Efficient removal of 2,4,6-trinitrotoluene (TNT) from industrial/military wastewater using anodic oxidation on boron-doped diamond electrodes.

With growing public concern about water quality particular focus should be placed on organic micropollutants, which are harmful to the environment and people. Hence, the objective of this research is to enhance the security and resilience of water resources by developing an efficient system for reclaiming industrial/military wastewater and protecting recipients from the toxic and cancerogenic explosive compound-2,4,6-trinitrotoluene (TNT), which has been widely distributed in the environment. This research used an anodic oxidation (AO) process on a boron-doped diamond (BDD) electrode for the TNT removal from artificial and real-life matrices: marine water and treated wastewater. During experiments, TNT concentrations were significantly decreased, reaching the anodic degradation efficiency of above 92% within two hours and > 99.9% after six hours of environmental sample treatment. The presented results show the great potential of AO performed on BDD anodes for full-scale application in the industry and military sectors for TNT removal.

Expression of the gene encoding blood coagulation factor VIII without domain B in E. coli bacterial expression system.

In this article, we have demonstrated the feasibility of generating an active form of recombinant blood coagulation factor VIII using an E. coli bacterial expression system as a potential treatment for hemophilia type A. Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. So far, all available recombinant FVIII formulations have been produced using eukaryotic expression systems. Mammalian cells can produce catalytically active proteins with all the necessary posttranslational modifications. However, cultivating such cells is time-consuming and highly expensive, and the amount of the obtained product is usually low. In contrast to eukaryotic cells, bacterial culture is inexpensive and allows the acquisition of large quantities of recombinant proteins in a short time. With this study, we aimed to obtain recombinant blood coagulation factor VIII using the E. coli bacterial expression system, a method not previously explored for this purpose. Our research encompasses the synthesis of blood coagulation factor VIII and its expression in a prokaryotic system. To achieve this, we constructed a prokaryotic expression vector containing a synthetic factor VIII gene, which was then used for the transformation of an E. coli bacterial strain. The protein expression was confirmed by mass spectrometry, and we assessed the stability of the gene construct while determining the optimal growth conditions. The production of blood coagulation factor VIII by the E. coli bacterial strain was carried out on a quarter-technical scale. We established the conditions for isolation, denaturation, and renaturation of the protein, and subsequently confirmed the activity of FVIII.

Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions.

BACKGROUND: Gene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called "trivial overlapping", there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome's novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family. RESULTS: A single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR' protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR' have opposing functions - RepR is crucial for initiation of pIGRK replication, while RepR' is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression. CONCLUSIONS: Regulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Reference Data on Neonatal Serum N-Acetyl-ß-hexosaminidase Activity.

BACKGROUND: Determination of neonate serum's N-acetyl-ß-hexosaminidase (HEX) activity and correlation results with Apgar scale and factors routinely determined in newborn serum. AIMS: Providing reference values of neonates serum HEX activities, and indicate their diagnostic significance. STUDY DESIGN: The study was performed using random serum samples of 111 infants (53 ♂/58 ♀), aged 1-30 days. The activity of HEX was determined colorimetrically and expressed in nKat/L. RESULTS: Serum HEX activity of 111 newborns was 360.5 ± 114.0 nKat/L and significantly positively correlated with gestation week at the day of delivery, birth weight, weight on day of blood collection, sex, and serum CRP. CONCLUSIONS: Reference values presented for neonatal serum activities of HEX may be used in neonatal diagnostics, for example, to detect inflammation and other diseases or for early assessment of the risk of Tay-Sachs and Sandhoff diseases.

Production of highly and broad-range specific monoclonal antibodies against hemagglutinin of H5-subtype avian influenza viruses and their differentiation by mass spectrometry.

BACKGROUND: The highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs. RESULTS: We applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished. CONCLUSIONS: Our work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.

Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

The radiometal makes a difference. Synthesis and preliminary characterisation of DOTA-minigastrin analogue complexes with Ga, Lu and Y.

