α-Mangostin-encapsulated PLGA nanoparticles inhibit colorectal cancer growth by inhibiting Notch pathway.

Colorectal cancer (CRC) is the fourth leading cause of cancer-related mortality. Recent studies have stated that Notch signalling is highly activated in cancer stem cells (CSCs) and plays an important role in the development and progression of CRC. Like normal colorectal epithelium, CRCs are organized hierarchically and include populations of CSCs. In order to enhance the biological activity of α-mangostin, we formulated α-mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which Mang-NPs inhibit CRC cell viability, colony formation, epithelial-mesenchymal transition (EMT) and induce apoptosis. Mang-NPs inhibited cell viability, colony formation and induced apoptosis. Mang-NPs also inhibited EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. As dysregulated signalling through the Notch receptors promotes oncogenesis, we measured the effects of Mang-NPs on Notch pathway. Mang-NPs inhibited Notch signalling by suppressing the expression of Notch receptors (Notch1 and Notch2), their ligands (Jagged 1 and DLL4), γ-secretase complex protein (Nicastrin) and downstream target (Hes-1). Notch receptor signalling regulates cell fate determination in stem cell population. Finally, Mang-NPs inhibited the self-renewal capacity of CSCs, stem cell markers (CD133, CD44, Musashi and LGR5) and pluripotency maintaining factors (Oct4, Sox-2, KLF-4, c-Myc and Nanog). Overall, our data suggest that Mang-NPs can inhibit CRC growth, EMT and CSCs' population by suppressing Notch pathway and its target. Therefore, Mang-NPs can be used for the treatment and prevention of CRC.

Role of Janus Kinase 3 in Predisposition to Obesity-associated Metabolic Syndrome.

Obesity, a worldwide epidemic, is a major risk factor for the development of metabolic syndrome (MetS) including diabetes and associated health complications. Recent studies indicate that chronic low-grade inflammation (CLGI) plays a key role in metabolic deterioration in the obese population. Previously, we reported that Jak3 was essential for mucosal differentiation and enhanced colonic barrier functions and its loss in mice resulted in basal CLGI and predisposition to DSS induced colitis. Since CLGI is associated with diabetes, obesity, and metabolic syndrome, present studies determined the role of Jak3 in development of such conditions. Our data show that loss of Jak3 resulted in increased body weight, basal systemic CLGI, compromised glycemic homeostasis, hyperinsulinemia, and early symptoms of liver steatosis. Lack of Jak3 also resulted in exaggerated symptoms of metabolic syndrome by western high-fat diet. Mechanistically, Jak3 was essential for reduced expression and activation of Toll-like receptors (TLRs) in murine intestinal mucosa and human intestinal epithelial cells where Jak3 interacted with and activated p85, the regulatory subunit of the PI3K, through tyrosine phosphorylation of adapter protein insulin receptor substrate (IRS1). These interactions resulted in activation of PI3K-Akt axis, which was essential for reduced TLR expression and TLR associated NFκB activation. Collectively, these results demonstrate the essential role of Jak3 in promoting mucosal tolerance through suppressed expression and limiting activation of TLRs thereby preventing intestinal and systemic CLGI and associated obesity and MetS.

Role of Janus kinase 3 in mucosal differentiation and predisposition to colitis.

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein ß-catenin. Jak3 expression in these cells was essential for AJ localization of ß-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of ß-catenin.

Developments in purification methods for obtaining and evaluation of collagen derived endogenous angioinhibitors.

Collagen constitutes one of the vital components of the basement membrane scaffolds. Non-collagenous domains (NC1) derived from collagens exhibit potent anti-angiogenic properties, thus attaining significance in regulation of angiogenesis promoted diseases. Individual NC1 domains essential for anti-angiogenic evaluations are generally obtained through purification of individual non-collagenous domains, which have undergone steady developments for enhancing the yields, purpose of biological evaluations and solubility based on the nature of different NC1 domains. This review focuses on the method developments in obtaining biologically active NC1 domains and for specific evaluations in different scenarios.

Role of Porphyromonas gingivalis phosphoserine phosphatase enzyme SerB in inflammation, immune response, and induction of alveolar bone resorption in rats.

