Genome-level insights into the operation of an on-site biological wastewater treatment unit reveal the importance of storage time.

On-site wastewater treatment systems are gaining popularity in areas where centralized wastewater treatment is not available. In the current case study a domestic activated sludge system was investigated, where treated effluent was stored in a short-term (1 week turn-over time) and a long-term (over 2-3 months) storage tank and was then used for irrigation. This design provided a unique opportunity to assess the chemical and microbial changes of the effluent upon storage. Long-term storage greatly improved both the chemical quality and the degradation efficiency of most organic micropollutants examined, including petroleum hydrocarbons and the pesticide diethyltoluamide. Taxonomic profile of the core microbiome of the effluent was also influenced upon storage. Relative abundance values of the members of Azoarcus and Thauera genera, which are important in degrading polycyclic aromatic hydrocarbons compounds, clearly indicated the biodegrading activity of these microbes across samples. The abundance of xenobiotics degradation functions correlated with the observed organic micropollutant degradation values indicating efficient microbial decomposition of these contaminants. Functions related to infectious diseases also had the highest abundance in the short-term storage tank corresponding well with the relative abundance of indicator organisms and implying to the significance of storage time in the elimination of pathogens. Based on these results, small, on-site wastewater treatment systems could benefit from long-term storage of wastewater effluent.

The complete genome sequence of Ensifer meliloti strain CCMM B554 (FSM-MA), a highly effective nitrogen-fixing microsymbiont of Medicago truncatula Gaertn.

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA.

Enhanced International Prognostic Index (NCCN-IPI), Charlson Comorbidity Index and absolute lymphocyte count as predictors for survival of elderly patients with diffuse large B cell lymphoma treated by immunochemotherapy.

Diffuse large B cell lymphoma (DLBCL) affects more commonly patients over 60 years. These patients have vast number of comorbidities which can modify survival as well as other clinical parameters. The aim of this study was to evaluate prognostic significance of the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI), absolute lymphocyte count (ALC), absolute monocyte count (AMC), lymphocyte-to-monocyte ratio (LMR) and comorbidities expressed with Charlson Comorbidity Index (CCI). A total of 182 DLBCL patients 60 years old and older were included, focusing on whole group and patients older than 70. All patients were treated with immunochemotherapy.Overall treatment response was achieved in 84.6% of patients. The NCCN-IPI was of highly prognostic value in the analyzed group (p<0.0001). Survival analysis showed that ALC>1.1x109/L, AMC≤0.59x109/L, and LMR>2.8 were associated with more favorable outcome (p=0.029, p=0.019, p=0.028, respectively). The patients with CCI≥2 had poorer outcome (p=0.008) compared to the patients with CCI 0-1. Multivariate analysis showed that among ALC, AMC, LMR, NCCN-IPI and CCI, the NCCN-IPI was the critical parameter that significantly affected survival (p<0.0001). Furthermore, comorbidities were also valuable independent factors which influenced survival (p=0.031) as well as the ALC (p=0.024). In elderly DLBCL patients, NCCN-IPI and ALC proved their prognostic validity, while poorer outcome could be expected in older patients with high CCI (≥2). Furthermore, mentioned prognostic parameters retained their prognostic value in the group of patients older than 70.

Responses of Acilius sulcatus (Coleoptera: Dytiscidae) to chemical cues from perch (Perca fluviatilis).

In this study we tested the hypothesis that the presence of chemical stimuli from a hungry predator would initiate anti-predator responses, while stimuli from a satiated predator would not. We used chemical stimuli released from starved perch (Perca fluviatilis) and from satiated perch (predator). As prey we used adult Acilius sulcatus (Coleoptera: Dytiscidae). The reaction of the beetles to different predator conditions was tested during daytime. We also tested the reaction to starved perch during the night. A. sulcatus activity decreased when it was exposed to stimuli released from starved perch during daytime when visibility was poor, due to the presence of artificial vegetation. There was, however, no reaction to satiated perch under the same experimental conditions. These results indicate that A. sulcatus can discriminate between chemical cues from hungry and satiated fish predators. When visibility was good and the concentration of chemical cues was constant, the beetles did not react to starved perch in the daytime, but their activity decreased at night in response to stimuli released from starved perch. Visual as well as chemical cues seem to be important for detecting a potential predator. When visibility is good, beetles seem to rely on visual stimuli, while in darkness they seem to use chemical stimuli to detect the presence of predators.

