Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

Introduction and objective: p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). Material and methods: This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. Results and discussion: No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Evaluation of the Treatment with Akkermansia muciniphila BAA-835 of Chemotherapy-induced Mucositis in Mice.

Mucositis is a high-incidence side effect in cancer patients undergoing chemotherapy. Next-generation probiotics are emerging as new therapeutic tools for managing various disorders. Studies have demonstrated the potential of Akkermansia muciniphila to increase the efficiency of anticancer treatment and to mitigate mucositis. Due to the beneficial effect of A. muciniphila on the host, we evaluated the dose-response, the microorganism viability, and the treatment protocol of A. muciniphila BAA-835 in a murine model of chemotherapy-induced mucositis. Female Balb/c mice were divided into groups that received either sterile 0.9% saline or A. muciniphila by gavage. Mucositis was induced using a single intraperitoneal injection of 5-fluorouracil. The animals were euthanized three days after the induction of mucositis, and tissue and blood were collected for analysis. Prevention of weight loss and small intestine shortening and reduction of neutrophil and eosinophil influx were observed when animals were pretreated with viable A. muciniphila at 1010 colony-forming units per mL (CFU/mL). The A. muciniphila improved mucosal damage by preserving tissue architecture and increasing villus height and goblet cell number. It also improved the integrity of the epithelial barrier, decreasing intestinal permeability and bacterial translocation. In addition, the treatment prevented the expansion of Enterobacteriaceae. The immunological parameters were also improved by decreasing the expression of pro-inflammatory cytokines (IL6, IL1ß, and TNF) and increasing IL10. In conclusion, pretreatment with 1010 CFU/mL of viable A. muciniphila effectively controlled inflammation, protected the intestinal mucosa and the epithelial barrier, and prevented Enterobacteriaceae expansion in treated mice.

Staged vs concurrent hardware removal in total ankle arthroplasty.

INTRODUCTION: Ankle osteoarthritis is more commonly posttraumatic. Consequently, dealing with hardware removal is quite frequent when performing a total ankle arthroplasty (TAA). The purpose of this study is to compare outcomes regarding either a staged or concurrent hardware removal when performing TAA. MATERIALS AND METHODS: 275 consecutive patients with TAA previously treated with internal fixation were retrospectively reviewed. Finally, 57 patients were enrolled based on exclusion criteria, and were differentiated into two groups considering the timing of hardware removal (staged-group A vs concurrent-group B) to compare: neurovascular and wound complications, time to recover full weight bearing, scar-tissue esthetic, and surgical time. Moreover, a subgroup comparison considering the surgical approach (single approach, minor additional approach, major additional approach) was performed between the group A and group B. RESULTS: No statistically significant difference other that longer surgical time (p < 0.05) was observed between group A and group B. When considering surgical approach subgroups, statistically significant higher surgical wound complications and revision rate were reported in group B (concurrent) major additional approach subgroup, and a statistically significant shorter time to full weight bearing was reported in group A (staged) major additional approach subgroup. CONCLUSIONS: When performing TAA requiring hardware removal, no clear superiority of staged over concurrent hardware removal was observed. However, when considering a subgroup of patients requiring a separate major incision, a staged approach has shown reduced surgical time, less risk of wound complications, and shorter recovery to full weight bearing. LEVEL OF EVIDENCE: III.

Pediatric vascular anomalies in Austria. Where are we at? A survey among primary care pediatricians.

