Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches.

The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

Toward a predictive theoretical model for osmolality rise with non-humidified incubation: a randomized, multivariate response-surface study.

STUDY QUESTION: What factors associated with embryo culture techniques contribute to the rate of medium osmolality change over time in an embryo culture incubator without added humidity? SUMMARY ANSWER: The surface area-to-volume ratio of culture medium (surface area of the medium exposed to an oil overlay), as well as the density and height of the overlaying oil, all interact in a quantitative way to affect the osmolality rise over time. WHAT IS KNOWN ALREADY: Factors such as medium volume, different oil types, and associated properties, individually, can affect osmolality change during non-humidified incubation. STUDY DESIGN, SIZE, DURATION: Several experimental designs were used, including simple single-factor completely randomized designs, as well as a multi-factor response surface design. Randomization was performed at one or more levels for each experiment. Osmolality measurements were performed over 7 days, with up to 8 independent osmolality measurements performed per treatment group over that time. For the multi-factor study, 107 independent combinations of factor levels were assessed to develop the mathematical model. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in a research laboratory setting. Commercially available embryo culture medium and oil was used. A MINC incubator without water for humidification was used for the incubation. Osmolality was measured with a vapor pressure osmometer after calibration. Viscometry and density were conducted using a rheometer, and volumetric flasks with an analytical balance, respectively. Data analyses were conducted with several commercially available software programs. MAIN RESULTS AND THE ROLE OF CHANCE: Preliminary experiments showed that the surface area-to-volume ratio of the culture medium, oil density, and oil thickness above the medium all contributed significantly (P < 0.05) to the rise in osmolality. A multi-factor experiment showed that a combination of these variables, in the form of a truncated cubic polynomial, was able to predict the rise in osmolality, with these three variables interacting in the model (P < 0.05). Repeatability, as measured by the response of identical treatments performed independently, was high, with osmolality values being ± 2 of the average in most instances. In the final mathematical model, the terms of the equation were significant predictors of the outcome, with all P-values being significant, and only one P-value > 0.0001. LIMITATIONS, REASONS FOR CAUTION: Although the range of values for the variables were selected to encompass values that are expected to be encountered in usual embryo culture conditions, variables outside of the range used may not result in accurate model predictions. Although the use of a single incubator type and medium type is not expected to affect the conclusions, that remains an uncertainty. WIDER IMPLICATIONS OF THE FINDINGS: Using this predictive model will help to determine if one should be cautious in using a specific system and will provide guidance on how a system may be modified to provide improved stability during embryo culture. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Cook Medical. The author is a Team Lead and Senior Scientist at Cook Medical. The author has no other conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

A rapid, low pH, nutrient stress, assay to determine the bactericidal activity of compounds against non-replicating Mycobacterium tuberculosis.

There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anti-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not.

Anthranilic amide and imidazobenzothiadiazole compounds disrupt Mycobacterium tuberculosis membrane potential.

A family of compounds typified by an anthranilic amide 1 was identified from a whole-cell screening effort targeted at identifying compounds that disrupt pH homeostasis in Mycobacterium tuberculosis. 1 demonstrated bactericidal activity against non-replicating M. tuberculosis in pH 4.5 buffer (MBC4.5 = 6.3 µM). Exploration of the structure-activity relations failed to simplify the scaffold. The antitubercular activity proved dependent on the lipophilicity and planarity of the molecule and directly correlated with mammalian cytotoxicity. Further studies revealed a pH-dependent correlation between the family's disruption of M. tuberculosis membrane potential and antitubercular activity, with active compounds causing a drop in membrane potential at concentrations below their MBC4.5. A second compound family, identified in the same screening effort and typified by imidazo(4,5-e)(2,1,3)benzothiadiazole 2, provided a contrasting profile. As with 1, structure-activity profiling of 2 (MBC4.5 = 25 µM) failed to minimize the initial scaffold, mammalian cytotoxicity was observed for a majority of the active compounds, and many of the active compounds disrupted M. tuberculosis membrane potential. However, unlike the anthranilic amide compounds, the benzothiadiazole compounds disrupted M. tuberculosis membrane potential primarily at concentrations above the MBC4.5 in a pH-independent fashion. These differences suggest an alternative mechanism of action for the benzothiadiazole compounds. As a result, while the cytotoxicity of the anthranilic amides limits their utility to tool compounds, benzothiadiazole 2 presents an attractive target for more focused SAR exploration.

Identification of Compounds with pH-Dependent Bactericidal Activity against Mycobacterium tuberculosis.

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.

The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents.

We identified a di-substituted triazolopyrimidine with anti-tubercular activity against Mycobacterium tuberculosis. Three segments of the scaffold were examined rationally to establish a structure-activity relationship with the goal of improving potency and maintaining good physicochemical properties. A number of compounds displayed sub-micromolar activity against Mycobacterium tuberculosis with no cytotoxicity against eukaryotic cells. Non-substituted aromatic rings at C5 and a two-carbon chain connecting a terminal aromatic at C7 were preferred features; the presence of NH at C7 and a lack of substituent at C2 were essential for potency. We identified compounds with acceptable metabolic stability in rodent and human liver microsomes. Our findings suggest that the easily-synthesized triazolopyrimidines are a promising class of potent anti-tubercular agents and warrant further investigation in our search for new drugs to fight tuberculosis.

