Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii

The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly used in yeast cultivation media. The yeast Komagataella phaffii (Pichia pastoris) is an important host for heterologous protein production and compliance with the REACH regulation is desirable. Boric acid and cobalt dichloride are used as boron and cobalt sources, respectively. Boron and cobalt support growth and productivity and a number of cobalt-containing enzymes exist. Therefore, depletion of boric acid and cobalt dichloride could have various negative effects, but knowledge is currently scarce. Herein, we provide an insight into the impact of boric acid and cobalt depletion on recombinant protein production with K. phaffii and additionally show how different vessel materials affect cultivation media compositions through leaking elements. We found that boric acid could be substituted through boron leakiness from borosilicate glassware. Furthermore, depletion of boric acid and cobalt dichloride neither affected high cell density cultivation nor cell morphology and viability on methanol. However, final protein quality of three different industrially relevant enzymes was affected in various ways.

Novel Cobalt Dichloride Complexes with Hindered Diphenylphosphine Ligands: Synthesis, Characterization, and Behavior in the Polymerization of Butadiene

Two novel cobalt diphenylphosphine complexes were synthesized by reacting cobalt(II) chloride with tert-butyl(diphenyl)phosphine (PtBuPh2) and (S)-(+)neomenthyldiphenylphosphine [(S)-NMDPP]. The crystal structure of the former was determined by single-crystal X-ray diffraction studies. The two complexes were then used in combination with methylaluminoxane (MAO) for the polymerization of 1,3-butadiene: crystalline highly syndiotactic 1,2 poly(1,3-butadiene)s were obtained, with a 1,2 content and a syndiotactic index (percentage of syndiotactic triads [rr]) up to 95% and 85%, respectively. The results obtained further support and confirm what was already observed in the polymerization of 1,3-butadiene with CoCl2(PRPh2)2−MAO (R = methyl, ethyl, normal-propyl, iso-propyl, and cyclohexyl): the nature of the phosphine ligand strongly affects the polymerization stereoselectivity, the polymer syndiotacticity increasing with increasing phosphine ligand steric hindrance.

Patch Testing in Lao medical students

BackgroundDermatological services in Laos, South East Asia are limited to the capital and patch testing is currently not available, so no data exists regarding the common cutaneous allergens in this population.ObjectivesThe aim of this study was to document common allergens in medical students in Laos. Patients/Materials/MethodsOne hundred and fifty medical students were patch tested using TRUE Test® panels 1 to 3 (35 allergens). Readings were taken at Days 2 and 4.ResultsThirty-eight students (25.3%) had a positive reaction to at least one allergen, accounting for 52 reactions in total. The proportion of the students with positive patch test reading was significantly higher in the female [33/96 (34%)] than in the male [5/54 (9%)], p<0.001. The most common allergens were: nickel (10%), gold (6.6%), thiomersal (6.6%), cobalt dichloride (2%) and p-tert-Butylphenol formaldehyde resin (2%). Balsam of Peru (0.66%), black rubber mix (0.66%), Cl+Me-Isothiazolinone (0.66%), fragrance mix 1 (0.66%), quinolone mix (0.66%), methyldibromo glutaronitrile (0.66%), mercapto mix (0.66%), epoxy resin (0.66%), paraben mix (0.66%), thiuram (0.66%) and wool alcohols (0.66%) accounted for all of the other positive reactions.ConclusionThis study represents the first documented patch test results in Lao medical students and in the adult Lao population. The results of this study will inform any future research into contact allergy in Laos and give an insight into the background level of contact sensitivity in this population.

Contact sensitivity in paediatric patients with atopic dermatitis in Laos

BackgroundDermatological services in Laos, South East Asia are limited mainly to the capital and patch testing is currently not available, so no data exists regarding the common cutaneous allergens in this population.ObjectivesThe aim of this study was to document common allergens in paediatric patients with atopic dermatitis attending the allergy clinic in the capital, Vientiane.Patients/Materials/MethodsFifty paediatric patients with atopic dermatitis were patch tested using TRUE Test® panels 1 to 3 (35 allergens). Readings were taken at Days 2 and 4.ResultsTwenty-six positive patch tests were recorded on Day 4 in 15 children (30%). The most common allergens were: gold (18%), nickel (10%), formaldehyde (6%) and p-Phenylenediamine (6%). Other positive allergens were potassium dichromate (2%), cobalt dichloride (2%), Bronopol (2%), paraben mix (2%), fragrance mix 1 (2%) and neomycin (2%). The majority of the patients with positive reactions were female.ConclusionsThis study represents the first documented patch test results in the Lao population. It is hoped that these findings will help clinicians to advise the families of children with atopic dermatitis on common allergens to avoid and inform future work on contact dermatitis in this population.

