resistant to resuscitative treatment. The majority of Authors in the literature agree on the usefulness of non-operative treatment, especially for children. Regarding surgical treatment, in the near future probably we'll see a larger application of laparoscopic techniques also in the field of abdominal traumatology.

Auricular reconstruction of congenital microtia: personal experience in 225 cases.

Microtia is a congenital disease with various degrees of severity, ranging from the presence of rudimentary and malformed vestigial structures to the total absence of the ear (anotia). The complex anatomy of the external ear and the necessity to provide good projection and symmetry make this reconstruction particularly difficult. The aim of this work is to report our surgical technique of microtic ear correction and to analyse the short and long term results. From 2000 to 2013, 210 patients affected by microtia were treated at the Maxillo-Facial Surgery Division, Head and Neck Department, University Hospital of Parma. The patient population consisted of 95 women and 115 men, aged from 7 to 49 years. A total of 225 reconstructions have been performed in two surgical stages basing of Firmin's technique with some modifications and refinements. The first stage consists in fabrication and grafting of a three-dimensional costal cartilage framework. The second stage is performed 5-6 months later: the reconstructed ear is raised up and an additional cartilaginous graft is used to increase its projection. A mastoid fascial flap together with a skin graft are then used to protect the cartilage graft. All reconstructions were performed without any major complication. The results have been considered satisfactory by all patients starting from the first surgical step. Low morbidity, the good results obtained and a high rate of patient satisfaction make our protocol an optimal choice for treatment of microtia. The surgeon's experience and postoperative patient care must be considered as essential aspects of treatment.

Determination of fosinopril in pharmaceutical formulations containing hydrochlorothiazide by multiwavelength UV spectrophotometry.

A sensitive, simple, rapid and precise method for the simultaneous determination of fosinopril (FOS) and hydrochlorothiazide (HCT) in pharmaceutical formulations is presented. These active ingredients are extracted in aqueous solution and measured by multiwavelength UV spectrophotometry using the program QUEST. HCT acts as an internal standard to verify the accuracy of the analysis. Some aspects of the chemical, spectroscopic and thermoanalytical behaviour of FOS are also reported.

Determination of choline containing phospholipids in serum, bile and amniotic fluids by the derivative enzymatic-spectrophotometric method.

A comparison was performed of the analytical results obtained by applying a well-known enzymatic-spectrophotometric method for the determination of choline containing phospholipids in biological human fluids (sera, bile and amniotic fluids) following the standard procedure method, or else using the first or second derivative methods. In terms of result reproducibility the comparison was extended to include also a biosensor-based method developed in recent years by the present authors. Some advantages are associated with using the first derivative method in the case of serum samples containing blood traces, especially via the 'Gran's plot' method.

Determination of lecithin drug specialties and diet integrators; by means of first or second derivative enzymatic-spectrophotometric analysis.

The possibility has been investigated of applying derivative analysis to a classical enzymatic-spectrophotometric method for lecithin determination for the purpose of developing an analytical direct method that does not require long pretreatment of the test sample even in the case of turbid samples. Several samples of drugs and food integrators containing lecithin were thus tested using both the standard and the derivative method and then comparing the results obtained. The RSD% values of measurements on real (food or drugs) samples were always <2.5. Using a first derivative spectrophotometric method, average recovery was always between 102 and 105%.