Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications.
In this study, the expression of λ Red genes in P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of λ gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the λ Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated.
The λ Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with λ Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.
Pantoea ananatis belongs to the Enterobacteriacea family. The P. ananatis strain AJ13355 (SC17) was isolated from soil in Iwata-shi (Shizuoka, Japan) as a bacterium able to grow at acidic pH and showing resistance to high concentrations of glutamic acid . These physiological features made this organism an interesting object for biotechnological studies, and for this reason its genome has been sequenced by Ajinomoto Co. (unpublished results). Nevertheless, up to the recent past, the absence of efficient genetic tools has hampered manipulations of this bacterium and retarded both basic research and applied investigations.
Over the last decade [2–7], the most powerful method for generating a wide variety of DNA rearrangements in E. coli has been termed "recombineering" (recombination-mediated genetic engineering) . The term generally refers to in vivo genetic engineering with DNA fragments carrying short homologies with a bacterial chromosome, using the proteins of a homologous recombination system of the bacteriophage λ (λ Red system). Lambda red operon includes only three genes encoding Exo, Beta and Gam proteins. Gam inhibits the host nucleases, RecBCD and SbcCD, thereby protecting the dsDNA substrate for recombination [9, 10]; Exo degrades linear dsDNA from each end in a 5'→3' direction, creating dsDNA with 3' single-stranded DNA tails [11–14]; and Beta stably binds a ssDNA greater than 35 nucleotides in length and mediates pairing the one with a complementary target [15–17].
The λ Red-mediated recombineering technology developed initially for modification of the genome of Escherichia coli K12 [2, 4–7], was later broadened to other E. coli strains including enteropathogenic ones , and to Salmonella [19, 20], Shigella [21, 22], Yersinia [23, 24], Pseudomonas , as well. Probably, one of the factors impeding application of this system in other hosts is the toxicity of expression of the λ Red genes for the cells.
In the present study, to overcome this toxicity for Pantoea ananatis, the special strain resistant to simultaneous expression of the λ Red genes was selected. Using this mutant, construction of all types of chromosomal rearrangements previously obtained in E. coli by recombineering was reproduced. The approach described may be used for adjusting the technology to other hosts.
To provide regulated expression of the λ Red genes in different bacteria, the plasmid pRSFRedTER [GenBank:FJ347161] based on the broad-host-range replicon of RSF1010  has been constructed (see Additional file 1). This plasmid is useful for λ Red-mediated recombineering because: 1) the replicon is stably maintained in many Gram-negative  and some Gram-positive bacteria ; 2) the λ Red genes are placed under the control of the Plac UV5promoter recognized by different bacterial RNA polymerases [29, 30]; 3) the auto-regulated element Plac UV5-lac I provides IPTG-inducible expression of the λ Red genes with low basal level ; 4) the plasmid contains the levansucrase gene from B. subtilis allowing rapid and efficient recovery of this plasmid from the cells in a medium containing sucrose.
To test recombineering efficiency, pRSFRedTER mediated disruption of galK gene in E. coli MG1655 chromosome by integration of the PCR-generated DNA fragment carrying the KmR gene from pUC4K flanked by attL λ and attR λ sites (attL λ - KmR -attR λ ) was performed. The constructed plasmid provided about 300 transformants per 108 survivors following electroporation. A similar frequency was obtained when pKD46 plasmid  was used as λ Red-expressing plasmid. In each case a chromosome structure of ten KmR colonies was confirmed with PCR analysis.
Another plasmid carrying λ Red genes and named pRSFRedkan [GenBank:FJ347162], has been constructed via substitution of CmR and B. subtilis sacB genes by the KmR gene from pUC4K .
Clones of P. ananatis SC17 strain  obtained after electroporation by pRSFRedTER and plated on a solid LB-medium supplemented with Cm (50 μg/ml) were of very small size. Bacteria from these colonies grew very slowly in comparison with bacteria carrying pRSFsacB plasmid (see Additional file 1), which served as a vector for cloning of λ Red genes. Addition of IPTG (1 mM) for induction of expression of λ Red genes, led to complete cessation of the growth of pRSFRedTER containing cells. This effect was not detected for the cells carrying pRSFsacB and was based on the toxicity of the expression of λ Red genes.
To establish which component of the λ Red system caused the toxic effect, we constructed the pRSFGamBet [GenBank;FJ347163] plasmid lacking the exo gene encoding 5'→3' exonuclease.
It is well known that exonuclease activity is necessary for integration of dsDNAs only; integration of ssDNAs, such as chemically synthesized oligos, requires only recombinase (product of the bet gene, see ). To test the functional activity of the constructed pRSFGamBet, the plasmid was used to promote recombination between the artificial ss-oligos comprising two 36-nt homologies to the galK sequences and chromosome of the E. coli MG1655galK::(attL λ - KmR-attR λ ) strain. As a result of recombination a native structure of galK gene was restored. Between (1.5–2.5) × 104 Gal+ integrants per 108 survivors following electroporation were obtained in three independent experiments.
When introduced into P. ananatis SC17 strain, the pRSFGamBet plasmid did not inhibit cell growth even under the induced conditions (in the presence of 1 mM IPTG). Hence, the detected toxicity of pRSFRedTER was apparently caused by exo expression, or by simultaneous expression of all λ Red genes in P. ananatis cells.
A mutant P. ananatis strain, SC17(0), resistant to concerted expression of all λ Red genes, and thus manifesting the properties of a suitable recipient strain for λ Red-mediated integration of the linear DNAs into the chromosome, was obtained as follows. About 10 clones from 106 transformants obtained after electroporation of P. ananatis SC17 strain by pRSFRedTER, were of larger size after being plated on LB-agar with Cm. In LB-broth, bacteria from the "large" clones had a growth rate similar to the control strain with the pRSFsacB plasmid, and induction of the λ Red genes by IPTG caused only slight retardation in the growth of these cells.
Several of the selected "large" clones were cured from the plasmid on LB-agar containing sucrose, and re-transformed with pRSFRedTER. All clones grown after this re-transformation were of large size, similar to the parental clones. Three of the pRSFRedTER transformants that grew well were used as recipient strains for λ Red-mediated disruption of hisD gene. A PCR substrate, containing (attL λ - KmR-attR λ )-marker flanked by 40-bp homologous to the hisD gene, was electroporated into these strains. From 100 to 150 KmRHis- clones per 108 survivors following electroporation were obtained for each tested recipient strain. The insertion of the marker in the desired point of hisD gene was confirmed by PCR-analysis of 10 independent KmRHis- clones in each case. The observed integration frequency was similar to that obtained in the corresponding experiments with E. coli [4–6]. One of the plasmid-less strain used as a recipient in this experiment was named as SC17(0), and the obtained hisD strain constructed on its basis – as SC17(0)hisD::(attL λ - KmR-attR λ ).
We tried to determine the nature of the mutation/mutations that provide resistance to concerted expression of λ Red genes to P. ananatis. There were no auxotrophic properties for SC17(0) strain growing on the M9 minimal media, supplemented with different carbon sources, in comparison with initial SC17 strain . One of the possible explanations for the SC17(0) resistance is the reduced level of accumulation of λ Red proteins in this strain. To test the level of accumulation of λ Red proteins in SC17 and SC17(0), we performed SDS-PAGE of extracts of both strains carrying pRSFRedTER plasmid. Unfortunately, bands of λ Red proteins were not detected among the total cellular proteins even in conditions of IPTG induction for the both plasmid-carrier strains. The reduction in level of accumulation of λ Red proteins in SC17(0) strain, also, can be caused by decreased copy-number of RSF1010-replicon carrying plasmids in this strain. However, no reliable change in copy-number of pRSFsacB plasmid extracted from the cells of P. ananatis SC17 or SC17(0) strains could be experimentally found. On the other hand, the toxicity of the expression of λ Red genes has been detected for the plasmid-carrier cells of SC17 strain grown even without IPTG addition to the medium. In this case the transcription of the operon mediated by auto-regulated Plac UV5-lacI genetic element has to be tightly repressed. According to Skorokhodova et al. addition of 1 mM IPTG to the culture medium provides an increase of transcriptional level up to 10–20 fold . But, the transcription level of λ Red genes under such conditions is not toxic for SC17(0). Hence, it is unlikely, that the synthesis of λ Red proteins in SC17 under the repressed conditions could be significantly higher than in SC17(0) after induction.
Perhaps, mutation/mutations that are present in the genome of the SC17(0) strain does not affect most of the genes encoding factors of global cellular regulation. At least, the patterns of total cellular proteins separated by 2D-PAGE in such a fashion that about 350 of individual polypeptides could be quantitatively analyzed , were not different for the SC17 and SC17(0) strains (data not shown).
Thus, the molecular mechanism of the resistance of the mutant SC17(0) strain to concerted expression of all λ Red genes is unknown as yet. Nevertheless, as will be shown below, various λ Red-driven modifications of bacterial chromosome could be provided on the basis of this selected strain.
Earlier, a λ Int/Xis-driven system for removing the markers from E. coli chromosome was constructed, similar to one developed by Peredelchuk & Bennet [34, 35]. It comprised of the plasmids carrying removable markers of KmR or CmR flanked by attL λ and attR λ sites and the plasmid pMW-intxis-ts with thermo-sensitive pSC101-like replicon. This plasmid provided thermo-inducible expression of the xis-int genes under the control of λ PR promoter regulated by the temperature sensitive λ CIts857 repressor. Even being partially induced at 37°C, this system provided a high frequency (about 30%) of marker excision in E. coli. The high frequency of marker eviction at +37°C was very important for use of the system in P. ananatis because this bacterium has a lower temperature optimum than E. coli and cannot grow at 42°C (the standard temperature for temperature sensitive λ CIts857 repressor inactivation).
The pMW-intxis-ts plasmid contains ApR gene as a selective marker that was not practicable for P. ananatis because of its high natural resistance to Ap. For this reason, we have substituted this marker with the CmR gene. The resulting plasmid (pMW-intxis-cat) was introduced to the SC17(0)hisD::(attL λ - KmR-attR λ ) strain described above by electroporation. More than 30% of the transformants grown at 37°C on the plates containing LB-agar with Cm had lost the Km resistance. Loss of the KmR cassette in the kanamycin-sensitive colonies was verified by PCR. Thus, the KmR cassette can be used in the next step of the chromosomal modifications of this strain (Fig. 1A). Curing of the KmS clones from the pMW-intxis-cat plasmid (with a frequency of 10%) was performed by re-streaking bacteria on LB-agar without antibiotic followed by cultivation at 37°C.
Scheme of a construction of multiple chromosomal modifications, using the combined λ Red-Int/Xis system. A) Selective marker M flanked by attL λ /attR λ is used for introduction of an appropriate mutation into the chromosome by λ Red-dependent recombination. Then the marker is eliminated from the chromosome by λ Int/Xis site-specific recombination. As a result, only the 31 bp long attB λ sequence linked to the mutation remains in the chromosome. Selective marker M can then be used in the next step of the introduction of multiple chromosomal modifications. B) The sequence of the attB λ site. One of the six ORFs provided by this sequence does not contain stop codons. Hence, it is possible to design an "in-frame" deletion of a gene. Asterisks mark stop codons.
The attB λ site (31 bp in length), remaining in the chromosome after marker excision, contains six possible reading frames. One of these reading frames does not contain stop codons (Fig. 1B). Therefore, usage of the removable markers flanked with attL λ and attR λ sites allows design and construction of "in frame" deletions.
Using the λ Red-driven chromosomal modification followed by λ Int/Xis-mediated excision of the selective marker, it was possible to provide, step-by-step, the multiple chromosomal modifications in P. ananatis SC17(0) strain. This approach was repeatedly used for different modifications. Among them were 1) combinations of the simple or "in frame" deletions of several genes/operons; 2) integration of the marked heterologous genes into the chromosome of P. ananatis and 3) modification of the regulatory regions of the genes of interest . Up to the present, several P. ananatis strains carrying more than 10 different modifications have been constructed using this strategy; the presence of multiple attB λ sites in their chromosomes did not hamper the repeated exploitation of this system.
General transduction is the most efficient and popular method for transfer of mutations between different E. coli strains. Although P. ananatis and E. coli are close relatives, the known E. coli transducing phages cannot infect P. ananatis cells. Therefore, development of another method for transfer of mutations between P. ananatis strains was necessary.
The electroporation of genomic DNA has been described for the transfer of genetic markers between different backgrounds of E. coli and Pseudomonas [37, 38]. We tried to apply this technique to P. ananatis. The SC17(0)hisD::(attL λ - KmR-attR λ ) strain was used as a donor of KmR marker. Wild type strain SC17 was used as a recipient for electro-transformation of chromosomal DNA.
Previously, it was shown that special electroporation conditions are needed for the transformation of E. coli cells with large DNA molecules (see  for details). Different cultivation conditions of recipient strain and parameters of electroporation (electric field strength – E, time constant – τ) were tested. For P. ananatis, the highest yield of integrants (about 100 KmR His- integrants per 108 survivors following electroporation) was obtained under the following conditions. Recipient strain was grown up to absorbance of 0.8 – 1.0. Then electro-competent cells were prepared using 10 ml of culture as described in the "Plasmid electro-transformation" section (see "Methods"). Electroporation was performed at E = 12.5 kV/cm and τ = 10 msec (resistance of 400 Ω and capacity of 25 μF). Electro-transformation with chromosomal DNA is very fast method: all procedures, including DNA isolation and electroporation, can be performed in one day.
We found that marked chromosomal modification, obtained in P. ananatis SC17(0) strain via λ Red-recombineering method, could be easily transferred into the wild type SC17 strain by electroporation of chromosomal DNA. At the time of writing up to ten different mutations had been combined in the chromosome of the SC17 strain by repeated electro-transformation with chromosomal DNA followed by λ Int/Xis-driven excision of selective marker. The frequency of marked mutation transfer varied from several up to several hundreds of integrants per trial.
A two-step λ Red-mediated procedure for the introduction of unmarked mutations was elaborated for P. ananatis SC17(0). It comprises: 1) λ Red-driven insertion of dual selective/counter-selective marker into the desired point; 2) elimination of the marker via λ Red-mediated integration of the short dsDNA fragment containing the mutation of interest flanked with sites homologous to the appropriate target. Such an approach based on ET-recombination or λ Red-recombination was previously exploited for introduction of the unmarked mutations in E. coli [40, 41]. One of the most popular counter-selective markers used for this purpose is the sacB gene from B. subtilis, whose introduction imparts sucrose sensitivity to the bacterium.
To create a template for the PCR amplification of the dual selective/counter-selective marker, cat/sacB, the pRSFPlacsacB plasmid was constructed. The cassette Plac UV5-sacB-cat was amplified with primers containing 36-nt homologies to the target on their 5'-ends, and integrated into Sma I recognition site located in hisD gene using SC17(0) harboring pRSFRedkan as a recipient and the cat gene in the cassette, as the selective marker. A short 170-bp long dsDNA fragment harboring an appropriate mutation and 82-bp long flanks homologous to the target region has been constructed (Fig. 2A). The desired modification of bacterial chromosome (substitution of the artificial Xho I for the native Sma I site) was finally achieved via the λ Red-mediated integration of the obtained DNA fragment accompanied by elimination of Plac UV5-sacB-cat, using sacB gene for counter-selection (Fig. 2B). The integrants of interest were found with a rather high frequency (25%) among the clones grown on LB-agar containing 30% sucrose.