Introduction: Vascular anomalies (VAs) are rare conditions and affected patients often experience a difficult patient journey. Timely diagnosis is only possible if primary caregivers are aware of the anomalies and are connected with dedicated specialists. Aim of our survey was to investigate the knowledge about diagnostic and therapeutic possibilities for children with VAs, and the existing networking among primary pediatric caregivers in Austria. Methods: Primary care pediatricians in Austria were invited to complete an online questionnaire consisting of 28 questions focusing on pediatric VAs. Results: Out of 373 invited pediatricians 93 (25%) returned the questionnaires, 86 of which were complete. Most physicians (39/93 42%) answered that they see between 15 and 30 patients with infantile hemangiomas per year. Vascular malformations are rarely treated in the primary care setting; most primary care pediatricians (58/86, 67%) reported that they currently treat fewer than 5 patients with such type of VAs. There was unequivocal agreement among the participants (84/86, 98%) on the need to establish a network of specialists and a registry dedicated to pediatric VAs. Conclusions: This survey represents the first study shedding light on the awareness of VAs among Austrian pediatricians and can serve as a basis for future investigations and advances in the management of these conditions in Austria and other countries with a similar healthcare setting.

Epidemiology of sepsis in Internal Medicine Units of Apulia: results of SEMINA (SEpsis Management in INternal medicine Apulia) study.

Background: The prevalence and mortality of sepsis in Internal Medicine Units (IMUs) is poorly understood as most of the data derive from studies conducted in Intensive Care Units. Aim of SEpsis Management in INternal medicine Apulia (SEMINA) study was to determine the prevalence of sepsis and the characteristics and outcomes of patients with Sepsis-3 criteria admitted in Apulia's Internal Medicine Units for over six months. Methods: The SEpsis Management in INternal medicine of Apulia study was a prospective, multicentre, observational study. Adult admissions to the 13 Apulia Region's Internal Medicine Units between November 15, 2018 and May 15, 2019 were screened for sepsis according to the Sepsis-3 criteria. Medical data were collected in electronic case report form. Results: Out of 7,885 adult patients of the Internal Medicine Units, 359 (4.55%) fulfilled the inclusion criteria, and 65 of them (18.1%) met the septic shock criteria. The patients enrolled were elderly, suffering from chronic poly-pathologies and from cognitive and functional impairment. The respiratory system was the most common site of infection and the most common pathogens isolated from blood cultures were Staphylococcus spp., E. coli, Klebsiella spp., Enterococcus spp. and Acinetobacter spp. The in-hospital fatality rate was 31.2% and was significantly higher for septic shock. Sequential Organ Failure Assessment score, dementia and infections from Acinetobacter spp. were independent risk factors for mortality. Conclusions: A high prevalence of sepsis and a high fatality rate were detected in Apulia Region's Internal Medicine Units. The high fatality rate observed in our study could be related to the underlying diseases and to the vulnerability of elderly patients admitted to our Internal Medicine Units.

Evaluation of a Functional Craft Wheat Beer Fermented with Saccharomyces cerevisiae UFMG A-905 to treat Salmonella Typhimurium infection in mice.

Functional foods containing probiotics are generally administered as dairy products. Non-dairy beverages are another possibility, but probiotic functionality must be confirmed in such vehicles. In the present study, a craft wheat beer brewed with the probiotic yeast Saccharomyces cerevisiae UFMG A-905 (905) was evaluated in a murine model of Salmonella Typhimurium infection. Unfiltered or filtered beer brewed with 905, a commercial wheat beer used as a negative control, or saline were administered orally to mice before and during oral S. Typhimurium challenge. High fecal levels of yeast were only counted in mice treated with the unfiltered 905 beer, which also had reduced mortality and body weight loss due to S. Typhimurium infection. Increased levels of intestinal IgA, translocation to liver and spleen, liver and intestinal lesions, pro-inflammatory cytokines in liver and ileum, and hepatic and intestinal myeloperoxidase and eosinophilic peroxidase activities were observed in animals infected with S. Typhimurium. All these parameters were reduced by the treatment with unfiltered 905 beer. In conclusion, the results show that a craft wheat beer brewed with S. cerevisiae UFMG A-905 maintained the probiotic properties of this yeast when administered orally to mice challenged with S. Typhimurium.

