Engineering of cell membranes with a bisphosphonate-containing polymer using ATRP synthesis for bone targeting

The field of polymer-based membrane engineering has expanded since we first demonstrated the reaction of N-hydroxysuccinimide ester-terminated polymers with cells and tissues almost two decades ago. One remaining obstacle, especially for conjugation of polymers to cells, has been that exquisite control over polymer structure and functionality has not been used to influence the behavior of cells. Herein, we describe a multifunctional atom transfer radical polymerization initiator and its use to synthesize water-soluble polymers that are modified with bisphosphonate side chains and then covalently bound to the surface of live cells. The polymers contained between 1.7 and 3.1 bisphosphonates per chain and were shown to bind to hydroxyapatite crystals with kinetics similar to free bisphosphonate binding. We engineered the membranes of both HL-60 cells and mesenchymal stem cells in order to impart polymer-guided bone adhesion properties on the cells. Covalent coupling of the polymer to the non-adherent HL-60 cell line or mesenchymal stem cells was non-toxic by proliferation assays and enhanced the binding of these cells to bone.     

Effect of stereochemistry,chain length and sequence pattern on antimicrobial properties of short synthetic β-sheet forming peptide amphiphiles

In the face of mounting global antibiotics resistance, the identification and development of membrane-active antimicrobial peptides (AMPs) as an alternative class of antimicrobial agent have gained significant attention. The physical perturbation and disruption of microbial membranes by the AMPs have been proposed to be an effective means to overcome conventional mechanisms of drug resistance. Recently, we have reported the design of a series of short synthetic β-sheet folding peptide amphiphiles comprised of recurring (X₁Y₁X₂Y₂)_n-NH₂ sequences where X: hydrophobic amino acids, Y: cationic amino acids and n: number of repeat units. In efforts to investigate the effects of key parameters including stereochemistry, chain length and sequence pattern on antimicrobial effects, systematic d-amino acid substitutions of the lead peptides (IRIK)₂-NH₂ (IK8-all L) and (IRVK)₃-NH₂ (IK12-all L) were performed. It was found that the corresponding D-enantiomers exhibited stronger antimicrobial activities with minimal or no change in hemolytic activities, hence translating very high selectivity indices of 407.0 and >>9.8 for IK8-all D and IK12-all D respectively. IK8-all D was also demonstrated to be stable to degradation by broad spectrum proteases trypsin and proteinase K. The membrane disrupting bactericidal properties of IK8-all D effectively prevented drug resistance development and inhibited the growth of various clinically isolated MRSA, VRE, Acinetobacter baumanni, Pseudomonas aeruginosa, Cryptococcus. neoformans and Mycobacterium tuberculosis. Significant reduction in intracellular bacteria counts was also observed following treatment with IK8-all D in the Staphylococcus. aureus infected mouse macrophage cell line RAW264.7 (P < 0.01). These results suggest that the d-amino acids substituted β-sheet forming peptide IK8-all D with its enhanced antimicrobial activities and improved protease stability, is a promising therapeutic candidate with potential to combat antibiotics resistance in various clinical applications.     

Development of a realistic in vivo bone metastasis model of human renal cell carcinoma

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastases. The incidence of RCC is increasing and bone metastatic RCC merits greater focus. Realistic preclinical bone metastasis models of RCC are lacking, hampering the development of effective therapies. We developed a realistic in vivo bone metastasis model of human RCC by implanting precision-cut tissue slices under the renal capsule of immunodeficient mice. The presence of disseminated cells in bone marrow of tissue slice graft (TSG)-bearing mice was screened by human-specific polymerase chain reaction and confirmed by immunohistology using human-specific antibody. Disseminated tumor cells in bone marrow of TSG-bearing mice derived from three of seven RCC patients were detected as early as 1 month after tissue implantation at a high frequency with close resemblance to parent tumors (e.g., CAIX expression and high vascularity). The metastatic patterns of TSGs correlated with disease progression in patients. In addition, TSGs retained capacity to metastasize to bone at high frequency after serial passaging and cryopreservation. Moreover, bone metastases in mice responded to Temsirolimus treatment. Intratibial injections of single cells generated from TSGs showed 100 % engraftment and produced X-ray-visible tumors as early as 3 weeks after cancer cell inoculation. Micro-computed tomography (μCT) and histological analysis revealed osteolytic characteristics of these lesions. Our results demonstrated that orthotopic RCC TSGs have potential to develop bone metastases that respond to standard therapy. This first reported primary RCC bone metastasis model provides a realistic setting to test therapeutics to prevent or treat bone metastases in RCC.     

UPLC-QTOF-MS-guided isolation of anti-COPD ginsenosides from wild ginseng

Four previously undescribed ginsenosides, along with five known analogues were isolated from wild ginseng by a UPLC-QTOF-MS-guided fractionation procedure. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, HR-ESI-MS). The isolated compounds could significantly inhibit the cigarette smoke extract (CSE)-induced inflammatory reaction in A549 cells. The HDAC2 pathway might be involved in the protective effect against the CSE-mediated inflammatory response in A549 cells.

Four previously undescribed ginsenosides, along with five known analogues were isolated from wild ginseng by a UPLC-QTOF-MS-guided fractionation procedure.     