Construction of the unmarked nucleotide exchange in the P. ananatis hisD gene. A) Construction of a dsDNA fragment with appropriate mutation in the center. First, his-XhoI-1 and his-XhoI-2 oligos are annealed to each other. The nucleotide exchange of interest included in the sequence of his-XhoI-1/his-Xho2 olig is indicated by asterisks. The resulting dsDNA fragment is then used as DNA-template for PCR-amplification with his-SL and his-SR oligos. As a result a linear dsDNA fragment, harboring a Xho I restriction site and 82 bp long arms homologous to the target region of hisD gene, was obtained. B) The cassette containing dual selective/contra-selective marker is integrated into the target point of hisD gene. The constructed in vitro linear dsDNA fragment or ssDNA harboring appropriate mutation in the center is then integrated into the chromosome of this strain by the λ Red recombination system. As a result, the dual selective/contra-selective marker is eliminated from the chromosome with simultaneous introduction of the desired mutation into the hisD gene. This mutation leads to substitution of the native Sma I restriction site by the Xho I restriction site and restoration of the amino-acid sequence of HisD protein. Integrants are selected as colonies resistant to sucrose. Such colonies are subsequently tested for Cm sensitivity, ability for growth without histidine and presence of Xho I restriction site in the target chromosome point.
This procedure could be applied for introduction of unmarked mutations to the genome of SC17(0) strain. Transfer of the obtained unmarked mutations to other strains by electro-transformation with chromosomal DNA is difficult because of impossibility of direct selection of integrants. Hence, to provide construction of unmarked mutations in the genome of the P. ananatis strains, sensitive to expression of all λ Red genes, we performed the method of λ Beta-driven integration of single-stranded oligos into the chromosome of SC17 strain.
As mentioned above, ssDNAs, e.g. oligos containing short flanks homologous to the target site, can be integrated into the E. coli chromosome using only the product of the λ bet gene . As the expression of gam and bet genes from pRSFGamBet plasmid was not toxic towards P. ananatis SC17 even under induced conditions, we tried to perform λ Beta-driven integration of oligos into the chromosome of this strain.
The hisD::Plac UV5-sacB-cat chromosomal modification was transferred into the SC17 strain by electro-transformation with genomic DNA isolated from SC17(0)hisD::Plac UV5-sacB-cat strain. The resulting strain, selected on LB medium supplemented with Cm, was named SC17hisD::Plac UV5-sacB-cat. The pRSFGamBet-kan [GenBank:FJ347164] plasmid was constructed by substitution of sacB and cat genes of pRSFGamBet for the KmR gene from pUC4K. This plasmid was introduced to the obtained SC17hisD::Plac UV5-sacB-cat strain. It is known from the literature [8, 42] that the efficiency of λ Red-dependent integration of the oligos depends on a direction of replication through the recombination site. The direction of replication at the hisD locus in the P. ananatis chromosome is not known. Hence, the plasmid-carrier cells were independently electroporated with hisD-XhoI-1 and hisD-XhoI-2 ss-oligos complementary to each other. Cells were plated on LB medium containing 30% sucrose. No colonies were observed after 24-hour cultivation when hisD-XhoI-2 olig was used for electroporation. About 200 clones were obtained after electro-transformation with hisD-XhoI-1 olig. To test the phenotype of the obtained transformants, 100 clones were replicated on the following solid mediums: LB medium supplemented with 30% sucrose, LB medium supplemented with Cm and M9 minimal medium. Seven of the tested clones had SucRCmSHis+ phenotype. These colonies were further verified by PCR and restriction of the amplified product. The presence of the expected mutation (substitution of the Sma I by Xho I recognition site) was confirmed in all of the seven SucRCmSHis+ clones.
Although the frequency of selection of the desired mutation was not high, in principle, λ Beta-dependent integration of ssDNAs allowed the construction of the unmarked mutations in the P. ananatis.
It is currently well established that high-frequency recombination between short homologies can be catalyzed in E. coli cells by the λ Red functions [4–7, 43–45]. Unfortunately, up to the present, the range of bacteria for which this system has been utilized is limited. The main goal of the present study was to widen use of the λ Red-recombineering technology to P. ananatis, a bacterium of interest in the field of metabolic engineering. A broad-host-range λ Red-expressing plasmid useful for P. ananatis was constructed. The observed general toxicity of simultaneous expression of the λ gam, bet, and exo genes for P. ananatis SC17 cells was overcome by selection of the special recipient, SC17(0).
It is known that expression of λ Red functions in E. coli cells can lead to toxic effects [46, 47]. Certainly, expression of λ Red genes has been investigated for E. coli in detail. In addition to its direct influence on traditional recombination pathways due to inhibition of RecBCD and SbcCD nucleases by λ Gam, the activity of λ Red proteins can interfere with the processes of replication and repair (see, [48, 49] for reviews). For example, prolonged expression of gam gene could lead to formation of linear multimers of high, medium and low copy-number plasmids, and, even, of minichromosomes [50–53]. Expression of exo gene in addition to gam enhances this effect [50, 52]. The plasmid linear multimers, also, may interfere with λ Red-recombination . Murphy and co-workers showed that extended expression of λ Red-recombination functions could significantly induce a spontaneous mutagenesis, probably caused by interfering with mismatch repair UvrD-dependent pathway of E. coli .
Toxicity of expression of λ Red genes for the bacteria closely related to E. coli, but differing in the enzymes of replication, recombination and reparation, could not be predicted in advanced, as in the case of P. ananatis SC17. It is difficult, as well, to give an univocal explanation of how λ Red-mediated toxicity could be overcome. It seems that the decrease of this toxicity could be based on (i) lower intracellular level of λ Red proteins in the mutant cell, caused by reduced level in the synthesis of λ Red proteins or by increased efficiency of the specific proteolysis of λ Red proteins, (ii) decreased affinity of specific targets for interaction with λ Red proteins or increased level of biosynthesis of these targets. As for the P. ananatis mutant strain obtained, both variants of the explanation may be possible. As mentioned in the Results section, it is not very probable that SC17(0) strain possesses reduced level of the synthesis of λ Red proteins. But this possibility can not be completely rejected.
Nevertheless, even without information concerning the nature of the SC17(0) mutation, it is possible to use the corresponding strain for the desired λ Red-mediated rearrangements of P. ananatis chtomosome. All types of chromosome modifications constructed in E. coli by the λ Red-mediated recombineering, have been successfully reproduced in SC17(0) with pRSFRedTER as λ Red-expressing plasmid. Typically, the yield of recombinants varied from several tens to several hundreds per trial.
Using SC17(0) as the initial recipient for λ Red-promoting modifications it is subsequently possible to transfer the marked mutation to other P. ananatis strains of interest using the method of electro-transformation with chromosomal DNA. This method is a unique way to combine the set of marked mutations constructed in different P. ananatis strains into a single strain. In addition, potentially, co-transfer of rather closed mutations could be prevented by digestion of the chromosomal DNA by the appropriate restriction endonucleases, whose recognition sites are located between the mutations.
As the number of combined mutations of interest exceeds the number of available antibiotic resistance markers, curing of the intermediate strains from the used selective markers is necessary. Moreover, the presence of antibiotic resistance genes in the genomes of the industrial strains is rigorously restricted by the legislations of different countries. A wide variety of systems for marker curing based on the site-specific recombination systems (Cre/lox, Flp/FRT) are well-known [54–57]. These systems provide "symmetrical" recombination reaction between two identical sites flanking the removing marker, e.g. FRTxFRT = 2FRT. In this case, after removing the marker, the active site remains in the chromosome. Repeated action of such systems can lead to inversion or deletion of extended chromosomal fragments caused by site-specific recombination between the sites remaining at different points in the chromosome. Therefore, use of systems providing "asymmetrical" recombination reaction would be preferable. One such system is the site-specific recombination system of λ phage. It includes the Int and Xis proteins encoded by int and xis genes that, together with the host factors (IHF, RecA and Fis), provide the following reaction: attL λ x attR λ = attP λ +attB λ . Peredelchuk & Bennet  were the first who used this system for removal of the selective markers flanked by attL λ and attR λ sites. The attB λ site remaining in the chromosome after marker excision cannot recombine with attL λ or attR λ site in the next steps of strain construction. Thus, repeated action of the system would not influence the strain stability. Certainly, residual attB λ sites could recombine with each other via host general recombination system or provoke replication errors, especially if many left over scars (attB λ ) are presented in chromosome and their positions were rather close to each other. Such events would lead to deletions of chromosomal fragments.
We adjusted the λ Int/Xis-dependent system for use in high-efficient marker excision in P. ananatis. Note that it is also possible, in particular, to design marker-less strains, carrying "in-frame" deletions.
Finally, the two-step procedure for introducing the unmarked mutations into the P. ananatis genome was demonstrated using B. subtilis sacB gene as a counter-selective marker. Desirable mutants were achieved at the second stage via λ Red-mediated integration of the short dsDNA in SC17(0) or ssDNA in any other P. ananatis strain carrying the dual selective/counter-selective marker in the target point of the genome.
Up to the present, the developed λ Red-mediated method has been used for deletion of more than 50 P. ananatis genes whose products were involved in central metabolism, respiration, transcription regulation, etc. Several marker-less P. ananatis strains carrying multiple (> 10) different chromosomal modifications including "in-frame" deletions, point mutations, rearrangements of regulatory regions, in particular, were constructed for basic research and applied purposes using combined application of the λ Red-Int/Xis systems and electro-transformation with chromosomal DNA.
The λ Red-mediated recombineering has been adjusted for rapid and efficient construction of genome rearrangements in P. ananatis. In combination with the established procedures of λ Int/Xis-dependent marker elimination and electro-transformation with chromosomal DNA, this method provides a simple route to obtaining marker-less strains carrying multiple mutations of different types (deletions, substitutions of regulatory regions, integration of heterologous genes, and point mutations). The described approach of selection of the recipient strain resistant to expression of λ Red genes could be useful in exploiting λ Red-recombineering in other bacteria.
Strains and plasmids used or generated in this study are listed in Table 1. A detailed description of plasmids obtained in this work is in Additional file 1.
Strains and plasmids used or generated in this study.
A P-element marks the auto-regulated element Plac UV5-lac I [see  for details].
E. coli and P. ananatis strains were cultivated with aeration in LB medium at 37°C and 34°C, respectively. The following antibiotic concentrations were used to select transformants and to maintain the plasmids: Km – 40 mg/l, Cm – 50 mg/l. The M9 salt medium supplemented with galactose (1 g/l) or glucose (1 g/l) was used to select Gal+ or His+ cells.
DNA manipulations were performed according to standard methods . Restrictases were provided by "Fermentas" (Lithuania). T4-DNA ligase was from Promega (USA). All reactions were performed according to the manufacturer's instructions. PCR was carried out with Taq-polymerase ("Fermentas"). Primers were purchased from "Syntol" (Russia). All primers used in this work are listed in Additional file 2.
To provide cassettes for λ Red-dependent integration, the appropriate selective marker was amplified by PCR with oligos containing on their 5'-ends 36-nt sequences homologous to the target region. To disrupt E. coli galK and P. ananatis hisD genes, a removable KmR marker flanked by attL λ and attR λ was amplified with galK-5/galK-3 and hisD-5/hisD-3 primers, respectively. The pMW-attL λ - KmR-attR λ plasmid was used as DNA template. To obtain insertion into the P. ananatis hisD gene, the Plac-sacB-cat cassette was amplified in PCR with his-Plac-5/his-cat-3 primers using pRSFPlacsacB plasmid as template.
First, his-XhoI-1 and his-XhoI-2 oligos complementary to each other were annealed. Both oligos contained sequences corresponding to the Xho I restriction site in the center and arms homologous to the sequence surrounding native Sma I site of the P. ananatis hisD. As a result, the short dsDNA fragment containing the Xho I recognition site in its center and 33 bp long arms homologous to the target region, was obtained. The obtained fragment was amplified and extended by PCR with the primers his-SL and his-SR. The resultant DNA fragment generated by PCR contained Xho I recognition site in its center flanked with 82 bp arms homologous to the appropriate site in P. ananatis hisD gene.
An overnight culture of P. ananatis strain grown at 34°C with aeration was diluted with fresh LB broth 100 times and the cultivation was continued up to the OD 600 = 0.5–0.8. Cells from ten millilitres were washed three times with an equal volume of deionized ice water followed by washing with 1 ml of 10% cold glycerol and resuspended in 35 μl of 10% cold glycerol. Just before electroporation, 10–100 ng of the plasmid DNA dissolved in 2 μl of deionized water was added to the cell suspension. The procedure of plasmid electro-transformation was performed using the GenePulser and Pulse Controller ("BioRad", USA). The applied pulse parameters were: electric field strength of 20 kV/cm, time constant of 5 msec. After electroporation, 1 ml of LB medium enriched with glucose (5 g/l) was immediately added to the cell suspension. Then the cells were cultivated under aeration at 34°C for 2 h and plated on LB-agar containing the appropriate antibiotic. This was followed by an overnight incubation at 34°C. A competence of P. ananatis cells, determined for RSF1010 plasmid, was 106 CFU per μg of DNA. Typically, 105–106 antibiotic resistant colonies were obtained per 108 survivors following electroporation.
Overnight cultures of P. ananatis or E. coli strains harbouring the plasmid, expressing appropriate λ Red genes, grown in LB broth with Cm (for pRSFRedTER, pRSFGamBet plasmids) or Km (for pRSFRedkan, pRSFGamBetkan plasmids) were diluted 100 times with the same fresh medium supplemented with 1 mM IPTG for induction of the λ Red genes. At culture density of 0.5–0.6, electro-competent cells were prepared as described above. From 200 to 500 ng of a PCR-generated linear dsDNA or 100 ng of a ss-oligos were used for transformation. Electroporation was carried out at electric field strength of 25 kV/cm and time constant of 5 msec for both types of DNA substrates. The chromosome structure of the obtained transformants was verified in PCR with galK-t1/galK-t2 primers for E. coli galK gene disruption, hisD-t1/hisD-t2 for P. ananatis hisD gene disruption and for insertion of the double selective/contra-selective marker into the P. ananatis hisD.
Gene disruption provided with pKD46 plasmid was performed as described in (4).
Cells were grown in LB medium up to OD 600 = 0.8–1.0. Electro-competent cells were prepared as described above. From 1 to 2 mg of a chromosomal DNA, isolated using a Genomic DNA Isolation Kit (Sigma), was used for transformation. Electroporation was carried out with an electric field strength of 12.5 kV/cm and time constant of 10 msec.
SDS-PAGE of cell extracts was performed according to Laemmli  with polyacrylamide gel with linear gradient of concentrations from 10% to 15%.
Authors acknowledge Prof. B.L. Wanner kindly provided us with BW25113/pKD46 strain. This study was carried out at the request of and with financial support from the Ajinomoto Co.
JIK and YH are the project leaders in AGRI and in Ajinomoto, respectively. JIK obtained SC17(0), designed the main experiments concerned with λ Red-driven modifications, and drafted the manuscript. YH developed the transfer of λ Red-driven mutations to P. ananatis strains differed from SC17(0). LIG constructed all recombinant plasmids, expressing λ Red genes, tested their function initially in E. coli, and edited the manuscript. TMK performed the λ Red-driven modifications of the chromosome of P. ananatis SC17(0) strain. IGA performed λ Red-driven oligos integration into chromosome of P. ananatis SC17 strain. SVM supervised and coordinated the work and edited the manuscript. All authors have read and approved the final version of the manuscript.


