TK Group Research Articles

Fermentation characteristics of tofu with kimchi seasoning

Tofu (T) was fermented with kimchi seasoning (K) at a ratio of 1:0.5 and 1:1 (w/w) without and with a starter (L) (designated 1:0.5 TK, 1:0.5 TK-L, 1:1 TK and 1:1 TK-L) to utilize as a new type of lactic acid fermented food. A starter strain KL-12 was isolated from kimchi and identified to belong to the Lactobacillus plantarum group. During fermentation of four TK groups for 16 weeks at 20 °C, pH decreased from initial 6.4–6.5 to 4.1–4.6 after 16 weeks, with a remarkable decrease after 1 week. A reversible trend was observed with LAB viable cells. Antioxidant activities of four TK groups increased with fermentation time. After 16 weeks, the 1:1 TK-L group showed higher antioxidant activities (total phenolic content, DPPH and NO scavenging activity) and free amino acids than other three groups. These results indicate the potential use of lactic acid fermented food as a valuable resource for development of functional foods.

Revised classification of kinases based on bioactivity data: the importance of data density and choice of visualization

Kinases are a major class of drug targets and are involved in a variety of diseases such as diabetes, cancer and inflammation. Still, understanding kinase inhibitor selectivity and promiscuity remains a major challenge. In order to improve upon the current situation, we analyzed a dataset comprising 157 compounds, tested at concentrations of 1 μM and 10 μM against a panel of 225 human protein kinases. Our bioactivity-based classification of kinases shows similarities with the Sugen sequence-based classification [1], where particularly kinases from the TK, CDK, CLK and AGC groups cluster together. However, 57% of all kinase pairs inhibited by 6 known inhibitors consist of kinases which lie far apart from each other in the Sugen tree (relative distance of 0.6 - 0.8 on a scale from 0 to 1), but are correctly located closer to each other in our bioactivity-based tree (distance 0 - 0.4). For 80% of all analyzed kinases, those classified as neighbors according to the bioactivity-based classification also show high similarity in shared active compounds. However, among the remaining ~20%, distant kinases did not necessarily show low SAR similarity, and neighboring kinases did not necessarily show high SAR similarity; i.e., the placement in the tree was misleading. We identified two reasons for this: firstly, 'misplaced' kinases exhibit inconsistent SAR, and secondly, these kinases had only a few shared activities with other kinases, making both the computation of their bioactivity-based distance and their place in the tree less accurate. In a follow-up analysis, we resolved both problems by visualizing inconsistent SAR more accurately using MDS plots, rather than phylogenetic trees, and by excluding kinases with 16 or fewer shared activities. Only 7 kinases (4% of the kinases analyzed) did not show a clear relationship between kinase bioactivity profile similarity and shared active compounds. Hence, this analysis improves on previous studies, where the influence of data density on kinase similarity was not considered, and leads to a more reliable placement of kinases into the kinome tree. Overall, our analysis suggests that bioactivity-based classification of kinases is indeed more useful than sequence-based classification for predicting kinase-inhibitor interactions. However, care needs to be taken with respect to data density (i.e., kinases with too few data points need to be omitted) and visualization of the data (i.e., phylogenetic trees imply a neighborhood relationship that is not consistently observed in every case).

Immune Reconstitution and Immune Correlates of Clinical Outcome IN Acute LEUKEMIA PATIENTS Treated with Haploidentical STEM CELL TRANSPLANTATION.

