Elastic modulus and hardness of cortical and trabecular bovine bone measured by nanoindentation

The elastic modulus and hardness of several microstructure components of dry bovine vertebrae and tibia have been investigated in the longitude and transverse directions using nanoindentation. The elastic modulus for the osteons and the interstitial lamellae in the longitude direction were found to be (24.7±2.5) GPa and (30.1±2.4) GPa. As it's difficult to distinguish osteons from interstitial lamellae in the transverse direction, the average elastic modulus for cortical bovine bone in the transverse direction was (19.8±1.6) GPa. The elastic modulus for trabecular bone in the longitude and transverse direction were (20±2) GPa and (14.7±1.9) GPa respectively. The hardness also varied among the microstructure components in the range of 0.41–0.89 GPa. Analyses of variance show that the values are significantly different.

Nano- and macro-scale characterisation of the mechanical properties of bovine bone

In the present study, nano- and macro-scale characterisations on the mechanical properties of bovine cortical bones have been performed by using nanoindentation and conventional compressive tests. Nanoindentation results showed that the elastic modulus for the osteons and the interstitial lamellae in the longitude direction were 24.7 ± 2.5 GPa and 30.1 ± 2.4 GPa. As it’s difficult to distinguish osteons from interstitial lamellae in the transverse direction, the average elastic modulus for cortical bovine bone in the transverse direction was 19.8 ± 1.6 GPa. Significant differences were found in the modulus values between different microstructures of bone tissue and in different testing direction. It was found that the elastic modulus of bone bovine material in nano-level was higher than that in macro-level. The elastic modulus and
ultimate stress of large bone samples were 12.5 ± 1.9 GPa and 195 ± 19 MPa respectively from the compression test.

Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria

The bovine Muc1 protein is synthesized by mammary epithelial cells and shed into milk as an integral component of the milk fat globule membrane; however, the structure and functions of this mucin, particularly in relation to lactation, are poorly defined. The objectives of this investigation were to investigate the Muc1 gene and protein structures in the context of lactation and to test the hypothesis that Muc1 has a role in innate immune defense. Polymerase chain reaction analysis of genomic DNA from 630 cattle revealed extensive polymorphism in the variable number of tandem repeats (VNTR) in the bovine Muc1 gene. Nine allelic
variants spanning 7 to 23 VNTR units, each encoding 20 AA, were identified. Three alleles, containing 11, 14, and 16 VNTR units, respectively, were predominant. In addition, a polymorphism in one of the VNTR units has the potential to introduce a unique site for N-linked glycosylation. Statistical analysis indicated weak associations between the VNTR alleles and milk protein and fat percentages in a progeny-tested population of Holstein-Friesian dairy cattle. No association with somatic cell count could be demonstrated. Bovine Muc1 was purified from milk fat globule membranes and characterized. The protein was highly glycosylated, primarily with O-linked sialylated T-antigen [Neu5Ac(α2–3)-Gal(β1–3)-GalNAcα1] and, to a lesser extent, with N-linked oligosaccharides, which together accounted for approximately 60% of the apparent mass of Muc1. Purified bovine Muc1 directly bound fluorescently labeled Escherichia coli BioParticles (Invitrogen, Mount Waverley, Australia) and inhibited their binding to bovine mammary epithelial cells grown in vitro.

Novel alginate-enclosed chitosan–calcium phosphate-loaded iron-saturated bovine lactoferrin nanocarriers for oral delivery in colon cancer therapy

Aim: To develop polymeric-ceramic nanocarriers (NCs) in order to achieve oral delivery of the anticancer neutraceutical iron-saturated bovine lactoferrin (Fe-bLf) protein.

Materials & methods: Fe-bLf or paclitaxel (Taxol®) were adsorbed onto calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate. The Fe-bLf or Taxol-loaded NCs indicated as AEC–CP–Fe-bLf or AEC–CP–Taxol NCs, respectively, were made by combination of ionic gelation and nanoprecipitation. Size distribution, morphology, internalization and release profiles of the NCs were studied along with evaluation of in vitro and in vivo anticancer activities and compared with paclitaxel.