text:
Home » AN2 Therapeutics Closes $80M Series B Financing
AN2 Therapeutics Closes $80M Series B Financing
Published on January 7, 2022 January 7, 2022 By FinSMEs
AN2 Therapeutics, Inc., a Menlo Park, CA-based clinical-stage biopharmaceutical company focused on developing treatments for rare, chronic, and serious infectious diseases, completed an $80m Series B redeemable convertible preferred stock financing.
The round was led by RA Capital Management, and included BVF Partners L.P., Surveyor Capital (a Citadel company), Avidity Partners, Monashee Investment Management, and Hatteras Venture Partners, who joined existing investors to complete this round.
The financing is expected to support advancement of AN2's development candidate epetraborole, a once-daily, orally administered investigational treatment for patients with chronic non-tuberculous mycobacterial lung disease (NTM).
Led by Eric Easom, President and Chief Executive Officer, AN2 Therapeutics is a clinical-stage biopharmaceutical company focused on developing novel therapies to treat rare, chronic, and serious infectious diseases in areas of high unmet need. The company is developing epetraborole, a once-daily oral investigational treatment for patients with chronic non-tuberculous mycobacterial lung disease (NTM).
In addition, AN2 Therapeutics also appointed two additional members to its Board of Directors, Patricia Martin and Stephanie Wong.
Ms. Martin has over 25 years of biopharmaceutical leadership experience across multiple therapeutic areas and disciplines. A former Eli Lilly and Company executive, Ms. Martin was the Chief Operating Officer of Lilly Diabetes as well as Lilly's corporate Chief Diversity Officer and Chief Alliance Officer. During her career at Lilly, she also held leadership roles in strategy, finance, clinical product development, human resources, and investor relations. Since 2019, Ms. Martin has served as the President and CEO of BioCrossroads, where she is focused on growing Indiana's life sciences sector, through research and education, seed funds, and economic development. Ms. Martin received a B.S. in Accounting from Indiana University, an M.B.A. from Harvard Business School, and is a Certified Public Accountant (inactive) in the State of Arizona.
Ms. Wong has over 20 years of finance and accounting experience in public and private companies within the biotechnology industry. Currently, Ms. Wong serves as Chief Financial Officer and Secretary of Calithera Biosciences (NASDAQ: CALA). Prior to joining Calithera, Ms. Wong served as Vice President, Finance and Controller at SciClone Pharmaceuticals and in senior finance roles at AcelRx Pharmaceuticals and Kosan Biosciences. Ms. Wong received a B.S. in Business Administration from the University of California, Berkeley and is a Certified Public Accountant (inactive) in the State of California.
Tagged with: AN2 Therapeutics, Avidity Partners, BVF Partners, Capital Management, Hatteras Venture Partners, Monashee Investment Management, Surveyor Capital
Previous storyRemilk Raises $120M In Series B Funding
Next storyAvataar Raises $45M in Series B Funding
ElevateBio Raises $525M in Series C Financing
By FinSMEs	Published on March 15, 2021
Strata Oncology Closes $90M Series C Financing
By FinSMEs	Published on July 29, 2021 July 29, 2021
fundingStart UpUSAventure capital
Hatteras Venture Partners Holds First Close Fund IV at $74M
By FinSMEs	Published on August 17, 2011