Abstract 46The broader application of haploidentical stem cell transplantation (haplo-HCT), is limited by the delayed immune reconstitution (IR) secondary to the procedures for GvHD prophylaxis. This ultimately results in a high-rate of infectious complications and non-relapse mortality. We dynamically analyzed immunoreconstitution (IR) in patients undergoing haplo-HCT for acute leukemias enrolled in two different phase I-II clinical trials aimed at improving IR. In the first trial (TK007), 28 patients (out of 50 enrolled) received suicide-gene transduced donor T cells at day +42 after a T-cell depleted graft, in the absence of post-transplant immunosuppression. In the second trial (TrRaMM), 40 patients received an unmanipulated graft and a rapamycin-based GvHD prophylaxis. T-cell immune reconstitution was more rapid in TrRaMM than in TK007 patients, with a threshold of CD3 cells>100/μl reached at days +30 and +90, respectively. In both trials IR was mainly composed of Th1/Tc1 lymphocytes with an inverted CD4/CD8 ratio. While in TrRaMM patients we observed an early expansion of naïve and central memory T cells, producing high amounts of IL-2, in TK patients IR was mainly composed of activated effectors. Furthermore, in TrRaMM patients we detected high levels of CD4+CD25+CD127- T regulatory cells (up to 15% of circulating T lymphocytes) that persisted after rapamycin withdrawal, and was significantly superior to that observed in TK patients and in healthy controls. Interestingly, in contrast to the different kinetics of T-cell reconstitution, no differences were observed in time required to gain protective levels of CMV-specific T cells, as shown by ψIFN ELISPOT analysis. Protective frequencies of CMV-specific lymphocytes were observed 3 months after HCT in both groups, a time-point that in TrRaMM patients corresponds to the average time of rapamycin withdrawal. In both trials the number of circulating CMV-specific T cells was inversely correlated to the number and severity of subsequent CMV reactivations and days of antiviral therapy. GvHD was diagnosed in 16 TrRaMM patients (40%) and in 10 TK patients (35% of patients who received TK cells). Severity of GvHD was different in the two cohort of patients with 5 TrRaMM patients (12,5%) and only 2 TK patients (7%) with grade III-IV GvHD. Of interest, in the TrRaMM group CMV-specific immunity was significantly hampered by the immunosuppressive treatment required to treat GvHD. On the contrary, in the TK group, the administration of ganciclovir was able to activate the suicide machinery and control GvHD without impairing viral-specific T-cell immunocompetence. These results matched with the kinetics of CMV reactivations. We observed that while in TrRaMM patients 80% of viral reactivations occurred after the immunosuppressive therapy, in TK patients no significant differences could be assessed before and after therapy. IFN-ψ ELISPOT might thus be a relevant and predictive test to guide patient-specific clinical monitoring and antiviral treatment. Overall, these results show that early immune reconstitution can be promoted in haplo-HCT by different strategies associated with a wide range of alloreactive potential. The risks and benefits associated with alloreactivity should guide the therapeutic choice tuned on patient disease status and co-morbidities. Disclosures:Bordignon:Molmed Spa: Employment.

Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

BackgroundCompared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors.MethodsA recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue.ResultsPlasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo.ConclusionOur results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for gene therapy in ovarian cancer.

Ngā Nekehanga o te Whakahua i te Reo Māori i roto i te Rautau kua Hipa nei

Māori and English have been in increasing contact within New Zealand for over 200 years. The impact of each language on the other in vocabulary, which has been borrowed in both directions, is clear. More subtle is the mutual influence in the area of pronunciation. The MAONZE project has been investigating changes in the pronunciation of Māori and of the English of speakers of Māori, using recordings of seven kaumātua born in the 19th century who were interviewed by the Mobile Unit of Radio New Zealand in the 1940s (referred to in the text as the TK group), of ten kaumātua alive today (K), and of ten younger speakers (T). This paper describes our methodology and reports the results of the investigation of the long and short vowels of Māori as well as of some diphthongs. These were all studied acoustically, that is to say, using computers to analyse the sound waves characteristic of each sound. For each vowel and diphthong, and for each speaker, we attempted to analyse thirty examples, though some sounds are relatively rare, such as /ī/ and /ao/, so that this target could not always be achieved. The analysis did indeed find that there had been shifts in the pronunciation of these sounds over the three generations of speakers in the study. Except for the distinction between /a/ and /ā/, the length and qualitative difference between the historical long and short vowels is decreasing. Most striking is the shift in /u/ and /ū/ to a much more forward position in the mouth, a change which parallels movement in English. The five diphthongs studied were /au, ou, ao, ai, ae/, all of which are clearly distinct in the speech of the TK and K groups, but which are tending to merge to three in the younger group, who no longer distinguish tae and tai or hau and hou so clearly. Within the T group, we distinguished between those who had been speaking Māori since birth (R1), and those who had acquired good Māori at school or later (R2). It turned out that in all the changes taking place, the R2-T group is leading the way, and is thus probably the direction the language will take into the future, particularly as the majority of younger speakers of Māori these days belong to this category.

