Pilot-Scale Lactic Acid Production via Batch Culturing of Lactobacillus sp. RKY2 Using Corn Steep Liquor As a Nitrogen Source

U ovom se istraživanju pokušala odrediti učinkovitost proizvodnje mliječne kiseline uzgojem soja Lactobacillus sp. RKY2 u pilot-postrojenju uporabom kukuruznog ekstrakta kao izvora dušika. Količina mliječne kiseline nakon fermentacije, rast stanica, prinos i produktivnost nisu se bitno promijenili dodatkom čiste glukoze pri povećanju obujma procesa s 2,5 na 30 i 300 L. U svim pokusima udio mliječne kiseline linearno se povećavao s porastom početne koncentracije glukoze. U pokusu s hidrolizatom drva smanjili su se produktivnost mliječne kiseline i rast stanica s povećanjem obujma procesne opreme, zbog toksičnih kemikalija iz hidrolizata. Međutim, u svim je pokusima prinos mliječne kiseline bio veci od 90 % bez obzira na utrošak glukoze. Zaključeno je da je mliječna kiselina uspješno proizvedena u pilot-postrojenju upotrijebljenom u ovom istraživanju.In this study, the determination of the efficiency of a pilot-scale fermentation process using corn steep liquor as a nitrogen source was attempted in order to produce lactic acid via batch culturing of Lactobacillus sp. RKY2. Using pure glucose, fermentation efficiency characteristics, such as final lactic acid, cell growth, yield, and productivity were not substantially influenced by the scale-up of the laboratory-scale fermentation from 2.5- to 30- and 300-litre scale fermentations. In all experiments, the content of lactic acid produced increased in a linear fashion with increases in the initial glucose concentration. In the experiments using wood hydrolyzate, both lactic acid productivity and cell growth were decreased as a result of the scaling-up of the fermentation. This might be attributed to the toxic chemicals contained in the wood hydrolyzates. However, in all experiments, lactic acid yields remained higher than 90 % with regard to the amount of glucose consumed. Therefore, lactic acid was successfully produced by the pilot-scale bioreactor scheme adopted in this study

Pilot-Scale Lactic Acid Production via Batch Culturing of Lactobacillus sp. RKY2 Using Corn Steep Liquor As a Nitrogen Source

U ovom se istraživanju pokušala odrediti učinkovitost proizvodnje mliječne kiseline uzgojem soja Lactobacillus sp. RKY2 u pilot-postrojenju uporabom kukuruznog ekstrakta kao izvora dušika. Količina mliječne kiseline nakon fermentacije, rast stanica, prinos i produktivnost nisu se bitno promijenili dodatkom čiste glukoze pri povećanju obujma procesa s 2,5 na 30 i 300 L. U svim pokusima udio mliječne kiseline linearno se povećavao s porastom početne koncentracije glukoze. U pokusu s hidrolizatom drva smanjili su se produktivnost mliječne kiseline i rast stanica s povećanjem obujma procesne opreme, zbog toksičnih kemikalija iz hidrolizata. Međutim, u svim je pokusima prinos mliječne kiseline bio veci od 90 % bez obzira na utrošak glukoze. Zaključeno je da je mliječna kiselina uspješno proizvedena u pilot-postrojenju upotrijebljenom u ovom istraživanju.In this study, the determination of the efficiency of a pilot-scale fermentation process using corn steep liquor as a nitrogen source was attempted in order to produce lactic acid via batch culturing of Lactobacillus sp. RKY2. Using pure glucose, fermentation efficiency characteristics, such as final lactic acid, cell growth, yield, and productivity were not substantially influenced by the scale-up of the laboratory-scale fermentation from 2.5- to 30- and 300-litre scale fermentations. In all experiments, the content of lactic acid produced increased in a linear fashion with increases in the initial glucose concentration. In the experiments using wood hydrolyzate, both lactic acid productivity and cell growth were decreased as a result of the scaling-up of the fermentation. This might be attributed to the toxic chemicals contained in the wood hydrolyzates. However, in all experiments, lactic acid yields remained higher than 90 % with regard to the amount of glucose consumed. Therefore, lactic acid was successfully produced by the pilot-scale bioreactor scheme adopted in this study

Microporation is a valuable transfection method for efficient gene delivery into human umbilical cord blood-derived mesenchymal stem cells

<p>Abstract</p> <p>Background</p> <p>Mesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.</p> <p>Results</p> <p>Using microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks <it>in vitro </it>and <it>in vivo</it>. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their <it>in vitro </it>differentiation into neural cells.</p> <p>Conclusion</p> <p>Taken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.</p

Levodopa-carbidopa-entacapone overdose presenting as altered mental status, xanthoderma, and yellowish sclera