redpajama_set_name: RedPajamaCommonCrawl


text:
Modální logika je oblast logiky zkoumající logické operace, tzv. modality (modální operátory jsou například "je možné, je nutné, je nemožné").

Historie a vývoj
První, kdo se začal zajímat o modální logiku, byl Aristotelés, který v rámci své sylogistiky objevil řadu principů modální logiky.

Ve středověku toto odvětví dále zkoumal W. Ockham. Analyzoval strukturu modálních výroků a modální sylogistiku.

Dalším podnětem pro hlubší zkoumání modální logiky se stala matematická logika, na jejímž základě byly vytvořeny modální logiky trojhodnotové (Jan Lukasiewicz), systém striktní implikace (C. I. Lewis), Lewisovy systémy s šesti neredukovatelnými modalitami, systémy relativní modality (G. H. Wright).

Další, kdo se zabýval různými formami modalit, byli například H. B. Curry, W. V. Quine nebo W. Ackermann, dosud však obecně teorii modální logiky nikdo nezpracoval.

Externí odkazy

Matematická logika
Filozofie

redpajama_set_name: RedPajamaWikipedia


text:
Turbulence, Firm Decentralization and Growth in Bad Times
See all articles by Philippe Aghion
Philippe Aghion
College de France and London School of Economics and Political Science, Fellow; Centre for Economic Policy Research (CEPR); National Bureau of Economic Research (NBER)
Nicholas Bloom
Stanford University - Department of Economics; National Bureau of Economic Research (NBER)
Brian Lucking
Raffaella Sadun
Harvard University - Strategy Unit; London School of Economics & Political Science (LSE) - Centre for Economic Performance (CEP); National Bureau of Economic Research (NBER); Centre for Economic Policy Research (CEPR)
John Van Reenen
London School of Economics - Centre for Economic Performance (CEP); Stanford Graduate School of Business; Institute for Fiscal Studies (IFS); Centre for Economic Policy Research (CEPR)
Number of pages: 61 Posted: 01 May 2017 Last Revised: 25 Apr 2021
What is the optimal form of firm organization during "bad times"? Using two large micro datasets on firm decentralization from US administrative data and 10 OECD countries, we find that firms that delegated more power from the Central Headquarters to local plant managers prior to the Great Recession out-performed their centralized counterparts in sectors that were hardest hit by the subsequent crisis. We present a model where higher turbulence benefits decentralized firms because the value of local information and urgent action increases. Since turbulence rises in severe downturns, decentralized firms do relatively better. We show that the data support our model over alternative explanations such as recession-induced reduction in agency costs (due to managerial fears of bankruptcy) and changing coordination costs. Countries with more decentralized firms (like the US) weathered the 2008–09 Great Recession better: these organizational differences could account for about 16% of international differences in post-crisis GDP growth.
Keywords: decentralization, growth, turbulence, Great Recession
JEL Classification: O31, O32, O33, F23
Aghion, Philippe and Bloom, Nicholas and Lucking, Brian and Sadun, Raffaella and Van Reenen, John Michael and Van Reenen, John Michael, Turbulence, Firm Decentralization and Growth in Bad Times. Available at SSRN: https://ssrn.com/abstract=2960495 or http://dx.doi.org/10.2139/ssrn.2960495
Philippe Aghion (Contact Author)
College de France and London School of Economics and Political Science, Fellow ( email )
Centre for Economic Policy Research (CEPR)
Stanford University - Department of Economics ( email )
Landau Economics Building, Room 231
579 Serra Mall
HOME PAGE: http://economics.stanford.edu/faculty/bloom
Stanford University ( email )
Harvard University - Strategy Unit ( email )
Soldiers Field Road
HOME PAGE: http://people.hbs.edu/rsadun
London School of Economics & Political Science (LSE) - Centre for Economic Performance (CEP) ( email )
London WC2A 2AE
HOME PAGE: http://cep.lse.ac.uk/_new/staff/person.asp?id=1758
John Michael Van Reenen
Stanford Graduate School of Business ( email )
London School of Economics - Centre for Economic Performance (CEP) ( email )
Institute for Fiscal Studies (IFS) ( email )
7 Ridgmount Street
London, WC1E 7AE