Deferoxamine but Not Dimethyloxalylglycine, L-Mimosine, or Cobalt Dichloride Can Interfere with the MTT Assay

Hypoxia mimetic agents (HMAs) have been shown to have a positive influence on cellular functions in a multitude of tissue regenerative strategies. Novel experimental approaches use biomaterials as carriers for controlled delivery of these HMAs. Here, the cytotoxic aspects of biocompatibility are of key relevance. The MTT assay is widely used to evaluate cytotoxicity and proliferation. Based on the implications from the proceeding research we hypothesized that specific HMAs such as deferoxamine at high concentrations can interfere with the MTT assay. Thus, the aim of this study was to test the repercussions of the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl2 on the validity of the MTT assay. Murine MC3T3-E1 cells were cultured in serum-free alphaMEM and in alphaMEM supplemented with 10 % fetal bovine serum with the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl2, respectively, at 3 mM-0.3 mM for 24 h (experimental groups). Cells without HMAs served as control (control groups). The same experiments were performed with medium and phosphate buffered saline (PBS) without cells. In all settings MTT solution was added to PBS-washed or unwashed culture plates for the last two hours of the incubation period. Then MTT solution was removed and dimethyl sulfoxide was added to dissolve the formazan crystals and absorption was measured. Our data show that the presence of deferoxamine can interfere with the MTT assay if not removed before the addition of MTT. This is particularly important when evaluating cell viability in setups where deferoxamine-loaded biomaterials are used.

Combined preconditioning with tetra-(1-vinylimidazole) cobalt dichloride

The aim of this study was to investigate the vinylimidazole derivative as pharmacological preconditioning in experimental acute hypoxia and cerebral ischemia. All experiments were conducted with mice and rats. Acute hypoxia with hypercapnia was modeled by placing the mice in a glass with a glass lid shtanglazy 250 ml. Acute hypobaric hypoxia was performed putting animals under a glass cope, where from the Kamovsky pump pumped up the air which is equal to the altitude of 5000 meters (moderate hypoxia) and 11 000 meters (severe hypoxia). Cerebral ischemia in rats was modeled by one-stage bilateral common carotid artery occlusion. Separate pharmacological and hypoxic preconditioning and combined preconditioning by alternately use of pharmacological and hypoxic factors were investigated. It was revealed that combined preconditioning (tetra-(1-vinylimidazole) cobalt dichloride 30 mg/kg + moderate hypobaric hypoxia) enhance the resistance of the animal organism to acute hypoxia in the early period (in 1 hr) and delayed period (in 48 hr) more effective than the separate preconditioning by moderate hypoxia or with the help of tetra-(1-vinylimidazole) cobalt dichloride. The combined preconditioning increase the survival of rats in the early periods and in the delayed periods of cerebral ischemia performance, but doesn’t reduce the severity of neurological deficit in the surviving animals registered with the Stroke Index McGraw scale.

Controlled polymerization of isoprene promoted by a type of hemilabile XPN3 (X = O, S) ligand supported cobalt(ii) complexes: the role of a hemilabile donor on the level of control

Cobalt dichloride complexes (Co1–Co9) carrying a novel type of hemilabile donor PN3 (L1–L3) or XPN3 (L4–L6, X = O; L7–L9, X = S) are reported as precursors for controlled polymerization of isoprene.

Oxidation of 4-nitro-o-xylene with nitric acid using N-hydroxyphthalimide under phase transfer conditions

Abstract4-Nitro-o-xylene was selectively oxidized to 2-methyl-4-nitrobenzoic acid using dilute nitric acid as the oxidizing agent under atmospheric pressure. The oxidation of 4-nitro-o-xylene was effectively promoted by an addition of radical initiators. Under reflux, 2-methyl-4-nitrobenzoic acid was afforded in high yield using nitric acid combined with N-hydroxyphthalimide, cobalt dichloride (CoCl