Anti-Mayaro virus activity of a hydroethanolic extract from Fridericia chica (Bonpl.) L. G. Lohmann leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Mayaro fever is a neglected tropical disease. The region of the most significant circulation of the Mayaro virus (MAYV) is the Amazon rainforest, situated in remote areas that are difficult to access and where medicine is scarce. Thus, the regional population uses plants as an alternative for the treatment of various diseases. Fridericia chica is an endemic plant of tropical regions used in traditional medicine to treat fever, malaise, inflammation, and infectious diseases such as hepatitis B. However, its antiviral activity is poorly understood. AIM OF THE STUDY: This study aimed to investigate the anti-MAYV activity of the hydroethanolic extract of the leaves of Fridericia chica (HEFc) in mammalian cells and its possible mechanism of action. MATERIALS AND METHODS: The antiviral activity of HEFc was studied using Vero cell lines against MAYV. The cytotoxicity and antiviral activity of the extract were evaluated by the 3-(4, 5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The overall antiviral activity was confirmed by the plaque forming units (PFU) method. Then, the effects of HEFc on MAYV multiplication kinetics, virus adsorption, penetration, and post-penetration, and its virucidal activity were determined in Vero cells using standard experimental procedures. RESULTS: HEFc exerted a effect against viral infection in Vero cells at a non-cytotoxic concentration, and no virion was detected in the supernatant in a dose-dependent and selective manner. HEFc inhibited MAYV in the early and late stages of the viral multiplication cycle. The extract showed significant virucidal activity at low concentrations and did not affect adsorption or viral internalization stages. In addition, HEFc reduced virions at all post-infection times investigated. CONCLUSIONS: HEFc has good antiviral activity against MAYV, acting directly on the viral particles. This plant extract possesses an excellent and promising potential for developing effective herbal antiviral drugs.

Prognostic Relevance of ZNF844 and Chr 19p13.2 KRAB-Zinc Finger Proteins in Clear Cell Renal Carcinoma.

BACKGROUND/AIM: Clear-cell renal cell carcinoma (ccRCC) is the most common and aggressive form of all urological cancers, with poor prognosis and high mortality. Despite growing evidence of involvement in carcinogenesis, the role of KRAB-ZFP in ccRCC has not been fully explored. KRAB Zinc finger proteins (KRAB-ZFPs) are the largest family of mammalian transcription regulators. They are differentially expressed in various tissues during cellular development and phenotypic differentiation. MATERIALS AND METHODS: In this study, the levels of transcripts of ccRCC from The Cancer Genome Atlas (TCGA) dataset were used to identify prognostic biomarkers in this disease. RESULTS: Using bioinformatics techniques, we demonstrate that approximately 60% of KRAB zinc finger proteins located on chromosome 19p13.2 are differentially expressed, with all but two being down-regulated in ccRCC. Moreover, ZNF844, a paralog of ZNF433, was the most down-regulated across all histological grades and pathological stages (p<0.001). In addition, the decrease in ZNF844 expression was associated with poor patient survival (HR=0.41; 95% CI=0.3-0.56; p<0.0001). Gene Set Enrichment Analysis of genes inversely co-expressed with ZNF844 revealed that enriched pathways were consistently related to immune and translation processes (p<0.05, FDR <0.05). Lastly, ZNF844 expression showed moderate, inverse correlation to Helper T-cell (CD4 or Th1) subtype 1 (R=-0.558, p=5.15×10-39) infiltration and with the exhausted T-cell phenotype (R=-0.37; p=4.1×10-21). CONCLUSION: Down-regulation of KRAB-ZFPs at 19p13.2 may represent a signature for ccRCC. Moreover, ZNF844 is a prognostic marker for ccRCC and may serve as a putative immune-related tumor suppressor gene.

Antarctic Strain of Rhodotorula mucilaginosa UFMGCB 18,377 Attenuates Mucositis Induced by 5-Fluorouracil in Mice.