Results: AEC–CP–Fe-bLf NCs obtained spherical morphology and showed enhanced endocytosis, transcytosis and anticancer activity in Caco-2 cells in vitro. AEC–CP–Fe-bLf NCs were supplemented in an AIN 93G diet and fed to mice in both prevention and treatment human xenograft colon cancer models. AEC–CP–Fe-bLf NCs were found to be highly significantly effective when given orally, as a pretreatment, 1 week before Caco-2 cell injections. None of the mice from the AEC–CP–Fe-bLf NC-fed group developed tumors or showed any signs of toxicity, while the mice fed the control AIN 93G diet showed normal tumor growth. Fe-bLf or Taxol, when given orally in a diet as nanoformulations post-tumor development, showed a significant regression in the tumor size with complete inhibition of tumor growth later, while intratumoral injection of Taxol just delayed the growth of tumors. The pharmacokinetic and bioavailability studies indicated that nanoformulated Fe-bLf was predominantly present on tumor cells compared to non-nanoformulated Fe-bLf. Fe-bLf-loaded NCs were found to help in absorption of iron and thus may have utility in enhancing the iron uptake during iron deficiency without interfering with the absorption of calcium.

Conclusion: With the promising results of our study, the future potential of NC-loaded Fe-bLf in chemoprevention and in the treatment of human colon cancer, deserves further investigation for translational research and preclinical studies of other malignancies.

The effect of oral administration of iron saturated-bovine lactoferrin encapsulated chitosan-nanocarriers on osteoarthritis

In this study, the therapeutic potentials of 100% iron saturated-bovine lactoferrin encapsulated in alginate-chitosan polymeric nanocarriers (AEC-CP-Fe-bLf-NCs) were examined in in vitro inflammatory OA model and in collagen-induced arthritis (CIA) mice. Oral administration of nanocarriers in mice were non-toxic and significantly induced disease modifying activity by reducing joint inflammation and downregulating the expression of catabolic genes, IL-1β, NO, JNK and MAPK. In addition, up-regulation of type II collagen, aggrecan and inflammation depleted iron and calcium metabolisms via inhibition of miRNA of iron transporting receptors was shown in AEC-CP-Fe-bLf-NCs treated mice.

Drying and denaturation characteristics of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in a convective drying process

Drying and denaturation kinetics of aqueous droplets of α-lactalbumin (α-lac), β-lactoglobulin (β-lg), and bovine serum albumin (BSA) were measured in a convective drying environment. Single droplets having an initial droplet diameter of 2 ± 0.1 mm and containing 10% (w/v) protein concentration were dried using conditioned air (65 and 80 °C, 2-3% RH, 0.5 m/s velocity) for 600 s. The denaturation of these proteins was measured by using reversed-phase HPLC. At the end of 600 s of drying 13.3 and 19.4% α-lac was found to be lost due to denaturation at 65 and 80 °C, respectively. Up to 31.0% of β-lg was found to be denatured, whereas BSA was not found to be significantly (p > 0.05) denatured in these drying conditions. The formation and strength of skin and the associated morphological features were found to be linked with the degree of denaturation of these proteins. The secondary structure of these proteins was significantly (p < 0.05) affected and altered by the drying stresses. The β-sheet and random coil contents were increased in α-lac by 6.5 and 4.0%, respectively, whereas the α-helix and β-turn contents decreased by 5.5 and 5.0%, respectively. The β-sheet and random coil contents in β-lg were increased by 7.5 and 2.0%, respectively, whereas the α-helix and β-turn contents decreased by 3.5 and 6.0%, respectively. In the case of BSA the β-sheet, α-helix, and random coil contents were found to increase, whereas the β-turn content decreased.

The microstructure and physicochemical properties of probiotic buffalo yoghurt during fermentation and storage: a comparison with bovine yoghurt

The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s-1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products. © 2013 Springer Science+Business Media New York.