Porphyromonas gingivalis secretes a serine phosphatase enzyme, SerB, upon contact with gingival epithelial cells in vitro. The SerB protein plays a critical role in internalization and survival of the organism in epithelial cells. SerB is also responsible for the inhibition of interleukin-8 (IL-8) secretion from gingival epithelial cells infected with P. gingivalis. This study examined the ability of a P. gingivalis SerB mutant to colonize the oral cavity and induce gingival inflammation, immune responses, and alveolar bone resorption in a rat model of periodontal disease. Both P. gingivalis ATCC 33277 and an isogenic ΔSerB mutant colonized the oral cavities of rats during the 12-week experimental period. Both of the strains induced significant (P < 0.05) systemic levels of immunoglobulin G (IgG) and isotypes IgG1, IgG2a, and IgG2b, indicating the involvement of both T helper type 1 (Th1) and Th2 responses to infection. Both strains induced significantly (P < 0.05) higher levels of alveolar bone resorption in infected rats than in sham-infected control rats. However, horizontal and interproximal alveolar bone resorption induced by the SerB mutant was significantly (P < 0.05) lower than that induced by the parental strain. Rats infected with the ΔSerB mutant exhibited significantly higher levels of apical migration of the junctional epithelium (P < 0.01) and polymorphonuclear neutrophil (PMN) recruitment (P < 0.001) into the gingival tissues than rats infected with the wild type. In conclusion, in a rat model of periodontal disease, the SerB phosphatase of P. gingivalis is required for maximal alveolar bone resorption, and in the absence of SerB, more PMNs are recruited into the gingival tissues.

Transition of a solitary to a biofilm community life style in bacteria: a survival strategy with division of labour.

Multicellularity is associated with higher eukaryotes having an organized division of labour and a coordinated action of different organs composed of multiple cell types. This division of different cell types and organizations to form a multicellular structure by developmental programming is a key to the multitasking of complex traits that enable higher eukaryotes to cope with fluctuating environmental conditions. Microbes such as bacteria, on the other hand, are unicellular and have flourished in diverse environmental conditions for a much longer time than eukaryotes in evolutionary history. In this review, we will focus on different strategies and functions exhibited by microbes that enable them to adapt to changes in lifestyle associated with transitioning from a unicellular solitary state to a complex community architecture known as a biofilm. We will also discuss various environmental stimuli and signaling processes which bacteria utilize to coordinate their social traits and enable themselves to form complex multicellular-like biofilm structures, and the division of labour operative within such communities driving their diverse social traits. We will also discuss here recent studies from our laboratory using a plant-associated bacterial pathogen as a model organism to elucidate the mechanism of bacterial cell-cell communication and the transition of a bacterial community to a multicellular-like structure driven by the complex regulation of traits influenced by cell density, as well as environmental sensing such as chemotaxis and nutrient availability. These studies are shedding important insights into bacterial developmental transitions and will help us to understand community cooperation and conflict using bacterial cell-cell communication as a model system.

Assessment of orthodontic treatment outcome using Peer Assessment Rating (PAR) index among patients with non-syndromic unilateral cleft lip and palate.

OBJECTIVE: To evaluate the orthodontic treatment outcome among patients with non-syndromic unilateral cleft lip and palate using the Peer Assessment Rating (PAR) index. MATERIALS AND METHODS: The retrospective study comprised a sample of 80 patients with unilateral cleft lip and palate (39 males and 41 females) with complete pretreatment and posttreatment orthodontic records. The patients were divided into two groups according to the treatment modalities. Group 1 (n = 55), nonsurgical (consisted of patients treated with comprehensive orthodontics) and Group 2 (n = 25), surgical (with presurgical orthodontics followed by maxillary advancement orthognathic surgery). PAR score was evaluated on pretreatment and posttreatment study models for both groups. RESULTS: The mean percentage change for the weighted PAR score of Group 1 and Group 2 was 76.79 ± 20.27% and 82.37 ± 11.38%, respectively. Out of the total sample of 80 cases; 62 (77.5%) cases were "greatly improved," 16 (20%) cases were "improved," and 2 (2.5%) showed "worse/no improvement." Nearly 72.5% of cases in Group 1 and 88% in Group 2 were greatly improved. CONCLUSIONS: The reduction in PAR score in both groups was satisfactory as more than 70% of the patients were in the greatly improved category. The results of the PAR index revealed a high occlusal outcome of orthodontic treatment rendered by the department for patients with unilateral cleft lip and palate.

Myxomavirus anti-inflammatory chemokine binding protein reduces the increased plaque growth induced by chronic Porphyromonas gingivalis oral infection after balloon angioplasty aortic injury in mice.