BACKGROUND: The minigastrin analogue - CP04: DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 has been developed for CCK2R targeting. This analogue can be radiolabelled with 111In or 68Ga for imaging, or with 90Y and 177Lu for therapy. However, affinity of the chelator-peptide conjugates to the cell membrane receptors may vary depending on the metal incorporated into the complex. So far, there are no such studies for the ligands of gastrin/cholecystokinin receptor CCK2R. It is supposed that the reason for the differentiation of receptor affinity to the respective receptors is in the changes of structure of chelating system and their influence on the bioactive conformations of the metal conjugated peptides. Herein, we report on the radiolabeling of CP04 with 90Y, 177Lu and 68Ga and synthesis of cold CP04 complexes with respective stable metals for further structural and physico-chemical and biological studies. MATERIALS AND METHODS: From 200 to 600 MBq of 90Y, 177Lu or 68Ga were used for radiolabelling of 20 µg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5). Non-radioactive complexes with Lu and Ga were synthesized in milligram amounts starting from 0.5 mg up to 5 mg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5) when using 2-molar excess of the metal ions. Complex formation needed 5 min in microwave oven or 12 min in thermo-block at 95°C. RP-HPLC isocratic method (Kinetex 150/4.6 mm; 25% AcN/0.1% TFA, 1 mL/min) with UV/Vis and radiometric detection was developed for investigation of the radiolabelled and "cold" complexes. For LC-MS investigations, HPLC method was modified replacing TFA by formic acid. RESULTS AND DISCUSSION: Yields of CP04 radiolabelling were greater than 90% for all three radionuclides. The HPLC method enabled identification of these radio-complexes based on comparison to their non-radioactive equivalents. In all cases, chromatograms revealed peaks that could be attributed to the metal-CP04 complexes and to impurities (including methionine oxidation). LC-MS analysis of Ga and Lu complexes revealed conformity of the observed molecular ions to the predicted formulas (m/z 2116 and 2220 Da for Ga and Lu, respectively). Different chromatographic behaviour observed for Ga-CP04 complex comparing to Lu- and Y- labelled peptide (relative retention to CP04: 1.08, 0.86 and 0.85, respectively) suggest different coordination of the metal ions. Therefore, further studies are planned using the non-radioactive complexes in order to assess their structural conformations.

pIGWZ12--A cryptic plasmid with a modular structure.

We studied the detailed structure of the cryptic plasmid pIGWZ12, which was isolated from an Escherichia coli strain. pIGWZ12 is composed of two structural modules of distinct evolutionary origin. The REP module, which contains all the features necessary for replication and stable maintenance in the bacterial cell, was assigned by genotyping to the IncF family. The MOB module, which is responsible for plasmid mobilization, shows significant homology to MOBQ modules from broad-host-range plasmids belonging to the RSF1010/R1162 family. We showed that iterons located in the origin of replication are the target for specific binding by the replication initiator protein RepApIGWZ12. Furthermore, we proved that the promoter for the repA gene overlaps with the iterons, and that the latter are the sole determinant of incompatibility. We performed a mutagenesis analysis of the MOBpIGWZ12 module and characterized the roles played by all identified genes (mobA and mobC), as well as the role played by oriT in mobilization. Finally, we showed that it was possible to remove the MOB module from pIGWZ12 without any loss in plasmid replication and stability. Furthermore, the MOBpIGWZ12 module was fully functional after subcloning into another plasmid. Therefore, pIGWZ12 is yet another example of modular structure in small cryptic plasmids.

Magnetic properties of a doped linear polyarylamine bearing a high concentration of coupled spins (S = 1).