Mucositis is one of the most strenuous side effects caused by chemotherapy drugs, such as 5-fluorouracil (5-FU), during the treatment of several types of cancers. The disease is so prevalent and aggressive that many patients cannot resist such symptoms. However, despite its frequency and clinical significance, there is no effective treatment to prevent or treat mucositis. Thus, the use of probiotics as an adjuvant for the treatment has gained prominence. In the present study, we evaluated the effectiveness of oral administration of the Antarctic strain of Rhodotorula mucilaginosa UFMGCB 18,377 as an alternative to minimize side effects of 5-FU-induced mucositis in mice. Body weight, food consumption, stool consistency, and presence of blood in the feces were assessed daily in mice orally treated or not with the yeast and submitted or not to experimental mucositis. Blood, bones, and intestinal tissues and fluid were used to determine intestinal permeability and immunological, microbiological, and histopathological parameters. Treatment with R. mucilaginosa UFMGCB 18,377 was able to decrease clinical signs of the disease, such as reduction of food intake and body weight loss, and also decreased the number of intestinal enterobacteria and intestinal length shortening. Additionally, treatment was able to decrease the levels of MPO and EPO activities and inflammatory infiltrates, as well as the histopathological lesions characteristic of mucositis in the jejunum and ileum. Results of the present study showed that the oral administration of R. mucilaginosa UFMGCB 18,377 protected mice against mucositis induced by 5-FU.

Splenic, hepatic, renal and pulmonary clearance dysfunction associated with high-energy X-rays.

PURPOSE: To verify the high-energy X-rays effects on the blood clearance of colloidal particles by the spleen, liver, kidneys, and lungs. MATERIALS AND METHODS: Seventeen male Wistar rats were distributed into three groups. Group 1 (n = 5) - control - non-irradiated animals, group 2 (n = 6) - irradiated animals studied 24 h after irradiation, and group 3 (n = 6) - irradiated animals studied 48 h after irradiation. The animals were anesthetized and irradiated with a non-fractionated 8 Gy dose in the abdominal region divided into two parallel and opposite fields, 4 Gy was given to the anteroposterior and 4 Gy to the posteroanterior. This high dose of high-energy X-rays causes extensive cell killing, tissue disorganization and break down cell to cell communication. According to the groups, 50 µCi of technetium-phytate were injected into the right internal jugular vein. After 30 minutes, the liver, spleen, kidneys, and lungs were removed. The clot was harvested from the abdominal cavity two minutes after the sectioning of the abdominal aorta and cava vein. The organs and clot were placed into plastic flasks to be weighed and studied for the emission of radioactivity in a gamma radiation detector. The uptake function of each organ was calculated based on the count of gamma rays emitted per minute and normalized with the organ mass, having as a reference the radioactivity count of a standard sample. The arithmetic mean of each organ uptake was calculated and compared among the groups. RESULTS: After irradiation, the spleen uptake of colloidal radiopharmaceutical was greater, while the hepatic, renal, and pulmonary uptake were lower. The renal uptake decreased slower than the hepatic and pulmonary uptake. CONCLUSIONS: A single high dose of high-energy X-rays enhances the splenic clearance function, while it reduces the hepatic, renal, and pulmonary clearance until 48 h after irradiation, with a rapid deterioration of the hepatic and pulmonary uptake function.

Lyophilized Symbiotic Mitigates Mucositis Induced by 5-Fluorouracil.

Mucositis is an adverse effect of cancer chemotherapies using 5-Fluorouracil (5-FU). It is characterized by mucosal inflammation, pain, diarrhea, and weight loss. Some studies reported promising healing effects of probiotic strains, when associated with prebiotics, as adjuvant treatment of mucositis. We developed a lyophilized symbiotic product, containing skimmed milk, supplemented with whey protein isolate (WPI) and with fructooligosaccharides (FOS), and fermented by Lactobacillus casei BL23, Lactiplantibacillus plantarum B7, and Lacticaseibacillus rhamnosus B1. In a mice 5-FU mucositis model, this symbiotic lyophilized formulation was able to reduce weight loss and intestinal permeability. This last was determined in vivo by quantifying blood radioactivity after oral administration of 99mTc-DTPA. Finally, histological damages caused by 5-FU-induced mucositis were monitored. Consumption of the symbiotic formulation caused a reduced score of inflammation in the duodenum, ileum, and colon. In addition, it decreased levels of pro-inflammatory cytokines IL-1ß, IL-6, IL-17, and TNF-α in the mice ileum. The symbiotic product developed in this work thus represents a promising adjuvant treatment of mucositis.

