Development of α+β-type biomedical Ti–Nb alloys with high oxygen content

Ti-(5–20)Nb-(0.5–1)O alloys (mass%) were investigated for developing low-cost biomedical α+β-type Ti alloy. Ti-(5, 10, 15, 20)Nb-(0.5, 0.75, 1)O alloys (mass%) were arc-melted and forged into bars. The forged alloy bars were heat-treated at 873 to 1373 K for 3.6 ks in an Ar atmosphere and quenched in iced water. β transus (Tβ) of the Ti-Nb-O alloys decreased with increasing Nb content. An increase in the oxygen content led to an increase in Tβ. After quenching, the formation of α′ martensite was observed in Ti-5Nb-yO alloys. An increase in the Nb content to 10 mass% led to the formation of α′ and α″ martensites. A further increase in the Nb content to 15 and 20mass% resulted in the formation of more α″ martensites. The boundary temperature for the formation of α′ and α″ martensite in the Ti-10Nb-yO alloys increased with increasing oxygen content, because oxygen enhances the Nb distribution to the β phase. The ultimate tensile strength of the Ti-xNb-0.75O alloys heattreated to obtain the α-phase fraction (fα) of 0.5 was over 1000 MPa, except for the Ti-15Nb-0.75O alloy. The total elongation decreased with increasing Nb content. The Ti-5Nb-0.75O alloy exhibited excellent strength-ductility balance as a low-cost α+β-type biomedical Ti alloy.

IDENTIFICATION OF HAZARDOUS CHEMICAL AND MICROBES CONTAMINATION IN FOODS OF STREET VENDORS AROUND IAIN SYEKH NURJATI CIREBON

This study aimed at identifying chemical and microorganism contamination in foods of street vendors around Institut Agama Islam (IAIN) Syekh Nurjati Cirebon. The samples are meatball, cilok, grilled fish cake, nuggets, rolade, sauce, crackers, and iced water drinks. The sample tests are formalin test, borax test, Rodhamine B test, Methanyl Yellow test, and Coliform & Escherichia coli MPN test. The results showed that some food samples like meatball, cilok, grilled fish cake, nuggets,and rolade were positive of formalin & borax contaminated. The iced water drinks are contaminated by Coliform & E. coli. The other samples like sauce and crackers were negative of Rhodamine B & Methanyl Yellow contaminated. In conclusion, according to this study, the foods of street vendors at IAIN Cirebon are not yet safe to be consumed.

Quenching induced hierarchical 3D porous g-C3N4 with enhanced photocatalytic CO2 reduction activity

Hierarchical 3D porous g-C3N4 (HCN) is synthesized by quenching in iced water and shows superior photocatalytic CO2 reduction performance.

L-Asparaginase Ex Vivo Activity Distorts Plasmatic Asparagine Depletion Assessment