Magnetic properties of a doped linear polyarylamine (PA2), whose chain includes alternating para-phenylene and meta-phenylene groups, and of two cyclic and linear model compounds (C2 and D2) were explored by pulsed-EPR nutation spectroscopy, SQUID magnetometry and DFT calculations. Stoichiometrically doped PA2 samples exhibit a pure S = 1 state (exchange coupling constant J = 18 K) with a high spin concentration (0.65) corresponding to 65% of mers bearing holes. Such properties were already observed for doped reticulated polyarylamines but are quite unusual for doped linear polyarylamines. In order to better understand the properties of PA2, model compounds C2 and D2 were also investigated: pure S = 1 spin states could also be obtained, but with higher J (respectively 57 K and 35 K) and, surprisingly, with high but still limited spin concentrations (respectively 0.77 and 0.65).

Optimization of aripiprazole synthesis.

A series of aripiprazole (AR3) syntheses were performed at laboratory scale (10 mmol of the ARI substrate) in order to optimize the amount of another substrate AR2, as well as Na2CO3, ethanol and varying the reaction time. The reaction parameters were chosen according to the D-optimal plans. A high conversion ratio, about 90-99%, was obtained. Purity of crude product (AR3) was determined by HPLC. Molar content of crude reaction product was predicted theoretically with the use of the mass balance and the corresponding HPLC parameters. The theoretical predictions were verified with the potentiometric and thermogravimetric analysis of selected samples. Based on the predicted molar content of reaction mixtures, a series of reaction response surfaces was calculated and optimal set of reaction parameters for aripiprazole synthesis was determined.

[Obstetric outcomes in oxytocin-related and spontaneous deliveries--analysis of 2198 cases]. / Wyniki poloznicze porodów wspomaganych oksytocyna oraz porodów przebiegajacych spontanicznie--analiza 2198 porodów.

AIM: The aim of the work was to compare labour courses, ways of delivery, condition of the newborns in spontaneous and oxytocin-related labours and to analyse the indications for oxytocin administration. MATERIAL AND METHODS: 2198 full-term deliveries (pregnant women qualified for elective caesarean section were excluded from the study) at the Provincial Hospital in Przemysl, Poland. STUDY GROUP: Labours with the adjunctive oxytocin infusion--1102 women. CONTROL GROUP: spontaneous labours (without oxytocin administration)--1096 women. The analysis of the compatibility of measured traits was carried out by the Chi2 test, p < 0.05 was assumed as statistically significant level. RESULTS: I. Indications for the oxytocin administration: secondary hypokinetic contractions of the uterus (642 labours--58.25%), premature rupture of membranes (176 labours - 15.97 %). II. Deliveries by caesarean section: 1. study group--187 women (16.97%). 2. control group--97 women (8.85%). Ch2 = 32.192; df = 1; p = 0.0000. III. Newborns after vaginal labours scored 7 or below according to the Apgar in the first minute after the delivery. 1. study group--35 newborns (8.7%); 2. control group--18 newborns (1.8%) ch2 = 5.493; df = 1; p = 0.0190. IV. Newborns hospitalised for over 48 hours: 1. study group--346 (31.39%); 2. control group--216 newborns (19.70%). Chi2 = 39.454; df = 1; p = 0.0000. CONCLUSIONS: (1) Hypokinetic uterine contractions were the most frequent indication for oxytocin administration during labour. (2) Oxytocin administration increases twice the risk of delivery by the caesarean section. (3) Newborns after vaginal oxytocin-related labours scored 7 or below on the Apgar score in the first minute after the birth when compared to the newborns after spontaneous labour. (4) Oxytocin administration during parturition elongates the time of newborns hospitalisation.

Structural studies on aryl-substituted enaminoketones and their thio analogues. Part II. Experimental and DFT calculated carbon-carbon coupling constants, (n)J(CC)'s (n = 1-3).