Trypsin complexed with alpha 1-proteinase inhibitor has an increased structural flexibility.

Mutant rat trypsin Asp189Ser was prepared and complexed with highly purified human alpha 1-proteinase inhibitor. The complex formed was purified to homogeneity and studied by N-terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with alpha 1-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg117-Val118 one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.

Paracrystalline assemblies of light meromyosins with various chain weights.

Paracrystals formed from well defined insoluble fragments of myosin rod: LMM-A, LMM-B, LMM-C, and LMM-D with apparent chain weights of 78 000, 72 000, 68 000, and 56 000, respectively (Nyitray et al., 1983) were studied in the electron microscope with a negative staining technique. All fragments formed tactoids with 14.3 and 43 nm periodicities as well as aperiodic tactoids and sheets. Tactoids and sheets described earlier with a 43 nm periodicity and a pattern of alternating light bands 10 nm wide and dark bands 33 nm wide were observed in LMM-A preparations only. LMM-B and LMM-C formed tactoids with a 43 nm periodicity but without the diversified band pattern. LMM-D formed sheets and tactoids with a newly observed band pattern of alternating light bands 23 nm wide and dark bands 20 nm wide. This pattern can be explained assuming the length of LMM-D molecules to be 66 nm which is fairly consistent with the chain weight of this fragment. A model for molecular arrangement in this type of paracrystal is presented. The model involves both parallel and antiparallel interactions with a parallel axial displacement of the molecules by 43 nm as suggested by Bennett (1981) for paracrystals formed from LMM molecules 90 nm long. It is deduced from the model that LMM-D is shorter than LMM-A by 15 nm at the NH2-terminal end and by 9 nm at the COOH-terminal end. LMM-D, like the other insoluble fragments of myosin rod, is also able to form square and hexagonal nets with an approximately 40 nm distance between lattice points. The structural features of the nets obtained from LMM-D can be explained assuming the same kinds of molecular interactions within the strands of the net as those in the sheets and tactoids with a 43 nm axial repeat. It is concluded that all insoluble fragments of myosin rod are able to form paracrystalline assemblies involving the same types of parallel and antiparallel interactions.

Localization of a new proteolytic site accessible in oxidized myosin rod.

We have compared the proteolysis pattern of reduced and oxidized myosin rods in which the five pairs of SH-groups were interchain crosslinked by employing CuCl2 or 5,5'-dithiobis-2-nitrobenzoate. In the tryptic digest of oxidized rod three new fragments appeared on SDS-polyacrylamide gel electrophoresis (chain masses of 100, 45, and 25 kDa). Based on the N-terminal sequences of the isolated peptides, it is concluded that oxidation creates a new cleavage site 102 residues away from the N-terminus of the rod, in the vicinity of one of the modified SH-groups (Cys-108). This observation indicates that S-S crosslinking of myosin rod leads to a local unfolding of the coiled-coil structure.

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1.