Oral administration of iron-saturated bovine lactoferrin-loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

We determined the anticancer efficacy and internalization mechanism of our polymeric-ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC50 values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Short communication: bovine-derived proteins activate STAT3 in human skeletal muscle in vitro

Bovine milk contains biologically active peptides that may modulate growth and development within humans. In this study, targeted bovine-derived proteins were evaluated for their effects on signal transducer and activator of transcription-3 (STAT3) phosphorylation in human skeletal muscle cells. Following an acute exposure, bovine-derived acidic fibroblast growth factor-1 (FGF) and leukemia inhibitory factor (LIF) activated STAT3 in differentiating myotubes. Chronic exposure to FGF and LIF during the proliferative phase reduced myoblast proliferation and elevated MyoD and creatine kinase (CKM) mRNA expression without altering apoptotic genes. In mature myotubes, neither FGF nor LIF elicited any action. Together, these data indicate that a reduction in proliferation in the presence of bovine-derived FGF or LIF may stimulate early maturation of myoblasts.

Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells

This study used the Eri silk nanoparticles (NPs) for delivering apo-bovine lactoferrin (Apo-bLf) (~2% iron saturated) and Fe-bLf (100% iron saturated) in MDA-MB-231 and MCF-7 breast cancer cell lines. Apo-bLf and Fe-bLf-loaded Eri silk NPs with sizes between 200 and 300 nm (±10 nm) showed a significant internalization within 4 hours in MDA-MB-231 cells when compared to MCF-7 cells. The ex vivo loop assay with chitosan-coated Fe-bLf-loaded silk NPs was able to substantiate its future use in oral administration and showed the maximum absorption within 24 hours by ileum. Both Apo-bLf and Fe-bLf induced increase in expression of low-density lipoprotein receptor-related protein 1 and lactoferrin receptor in epidermal growth factor (EGFR)-positive MDA-MB-231 cells, while transferrin receptor (TfR) and TfR2 in MCF-7 cells facilitated the receptor-mediated endocytosis of NPs. Controlled and sustained release of both bLf from silk NPs was shown to induce more cancer-specific cytotoxicity in MDA-MB-231 and MCF-7 cells compared to normal MCF-10A cells. Due to higher degree of internalization, the extent of cytotoxicity and apoptosis was significantly higher in MDA-MB-231 (EGFR+) cells when compared to MCF-7 (EGFR-) cells. The expression of a prominent anticancer target, survivin, was found to be downregulated at both gene and protein levels. Taken together, all the observations suggest the potential use of Eri silk NPs as a delivery vehicle for an anti-cancer milk protein, and indicate bLf for the treatment of breast cancer.

4-Hydroxy-N-propyl-1,8-naphthalimide esters: new fluorescence-based assay for analysing lipase and esterase activity

Research using 1,8-naphthalimide derivatives has expanded rapidly in recent years owing to their cell-permeable nature, ability to target certain cellular locations and fluorescent properties. Here we describe the synthesis of three new esters of 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) and the development of a simple and sensitive assay protocol to measure the activity of carboxylester hydrolases. The NAP fluorophore was esterified with short (butyrate), medium (octanoate) and long (palmitate) chain fatty acids. The esters were spectroscopically characterised and their properties investigated for their suitability as assay substrates. The esters were found to be relatively stable under the conditions of the assay and levels of spontaneous hydrolysis were negligible. Non-specific hydrolysis by proteins such as bovine serum albumin was also minimal. A simple and rapid assay methodology was developed and used to analyse a range of commercially available enzymes that included enzymes defined as lipases, esterases and phospholipases. Clear differences were observed between the enzyme classes with respect to the hydrolysis of the various chain length esters, with lipases preferentially hydrolysing the medium chain ester, whereas esterases reacted more favourably with the short ester. The assay was found to be highly sensitive with the fluorophore detectable to the low nM range. These esters provide alternate substrates from established coumarin-based fluorophores, possessing distinctly different excitation (447 nm) and emission (555 nm) optima. Absorbing at 440-450 nm also offers the flexibility of analysis by UV-visible spectrophotometry. This represents the first instance of a naphthalimide-derived compound being used to analyse these enzymes.