Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter

Background

It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O₂ and energetic metabolism, and its impact over cardiac contractility.

Methods

Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O₂ consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.

Results

Three hours after ROFA exposure, tissue O₂ consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O₂ consumption rates positively correlated with cardiac contractile state in controls (r² = 0.8271), but not in treated mice (r² = 0.1396).

General Significance

The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.     

Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1 M dimethyl sulfoxide (Me₂SO), 1 M ethylene glycol (EG), 1 M methanol (MeOH) and 0.1 M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and −2 °C for 5 min, 1 h and 3 h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and −2 °C at 1 h and 3 h exposure in each treatment. Heartbeat stage was more tolerant against chilling at −2 °C for 3 h exposure in Me₂SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

The association between Interleukin (IL)-4 gene intron 3 VNTR polymorphism and alopecia areata (AA) in Turkish population

Objective

Alopecia areata (AA) is hypothesized to be an organ-specific autoimmune disease of hair follicles mediated by T cells. As immunological and genetic factors have been implicated in the pathogenesis of AA, the purpose of the present study was to investigate possible associations between the functional Interleukin (IL)-4 gene intron 3 VNTR polymorphism and AA susceptibility and disease progression in Turkish population.

Methods

The study group consisted of 116 unrelated patients with AA and 125 unrelated healthy controls. Genomic DNA was isolated and IL-4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers.

Results

No association was observed between AA patients and controls according to genotype distribution (p = 0.051). The allele distribution of IL-4 gene intron 3 VNTR polymorphism was statistically different between AA patients and control group (p = 0.026). The frequency of P1 allele in patients was significantly higher than that in the control group. When the P2P2 genotype was compared with P1P2 + P1P1 genotypes, a statistically significant difference was observed between patients and controls (p = 0.036). Intron 3 VNTR polymorphism in the IL-4 gene was found to be associated with AA susceptibility in Turkish population.

Conclusion

The results suggest that IL-4 VNTR polymorphism in the intron 3 region may be a risk factor for the development of AA among Turkish population. This is the first to report that intron 3 VNTR polymorphism in the IL-4 gene is associated with AA susceptibility.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Association between fibromyalgia syndrome and polymorphism of the IL-4 gene in a Turkish population

Purpose

Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points. The etiology of FM is still obscure. Genetic factors may predispose individuals to FM. Cytokines may play a role in the pathophysiology of FM. The aim of this study was to investigate the interleukin-4 (IL-4) 70 bp VNTR variations in Turkish patients with FM and evaluate if there was an association with clinical features, especially between these polymorphisms.

Methods

The study included 300 patients with FM and 270 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) for the IL-4 gene 70 bp VNTR polymorphisms.

Results

There was statistically significant difference between the groups with respect to IL-4 genotype distribution and allele frequencies (p < 0.0001). The homozygous P₁P₁ genotype and P₁ allele were significantly higher in FM patients than in healthy controls (p = 0.04; OR: 3.25, 95% CI: 1–10, p < 0.0001; OR:4.84, 95% CI:3–7.7). There was not any difference between the groups respect to IL-4 genotype distribution and allele frequencies (p > 0.05) and clinical characteristics.

Conclusion

Our findings suggest that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person for development of FM. As a result, further studies are necessary to determine whether IL-4 may be a genetic marker for FM in the Turkish population.     

Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years

Objective

To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs).

Methods

Samples of human BMCs that were cryopreserved for 23–25 years (n = 20) were thawed to obtain an initial culture and a primary culture (P₀) that was propagated through five passages (P₁–P₅) to obtain MSCs. Freshly collected human bone marrow samples (n = 20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P₃ cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing.

Results

In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p < 0.05) while the relative rate of recovery of MSCs was only 48.5 ± 8.6% in P₀. At the end of P₃, fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p > 0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P₃ from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells.