text:
Russian lawmakers to ratify treaty on common security space with Abkhazia Jan 23
The treaty of alliance and strategic partnership with Abkhazia envisages creation of a common space of defense and security, including a common group of the Russian and Abkhazian armed forces
Russia's State Duma
© Mikhail Japaridze/TASS
Security and recognition top priorities as Abkhazia parliament ratifies Russia treaty
MOSCOW, January 22. /TASS/. Russia's State Duma plans to ratify a treaty of alliance and strategic partnership with Abkhazia on January 23, the speaker of the lower house of parliament, Sergey Naryshkin, told the media after a meeting of the State Duma's Council on Thursday.
"The ratification of the treaty of alliance and strategic partnership with Abkhazia is on the agenda of tomorrow's session," he said.
Naryshkin believes that "the treaty will take relations between Abkhazia and Russia to a qualitatively new level."
The treaty's main feature is creation of a common space of defense and security.
Russian-Abkhazian Treaty to help solve security problem — Abkhazian president
Relations of Russia, Abkhazia, South Ossetia are those between sovereign states — Lavrov
Russia, Abkhazia sign Treaty on Allied Relations and Strategic Partnership
Russia to provide $110 million to Abkhazia in 2015 — Putin
Russia's bilateral agreements with Abkhazia should not be subject of discussion at UN
5 more materials
Also, as Naryshkin said, "the treaty will create conditions for Abkhazia's more active involvement in international economic cooperation with Russia."
The chairman of the State Duma's CIS affairs, Eurasian integration and diaspora relations committee, Leonid Slutsky, explained that the treaty would in the first place systematize the legal framework of inter-state relations, already based on nearly 80 bilateral agreements.
Russian President Vladimir Putin and Abkhazia's leader Raul Khadjimba concluded the treaty in Sochi on November 24, 2014. It envisages creation of a common space of defense and security, including a common group of the Russian and Abkhazian armed forces.
"Should either side be attacked by some other state or a group of states, this will be regarded as an act of aggression against the other side," the treaty says. Russia and Abkhazia will be jointly guarding the Georgian-Abkhazian border and also sea areas to which the sovereignty of the Republic of Abkhazia applies.
Results of Dmitry Medvedev's work as Russian prime minister
During Dmitry Medvedev's tenure as Prime Minister, the gross domestic product (GDP) of the Russian Federation at current prices increased by 53%


text:
Le Cannonau di Sardegna Jerzu est un vin DOC, depuis le décret du . Il est produit dans la province de Nuoro, dans la commune de Jerzu, à partir de raisins du cépage cannonau.

Caractéristiques organoleptiques
 couleur : rouge rubis plus ou moins intense, tendant à l'orange avec le vieillissement
 odeur : fruits rouges, agréable, caractéristique de ce vin
 saveur : sèche, tannique, sapide, caractéristique

Détails historiques
La Coopérative vinicole de Jerzu a été créée par le Dr Josto Miglior.

Mariage conseillé
 Venaison, fromages affinés (Brebis).

Production
Province, saison, volume en hectolitres :
 Nuoro  (1992/93)  3919,43
 Nuoro  (1993/94)  3621,07
 Nuoro  (1994/95)  3621,07
 Nuoro  (1995/96)  3319,4
 Nuoro  (1996/97)  1810,41

Notes et références

Bibliographie

Liens externes
  - Josto Miglior propose une biographie du Dr Josto Miglior

Vin de la province de Nuoro


Shop for Scholastic Reader - top sales, deals, offers, and Reader Scholastic discounts are available 24/7/365 at AllSalion.com.
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Scholastic Reader - Last checked shopping results - April 24, 2019.


text:
HC at BU
David Guarin
BU Senior David Guarin On His Identities as a Latino and a Scientist
By Madeleine Salman • BU Contributor • Culture December 13, 2019 at 4:00pm
When you first meet David Guarin you will most likely see him surrounded by a group of friends in the Howard Thurman Center. David, with his laid-back smile, makes everyone in the group feel at ease and they all laugh as he delivers the punchline to a funny anecdote. I met David Guarin in a setting just like this during my freshman year at an Alianza Latina meeting (Alianza Latina is BU's Latinx cultural club). Initially, I didn't understand what drew so many different people to David. I would see him at Alianza Latina (AL) meetings, and although he was a freshman, everybody already knew his name the moment he walked in. It kind of reminded me of an episode of Cheers.
I soon got a chance to uncover that mystery when I became David's "twin" in AL's "Hermanos" program that matches freshmen with upperclassmen mentors. Being his "twin" showed me the many different sides of David Guarin. He is more than just the guy who knows everyone—David is also incredibly smart, a compassionate friend, a talented researcher, and an active member of the Latinx community here at BU. So, this past Friday, I interviewed David about his identities as a Latino and as a scientist, so more people can get to know the deeper David beyond his winning smile
As the son of two Colombian immigrants, AL is very important to David. He makes it a point to attend every meeting, even if he has to do homework during it. When I asked him about why he puts so much importance on AL's weekly meetings, he said it's important that people from "different Latin countries are fostering a bond" despite growing divisions in Latin culture. For David, it's more important for Latinos to focus on what brings them together rather than what sets them apart. AL is the perfect vehicle to "take a step towards collaboration and unity in the Latinx community."
Photo Caption: David and I bonding over our mutual appreciation for bucket hats during an official Alianza Latina Outing
Photo Credit: David Guarin
David is also the first person in his family to go to college in the U.S. AL allows him to connect with other first-generation students, like the third sibling in our family, Nicolle Varadalles. Being a first-generation student at a school like BU can be an isolating experience and it can be hard to meet anyone who has gone through the same things. But at AL, there is a community of fellow BU students who he can relate to and become friends with. Ultimately, for David, Alianza Latina is a place where he can feel at home in a massive school like BU.
Being a first-generation student and Latino also connects to his medical research. David, who is a biochemistry and molecular biology major, does epidemiological research that explores the link between neurodegenerative disorders and certain types of cancers. This basically means that his research focuses on why diseases like Parkinson's put patients at a lower risk for certain types of cancer, like melanoma. Understanding this relationship, which is rarely studied, can lead to possible groundbreaking treatments in the neurodegenerative field. David just published a collaborative paper on his findings at just 19 years old. He reflected on all this by saying "who would have thought that anyone in my family would be a college grad and on top of that a published scientist." To David, his accomplishments mean so much more because of everything he had to overcome to even get the opportunity to work in the lab where he did all of his research.
Photo Caption: David presenting his research at a medical research conference in California.
David—who never considered this type of research until his father was diagnosed with Parkinson's—is committed to "being a leader in this budding field," not only because of the personal connection to his father but also to advance the visibility of Latinos in STEM. "How many Latino scientists are there…And how many Latino scientists own their own companies." David's goals of getting his Ph.D., publishing more research in his field as a principal investigator, and eventually starting his own business, is not just for his gain. As he sees it, his biggest goals in life contribute to both sides of his identity; one side is as a scientist who is committed to bettering the lives of those closest to him, and the other is as a Latino who wants to elevate and inspire a wider Latinx community to get involved in science.
Photo Caption: David (back left) and his research team in California
And that's why David is such a unique person. His path in life is defined by how he can use his talents to give back to the community which is most important to him. Almost every student at BU is motivated by goals that will benefit their quality of life in terms of money or fame. But David is the rebel who works hard so that others can achieve after him.
This may be the first article about David Guarin and his impressive accomplishments, but ten years from now it will just be one of many.
Want to keep up with HCBU? Make sure to like us on Facebook, follow us on Instagram, check out our Pinterest board, and read our latest Tweets!
Madeleine Salman (BU '22)
Madeleine Salman is a freshman in the College of General Studies and the College of Communication. She was born and raised in Miami, Florida and can't wait to explore Boston!
What English Majors Wish You Knew


This is a rotary clothes 6-color manual screen printing machine, suitable for printing 500mm * 500mm about the format of the fabric, paper, clothes; T-shirts and other products.
Running smoothly, cost-effective. Widely used in multicolor printed clothing and textile sectors.
Screen rotation arrangement, the operator independent of each other and co-ordination, printing and high efficiency.
Total-steel rotating-axis system ensure whirling free and Adaptability.
High-strength support pole make the machine structure rigidity.
Six-color screen printing one time, overprint is precision .
Screen frame can be adjustable according to different objects to be printed.
Screen frame can be rise up after printing so that printing quality.