L-asparaginase (L-ASP) is a well-established and important agent having demonstrated significant clinical efficacy in the treatment of acute lymphoblastic leukemia (ALL) Many cancerous cells are deficient in asparagine synthetase (ASNS), requiring endogenous plasma asparagine (ASN) for proliferation. Without this essential amino acid, the malignant cells undergo apoptosis. Plasma levels of ASN have been considered a relevant surrogate biological parameter to assess L-ASP efficacy. The aim is to maintain ASN levels below a threshold varying from 0.1 to 3µM. However, the measurement of ASN in presence of L-ASP is problematic, considering that 1 International Unit (IU) of L-ASP is able to cleave 1µmol of ASN per minute at 37°C. As the normal value of plasma ASN is around 50µM in humans, it requires about 30 seconds for 100 IU/L of L-ASP in the blood stream to fully deplete L-ASN, also considering there is no de novo ASN production., Plasma ASN is provided by normal cells expressing ASNS and by food intake. Once blood is drawn, no endogenous source of ASN is present and in the presence of active L-ASP a rapid ASN ex vivodepletion is expected to occur in the sampling tube. L-ASP activity can be reduced by low temperature (iced water) and/or by lowering the pH through addition of sulfosalicylic acid (Pieters et al, Blood, 2008). It is recommended to cool down the sample for 15 minutes on iced water immediately after the patient’s blood is drawn, and to centrifuge at 1000g for 15 minutes at +4°C to recover the plasma. A solution of 10% sulfosalicylic acid is added to the plasma 1:4 (v/v) before centrifugation at 3000g for 5 minutes to extract the proteins, including L-ASP. Here we report our investigation on the impact of the duration between blood draw and immersion in “iced water” and the impact of the sulfosalicylic acid concentration when “free” E. coliL-ASP (L-ASP) or L-ASP encapsulated in erythrocytes (L-ASP/RBC) is present. L-ASP or L-ASP/RBC was added to human blood, in concentrations comparable to patients receiving L-ASP treatment (0-200 IU/L). The sampling process used in clinical practice was strictly applied to the sample, except that different times (5 sec to 30 min) at room temperature (RT) between the addition of L-ASP (considered as the moment of the blood draw) and the step of cooling were tested. Two quantities (100µL and 500µL) of sulfosalicylic acid were also tested. ASN was quantified in the samples after the completion of the whole process to assess whether variations in these parameters could impact the measurement. When 20 IU/L of L-ASP was present in the sample, ASN was depleted by 75% after a 5-sec delay at RT, and below the limit of quantification (BLLQ=2µM) after 10 min. When reducing sulfosalicylic acid to a suboptimal quantity (100µL), complete depletion (BLLQ) of ASN was obtained after 5 sec at RT with 20 IU/L of L-ASP. These results confirmed the necessity of the acidic deproteinization of the samples but also revealed a significant ex vivo activity of L-ASP leading to overestimation of ASN depletion in plasma. Considering L-ASP/RBC, 25% ASN depletion was observed at 200IU/L after 3 min. at RT, and ex vivocomplete depletion was reached with 2000IU/L and a 30-minute delay at RT. In conclusion, considering current methods, due to inability to control ex vivo L-ASP metabolism, it is practically impossible to have a measurement of plasmatic ASN which reflects the in vivo reality. Indeed, in the presence of low concentrations of free L-ASP, a very rapid depletion of ASN is observed ex vivo. In the time needed to cool down and centrifuge samples further “artificial” depletion occurs rapidly and the patient can be erroneously considered ASN depleted. However, the comparison of the phamacodynamics between “free” asparaginases may be still considered valuable because the error is probably equal in both sides. On the contrary, “artificial” depletion is much less when the L-ASP is encapsulated in RBCs (thanks to the delay of asparagine to pass through the RBC membrane), leading to a potential bias to compare L-ASP or L-ASP/RBC. Finally, L-ASP activity is believed to be a more relevant marker than the measurement of ASN depletion. This is consistent with the approach taken by regulatory agencies in recent years, favoring the duration of L-ASP activity over 100IU/L as endpoint over depletion endpoints. Disclosures Berlier: ERYTECH: Employment, Equity Ownership. Godfrin:ERYTECH Pharma: Employment, Equity Ownership.

Preanalytical Aspects and Sample Quality Assessment in Metabolomics Studies of Human Blood

BACKGROUND Metabolomics is a powerful tool that is increasingly used in clinical research. Although excellent sample quality is essential, it can easily be compromised by undetected preanalytical errors. We set out to identify critical preanalytical steps and biomarkers that reflect preanalytical inaccuracies. METHODS We systematically investigated the effects of preanalytical variables (blood collection tubes, hemolysis, temperature and time before further processing, and number of freeze–thaw cycles) on metabolomics studies of clinical blood and plasma samples using a nontargeted LC-MS approach. RESULTS Serum and heparinate blood collection tubes led to chemical noise in the mass spectra. Distinct, significant changes of 64 features in the EDTA-plasma metabolome were detected when blood was exposed to room temperature for 2, 4, 8, and 24 h. The resulting pattern was characterized by increases in hypoxanthine and sphingosine 1-phosphate (800% and 380%, respectively, at 2 h). In contrast, the plasma metabolome was stable for up to 4 h when EDTA blood samples were immediately placed in iced water. Hemolysis also caused numerous changes in the metabolic profile. Unexpectedly, up to 4 freeze–thaw cycles only slightly changed the EDTA-plasma metabolome, but increased the individual variability. CONCLUSIONS Nontargeted metabolomics investigations led to the following recommendations for the preanalytical phase: test the blood collection tubes, avoid hemolysis, place whole blood immediately in ice water, use EDTA plasma, and preferably use nonrefrozen biobank samples. To exclude outliers due to preanalytical errors, inspect the biomarker signal intensities reflecting systematic as well as accidental and preanalytical inaccuracies before processing the bioinformatics data.