Conclusion

Using the methods described here, long-term (23–25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Higher frequency of septic shock in septic patients with the 47C allele (rs4880) of the SOD2 gene

Aim

To analyze the effect of the two different versions of the manganese superoxide dismutase gene (SOD2) on sepsis. The SOD2 gene presents the 47C > T single nucleotide polymorphism (SNP; ID: rs4880) which produces MnSOD with different activities. The − 9Val MnSOD (47T allele) is less efficient than the − 9Ala version (47C allele). During sepsis there are abundance of ROS, high SOD2 expression and excess of H₂O₂ synthesis. High concentrations of H₂O₂ could affect the sepsis scenario and/or the sepsis outcome.

Methods

We determined the 47C > T single nucleotide polymorphism (SNP) frequencies in 529 critically ill patients with or without sepsis, facing outcome. To collect information on population frequencies, we obtained a pilot 47C > T genotypic and allelic frequencies in a random group of 139 healthy subjects.

Results

We compared the 47C allele carriers (47CC + 47CT genotypes) with 47TT homozygotes and noticed a significant association between 47C allele carriers and septic shock in septic patients (P = 0.025). With an adjusted binary multivariate logistic regression, incorporating 47C > T SNP and the main clinical predictors, we showed high SOFA scores [P < 0.001, OR = 9.107 (95% CI = 5.319–15.592)] and 47C allele [P = 0.011, OR = 2.125 (95% CI = 1.190–3.794)] were significantly associated with septic shock outcome. With this information we presented a hypothesis suggesting that this negative outcome from sepsis is possibly explained by effects on cellular stress caused by 47C allele.

Conclusion

In our population there was a significant higher frequency of septic shock in septic patients with the 47C allele of the SOD2 gene. This higher 47C allele frequency in septic patients with negative outcome could be explained by effects of higher activity MnSOD on cellular stress during the sepsis.     

The Salmonella enterica ZinT structure,zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Background

In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT–ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.

Methods

ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).

Results

The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K_D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K_D < 20 nM) and one low affinity Zn(II) binding site (K_D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT–ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.

Conclusions

ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.

General significance

Knowledge of the ZinT–ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.     

Tolerance of apexes of coral Pocillopora damicornis L. to cryoprotectant solutions

In this study, we investigated the tolerance of Pocillopora damicornis apexes to treatments with solutions containing penetrating and non-penetrating cryoprotective agents (CPAs). CPAs were employed individually or in binary, tertiary or quaternary solutions. In some experiments apexes were treated successively with two CPA solutions with increasing total concentration. P. damicornis apexes withstood exposure for up to 30 min to solutions containing 0.6–0.8 M sucrose (Suc) or trehalose (Tre). When apexes were treated with binary cryoprotectant solutions containing Suc and ethylene glycol (EG), methanol (Meth), dimethyl sulfoxide (Me₂SO) or glycerol (Gly), the CPAs employed in combination with Suc could be ranked in the following order of decreasing tolerance: EG > Meth > Me₂SO > Gly. P. damicornis apexes tolerated exposure to complex CPA solutions containing Suc, Me₂SO, EG and/or Meth with a total molarity of 2.45 M. In experiments where two successive CPA solutions were employed, apexes withstood treatment with the second, more concentrated solution at 0 °C for up to 10 min. These preliminary results pave the way to the development of a cryopreservation protocol for P. damicornis apexes.     

Tetracycline nanoparticles loaded calcium sulfate composite beads for periodontal management

Background

The objective of this study was to fabricate, characterize and evaluate in vitro, an injectable calcium sulfate bone cement beads loaded with an antibiotic nanoformulation, capable of delivering antibiotic locally for the treatment of periodontal disease.

Methods

Tetracycline nanoparticles (Tet NPs) were prepared using an ionic gelation method and characterized using DLS, SEM, and FTIR to determine size, morphology, stability and chemical interaction of the drug with the polymer. Further, calcium sulfate (CaSO₄) control and CaSO₄-Tet NP composite beads were prepared and characterized using SEM, FTIR and XRD. The drug release pattern, material properties and antibacterial activity were evaluated. In addition, protein adsorption, cytocompatibility and alkaline phosphatase activity of the CaSO₄-Tet NP composite beads in comparison to the CaSO₄ control were analyzed.