text:
Q: How to send an animated GIF in a card like this in Facebook messenger API? I tried using the generic template, but it does not animate the GIF - this is needed for showing a 'Powered by Giphy' banner in the bottom of the card - like image below.
 animated GIF received in a messenger card/bubble

A: You have to send an attachment of image instead of a generic template, like this:
curl -X POST -H "Content-Type: application/json" -d '{
  "recipient":{
    "id":"USER_ID"
  },
  "message":{
    "attachment":{
      "type":"image",
      "payload":{
        "url":"https://petersapparel.com/img/shirt.png"
      }
    }
  }
}' "https://graph.facebook.com/v2.6/me/messages?access_token=PAGE_ACCESS_TOKEN"

Check out this page: https://developers.facebook.com/docs/messenger-platform/send-api-reference/image-attachment

A: Two bubbles. First is the image, the second is the advert.

redpajama_set_name: RedPajamaStackExchange


text:
Sporazoom: Ten Years of the Brussels Dialogue
17. March. 2021. | 11:51
The dialogue between Pristina and Belgrade, which goal was to solve the problems of the citizens, begun in Brussels in March 2011. Ten years later, today in Sporazoom, we are talking about the results of the dialogue and the prospects of its continuation after the early parliamentary elections in Kosovo. The show is hosting Bodo Weber, a senior associate of the Policy of Democratization Council in Berlin, Nenad Đurđević, an advisor to the President of Serbian Chambre of Commerce, and Veton Surroi, a publicist and journalist.
Sporazoom is broadcast on Wednesday, March 17, at 9 pm on TV Mir, TV Kim, TV Herc and TV Plus, as well as on our Facebook page.
Most reading
New Grant Opportunity for Local NGOs
28. July. 2015. | 03:09
12. February. 2018. | 03:20
We're Hiring! Open call for the position of (2) Legal Advisors with NGO Aktiv
21. September. 2018. | 12:51
Re-advertised position for finance and admin intern!
10. January. 2017. | 12:11
The countdown before the counting
Trend Analysis 2021: Attitudes of the Serbian Community in Kosovo - Belgrade conference
17. December. 2021. | 09:02
Trend Analysis 2021: Attitudes of the Serbian Community in Kosovo
Promoting cooperation among civil society in Kosovo and Serbia
Presentation of the Aber app in Gračanica/Graçanicë
9. December. 2021. | 03:34
Aktiv was founded in the divided city of Mitrovica. Since its inception in 2009, Aktiv has successfully implemented an array of projects and has become an active part of local civil society. It has cooperated with a number of both regional and international NGO's and has played a vital role in the political and social development of N. Kosovo. With a growing presence in the local community and with an expanding network of partners, Aktiv has been able to increase the scope and scale of its projects.