Mapping Pectic-Polysaccharide Epitopes in Cell Walls of Forage Chicory (Cichorium intybus) Leaves.

The cell walls of forage chicory (Cichorium intybus) leaves are known to contain high proportions of pectic polysaccharides. However, little is known about the distribution of pectic polysaacharides among walls of different cell types/tissues and within walls. In this study, immunolabelling with four monoclonal antibodies was used to map the distribution of pectic polysaccharides in the cell walls of the laminae and midribs of these leaves. The antibodies JIM5 and JIM7 are specific for partially methyl-esterified homogalacturonans; LM5 and LM6 are specific for (1→4)-ß-galactan and (1→5)-α-arabinan side chains, respectively, of rhamnogalacturonan I. All four antibodies labelled the walls of the epidermal cells with different intensities. JIM5 and JIM7, but not LM5 or LM6, labelled the middle lamella, tricellular junctions, and the corners of intercellular spaces of ground, xylem and phloem parenchyma. LM5, but not LM6, strongly labelled the walls of the few sclerenchyma fibres in the phloem of the midrib and lamina vascular bundles. The LM5 epitope was absent from some phloem parenchyma cells. LM6, but not LM5, strongly labelled the walls of the stomatal guard cells. The differential distribution of pectic epitopes among walls of different cell types and within walls may reflect the deposition and modification of these polysaccharides which are involved in cell wall properties and cell development.

The Identification of Zinc-Finger Protein 433 as a Possible Prognostic Biomarker for Clear-Cell Renal Cell Carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is the most common and aggressive form of all urological cancers, with poor prognosis and high mortality. At late stages, ccRCC is known to be mainly resistant to chemotherapy and radiotherapy. Therefore, it is urgent and necessary to identify biomarkers that can facilitate the early detection of ccRCC in patients. In this study, the levels of transcripts of ccRCC from The Cancer Genome Atlas (TCGA) dataset were used to identify prognostic biomarkers in this disease. Analyzing the data obtained indicated that the KRAB-ZNF protein is significantly suppressed in clear-cell carcinomas. Furthermore, ZNF433 is differentially expressed in ccRCC in a stage- and histological-grade-specific manner. In addition, ZNF433 expression was correlated with metastasis, with greater node involvement associated with lower ZNF433 expression (p < 0.01) and with a more unsatisfactory overall survival outcome (HR, 0.45; 95% CI, 0.33-0.6; p = 8.5 × 10-8). Since ccRCC is characterized by mutations in proteins that alter epigenetic modifications and /or chromatin remodeling, we examined the expression of ZNF433 transcripts in ccRCC with wildtype and mutated forms of BAP1, KDMC5, MTOR, PBRM1, SETD2, and VHL. Analysis revealed that ZNF433 expression was significantly reduced in ccRCC with mutations in the BAP1, SETD2, and KDM5C genes (p < 0.05). In addition, the ZNF433 promoter region was highly methylated, and hypermethylation was significantly associated with mRNA suppression (p < 2.2 × 10-16). In silico analysis of potential ZNF target genes found that the largest group of target genes are involved in cellular metabolic processes, which incidentally are particularly impaired in ccRCC. It was concluded from this study that gene expression of ZNF433 is associated with cancer progression and poorer prognosis, and that ZNF433 behaves in a manner that suggests that it is a prognostic marker and a possible tumor-suppressor gene in clear-cell renal cell carcinoma.