Spin-spin carbon-carbon coupling constants across one, two and three bonds, J(CC), have been measured for a series of aryl-substituted Z-s-Z-s-E enaminoketones and their thio analogues. As a result, a large set, altogether 178, of J(CC)s has been obtained. It consists of 82 couplings across one bond, 31 couplings across two bonds and 65 couplings across three bonds. Independently, the DFT calculations at the B3PW91/6-311++G(d,p)//B3PW91/6-311++G(d,p) level yielded a set of theoretical J(CC) values. A comparison of these two sets of data gave an excellent linear correlation with parameters a and b close to ideal; a = 0.9978 which is not far from unity and b = 0.22 Hz which is close to zero. The (1)J(CC) couplings determined for the crucial fragment of the molecules, i.e. -C=C-C=O (or -C=C-C=S), are: (1)J(C=C) approximately 68 Hz (67 Hz) and (1)J(C-C) = 60.5 Hz (60.0 Hz). The corresponding couplings found for the Z-s-Z-s-E isomer of the parent enaminoketone, 4-methylamino-but-3-en-2-one are 64.1 and 59.3 Hz, respectively. The most sensitive towards substitution of the oxygen atom by sulfur are two-bond couplings between the alpha-vinylic and aromatic C(ipso) carbon atoms, which attain 12 Hz in the enaminoketone derivatives and decrease to 5 Hz in their thio analogues.

Structural studies on aryl-substituted enaminoketones and their thio analogues. Part I. Analysis of high-resolution (1)H, (13)C NMR and (13)C CP MAS spectra combined with GIAO-DFT calculations.

A series of aryl-substituted enaminoketones and their thio analogues in CDCl(3) solution and in the solid state were studied by the use of high-resolution (1)H and (13)C as well as (13)C cross polarization magic angle spinning (CP MAS) NMR spectra in combination with gauge including atomic orbitals-density functional theory (GIAO-DFT) calculations performed at the B3PW91/6-311 + + G(d,p) level of theory using the B3PW91/6-311 + + G(d,p)-optimized geometries. The analysis of the (13)C NMR spectra in solution was done by using the Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) technique, whereas trends observed in the (13)C shielding constants, calculated for the compounds studied, were a great help in assigning most of the signals in the (13)C CP MAS NMR spectra. It was established on the basis of the experimental and theoretical NMR data that both groups of compounds exist in the form of Z-s-Z-s-E isomers in CDCl(3) solution as well as in the solid state, with the NH hydrogen atom involved in intramolecular hydrogen bonding. This conclusion is in agreement with the fact that some of the compounds studied reveal liquid-crystalline properties. Three-bond H, H and C, H coupling constants measured in solution played a crucial role in the structure elucidation.

Hemodynamic parameters following bilateral internal iliac arteries ligation as a treatment of intrapartum hemorrhage.

OBJECTIVE: The internal iliac arteries ligation (IIAL) is a particularly effective method, maintaining fertility, of dealing with intrapartum hemorrhage. AIM: Hemodynamic evaluation of the ovarian arteries(OA) and uterine arteries (UA) in patients after IIAL. MATERIAL: Study Group consisted of 6 women who underwent IIAL to treat intrapartum hemorrhage--without hysterectomy. Control Group consisted of 6 women, at the same age group, parity and time after delivery, who did not undergo IIAL. METHOD: Perfusion characteristics were studied by means of a transvaginal Doppler system. Resistance index (RI), pulsatility index (PI) and systolic/diastolic ratio (S/D) were measured in the uterine and ovarian arteries. Nonparametric comparison of the two groups was performed with the help of Two-sample Wilcoxon rank-sum (Mann-Whitney) test. RESULTS 1. Change of perfusion in OA-PI: 1.40 vs. 3.76 Prob < 0.05; RI: 0.86 vs. 0.91 Prob > 0.05; S/D: 3.25 vs. 18.2 Prob < 0.05. 2. Change of perfusion in UA-PI: 2.20 vs. 2.75 Prob > 0.05; RI 0.82 vs. 0.86 Prob > 0.05; SID: 5.28 vs. 7.81 Prob > 0.05. CONCLUSIONS: 1. IIAL as a way of treating intrapartum haemorrhage, causes the decrease of pulsatility index (PI) and systolic/diastolic ratio (S/D) in ovarian arteries. 2. Characteristic changes of PI, RI and S/D parameters in uterine arteries after IIAL have not been observed. 3. Changes of ovarian flow velocity parameters suggest the possibility of changes in the ovarian function.