Thrombotic occlusion of inflammatory plaque in coronary arteries causes myocardial infarction. Treatment with emergent balloon angioplasty (BA) and stent implant improves survival, but restenosis (regrowth) can occur. Periodontal bacteremia is closely associated with inflammation and native arterial atherosclerosis, with potential to increase restenosis. Two virus-derived anti-inflammatory proteins, M-T7 and Serp-1, reduce inflammation and plaque growth after BA and transplant in animal models through separate pathways. M-T7 is a broad spectrum C, CC and CXC chemokine-binding protein. Serp-1 is a serine protease inhibitor (serpin) inhibiting thrombotic and thrombolytic pathways. Serp-1 also reduces arterial inflammation and improves survival in a mouse herpes virus (MHV68) model of lethal vasculitis. In addition, Serp-1 demonstrated safety and efficacy in patients with unstable coronary disease and stent implant, reducing markers of myocardial damage. We investigate here the effects of Porphyromonas gingivalis, a periodontal pathogen, on restenosis after BA and the effects of blocking chemokine and protease pathways with M-T7 and Serp-1. ApoE-/- mice had aortic BA and oral P. gingivalis infection. Arterial plaque growth was examined at 24 weeks with and without anti-inflammatory protein treatment. Dental plaques from mice infected with P. gingivalis tested positive for infection. Neither Serp-1 nor M-T7 treatment reduced infection, but IgG antibody levels in mice treated with Serp-1 and M-T7 were reduced. P. gingivalis significantly increased monocyte invasion and arterial plaque growth after BA (P<0.025). Monocyte invasion and plaque growth were blocked by M-T7 treatment (P<0.023), whereas Serp-1 produced only a trend toward reductions. Both proteins modified expression of TLR4 and MyD88. In conclusion, aortic plaque growth in ApoE-/- mice increased after angioplasty in mice with chronic oral P. gingivalis infection. Blockade of chemokines, but not serine proteases significantly reduced arterial plaque growth, suggesting a central role for chemokine-mediated inflammation after BA in P. gingivalis infected mice.

Whole-lung lavage for pediatric patients with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a chronic disorder of surfactant clearance from the alveoli. Its prevalence is rare, especially in the pediatric population. Although there is no cure for this condition, symptoms of PAP are managed most effectively through whole-lung lavage (WLL). Perioperative RNs caring for children with PAP undergoing WLL in the OR should implement patient interventions to maintain vital signs and normothermia and preserve skin integrity. Additionally, perioperative RNs often are responsible for assembling closed-drainage systems for WLL. Detailed procedural preference cards, targeted education sessions, and multidisciplinary collaboration are crucial for establishing a comprehensive plan of care for the pediatric patient with PAP undergoing WLL in the OR.

Extra cellular matrix derived metabolite regulates angiogenesis by FasL mediated apoptosis.

OBJECT: Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of α1(IV)NC1 and its N- and C- peptides α1S1(IV)NC1, α1S2(IV)NC1 were investigated in-vitro and in-vivo. STUDY METHOD: Endothelial cells (ECs) were treated with α1(IV)NC1, α1S1(IV)NC1, α1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using α1(IV)NC1 in a mice model. RESULTS: Like α1(IV)NC1, its N- and C- terminal α1S2(IV)NC1 and α1S1(IV)NC1 domains posses anti-proliferative, pro-apoptotic activity and inhibit ECs migration and tube formation in-vitro. Both α1S1(IV)NC1 and α1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon α1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of α1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of α1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3. SIGNIFICANCE: α1(IV)NC1 and its N- and C- terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. α1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that α1(IV)NC1 and its peptides inhibit neo-vascular diseases.

Porphyromonas gingivalis and Treponema denticola Mixed Microbial Infection in a Rat Model of Periodontal Disease.

Porphyromonas gingivalis and Treponema denticola are periodontal pathogens that express virulence factors associated with the pathogenesis of periodontitis. In this paper we tested the hypothesis that P. gingivalis and T. denticola are synergistic in terms of virulence; using a model of mixed microbial infection in rats. Groups of rats were orally infected with either P. gingivalis or T. denticola or mixed microbial infections for 7 and 12 weeks. P. gingivalis genomic DNA was detected more frequently by PCR than T. denticola. Both bacteria induced significantly high IgG, IgG2b, IgG1, IgG2a antibody levels indicating a stimulation of Th1 and Th2 immune response. Radiographic and morphometric measurements demonstrated that rats infected with the mixed infection exhibited significantly more alveolar bone loss than shaminfected control rats. Histology revealed apical migration of junctional epithelium, rete ridge elongation, and crestal alveolar bone resorption; resembling periodontal disease lesion. These results showed that P. gingivalis and T. denticola exhibit no synergistic virulence in a rat model of periodontal disease.

Antigen-specific clonal expansion and cytolytic effector function of CD8+ T lymphocytes depend on the transcription factor Bcl11b.

CD8(+) T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8(+) T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8(+) T cells, CD8 coreceptor and Plcgamma1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8(+) T cells through association with specific enhancers. Short-lived Klrg1(hi)CD127(lo) effector CD8(+) T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.