IGF‐I response to tourniquet‐induced injury in young and old mice

The age-associated decline in skeletal muscle’s ability to regenerate is a concern among elderly patients of limb injury or orthopedic surgery. Changes in insulin-like growth factor-I (IGF-I) response have been implicated in this phenomenon. Previously, our laboratory has shown a drastic increase in IGF-I mRNA 7 days following tourniquet (TK) induced damage in adult rats, a response not seen in elderly rats. This study investigated the short-term time course of IGF-I expression in adult (6 month-old) and elderly (24 month-old) mice following 1, 3, 5, and 7-day recovery from 2 hour TK induced ischemia/reperfusion injury. RT-PCR analysis reveals an increase in IGF-I expression in both TK groups. Levels peaked ~20-fold 3 days post-injury in adult and ~8-fold 5 days post-injury in elderly subjects. Adult expression decreased sharply at day 5 and rose by day 7, suggesting the differential, temporal-specific expression of the IGF-I Ea and Eb isoforms in injury repair described by Goldspink et al. The early IGF-I response seen in adult subjects was not present in elderly subjects.

Convection-Enhanced Delivery of Adeno-Associated Virus Type 2 (AAV2) into the Striatum and Transport of AAV2 Within Monkey Brain

Adeno-associated virus type 2 (AAV2)-based vectors are promising transgene carriers for experimental gene therapy treatments of brain diseases. However, detailed evaluation of transgene distribution, trafficking, and transport within the brain is of the utmost importance before applying any type of gene therapy in humans. We examined the distribution of AAV2-thymidine kinase (AAV2-TK) and AAV2-aromatic L-amino acid decarboxylase (AAV2-AADC) in monkey brain after convection-enhanced delivery (CED). The AADC group consisted of two 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys that received unilateral infusions of AAV2-AADC into six sites in the right hemisphere. The TK group consisted of three monkeys that received bilateral CED infusion of AAV2-TK into the putamen; one side in all three monkeys was coinfused with heparin. Six weeks after AAV delivery, the brains were collected and processed for immunohistochemical staining. Volumetric measurement of TK distribution showed that at least 75% of the putamen could be covered by a single infusion of the vector; however, no effects of heparin coadministration were found, most likely because of the already robust gene transfer achieved by CED. Interestingly, TK- and AADCimmunoreactive cells were also present outside the striatum, in the globus pallidus, subthalamic nucleus, thalamus, and substantia nigra. CED proved to be an efficient method for delivery of the AAV2 vector. Detection of the transgenes in brain structures distant from the site of injection emphasizes the potential for gene transport, and the advantages and disadvantages of CED for gene therapy deserve further study.

Functional identification of kinases essential for T-cell activation through a genetic suppression screen

Activation of T-cells by antigens initiates a complex series of signal-transduction events that are critical for immune responses. While kinases are key mediators of signal transduction networks, several of which have been well characterized in T-cell activation, the functional roles of other kinases remain poorly defined. To address this deficiency, we developed a genetic screen to survey the functional roles of kinases in antigen mediated T-cell activation. A retroviral library was constructed that expressed genetic suppressor elements (GSEs) comprised of peptides and antisense nucleotides derived from kinase cDNAs including members of the STE, CAMK, AGC, CMGC, RGC, TK, TKL, Atypical, and Lipid kinase groups. The retroviral library was expressed in Jurkat T-cells and analyzed for their effect on T-cell activation as monitored by CD69 expression. Jurkat cells were activated by antigen presenting cells treated with superantigen, and sorted for a CD69 negative phenotype by flow cytometry. We identified 19 protein kinases that were previously implicated in T-cell signaling processes and 12 kinases that were not previously linked to T-cell activation. To further validate our approach, we characterized the role of the protein kinase MAP4K4 that was identified in the screen. siRNA studies showed a role for MAP4K4 in antigen mediated T-cell responses in Jurkat and primary T-cells. In addition, by analyzing multiple promoter elements using reporter assays, we have shown that MAP4K4 is implicated in the activation of the TNF-α promoter. Our results suggest that this methodology could be used to survey the function of the entire kinome in T-cell activation.

Wear of tool electrodes in electromachining

1. A study was made of the wear of VK and TK type sintered hard alloys during the electromachining of Armco iron and steel 3, 45, U8, and ShKh15 steels. 2. It was established that, generally speaking, the highest wear resistance is exhibited by the VK group of hard alloys, whose wear is essentially due to adhesion. It is postulated that TK group hard alloys wear out as a result of interdiffusion between the tool electrode material and the steels being machined. 3. The relationship was examined between tool electrode wear and the ratios of the elastic moduli and thermal conductivities of the tool and workpiece materials. It was established that, as the ratios of these characteristics increase, the wear of hard alloys becomes greater. 4. A study was made of the wear of tool plates as a function of the carbon content of steels. It was found that the wear surface area appreciably increases only up to a certain carbon content (0.8% C), markedly decreasing for ShKh15 steel.