The kinetics of tryptic breakdown of the heavy chain of chymotryptic myosin subfragment 1 (S1) according to the following scheme (where the numbers represent approximate masses in kDa) are altered at 21 degrees C by divalent (Formula: see text) cations (Me2+) and by ATP, ADP, adenosine 5'-[beta, gamma-imino]triphosphate or PPi, with or without Me2+. ATP or its analogs slow step 2 and accelerate steps 3 and 4, while Me2+ accelerates step 2. ATP and its analogs decrease the amount of a transient 27-kDa peptide [Hozumi, T. & Muhlrad, A. (1981) Biochemistry 20, 2945-2950]. We have found direct evidence for the suggestion in this reference that the 27-kDa peptide is not an obligatory precursor of the 25-kDa fragment and that ATP or ADP suppresses the formation of the larger N-terminal fragment rather than accelerates its breakdown. Cross-linking of sulfhydryl groups located in the 20-kDa fragment leads to trapping of MgADP in the N-terminal 25-kDa peptide [Wells, J.A. & Yount, R.G. (1980) Biochemistry 19, 1711-1717]; this process affects the tryptic fragmentation of S1 similarly to, but less effectively than, nucleotides. Acts-S1 formation prevents the effect of ATP on fragmentation. At 37 degrees C S1 loses ATPase activity; tryptic digestion proceeds more rapidly and the 50-kDa and 25-kDa fragments are degraded to small peptides. Nucleotides protect against the effects of higher temperature by producing conformational changes not only in the 27-kDa N-terminal portion (containing the putative nucleotide binding site) of the heavy chain of S1 but also in the 50-kDa peptide.

Use of cationic detergents for polyacrylamide gel electrophoresis in multiphasic buffer systems.

An improved system for polyacrylamide gel electrophoresis in the presence of cationic detergents, cetyltrimethylammonium bromide and cetylpyridinium chloride, respectively, is described. An acidic discontinuous buffer system generated according to the theory of multiphasic zone electrophoresis developed by T. M. Jovin (1973, Biochemistry 12, 871-904) was used. It was optimized with respect to the operational conditions and to the desirable range of relative mobility values for the proteins that have molecular weights from 16,500 to 90,300. Also presented is a procedure for the elimination of interference from cationic detergents frequently encountered during staining of gels. The electrophoretic system was suitable for fractionating a wide variety of proteins. The technique can also be used to provide an alternative estimate of molecular weight. To fully account for accurate estimations, the Ferguson relationship between mobility and gel concentration and the relation of molecular weight to mobility at a single gel concentration were both considered. Examples reported in this paper include the separation and/or molecular weight determination of several common proteins, histones, and microfibrillar and myofibrillar proteins. The results suggest that electrophoresis in the presence of cationic detergents offers the same degree of reliability in analysis of most proteins as is provided by the anionic detergent sodium dodecyl sulfate electrophoresis.

The proteolytic substructure of light meromyosin. Localization of a region responsible for the low ionic strength insolubility of myosin.

Light meromyosin (LMM), prepared by limited tryptic digestion of myosin, usually contains several polypeptide chains, LMM-A, LMM-B, and LMM-C in decreasing order of molecular weight estimated from sodium dodecyl sulfate-gel electrophoresis. Further limited tryptic digestion of LMM produces well defined fragments (Balint, M., Szilagyi, L., Fekete, Gy., Blazso, M., and Biro, E. N. A. J. Mol. Biol. (1968) 37, 317-330). Fragments LF-1, LMM-D, LF-2, and LF-3, with chain masses equal to 63, 56, 47, and 30 kDa, respectively, have been isolated by column chromatography. Based on the time course of the changes in the sodium dodecyl sulfate-gel pattern of the digests, chain masses estimated from sodium dodecyl sulfate-gel electrophoresis, and the NH2- and COOH-terminal sequences of the isolated peptides, the following scheme can be deduced. Formula; see text. C and N over the arrows indicate removal of residues from the COOH and NH2 terminus, respectively. The positions of the peptides along the myosin heavy chain have been established by comparison with the published primary structures of rabbit skeletal (Elzinga, M., Behar, K., Walton, G., and Trus, B. L. (1980) Fed. Proc. 33, 1579) and nematode myosin (McLachlan, A. D., and Karn, J. (1982) Nature (Lond.) 299, 226-231). LMM and fragment LMM-D are insoluble, whereas LF-1, LF-2, and LF-3 are soluble at low ionic strength. Their solubility properties, in conjunction with their locations along the myosin heavy chain, suggest that a relatively small stretch of peptide (chain weight, 5,000 Da) located about 100 residues from the COOH terminus of myosin heavy chain is responsible for the insolubility of LMM at low ionic strength.

Crosslinking by thiol disulfide interchange of 5,5'-dithiobis(2-nitrobenzoic acid)-treated light chain and heavy chain of rabbit skeletal myosin.