As published in the journal of the Heraldry and Genealogy Society of Canberra Inc, Ancestral Searcher, Vol 2 No 6 December 1979 pp120-12. Please also see Margaret Beynon's (nee Llewellyn) story by clicking on link below.
With John on board, the 'Royal Admiral' sailed for Sydney on 23 May 1800 leaving behind memories of a hard life in London and heading for an unknown life beyond the seas. The 'Royal Admiral', built in 1777 of 914 tons, was making her second voyage to New South Wales as a convict transport. Her master was William Wilson and her surgeon Samuel Turner. This voyage had its moments with the first occurring before sailing. On 7 May a disturbance broke out amongst the convicts; two were found attempting to rob others of money by menacing them with knives. A search on 8 May found four knives and a pair of razors and the culprits were flogged for their efforts. Again on 17 May seven convicts were flogged for stealing biscuits.
As was common in those days gaol fever was carried on board by the convicts and by 21 May thirteen were sick with the fever with the first death occurring on 24 May. So the 300 male convicts, 6 wives, 8 children, 67 crew and several passengers, including 11 missionaries bound for the South Sea Islands, were in for a long and hard voyage made more difficult by the death on board of the surgeon, Samuel Turner, on 3 June 1800. One of the missionaries, Mr. Elder, having had some experience in medicine, attempted to assist in stopping the spread of fever by fumigating with pitch and gunpowder, as 30 convicts were by this time extremely sick.
By 22 June the passengers and master had become concerned about a reported plot to seize the ship. As the passengers had doubts about the capacity of the crew to control the convicts, they, with the approval of the master, divided themselves into watches for the rest of the voyage. About this time the 'Royal Admiral' parted company with the fleet (19 ships having set out from England) and on 4 August strange sails were sighted and cannon fire could be heard. This must have caused the convicts below deck to wonder if they would ever see land again.
What part John Manning played in the events of the voyage are not known. However it would seem he was not one of those badly affected by the fever as he was soon active in the life of the colony.
What of John Manning's future wife, Margaret Baynon?
Margaret Llewellyn was born about 1777 probably in Cardiff, Wales. By 1797 she had married Thomas Baynon (here I use the most used spelling, others being Beynon, Baynham, Baynange) and on 28 March 1797, when aged 20 years Margaret was convicted at Glamorgan for an unknown crime, receiving a sentence of transportation for life.(6) From searches made in London it appears the Assize records for Glamorgan are no longer in existence. (7) What happen to Thomas is not known as the various records consulted only indicate that Margaret came to New South wales.
From a memorial by Margaret in 1810 it is known that she lived from the time of her arrival at No. 14 The Sign of the Compasses, South Row (O'Connell Street), Sydney. (9) From this and later events it can be assumed that this was also the residence of John Manning.
As yet it has not been established how John served out his sentence. But from the tenor of his life it would be reasonable to assume that he was allowed to work on his own account because of his profession as a carpenter and because he was a well behaved convict.
Only 10 months after arriving in the colony Margaret gave birth to a child on 18 October 1802; the father being John Manning. So it would seem Margaret went straight from the ship to the same house where John lived and co-habited with him soon after. The child was baptised Mary on 25 December 1805 at St. Phillips, Sydney. (10) Why John and Margaret delayed the baptism until then is not known but it could be that they waited until John's 7 year sentence had expired on 15 September 1805. Also on 4 June 1804 Margaret had been granted a conditional pardon by Governor King. This gave her back all her rights as a citizen except that she must remain within New South Wales.
The 1806 muster shows John Manning and Margaret Banyon living together with John being described as a self-employed carpenter. (12) With the rise in importance of wine and spirits as a medium of exchange it is possible that John, with the assistance of his wife ran an inn The Sign of the Compasses at 0'Connell Street, while carrying out jobs as a carpenter for in 1809 the Sydney Gazette states that John held a wine and spirit licence. (13) However, it would seem that not long after 1810 John found he was earning enough as a tradesman for there are no further references to him holding such a licence. Also with the arrival of Governor Macquarie in 1809 trade in wine and spirits became better controlled and this may have influenced John to cease trading.
This paragraph has been added in 2006. In view of the fact that their first child was born in 1802 and Eliza was born in 1808 (see below) it is likely that John and Margaret had other children during this period. The NSW Registery of Births, Deaths and Marriages has a record of a child named Margaret Manning being buried on 10 September 1806; no other details are recorded in the Church of England register (13a). It is likely that this child did not live long after birth and that she is the daughter of John and Margaret. This would mean that they had six children.
This was one of the many recorded events involving the Mannings to occur over the next few years. No doubt in recognition of John having lived at South Row since 1801 Governor Paterson granted John a lease of 45.5 rods on 22 August 1809 for 14 years at 5/- annual rent. (17) So Margaret had her absolute pardon, John his lease of land, and together they had two children and a reasonable future.
1. Ho 10/1 (AJCP 59). Ho 11/1 (AJCP 87) - National Library and OBSR 12.9.1798 No. 48 - Greater London Record Office; Mary Flower's letter 22.7.1971 to author.
4. St. Mary. Whitechapel Baptisms P 93/MRY 1/10 - Mary Flower's letter 4.9.1971 to author.
5. Diary of Jas. Wilshire - The 1788-1820 Gazette, Issues 44 to 46 and The Convict Ships 1787-1868, Charles Bateson, 1974.
6. HO 11/1 (AJCP 87) - National Library and CO 207/1 (AJCP 57) National Library. Since writing the Manning story the National Library of Wales has provided the following information - Thomas Baynon and Margaret Llewellyn were married on 14 May 1795 in the parish of Llansamlet, Wales. On 3 January 1797 Margaret Baynon, aged 22, was charged with burglary with her accomplice Ann David, having stolen a number of specified items from a shop at North Cornely where the Baynons lived. Both were sentenced to death on 1 April 1797 but, as their behaviour was orderly their sentences were commuted to transportation for life to Sydney.
7. Mary Flower's letter 22.7.1971 to author.
8. The Convict Ships, 1787-1868, Charles Bateson, 1974.
9. Card Index - Petitions - Mitchell Library, Sydney.
10. Certificate issued by the Principal Registrar, Registry of Births, Deaths & Marriages, Sydney, No. 1548 Vol 1 on 5.5.1978 to author. Governor Hunter laid the foundation of St. Phillip's Church (with two I's ) on Church Hill on 7 October 1798; it is understood one ''l" was dropped from the name when a new church was built after 1856.
11. Register of Absolute and Conditional Pardons 1800-1809, SZ 76, the Archives Authority of New South Wales .
12. HO 10/37 (AJCP 72) - National Library.
13. Sydney Gazette 26.2.1809 - mfm National Library.
13a. Certificate issued by the Principal Registrar, Registry of Births, Deaths and Marriages, Sydney, No 2100, Vol 2, issued on 11.9.1981 to the author.
14. Certificate issued by the Principal Registrar, Registry of Births, Deaths and Marriages, Sydney, No. 2350 Vol. 1 issued on 5.5.1978 to the author.
15. Ibid. No. 2351 Vol. 1 on 5.5.1978 to author.
16. Register of Absolute Pardons 1791-1825 - The Archives Authority of New South Wales A.O. 414486.
17. Land Grants 1788-1809, K. A. Johnson and M, R. Sainty, 1974, p226.
After his arrival in December, 1809, Governor Macquarie rescinded their pardons and lease, along with many others given by the previous administration. Margaret handed in her pardon on 12 February 1810. (18) The lease was re-granted by Macquarie on 18 October 1811, back dated to 1 January 1810 under the same conditions. (19) Before any pardons were reissued Macquarie decided that he had to be satisfied the pardons should be granted.
The petition and/or Margaret's good record must have had some effect, for on 10 April 1810 Macquarie reissued the absolute pardon. (21) Unfortunately, both absolute pardons do not state why she received a life sentence.
In partnership with Thomas Boulton, stonemason, John obtained a contract sometime around 1815 to build the Governor's Secretary's house for three thousand pounds stg. This must have been the highlight of John's work as this event is mentioned in his daughter Charlotte's obituary in 1904. (32) Soon after the work was completed the prominent convict architect Francis Greenway was very critical of the high cost, saying that he could have built the house for a third of the price. (33) This criticism may have caused a downturn in John's ability to attract building contracts as later events indicate he fell on bad times to some extent.
Issues of the Sydney Gazette for 26 August 1820 and 24 February 1821 reported that John Manning's lease, amongst others, would be ready for delivery on 5 March 1821. John cannot have been in a hurry (or was he overseas?) to pick up the document for on 5 July 1822 the Sydney Gazette listed leases still waiting for collection, including John Manning's town lease.
Then John ran into further difficulties. Included in a list of receipts credited to the Colonial Fund for the quarter ended 31 December 1823 was an amount of 10/- being for payment of a fine imposed by the Supreme Court on John Manning. (40) It is not known why he was fined and further trouble awaited him. In the Sydney Gazette of 22 March 1826 a police report stated that John Manning was a prisoner of the Crown, having offered stolen property for sale in the Market. On being taken into custody John was unable (or unwilling) to explain why he was in possession of stolen property. He was remanded until such time as the owner came forward or other evidence was obtained. What eventuated is not know, but John probably did not remain in custody for long.
The 1828 Census lists John, Margaret and Charles as living at O'Connell Street, Sydney. (41) We know by now that Mary was married, but it is not known what happened to Eliza or James.
Charlotte was probably living with Thomas Anderson, as both are not listed and then on 11 May 1829 Charlotte married Thomas at St. James. (42) In the Census John's age is given as 62 years and Margaret's as 52 years. In 1832 and 1834 John is again working as a carpenter living in 0'Connell Street ( 43).
I would assume that by the 1830's John would have either retired or was only doing a small amount of work for little is recorded about the Mannings until their deaths. In 1842 John Manning of O'Connell Street, Sydney is listed as an eligible elector for Bourke Ward in the first elections for the Municipality of Sydney. (44) The Sydney Morning Herald of 1 November 1842 published a list of electors supporting candidate A. Foss; one of the signatories being John Manning.
Then on 6 September 1849 John and Margaret's son, Charles, married Eliza McCutcheon at St. Andrews Church; one of the witnesses being Charles' sister, Charlotte Anderson. (45) It is possible that Charles had lived with his parents at 0'Connell Street and had supported them in their old age. Also it was most likely that this marriage was arranged at short notice as a special licence was issued for the ceremony. Charles' parents may have been ill and because of this and their age Charles and Eliza may have decided to bring their marriage forward while John and Margaret were still alive.
So passed on a pioneering couple, although not prominent in the early affairs of Sydney but truly the type of people who helped make this country what it is today. One of their great-grandchildren, Roland J. Anderson, became the Member for Botany in the New South Wales Parliament in the early 1900's - convict to parliamentarian. One final interesting point is that there is no record of a marriage between John and Margaret. In fact, various records examined indicate that they did not marry.
18. Returns of Pardons and Certificates of Freedom Surrendered, 29.1.1810- 18.2.1811 - The Archives Authority of New South Wales A.O. 4/4427.
19. Historical Records of Australia, Volume 7, p .315, p 440.
20. Card Index - Petitions - Mitchell Library, Sydney.
21. Copies of Returns of Absolute and Conditional Pardons granted 1810-1819, The Archives Authority of New South Wales, A.O. 4/4427.
22. Sydney Gazette, 21.4.1810, mfm, National Library.
23. A Century of Sydney Cove and The Genesis of the Circular Quay, Norman Selfe, Royal Australian Historical Society Journal, Vol 1, p 59.
24. Sydney Gazette, 20.4.1811, mfm National Library.
25. The Sydney Traders, D.R. Hainsworth, pp 207-8. 26. Sydney Gazette, 25.7.1812, mfm National Library.
27. Sydney Gazette, 20.10.1812, mfm National Library.
28. Sydney Gazette, 24.4.1813, mfm National Library.
29. Certificate issued by the Principal Registrar, Registry of Births, Deaths and Marriages, Sydney, No. 3048, Vol 1 issued on 7.11.1969 to author.
30. 1814 Muster, Society of Australian Genealogists, Sydney.
31. Certificate issued by the Principal Registrar, Registry of Births, Deaths and Marriages, Sydney, No. 3922 issued on 18.2.1972 to author.
32. The Windsor and Richmond Gazette, 27.8.1904, State Library of New South Wales, Sydney.
33. Francis Greenway, His Life and Times, M.H. Ellis, p 41, p 256.
34. Sydney Gazette, 12.6.1819, mfm National Library. 35. H010/10-11 (AJCP 63), National Library.
36. HO 10/14 (AJCP 64), National Library.
37. HO 10/16-17 (AJCP 65), National Library.
38. HO 10/36 (AJCP 72), National Library.
39. Card Index, Society of Australian Genealogists.
40. Sydney Gazette 24.2.1825, mfm National Library.
41. HO 10/21, 24 (AJCP 67-68), National Library.
42. Certificate issued by the Principal Registrar, Registry of Births, Deaths and Marriages, Sydney, No. 4529, Vol 3, issued on 9.4.1970 to author.
43. The New South Wales Calendar and General Post Office Directory 1832, 1834, National Library.
44. Sydney Morning Herald 14.9.1842, National Library.
45. Certificate issued by the Principal Registrar, Registry or Births, Deaths and Marriages, Sydney, No. 635, Vol. 79, issued on 9.11.1971, copy held by author .
46. Certificate issued by the Principal Registrar, Registry or Births, Deaths and Marriages, Sydney, No. 612, Vol 34B, issued on 16.7.1970 to author .
47. Certificate issued by the Principal Registrar, Registry or Births, Deaths and Marriages, Sydney, No. 780, Vol 36A, issued on 22.9.1970 to author.
JOHN MANNING, theft : simple grand larceny, 12th September, 1798.
528. JOHN MANNING was indicted for feloniously stealing on the 26th of May, a cloth coat, value 20s. the property of James Gibbons.
JONATHAN HAMMOND sworn. - I live at Mr. Gibbons's, woollen-draper, No. 84, Oxford-street; I was sent home with a coat to Mr. Webb's, in Bolton-street, Piccadilly, on Saturday evening, the 26th of May, we did not know the gentleman, and I was to wait for the money for it, and turning round the corner of Bolton-street, the prisoner met me, and said, you are going to Mr. Webb's, are you not? I told him, yes, I was; he said, he was come in a very great hurry from Mr. Webb, he had a key in his hand; he said, he was to take the coat, and I was to go to the great hotel place, where a great many coaches came to, facing the Green-park, I believe they call it the White-horse Cellar, I was to go there to ask for a parcel for Mr. Webb, and he was to take the coat to Mr. Webb; I gave him the coat wrapped up in a black wrapper; he asked me for the bill, and I gave it to him; I went to the White-horse Cellar, but there was no such parcel; then I came back to No. 10, Bolton-street, and Mr. Webb was not at home; I waited there, and was crying about the street, and asking the people; I went to No. 10, Bolton-row, to see if it was there, I was afraid to go home; a great while after that I called at No. 10, Bolton-street, again, and Mr. Webb was come home, and he went with me to Mr. Gibbons's; Mr. Webb had another coat made, and about a fortnight ago I was going with Mr. Gibbons into the country, Mr. Gibbons sent me home with a whole piece of woollen cloth, as much as I could carry; I went with it as far as the corner of Newman-street, in Oxford-road, and this John Manning came up to me, and said, that he was sent from Mr. Gibbons's shop to take the cloth; it struck me directly that that was the man that took the coat from me; I was very near Mr. Gibbons's, and he said I was to go back again to the corner of Hanway-yard for a parcel, where the coaches stop; I said to him, should I take the cloth along with me for the parcel, and he seemed quite in a flurry; he said, oh, no, I must take the cloth, and you must go for the parcel; I had put the cloth down from my shoulders, and he had his hands upon it to take it, but I did not let it go; then I took hold of him by the collar, and told some gentlemen to stop him; with that he got away from me in a minute, and pulled his apron off and buttoned his blue lappelled coat; he did that just as he began to run, he run as bard as he could; I called out stop thief, and I ran after him as well as I could; he ran down Oxford-street as far as Berner's-street, he was stopped at the top of Berner's-street by this young man; I am sure he is the man that took the coat, I knew him directly when he came up to me when I had the cloth, I am sure he is the same man; I asked the people that had got him to bring him to No. 84, Oxford-street, facing Poland-street, and just by the door he was rescued; he ran down Poland-street, and there was a constable delivering a summons there, and he took him again; he was brought back to Mr. Gibbons's shop, and searched, they tied his hands with a piece of lift, and took him to the office, there was a rule found in his pocket, and some papers of his own, he was a carpenter.
Prisoner. What kind of a coat had I on? - A. In Oxford-road he had a blue coat on, when I saw him before he had no coat on, he had a jacket and an apron on; there was a boy saw him talking to me at Newman-street, and another boy, and I asked them to go to the office, and they said, no, they would be out of the mess.
Q. Are you sure he is the man that took the coat? - A. Yes; it was a good while ago, but I knew him as soon as ever he came up to me when I had the cloth.
BARNETT- HENRY BROWN sworn. - I live in London-street, Fitzroy-square.
Q. What are you? - A. A gentleman's son; I was coming down Oxford-road, I heard a cry of stop thief, I stopped this man, who was running, and told him it was for a coat, I searched his pocket for his apron, and he had not got it; he said, he would go back with me; the boy told me he had an apron on, but when I stopped him he had not.
Prisoner. Q. What coloured coat did the boy say I had on? - A. He said you had a brown coat on, he made several hesitations about it, he was not quite certain.
Q. What are you yourself? - A. A gentleman's son; my father is a Portugal merchant, and generally resides there, he has no business here, he transacts for Mr. Duff, in Finsbury-square.
Court. Q. The man was running when you stopped him? - A. Yes.
Q. How long has your father lived in London-street? - A. Seven years and a half.
Q. Who is your father's partner in Portugal? - A. I do not know, all his letters come in Portuguese, I do not know any thing of him, he is a steward, I believe.
Q. Steward to a merchant? - A. Yes.
Q. What part of Purtugal is it? - A. Somewhere in the North.
Q. What province in the North? - A. I cannot tell, I never heard the name mentioned.
Q. Mr. Duff is the correspondent? - A. Yes.
Q. Did you never hear the name of your father's partner there? - A. No.
Q. How old are you? - A. Eighteen.
Q. Where did you stop the prisoner? - A.At the corner of Castle-street, in Berner's-street.
Q. You did not stop him in Poland-street? - A. No.
Q. You have not told us a word of his running away? - A. A young man laid hold of him from me, and asked him what business he had to stop or be taken, and he ran away down Poland-street, and there he was stopped by a constable, and then I went back with him to the shop.
Q. (To Hammond.) Did you ever say any thing about his having a brown coat? - A. No; I went into the crowd, and said, it was a blue coat; this Mr. Brown has been to see him in Clerkenwell prison.
Brown. No, I have not; I went to see another person there, Bill Doyle, and then I saw him.
Hammond. At the time I was getting the bill, I could not find him, and he did not answer, and when he came back, he said he had been to see poor Manning, that he had got five children.
Court. (To Brown.) Q. I shall enquire your description - what is your father's name? - A. Bernardino Escoffia , No. 40, London-street.
Q. How came you by the name of Brown? - A. That is my mother's name, and I put my own name besides, Bernard-Henry Brown, my mother lives there too.
Q. And she goes by the name of Escoffia? - A. No, Mrs. Brown, he is my second father, he is not my first father.
Q. How came she to go by her first husband's name? - A. I was too young to know any thing about it; my first father was killed in a duel.
Q. What was your own father? - A. I do not know, I never heard.
Q. And you never enquired what he was? - A. No.
Q. Then of course you never enquired whether he died worth any money? - A. I did not.
Q. You may have a very large fortune for any thing you know? - A. I do not know indeed.
Q. And you are eighteen years of age? - A. Yes.
Q. Does your father speak English? - A. He can speak a little English, but he speaks more French; Mr. Gordon, No. 29, Percy-street, comes to him very often.
Prisoner's defence. I am an innocent man of this fact, I was going that way about some business, I am entirely innocent of it, I have a wife and five small children; I know nothing of it more than the child unborn.
The prisoner called two witnesses, who gave him a good character.
Tried by the second Middlesex Jury before Mr. COMMON SERJEANT.


