Use Of BSA Research Articles

A Neutron Beam Shaping Assembly for Boron Neutron Capture Therapy of Superficial Tumors

The Vacuum Insulated Tandem accelerator have been developed in Budker Institute of Nuclear Physics. Neutrons are generated in 7Li(p,n)7Be reaction. Neutron beam shaping assembly is used for therapeutic beam forming. It consists of the moderator, reflector and filter. Magnesium fluoride is considered optimal material for neutron slowing down because of noticeable cross section of inelastic neutron scattering. Previously, we showed that it is optimal to use proton beam at energy 2.3 MeV for neutron generation.As a result of a critical analysis of our earlier decisions on the methods used to form a therapeutic neutron beam and decisions of other research groups, as well as successful experiments on the irradiation of laboratory pets and cell cultures carried out at our experimental facility, we noticed that with the recent trend towards a decrease in proton energy the process of inelastic scattering in MgF2 is no longer decisive in neutron moderation, and it was decided to consider materials based on plexiglass as a moderator material.In this work we considered Poly-Biz as moderator material and get the neutron beam the same quality as with MgF2 moderator and proton energy 2.3 MeV but at lower proton energy and current that can cause treatment time reducing and allows more reliable neutron generation.For a long time, the development of BNСT was hindered by the lack of charged particle accelerators that provide stable production of a stationary 2.5 MeV 10 mA proton beam. The use of BSA with Poly-Biz allows the use of these accelerators at lower energy, which improves the reliability of their operation, and reduces the therapy time by more than 2 times, which is also important for therapy. Also, the use of such an BSA simplifies the requirements for a charged particle accelerator and this opens up both the possibility of optimizing the developed accelerators and the use of other accelerators that have not yet reached the required parameters.

Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma.

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen-thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post-breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non-permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin-nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low-M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post-thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF.

Optimal Wavelet Neural Network-Based Intrusion Detection in Internet of Things Environment

As the Internet of Things (IoT) endures to develop, a huge count of data has been created. An IoT platform is rather sensitive to security challenges as individual data can be leaked, or sensor data could be used to cause accidents. As typical intrusion detection system (IDS) studies can be frequently designed for working well on databases, it can be unknown if they intend to work well in altering network environments. Machine learning (ML) techniques are depicted to have a higher capacity at assisting mitigate an attack on IoT device and another edge system with reasonable accuracy. This article introduces a new Bird Swarm Algorithm with Wavelet Neural Network for Intrusion Detection (BSAWNN-ID) in the IoT platform. The main intention of the BSAWNN-ID algorithm lies in detecting and classifying intrusions in the IoT platform. The BSAWNN-ID technique primarily designs a feature subset selection using the coyote optimization algorithm (FSS-COA) to attain this. Next, to detect intrusions, the WNN model is utilized. At last, the WNN parameters are optimally modified by the use of BSA. A widespread experiment is performed to depict the better performance of the BSAWNN-ID technique. The resultant values indicated the better performance of the BSAWNN-ID technique over other models, with an accuracy of 99.64% on the UNSW-NB15 dataset.

Use of Bulk Segregant Analysis for Determining the Genetic Basis of Azole Resistance in the Opportunistic Pathogen Aspergillus fumigatus.

A sexual cycle was described in 2009 for the opportunistic fungal pathogen Aspergillus fumigatus, opening up for the first time the possibility of using techniques reliant on sexual crossing for genetic analysis. The present study was undertaken to evaluate whether the technique ‘bulk segregant analysis’ (BSA), which involves detection of differences between pools of progeny varying in a particular trait, could be applied in conjunction with next-generation sequencing to investigate the underlying basis of monogenic traits in A. fumigatus. Resistance to the azole antifungal itraconazole was chosen as a model, with a dedicated bioinformatic pipeline developed to allow identification of SNPs that differed between the resistant progeny pool and resistant parent compared to the sensitive progeny pool and parent. A clinical isolate exhibiting monogenic resistance to itraconazole of unknown basis was crossed to a sensitive parent and F1 progeny used in BSA. In addition, the use of backcrossing and increasing the number in progeny pools was evaluated as ways to enhance the efficiency of BSA. Use of F1 pools of 40 progeny led to the identification of 123 candidate genes with SNPs distributed over several contigs when aligned to an A1163 reference genome. Successive rounds of backcrossing enhanced the ability to identify specific genes and a genomic region, with BSA of progeny (using 40 per pool) from a third backcross identifying 46 genes with SNPs, and BSA of progeny from a sixth backcross identifying 20 genes with SNPs in a single 292 kb region of the genome. The use of an increased number of 80 progeny per pool also increased the resolution of BSA, with 29 genes demonstrating SNPs between the different sensitive and resistant groupings detected using progeny from just the second backcross with the majority of variants located on the same 292 kb region. Further bioinformatic analysis of the 292 kb region identified the presence of a cyp51A gene variant resulting in a methionine to lysine (M220K) change in the CYP51A protein, which was concluded to be the causal basis of the observed resistance to itraconazole. The future use of BSA in genetic analysis of A. fumigatus is discussed.

Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding.

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

Detection of Cortisol in Pbs Buffer and Fish Blood Plasma By Electrochemical Impedance Spectroscopy

Cortisol (MW = 362.5) is a steroid hormone produced in the human body by the adrenal gland in response to elevated levels of stress. Normal levels of cortisol in the human body range from 6-23 microgram per deciliter. Cortisol has also been widely studied in order to better understand fish physiology, with applications to fish ecology and fish farming, since cortisol levels can also be employed as a biomarker for reproductive activity. Following immobilization of the polyclonal antibody to cortisol atop an Au electrode, we have detected cortisol in both PBS buffer solution and fish blood plasma, with a detection range up to 0.5 microgram/ml. Binding of cortisol approximately follows the Langmuir adsorption isotherm, and the detection range is limited at high concentrations by saturation of the antibody film. This allows the use of sample dilution to reduce the physiological concentration of cortisol down to the range in which it can be detected by impedance biosensing. The advantage of this approach is that non-specific adsorption is also dramatically reduced by sample dilution. Non-specific adsorption in this system is unmeasurable, due to both the use of sample dilution and the use of BSA for surface blocking.

A novel liposomal nanomedicine for nitric oxide delivery and breast cancer treatment

Breast cancer is the most common type of cancer occurring among women in the United States. Nitric oxide (NO) is endogenous signaling molecules that regulate biological processes. NO has the potential to induce either cancer progression or cancer cell apoptosis depending on intra-tumoral NO concentration. High levels of NO have a cytotoxic effect on cancer cells. A novel cytotoxic gas delivery system has been developed using NO-loaded echogenic liposomes (ELIP) for breast cancer treatment. Empty ELIP and NO-ELIP were prepared using the previously developed freezing-under-pressure method with modified lipid composition. Echogenicity of NO-ELIP was measured to determine the stability of NO-ELIP. Two types of breast cancer cell (BCC) lines, MDA-MB-231 and MDA-MB-468, were utilized. MTT assay was performed after NO-ELIP treatment to determine BCC viability. Echogenicity data demonstrated improved stability of NO-ELIP with the use of BSA for resuspension of NO-ELIP. Cell death induced by NO-ELIP was not from lipid cytotoxicity but from NO. The cytotoxic effect of NO-ELIP on BCC was highly dependent on NO-ELIP concentration. NO-ELIP in concentration of 1.0-2.0 mg/ml induced dramatically decreased BCC viability. This novel cytotoxic gas delivery nanomedicine using liposomal carriers, NO-ELIP, has the potential to provide improved therapeutic effect for breast cancer treatment.

Effect of albumin and casein supplementation on the common carp Cyprinus carpio L. sperm motility parameters measured by CASA

Adhesion of sperm to a glass surface can be manifested by the low percentage of motile spermatozoa and lower speed. This crucial factor could disprove the sperm quality prediction from computer-assisted sperm analysis (CASA) results. In our study, this was particularly observed when solution L was used. However, protein supplementation was effective in the reduction in sperm adhesion. We found that albumin increased the sperm motility at concentrations of 0.25 % in solution L and 0.5 % in solution P. Casein was effective in both solutions at 0.25 % concentration. However, in solution L, the motility was lower than that measured in solutions P and L with the supplementation of BSA at 0.25 and 0.5 %. BSA supplementation was especially beneficial in terms of higher speed values regardless of an activation solution. Casein used with solution P allows us to obtain similar results as in the samples activated with BSA supplementation. Casein supplementation to solution L did not increase CASA results to the level observed in the samples activated with BSA added to the solution L. Our data suggest that the selection of an activation solution is one of the most crucial steps in the experiments requiring CASA data. Depending on the composition, different protein supplements should be used to reduce adhesion of sperm to a glass surface. We recommend the use of BSA at the 0.5–1 % concentration because at this level we obtained the highest CASA parameters in both activation media.