Interchain disulfide crosslinks between the heavy-chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5'-dithiobis(2-nitrobenzoic acid (Nbs2), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2-nitro-5-thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs2-modified light chain 2 by 32 - 39%, if Ca2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs2-modified light chain 2 attaches to the heavy chain in the region of the 21-kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/subfragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190, 793-799]. Modification of thiol-1 groups with iodoacetamide as well as crosslinking the thiol-1 and thiol-2 groups by the bifunctional reagent p-N,N'-phenylenedimaleimide prior to incubation with Nbs2-modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs2-modified light chain 2 to the heavy chain. An examination of K+, Ca2+, Mg2+ and actin-activated Mg2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs2-modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs2-modified light chain 2 to light-chain-2-deficient heavy meromyosin restored the original light-chain-2-dependent Ca2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light 2.

The mechanism of limited tryptic proteolysis of heavy meromyosin as revealed by peptide analysis.

To elucidate some ambiguous details in the tryptic fragmentation scheme of HMM as given by Bálint et al. (J. Biol. Chem. 250 (1975) 6168; Arch. Biochem. Biophys. 190 (1978)793), the peptide fragments were isolated by a milligram scale preparative gel electrophoresis procedure. The dansyl-peptide map of the 20 kDal tryptic fragment obtained from tryptic heavy meromyosin (HMM) and that of a similar fragment from papainic subfragment-1 (S-1) were found to be nearly identical. This finding gives unequivocal proof of the location of the 17 kDal peptide stretch lost during digestion in the form of small peptides, at the C terminal part of the heavy chain backbone of HMM. The N terminals of the 150, 74, and 25 kDal fragments of the heavy chain isolated from HMM digested by trypsin under widely differing conditions were shown to be acetylated. The N terminal amino group of the other peptide fragments of HMM remains the same under widely differing conditions of digestion. We conclude that all the fragments are well defined polypeptides and digestion progresses by splitting from the C terminals formed by the primary splits.

The substructure of heavy meromyosin. The effect of Ca2+ and Mg2+ on the tryptic fragmentation of heavy meromyosin.

Heavy meromyosin, obtained by tryptic digestion of myosin, containing two main polypeptides whose masses were estimated as 81,000 and 74,000 dlatons from Na dodecyl-SO4 polyacrylamide gel electrophoresis, was further digested with trypsin. The Ca2+-activated ATPase activity remainded unchanged and the K+-EDTA activity increased while various smaller fragments were formed. The formation of some of these fragments is affected by Ca2+ or Mg2+ as first shown by Bálint et al. (Bálint, M., Schaefer, A., Biro, N. A., Menczel, L., AND Fejes, E. (1971) J. Physiol. Chem. Phys. 3, 455). On the basis of the time course of the appearance of fragments the following relationship emerges: see article. The 64K leads to 60K step is inhibited by divalent cations, while the breakdown of the 74K fragment is accelerated. The effect of Ca2+ was maximal at 0 similar to 0.1 muM, that of Mg2+ at 10 muM. The original light chains of myosin are not present in the heavy meromyosin serving as the starting material, but peptide material appears on electrophoresis in positions starting material, but peptide material appears on electrophoresis in positions where the light chains would be found. The fragments marked by an asterisk are considered to ba alpha-helical on the basis of their solubility at low ionic strength after precipitation with ethanol (Bálint et al.). The fact that alpha helical fragments are derived from the 60,000-dalton fragment indicateds that it is adjacent to the light meromyosin in the intact myosin while the 74,000- dalton fragment would be part of heavy meromysoin subfragment 1. Chromatography of Sephadex G-200 separates fractions with ATPase activity corresponding to heavy meromyosin and heavy meromyosin subfragment 1. Electrophoresis of these Sephadex fractions suggests that the main peptide constituting heavy meromysoin subfragment 1 is connected by noncobalent forces to a portion of the rod that is not immediately adjacent to it in the primary sequence. The significance of this finding is discussed in terms of the flexibility of the myosin head.