The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5 degrees and 22 degrees C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 degrees C than 22 degrees C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC's to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.

Towards cryopreservation of Greenshell mussel (Perna canaliculus) oocytes.

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshelltrade mark mussel (Perna canaliculus), New Zealand's main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2-0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to -10 degrees C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to -10 degrees C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

Reversible disassembly of the actin cytoskeleton improves the survival rate and developmental competence of cryopreserved mouse oocytes.

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method.

Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification.

OBJECTIVE: To determine the permeability of human metaphase II oocytes to ethylene glycol and water in the presence of ethylene glycol, and to use this information to develop a method to vitrify human oocytes. DESIGN: An incomplete randomized block design. SETTING: A university-affiliated assisted reproductive center. PATIENT(S): Women undergoing assisted reproduction in the Center for Reproductive Medicine at Shandong University. INTERVENTION(S): Oocytes were exposed to 1.0 molar ethylene glycol in a single step and photographed during subsequent volume excursions. MAIN OUTCOME MEASURE(S): A two-parameter model was employed to estimate the permeability to water and ethylene glycol. RESULT(S): Water permeability ranged from 0.15 to 1.17 microm/(min.atm), and ethylene glycol permeability ranged from 1.5 to 30 microm/min between 7 degrees C at 36 degrees C. The activation energies for water and ethylene glycol permeability were 14.42 Kcal/mol and 21.20 Kcal/mol, respectively. CONCLUSION(S): Despite the lower permeability of human metaphase II oocytes to ethylene glycol compared with previously published values for propylene glycol and dimethylsulfoxide, methods to add and remove human oocytes with a vitrifiable concentration of ethylene glycol can be designed that prevent excessive osmotic stress and minimize exposure to high concentrations of this compound.

Osmotic tolerance of in vitro produced porcine blastocysts assessed by their morphological integrity and cellular actin filament organization.

This experiment investigated the osmotic tolerance limits of the morphology and the cellular actin filament organization of porcine blastocysts. In vitro produced Day 6 blastocysts were subjected to osmotic treatments with sucrose solutions of different osmolalities (75, 150, 210, 600, 1200, and 2400 mOsm) and one isotonic solution (NCSU-23, 285 mOsm). Blastocysts were then either fixed immediately, or cultured for 18 h and subsequently fixed with formalin. The morphology of the treated blastocysts was examined under a stereomicroscope and the integrity of the cellular actin filaments of the blastocysts was examined by confocal microscopy after staining with Alexa Fluor 488 phalloidin. The results indicated that there was a significant relationship between the osmotic levels and the probability of blastocysts exhibiting disrupted cellular actin filaments. In addition, blastocysts also collapsed in proportion to the levels of osmotic treatments. The osmotic tolerance limits which would maintain 70% of the blastocysts with their original morphology immediately after the treatment were 90 and 170%, respectively, of isotonicity. After 18 h of culture, the osmotic tolerance limits were 61 and 163%, respectively, of isotonicity. Similarly, the osmotic conditions relative to isotonicity which would maintain the integrity of cellular actin filaments in 70% of treated blastocysts had to be within the range of 87 and 147% immediately after the treatment and 87 and 169% after 18 h of culture. Collectively, these data indicate that in vitro produced porcine blastocysts are very sensitive to osmotic stress. This information can be used to optimize cryopreservation procedures for porcine embryos.

Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility.

All cells have an intrinsic biophysical property related to their ability to undergo osmotically driven volume changes. This project is of fundamental importance to our understanding of the basic cryobiology of mouse spermatozoa. The objectives of this study were to determine the osmotic tolerance limits for (1) motility, (2) acrosome integrity, and (3) membrane integrity of mouse spermatozoa from multiple genetic backgrounds including: C57BL/6, BALB/c, FVB, C3H, 129/SVS2 hsd B6C3F1, CB6F1, and ICR. The maintenance of acrosomal and plasma membrane integrity was not affected by genetic background (p=0.13), however, there was an interaction between genetic background and osmolality. In addition, acrosome and plasma membrane integrity was highly correlated within each strain (p<0.01). In contrast to acrosome and plasma membrane integrity, the motility of spermatozoa from different genetic backgrounds fell sharply on both sides of isosmolality, both with and without return to isosmotic conditions. Exposure to hyposmotic conditions caused morphological changes in the spermatozoa, which inhibited motility. However, this morphological change was not reversible in all cases when returned to isosmotic conditions. The ability to maintain motility in an anisosmotic media was affected by genetic background, osmolality as well as the interaction between genetic background and osmolality (p<0.05). In conclusion, mice with different genetic backgrounds appear to have similar tolerance to osmotic changes in terms of sperm acrosome and plasma membrane integrity; however, the ability to maintain motility differs between genetic backgrounds.