Tris(hydroxymethyl)aminomethane‐functionalized agarose particles: Parameters affecting the binding of bovine serum albumin

A new protein adsorbent is introduced based on the coupling of the common buffer ion, tris(hydroxymethyl)aminomethane, to the agarose gel Sepharose HP from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. The article describes the synthesis of the new adsorbent and the use of BSA as a model in a binding study. By optimization of the coupling procedure, a maximum ligand density of 63.5 μmol/mL gel could be obtained. Adsorption equilibria were investigated in the pH range 5.0-8.0 and at salt concentrations of 0-0.4 mol/L. Binding of BSA under different conditions indicated that both electrostatic interaction and hydrogen bonding were involved in the adsorption process where the former played a major role.

Encapsulation of BSA in polylactic acid–hyperbranched polyglycerol conjugate nanoparticles: preparation, characterization, and release kinetics

Nanoparticles based on an amphiphilic copolymer with polylactic acid (PLA) grafted onto hyperbranched polyglycerol (HPG) were prepared by the use of BSA as a model protein. The characteristics of the nanoparticles were evaluated using particle size analyzer, transmission electron microscopy, and X-ray photoelectron spectroscopy. The secondary structure of BSA released from nanoparticles were analysed by circular dichroism experiments. Cell viability of nanoparticles was also evaluated by using NIH 3T3 cells. The mechanism of BSA release was studied by fitting experimental data to three model equations. Results indicated that BSA diffusion and the polymeric relaxation jointly governed the overall release process. The detailed analysis of BSA release was performed using the first-order kinetic model equation, which gave a good fit to the experimental release data. The influence of different copolymer structures and BSA loading capacities on release profiles were also evaluated for the potential of using nanoparticles as controlled release protein delivery systems.

The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Population pharmacokinetics of troxacitabine

Purpose Troxacitabine (TROX) is a L-cytidine analogue anticancer agent currently in phase II/III trials. The study's objective is to develop & validate a population pharmacokinetic (PPK) model for TROX. Methods Plasma samples from 111 cancer patients receiving IV doses of 0.12 – 12.5 mg/m2 were used to develop the PPK model with NONMEM. About 13 samples per patient were obtained from the 1st dose. 2 covariate groups (I: BSA, SEX, AGE, SCR; II: WT, HT, SEX, AGE, SCR) & PK parameters were evaluated by linear multiple regression. The 2 final PPK models were validated by internal & external methods. Results TROX PPK was characterized by a 3-compartment model, exponential interpatient variability (IPV) error model, combination residual error model, & FOCE INTER estimator method. Clearance was influenced by BSA (27% decrease IPV) or WT (20% decrease IPV). Central compartmental volume was influenced by BSA (12% decrease IPV). Model validations reveal both final models accurate in predicting plasma TROX concentrations with improved PK parameter predictions with the addition of covariates. Conclusion Covariate modeling supports the use of BSA in current dosing strategies for TROX. Clinical Pharmacology & Therapeutics (2004) 75, P82–P82; doi: 10.1016/j.clpt.2003.11.312