Blood pressure regulation VII. The “morning surge” in blood pressure: measurement issues and clinical significance

In keeping with this review-series theme, we question whether the morning surge in blood pressure (MSBP) is a benign response to the physiological challenges during the first 3 h after waking, or is it clinically important? Therefore, we scrutinise the circadian-related mechanisms, the measurement methods and the prognostic value of the MSBP. The MSBP is relatively small (<2 mmHg) under constant routine conditions. Nevertheless, the blood pressure response to exercise can be 8–14 mm Hg greater in the morning vs. afternoon, even when prior sleep is controlled. Systematic bias between MSBP methods can be >10 mmHg. The “sleep-trough” method provides the largest MSBP (≈25 mmHg), but the sensitivity of MSBP to a treatment/intervention depends largely on its repeatability. The repeatability standard deviation (SD) for most MSBP methods is ≈8 mm Hg. While the magnitude of this SD precludes the use of MSBP for diagnostic decisions on individual patients, sample sizes for future intervention studies may be feasible, depending on the minimal clinically important difference in MSBP. This difference is somewhat unclear given that a large MSBP has recently been reported to predict a reduced, rather than a higher, risk of cardiovascular disease, although this particular study has been criticised. The MSBP is also naturally correlated to changes in physical activity and nocturnal “dipping” status. Therefore, it is important to account for these potential confounders of the MSBP, so that more precise knowledge about its clinical significance is gained, thereby providing a sound rationale for physiological investigation and translational research.     

High-intensity intermittent cycling increases purine loss compared with workload-matched continuous moderate intensity cycling

Purpose

Exercise at 50–60 % of peak oxygen consumption (VO_{2 peak}) stimulates maximal fat oxidation rates. Despite a lower estimated work performed; high-intensity intermittent exercise (HIIE) training produces greater fat mass reductions when compared with workload-matched continuous (CON) steady state exercise. No metabolic basis has been documented nor mechanisms offered to explain this anomaly. This study investigated the physiological and metabolic responses of two different workload-matched exercise protocols.

Methods

On separate occasions and at least 1 week apart, eight apparently healthy males cycled for 30 min at either 50 % VO_{2 peak} (CON) or performed repeated 20 s bouts of supramaximal exercise at 150 %VO_{2 peak} separated by 40 s rest (HIIE).

Results

The average heart rate, oxygen consumption, plasma glycerol and free fatty acid concentrations were not different during exercise and recovery between the trials. Plasma lactate and hypoxanthine (Hx) concentrations were elevated and urinary excretion rates of Hx and uric acid were greater following HIIE as compared to CON (P < 0.05).

Conclusion

Exercise-induced plasma Hx accumulation and urinary purine excretion are greater following HIIE and indirectly represents a net loss of adenosine triphosphate (ATP) from the muscle. The subsequent restorative processes required for intramuscular de novo replacement of ATP may contribute to a negative energy balance and in part, account for the potential accelerated fat loss observed with HIIE when compared with CON training programs.     

Serum Bactericidal Assays To Evaluate Typhoidal and Nontyphoidal Salmonella Vaccines

Invasive Salmonella infections for which improved or new vaccines are being developed include enteric fever caused by Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B and sepsis and meningitis in young children in sub-Saharan Africa caused by nontyphoidal Salmonella (NTS) serovars, particularly S. enterica serovars Typhimurium and Enteritidis. Assays are needed to measure functional antibodies elicited by the new vaccines to assess their immunogenicities and potential protective capacities. We developed in vitro assays to quantify serum bactericidal antibody (SBA) activity induced by S. Typhi, S. Paratyphi A, S. Typhimurium, and S. Enteritidis vaccines in preclinical studies. Complement from various sources was tested in assays designed to measure antibody-dependent complement-mediated killing. Serum from rabbits 3 to 4 weeks of age provided the best complement source compared to serum from pigs, goats, horses, bovine calves, or rabbits 8 to 12 weeks of age. For S. Enteritidis, S. Typhimurium, and S. Typhi SBA assays to be effective, bacteria had to be harvested at log phase. In contrast, S. Paratyphi A was equally susceptible to killing whether it was grown to the stationary or log phase. The typhoidal serovars were more susceptible to complement-mediated killing than were the nontyphoidal serovars. Lastly, the SBA endpoint titers correlated with serum IgG anti-lipopolysaccharide (LPS) titers in mice immunized with mucosally administered S. Typhimurium, S. Enteritidis, and S. Paratyphi A but not S. Typhi live attenuated vaccines. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccine candidates.     

Diversity of Antibody Responses to Borrelia burgdorferi in Experimentally Infected Beagle Dogs

Lyme borreliosis (LB) is a common infection of domestic dogs in areas where there is enzootic transmission of the agent Borrelia burgdorferi. While immunoassays based on individual subunits have mostly supplanted the use of whole-cell preparations for canine serology, only a limited number of informative antigens have been identified. To more broadly characterize the antibody responses to B. burgdorferi infection and to assess the diversity of those responses in individual dogs, we examined sera from 32 adult colony-bred beagle dogs that had been experimentally infected with B. burgdorferi through tick bites and compared those sera in a protein microarray with sera from uninfected dogs in their antibody reactivities to various recombinant chromosome- and plasmid-encoded B. burgdorferi proteins, including 24 serotype-defining OspC proteins of North America. The profiles of immunogenic proteins for the dogs were largely similar to those for humans and natural-reservoir rodents; these proteins included the decorin-binding protein DbpB, BBA36, BBA57, BBA64, the fibronectin-binding protein BBK32, VlsE, FlaB and other flagellar structural proteins, Erp proteins, Bdr proteins, and all of the OspC proteins. In addition, the canine sera bound to the presumptive lipoproteins BBB14 and BB0844, which infrequently elicited antibodies in humans or rodents. Although the beagle, like most other domestic dog breeds, has a small effective population size and features extensive linkage disequilibrium, the group of animals studied here demonstrated diversity in antibody responses in measures of antibody levels and specificities for conserved proteins, such as DbpB, and polymorphic proteins, such as OspC.