In vitro effects of bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 on the physiology and interaction process of Leishmania amazonensis.

Leishmaniasis is one of the most relevant neglected tropical diseases in the world, affecting 14 million people. Despite the high morbidity, mortality and socio-economic impact, few therapeutic options are available for this disease. To make matters worse, the available molecules have several limitations such as limited efficacy, high cost, side effects and increased resistance. In this context, our group previously synthesized new compounds with anti-leishmania potential being the bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 (complex 4) the most promising one. Therefore, this present work revealed some morphological and physiological changes promoted by complex 4 on Leishmania amazonensis promastigotes as well as it was evidenced its potential against intramacrophage amastigotes. Complex 4 promoted a progressive reduction on the promastigotes size and a remarkable increase on the granularity/complexity as judged by flow cytometry. Transmission electron microscopy (TEM) analysis revealed extensive mitochondrial and plasma membrane alterations, although plasma membrane integrity remained. The mitochondrial changes observed by TEM were accompanied by a decrease in the activity of mitochondrial dehydrogenases with increased production of reactive oxygen species. Interestingly, promastigotes also showed changes in lipid metabolism. Besides the very low toxicity to macrophages, complex 4 had a great effect on intramacrophage amastigotes, displaying an IC50 of 3.91 µM. Collectively, the data presented here reinforce the potential of aminopyridyl compounds complexed to zinc against L. amazonensis. Thus, our work serves as a basis for in vivo assays to be designed or even the synthesis of more selective/effective compounds with lower cost.

Use of chitosan as pharmaceutical excipient in ocular drug delivery systems: Sterilization and pharmacokinetics.

The use of chitosan as a pharmaceutical excipient in the ocular field is already established. Nevertheless, some aspects related to its ocular administration, such as sterilization and excipient's pharmacokinetics, remain unclear. So, in this study, we evaluated those two relevant aspects, related to chitosan administration in eye. We used chitosan-based ocular inserts (CI) as formulation model. CI were produced by solvent/casting method and sterilized by saturated steam. Sterilization was confirmed by direct inoculation of inserts in suitable microbiological growth media. Physicochemical characterization of inserts before and after sterilization was performed. Results suggested that, although steam sterilization changed the arrangement of the matrix, the heat and the humidity did not modify the structure of the main polymeric chain. Pharmacokinetics of CI radiolabeled with technetium-99m (99m Tc) was assessed by scintigraphic images and ex vivo biodistribution study, after ocular administration in male Wistar rats. Scintigraphic and images analysis and ex vivo biodistribution study showed that the insert remained mainly in the eye until 6 hr after administration and its degradation products began to migrate to the abdominal cavity after 18 hr. Together, these data represent an important step forward the manufacturing and the clinical application of CI in the ophthalmic field.

Sheep Rumen Fermentation Characteristics Affected by Feeding Frequency and Feeding Level When Fed Fresh Forage.

Feeding frequency and feeding level are two important factors affecting rumen fermentation characteristics, but few studies on these have been conducted on fresh forage. Eight rumen-fistulated sheep were fed either fresh chicory or perennial ryegrass hourly in the first period (d 14 to 21) of the experiment and twice-daily in the second period (d 22 to 27) at 1.3 or 2.2 times the requirement of metabolizable energy for maintenance. When fed hourly, but not twice-daily, rumen fluid pH value was affected by forage species and feeding level. The total concentrations of short-chain fatty acid (SCFA) were similar at both feeding levels when fed chicory hourly, but they were greater at the higher feeding level in comparison with the lower feeding level when fed perennial ryegrass. However, forage species and feeding level did not affect rumen fluid total SCFA concentration when sheep were fed twice-daily. Therefore, rumen fermentation characteristics were affected by forage species, feeding frequency, feeding level and their interactions and the differences in fermentation characteristics were more apparent when feeding was performed hourly rather than twice-daily. This study highlighted the importance of feeding frequency on manipulating sheep ruminal metabolism when fed fresh forage.