314EFFECT OF MACROMOLECULE SUPPLEMENTATION DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF GOAT OOCYTES

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20–22h (6.0% CO2 in air, 38.7°C) in modified SOF medium (1.5mM glucose, 3.0mM L-lactate, 0.1mM pyruvate, 1.0mM glutamine, 0.1mM taurine) supplemented with 1×MEM nonessential amino acids, 0.5×MEM essential amino acids, 1×MEM vitamins, 0.1mM cysteamine, 5μg mL−1 insulin, 5μgmL−1 transferrin, 5ng mL−1 selenium, 50ngmL−1 EGF, 0.01U mL−1 LH and FSH, and 50μgmL−1 gentamicin. Treatments were: (1) 1mgmL−1 PVA (protein-free, defined); (2) 4mgmL−1 BSA (semi-defined); (3) 0.5mM citrate and 0.5mgmL−1 hylauronate (C+H, defined); and (4) 0.5mM citrate and 0.5mgmL−1 hylauronate with 4mgmL−1 BSA (C+H+BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant’s medium with 7.7mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50μL) containing COC at a final concentration of 14–15×106 spermmL−1. Gametes were coincubated in the presence of heparin (25μgmL−1) for 22–24h in 7% CO2 in air at 38.7°C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7°C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented ±SEM and probability values P<0.05 were considered significant. The use of BSA did not improve (P>0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P>0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions. Table 1 Development of goat oocytes following IVM with different macromolecules.

The interaction of human and bovine serum proteins with CYP3A in human liver microsomes: inhibition of testosterone 6β-hydroxylation by albumin, α-globulins, α 1-acid glycoprotein and γ-globulins

The effects of human and bovine serum proteins on CYP3A activity, using testosterone as the probe substrate, were investigated in human liver microsomes. Serum albumin, α-globulins, and α 1-acid glycoprotein (α 1-AGP) of both species significantly inhibited testosterone 6β-hydroxylation. When the inhibitory effects of serum proteins were compared with serum protein binding data, human α-globulins, with a ratio (relative metabolic activity/unbound fraction) of 0.3, showed higher, and bovine α 1-AGP, with the ratio of 1.4, showed lower inhibitory effects than those expected from protein binding of testosterone. The effects of the other serum proteins were close to those expected from protein binding, according to the free drug hypothesis. The K i values obtained from the Dixon plots were 0.32% (w/v, 48 μM) for human serum albumin (HSA), 0.48% for human α-globulins, and 0.23% (52 μM) for human α 1-AGP. K i values of bovine serum albumin, bovine α-globulins and bovine α 1-AGP were 3–5 times higher than those of the respective human proteins. The results suggest a direct interaction of some of these serum proteins with the active site of the CYP3A isoform. Since the bovine serum proteins showed weaker inhibitory effects than human serum proteins, the wide use of BSA, which is viewed as interchangeable with HSA, needs to be cautioned.

Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm

The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchusmykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchusmykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo-and isoosmotic solutions of 10 and 300mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 × 107 spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the “in vitro” tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760® alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.

A review of factors affecting the performances of latex agglutination tests

The present review describes the different strategies followed to improve the performance of latex agglutination tests. The analysis is mainly focused on the diverse parameters that affect the final colloidal stability of the immunoprotein-latex system. These parameters include: the surface properties of polymer carriers; the different kind of antibodies usually employed; the use of BSA as stabilizer; the co-adsorption of various macromolecules (BSA, surfactants and lipids) and antibodies; recent approaches to colloidal stability at high ionic strengths due to hydration forces; and the covalent coupling of antibodies on functionalized latex particles. Special emphasis is given to the relation between electrophoretic mobility and the colloidal stability of the sensitized particles and how this knowledge can be utilized for a better understanding of the immunoagglutination kinetic.

Only low levels of spermadhesin AWN are detectable on the surface of live ejaculated boar spermatozoa.