Levodopa-carbidopa-entacapone is a single combination drug consisting of levodopa (aromatic amino acid), carbidopa (dopa-decarboxylase inhibitor), and entacapone (catechol-O-methyltransferase inhibitor). The Food and Drug Administration approved levodopa-carbidopa-entacapone in 2003, as treatment for idiopathic Parkinson’s disease in patients experiencing signs and symptoms of wearing-off. Although various adverse drug reactions of levodopa-carbidopa-entacapone have been recorded, there has been no reported case of levodopa-carbidopa-entacapone overdose. We report the first case of signs and symptoms of an overdose of levodopa-carbidopa-entacapone (levodopa: 3000 mg; carbidopa: 750 mg; entacapone: 6000 mg) in a suicide attempt, presenting as altered mentality, xanthoderma, and yellowish sclera without hyperbilirubinemia

Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells

Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion

Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2 kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37 kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle

The Korean Organ Transplantation Registry (KOTRY): an overview and summary of the kidney-transplant cohort

Background As the need for a nationwide organ-transplant registry emerged, a prospective registry, the Korean Organ Transplantation Registry (KOTRY), was initiated in 2014. Here, we present baseline characteristics and outcomes of the kidney-transplant cohort for 2014 through 2019. Methods The KOTRY consists of five organ-transplant cohorts (kidney, liver, lung, heart, and pancreas). Data and samples were prospectively collected from transplant recipients and donors at baseline and follow-up visits; and epidemiological trends, allograft outcomes, and patient outcomes, such as posttransplant complications, comorbidities, and mortality, were analyzed. Results From 2014 to 2019, there were a total of 6,129 registered kidney transplants (64.8% with living donors and 35.2% with deceased donors) with a mean recipient age of 49.4 ± 11.5 years, and 59.7% were male. ABO-incompatible transplants totaled 17.4% of all transplants, and 15.0% of transplants were preemptive. The overall 1- and 5-year patient survival rates were 98.4% and 95.8%, respectively, and the 1- and 5-year graft survival rates were 97.1% and 90.5%, respectively. During a mean follow-up of 3.8 years, biopsy-proven acute rejection episodes occurred in 17.0% of cases. The mean age of donors was 47.3 ± 12.9 years, and 52.6% were male. Among living donors, the largest category of donors was spouses, while, among deceased donors, 31.2% were expanded-criteria donors. The mean serum creatinine concentrations of living donors were 0.78 ± 0.62 mg/dL and 1.09 ± 0.24 mg/dL at baseline and 1 year after kidney transplantation, respectively. Conclusion The KOTRY, a systematic Korean transplant cohort, can serve as a valuable epidemiological database of Korean kidney transplants

Neurotoxicity Screening in a Multipotent Neural Stem Cell Line Established from the Mouse Brain

Neural stem cells (NSCs) have mainly been applied to neurodegeneration in some medically intractable neurologic diseases. In this study, we established a novel NSC line and investigated the cytotoxic responses of NSCs to exogenous neurotoxicants, glutamates and reactive oxygen species (ROS). A multipotent NSC line, B2A1 cells, was established from long-term primary cultures of oligodendrocyte-enriched cells from an adult BALB/c mouse brain. B2A1 cells could be differentiated into neuronal, astrocytic and oligodendroglial lineages. The cells also expressed genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. B2A1 cells treated with hydrogen peroxide and L-buthionine-(S,R)-sulfoximine underwent 30-40% cell death, while B2A1 cells treated with glutamate and kainate showed 25-35% cell death. Cytopathologic changes consisting of swollen cell bodies, loss of cytoplasmic processes, and nuclear chromatin disintegration, developed after exposure to both ROS and excitotoxic chemicals. These results suggest that B2A1 cells may be useful in the study of NSC biology and may constitute an effective neurotoxicity screening system for ROS and excitotoxic chemicals

Translational and transcriptional control of Sp1 against ischaemia through a hydrogen peroxide-activated internal ribosomal entry site pathway

The exact mechanism underlying increases in Sp1 and the physiological consequences thereafter remains unknown. In rat primary cortical neurons, oxygen-glucose deprivation (OGD) causes an increase in H2O2 as well as Sp1 in early ischaemia but apparently does not change mRNA level or Sp1 stability. We hereby identified a longer 5′-UTR in Sp1 mRNA that contains an internal ribosome entry site (IRES) that regulates rapid and efficient translation of existing mRNAs. By using polysomal fragmentation and bicistronic luciferase assays, we found that H2O2 activates IRES-dependent translation. Thus, H2O2 or tempol, a superoxide dismutase-mimetic, increases Sp1 levels in OGD-treated neurons. Further, early-expressed Sp1 binds to Sp1 promoter to cause a late rise in Sp1 in a feed-forward manner. Short hairpin RNA against Sp1 exacerbates OGD-induced apoptosis in primary neurons. While Sp1 levels increase in the cortex in a rat model of stroke, inhibition of Sp1 binding leads to enhanced apoptosis and cortical injury. These results demonstrate that neurons can use H2O2 as a signalling molecule to quickly induce Sp1 translation through an IRES-dependent translation pathway that, in cooperation with a late rise in Sp1 via feed-forward transcriptional activation, protects neurons against ischaemic damage