Chitosan/hydroxyethyl cellulose inserts for sustained-release of dorzolamide for glaucoma treatment: In vitro and in vivo evaluation.

Eye drops containing hydrophilic drugs are commonly used to reduce intraocular pressure (IOP) in glaucoma patients, but compliance to the treatement is commonly reduced by frequent dosing and eventual systemic side effects. Sustained-release drug delivery systems, such as ocular inserts, can reduce dosing, limit systemic exposure, reduce side effects, and, then, improve patient adherence to therapy. Here, we developed and evaluated chitosan/hydroxyethyl cellulose-based ocular inserts for sustained release of dorzolamide, a hydrophilic drug. Dorzolamide inserts (DI) were produced by solvent/casting method and characterized by various physicochemical techniques. Pharmacokinetics studies were performed using scintigraphic images and ex vivo biodistribution. The effectiveness of inserts was tested in glaucomatous rats. Characterization studies showed that the drug strongly interacted with the polymeric matrix, but in vitro results showed that DI took only 3 h to release 75% of dorzolamide entraped. However, scintigraphic images and ex vivo biodistribution studies revealed that more than 50% of 99mTc-dorzolamide remained in the eye after 18 h of DI administration, while only about 30% of the drug remained in the eye after drops instilation. DI exerted significant hypotensive effect for two weeks, after single administration, while IOP values remained high in placebo and untreated groups. Eye drops were effective only during the treatment period. Only DI treatment prevented retinal ganglion cells death. Altogether, these findings evidenced the potential application of polymeric-based inserts for sustained release of dorzolamide in glaucoma management.

New mesalamine polymeric conjugate for controlled release: Preparation, characterization and biodistribution study.

Mesalamine (5-ASA) consists of the first-line therapy for the treatment of ulcerative colitis; however, it has low bioavailability, can cause several systemic adverse events, and has low treatment adherence due to the inconvenient dosing scheme. In this work, a new drug delivery system consisting of chondroitin sulfate linked to 5-ASA was synthesized using a carbodiimide as conjugating agent. The system was characterized by spectroscopic techniques (UV, ATR-FTIR, XRD, and NMR 1H) and thermal analysis (TG/DTG and DSC), suggesting the conjugation between the drug and the polymer. The in vitro release and the corresponding kinetics were also evaluated, revealing that approximately 40% of the drug linked was released at pH9 for up to 50h, following Higuchi's model. The conjugate did not show cytotoxicity for the human monocytic cell line at the doses tested, and an in vivo biodistribution study showed that the conjugate remained in the lower GIT for up to 8h with no uptake in the upper GIT. These data corroborate with the radiation found per segment of GIT and in blood. For this last test the conjugate was radiolabeled with Technetium-99m to allow the scintigraphy evaluation and radiation quantification. In conclusion, the polymeric conjugate was successfully synthesized and demonstrated a mucoadhesiveness on the colon as desired, thus supporting its potential use in the treatment of ulcerative colitis.

Mandible protraction alters Type I collagen, osteocalcin and osteonectin gene expression in adult mice condyle.

Mandible condyle remodeling is a great challenge on craniofacial growth studies. The great majority of the reports deals with growing period. However, there is a great necessity to clarify the importance of functional stimulation on adult mandible condyle remodeling. By using an adult mouse model, we investigated the influence of mandible forwarding on condyle remodeling and gene expression by bone forming cells. Tomographic and scintigraphic evaluations showed sagittal growth and cell activity enhancement. RT-PCR showed that Type I collagen, osteocalcin and osteonectin expression level can be altered. We showed that functional stimulation is necessary to maintain the regular gene expression by condyle bone forming cells in adult mice. It opens new frame for further investigations aiming new clinical approaches to temporomandibular joint problems treatment, as well as mandible retrusion treatment.

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles.

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and (1)H-Nuclear Magnetic Resonance Spectroscopy ((1)H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.