The zona-binding protein family of spermadhesins are constituents of boar seminal plasma that are generally believed to attach to the acrosomal region of spermatozoa and thereby assist sperm interaction with the zona pellucida at fertilization. However, previous studies have yielded conflicting results with respect to amounts of adhesin bound to ejaculated cells, to the distribution of bound adhesin within the sperm population, and the regionalization of binding on the sperm surface. In the present study, spermadhesin AWN in unfixed living suspensions of boar spermatozoa was assessed by means of flow cytometry and immunocytochemistry, using a polyclonal antibody raised in chicken. Direct probing with an Oregon Green conjugate of the antibody was compared with indirect probing using Alexa Fluor-conjugated goat anti-chicken IgG as second antibody. Regardless of staining procedure, the live sub-population showed homogeneously low levels of staining, whereas the dead sub-population showed high (more than 5-fold greater) levels of staining. The live cells were stained about 2-fold more intensely by anti-AWN than by preimmune immunoglobulin, indicating the presence of small amounts of AWN. Immunocytochemistry showed the live cells to be faintly stained all over their surface, whereas staining of the dead cells was largely localized to the acrosomal region. This latter staining was non-specific, preimmune immmunoglobulin resulting in as much bound fluorescence as anti-AWN. Attempts to block non-specific staining with appropriate pretreatment with chicken or goat serum (as compared with routine use of BSA) met with variable and incomplete success, and did not increase staining by anti-AWN relative to preimmune serum in either live or dead cells. It is concluded that limited amounts of spermadhesin AWN bind tightly over the whole surface of live ejaculated boar sperm. However, the acrosomal region of disrupted sperm has an alarming tendency to bind fluoro-conjugates of immunoglobulins non-specifically.

Dose calculation of anticancer drugs: a review of the current practice and introduction of an alternative.

To review the current dose-calculation practice and propose a non-body-surface area (BSA)-based dose-calculation method. Data that supported the introduction of BSA-based dose calculation in the late 1950s were reviewed. Data on 18 drugs that correlated pharmacokinetic (PK) variables for cytotoxic drugs with BSA were examined. Other methods of dose calculation, such as therapeutic drug monitoring (TDM) and adaptive control, were also examined. The BSA-based method of dose calculation was adopted without adequate investigation of its accuracy. BSA fails to standardize the marked interpatient variation in PK for most cytotoxic drugs. A definite correlation was found between PK variables and BSA for only one drug (docetaxel). PK parameters correlate with toxicity, as well as response in some tumors, but do not completely explain the variation in drug effect between individuals. The complexities of TDM may make its universal use impractical. A non-BSA-based dose calculation method is proposed that defines three mandatory steps: prime dose, modified dose, and toxicity-adjusted dose (PMT dosing). Prime dose is the fixed dose of a drug used alone or in combination and is derived from the reanalysis of phase I/II studies and from clinical practice. Modified dose is an adjustment of the prime dose before administration, based on dose-adjustment guidelines that predict the drug-handling ability of an individual. Population pharmacodynamic studies may be used for the development of these guidelines. Subsequent doses are adjusted in each patient according to a target toxicity, such as nadir neutrophil count or other objective toxicity, that serves as a surrogate marker for potential antitumor effect (toxicity-adjusted dose). Patients who are predicted to have very abnormal drug handling should be excluded from such a dosing scheme and TDM may be more suitable. The routine use of BSA for dose calculation should be reevaluated. Other methods of dose calculation should be investigated. TDM may be impractical in all patients and remains unvalidated. PMT dosing ensures that the condition of each individual is considered, to predict drug effects better. Clinical dose-calculation systems such as PMT dosing should be evaluated prospectively.

The effect of sera, bovine serum albumin and follicular cells on in vitro maturation and fertilization of porcine oocytes

The effects of fetal calf serum (FCS), estrus gilt serum (EGS) BSA, dispersed granulosa cells, hemi-sections of follicular wall, and replacement of medium after 24 hours on in vitro maturation and fertilization of porcine oocytes were studied. The results indicate that the use of BSA for 24 or 48 hours inhibited the expansion of cumulus cells and the maturation of oocytes. An incubation of 24 hours culture in FCS followed by a second 24 hours in BSA containing medium did not decrease the rate of maturation but significantly decreased the polyspermy and mean number of spermatozoa penetrated/oocyte. Renewing the medium with or without removal of cumulus cells during the second incubation increased the maturation rate. Removal of cumulus cells decreased the penetrability, the polyspermy rates of the oocyte and the mean number of spermatozoa/oocyte penetrated. The EGS-supplemented medium, dispersed granulosa cells or hemi-sections of follicular wall did not affect the maturation or fertilization rates. In conclusion, BSA, a protein supplement in maturation medium, inhibited cumulus cell expansion and maturation of porcine oocytes. After resumption of meiosis triggered by FCS, BSA did not influence maturation. The FCS-BSA treatment reduced the incidence of polyspermy and the mean number of spermatozoa penetrated/oocyte without decreasing the rate of maturation and fertilization.