The spermatozoa of the dasyurid marsupial, Sminthopsis crassicaudata, are highly susceptible to cold shock

Since the late 1970s research has suggested that marsupial spermatozoa did not suffer cold shock. We have re-examined cold shock to investigate problems with freezing of spermatozoa from a dasyurid marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). Epididymal spermatozoa were rapidly cooled to 0.5°C in a pre-cooled tube held in an iced-water slurry. Upon re-warming all spermatozoa were immotile and the addition of 10% or 20% egg yolk to the sperm medium had no beneficial effect. Spermatozoa that were rapidly cooled to 4°C maintained only 2% motility when re-warmed but the addition of at least 10% egg yolk was beneficial and upon re-warming greater than 65% of the initial motility was maintained. In order to achieve motile spermatozoa at 0°C, controlled-rate cooling at 0.5°C min–1 was examined. In the absence of egg yolk there was a significant decline in the percentage of motile spermatozoa below 4°C. However, the inclusion of at least 10% egg yolk resulted in no loss of motility in spermatozoa cooled to 0°C. This is the first experimental study indicating that spermatozoa from a marsupial are highly susceptible to cold shock and that the impact of rapid chilling can be mitigated by the addition of 10% egg yolk. The ability to successfully cool the spermatozoa of S. crassicaudata to 0°C may have an important role in future studies examining dasyurid sperm cryopreservation.

143. THE FIRST EVIDENCE OF HIGH SUSCEPTIBILITY TO COLD SHOCK BY THE SPERMATOZOA OF A MARSUPIAL, THE FAT TAILED DUNNART (SMINTHOPSIS CRASSICAUDATA)

Carnivorous marsupials are native Australian predators including the highly threatened northern quoll (Dasyurus hallucatus) and Tasmanian devil (Sarcophilus harrisii). These species are currently actively managed in captive populations but assisted reproductive techniques such as gamete banking may also contribute to their conservation. Previous studies on a model dasyurid, the fat tailed dunnart (Sminthopsis crassicaudata), have found that spermatozoa do not survive freezing and thawing using a variety of freezing protocols and cryoprotectants. We have re-examined cold shock to investigate problems with sperm cryopreservation in S. crassicaudata. Epididymal spermatozoa were rapidly cooled to 0.5ºC in a pre-cooled tube held in an iced water slurry and upon re-warming the spermatozoa were non-motile (n=6). The addition of up to 20% egg yolk, which is considered protective to the spermatozoa of cold shock sensitive eutherians, did not improve the outcome (n=6). Similarly when S. crassicaudata spermatozoa were rapidly cooled to 4ºC, just 2% remained motile upon re-warming (n=10). However when spermatozoa were combined with at least 10% egg yolk and rapidly cooled to 4ºC only small reductions in motility were observed upon rewarming (n≥8). In order to achieve motile spermatozoa at 0ºC, controlled rate cooling at 0.5ºC/minute was examined. In the absence of egg yolk there was a decline in the percentage of motile spermatozoa below 4ºC (n=6). However if spermatozoa were combined with at least 10% egg yolk there was no significant loss of motility at temperatures as low as 0ºC (n=6). This study has revealed that at least one species of marsupial is highly susceptible to cold shock. These paradigm shifting findings give direction to future experiments aiming to develop a robust technique for sperm preservation in endangered dasyurids.