Results

Tet NPs showed a size range of 130 ± 20 nm and the entrapment efficiency calculated was 89%. The composite beads showed sustained drug release pattern. Further the drug release data was fitted into various kinetic models wherein the Higuchi model showed higher correlation value (R² = 0.9279) as compared to other kinetic models. The composite beads showed antibacterial activity against Staphylococcus aureus and Escherichia coli. The presence of Tet NPs in the composite bead didn't alter its cytocompatibility. In addition, the composite beads enhanced the ALP activity of hPDL cells.

Conclusions

The antibacterial and cytocompatible CaSO₄-Tet NP composite beads could be beneficial in periodontal management to reduce the bacterial load at the infection site.

General significance

Tet NPs would deliver antibiotic locally at the infection site and the calcium sulfate cement, would itself facilitate tissue regeneration.     

Investigation of roles of divalent cations in Shewanella oneidensis pellicle formation reveals unique impacts of insoluble iron

Background

Bacteria adopt a variety of lifestyles in their natural habitats and can alternate among different lifestyles in response to environmental changes. At high cell densities, bacteria can form extracellular matrix encased cell population on submerged tangible surfaces (biofilms), or at the air–liquid interface (pellicles). Compared to biofilm, pellicle lifestyle allows for better oxygen access, but is metabolically more costly to maintain. Further understanding of pellicle formation and environmental cues that influence cellular choices between these lifestyles will definitely improve our appreciation of bacterial interaction with their environments.

Methods

Shewanella oneidensis cells were cultured in 24-well plates with supplementation of varied divalent cations, and pellicles formed under such conditions were evaluated. Mutants defective in respiration of divalent cations were used to further characterize and confirm unique impacts of iron.

Results and conclusions

Small amount of Fe^2 + was essential for pellicle formation, but presence of over-abundant iron (0.3 mM Fe^2 + or Fe^3 +) led to pellicle disassociation without impairing growth. Such impacts were found due to S. oneidensis-mediated formation of insoluble alternative electron acceptors (i.e., Fe₃O₄) under physiologically relevant conditions. Furthermore, we demonstrated that cells preferred a lifestyle of forming biofilm and respiring on such insoluble electron acceptors under tested conditions, even to living in pellicles.

General significance

Our finding suggests that bacterial lifestyle choice involves balanced evaluation of multiple aspects of environmental conditions, and yet-to-be-characterized signaling mechanism is very likely underlying such processes.     

Association between interleukin 4 gene intron 3 VNTR polymorphism and recurrent aphthous stomatitis in a cohort of Turkish patients

Objective

Recurrent aphthous stomatitis (RAS) is one of the most common oral mucosal diseases, with a multifactorial etiopathogenesis, an interaction between predisposing factors and/or systemic conditions and immunological components in genetically predisposed subjects. Although there is no clear genetic mode of inheritance, there is evidence that inheritance of specific gene polymorphisms may predispose individuals to RAS. The purpose of the present study was to investigate a possible association between the functional interleukin 4 (IL4) VNTR genetic polymorphism and RAS in a sample of Turkish patients.

Methods

The study included 145 unrelated patients with a clinical diagnosis of RAS and 150 unrelated healthy controls. Genomic DNA was isolated and IL4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers.

Results

The distribution of genotype and allele frequencies of IL4 gene intron 3 VNTR polymorphism was statistically different between RAS patients and control group (p < 0.0001 and p < 0.0001, respectively) P2P2 genotype and P2 allele were also found to be protective with a lower risk for susceptibility to RAS (p < 0.0001).

Conclusion

The results of this study suggest that intron 3 VNTR polymorphism in the IL4 gene is associated with RAS susceptibility in Turkish population.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.