text:
import {nanoid} from 'nanoid'

// `WeakMap`s require the first type param to extend `object`
// eslint-disable-next-line @typescript-eslint/ban-types
const randomIdCache = new WeakMap<object, string>()

/**
 * given an object, this function randomly generates an ID and returns it. this
 * result is then saved in a WeakMap so subsequent requests for the same object
 * will receive the same ID
 */
// eslint-disable-next-line @typescript-eslint/ban-types
export function assignId(obj: object): string {
  const cachedValue = randomIdCache.get(obj)
  if (cachedValue) return cachedValue

  const id = nanoid()
  randomIdCache.set(obj, id)
  return id
}

redpajama_set_name: RedPajamaGithub


text:
The Print Club of New York is proud to announce Donald Teskey as the Artist for our 2015 Presentation Print. The print will be presented by the artist to Club members at a reception in the fall of 2015. This Print Club of New York commission will be a 3 plate carborundum and one plate polymer print of the Mayo seascape, an area located off the north west coast of Ireland
The Print Club of New York was founded in 1991 as an organization for enthusiasts who appreciate, study and collect all manner of prints. Numerous events each year give our members the opportunity to interact with and learn from artists, curators, and other important players in the print world. From the start, the Club has commissioned an annual print for its membership (now 200); for many this is the highlight of the year. The first artist chosen in 1992 was Fred Mershimer, an outstanding master of the mezzotint. Subsequent artists have included Alex Katz, April Gornik, Will Barnet, Elizabeth Catlett, Joan Snyder, Richard Segalman, Chakaia Booker and in 2014 Faith Ringgold. Many of these PCNY presentation prints have been accepted by major museum collections, including the Metropolitan Museum of Art.
Although the majority of our artists are American, this is not the first time the PCNY has reached out to an artist from abroad for the presentation print. We are delighted to introduce Donald Teskey to our members and to many more American collectors and curators. Donald's work, whether painting, watercolor or print, presents a rugged exhilaration of land, sea and sky and a dramatic vision of the power and beauty of landscape. Much of his focus has been dedicated to the relentless and elemental nature of the Irish landscape and seascape.
The edition of 200 for PCNY members plus one archive print will be printed by Stoney Road Press, www.stoneyroadpress.com. Based in Dublin, Stoney Road Press is the only independent commercially run fine art print studio in Ireland. It is also the only Irish member of the International Fine Print Dealers Association, a body committed to promoting the highest standards of quality, connoisseurship and appreciation of fine art prints.
Combining traditional printing techniques with innovative digital technology, Stoney Road Press is transformed with every artist's collaboration, producing a body of work that reflects the individual skill of each artist.
Donald Teskey is also represented by Oliver Sears Gallery, Dublin, Ireland www.oliversearsgallery.com .
Dolan/Maxwell 2046 Rittenhouse Square Street
Philadelphia PA 19103 USA www.dolanmaxwell.com
Print Club of New York Award 2015


There's no doubt about it: August Monday is definitely a celebration for the books in Anguilla. Locals and tourists alike come out en masse and 2013's version drew even greater numbers from neighboring St. Martin and St. Barths.
Anyone else in Anguilla on August Monday? Share your thoughts from the festivities below!


text:
Zarqawi's Dead
Although I'm not sure at this point it matters as much as had it happened a year ago, I still think it's great news.
BAGHDAD, Iraq (CNN) — Terrorist leader Abu Musab al-Zarqawi, the most wanted man in Iraq, was killed in a coalition airstrike near Baquba, jubilant U.S. and Iraqi authorities announced Thursday.
And you know, for once I find myself agreeing with al-Qaeda:
"We want to give you the joyous news of the martyrdom of the mujahed sheik Abu Musab al-Zarqawi," said the statement, signed by "Abu Abdel- Rahman al-Iraqi," identified as the deputy "emir" or leader of al- Qaida in Iraq.
Yep, pretty joyous indeed.
Realistically, it seems are so fractured there that I'm not sure how much influence he really had, but taking him out is a good thing.
A great thing.
World Stuff | Mr. Bingley | June 8, 2006 7:22 am
7 Responses to "Zarqawi's Dead"
Oh, HAPPY day! I'll drink to that! We just got moving around here, I got the 'puter fired up after making coffee and there was a lovely email from John with a very uncharacteristic "woo hooo!" subject line. "Hmmm", I thought. "Must be good news." It was. Woo HOOO!! Thank you, John.
Cullen says:
Joyous news indeed.
Nightfly says:
If Abdel-Rahman thinks it's so joyous, he can volunteer next. My tax dollars at work!
My wish right now is that they don't bury him within the Koran's proscribed time line. F*ck him. I'd leave the body in the street for the dogs. And, if there are 72 virgins waiting somewhere, may their nether regions hold razor blades.
The_Real_JeffS says:
Ken Summers says:
See my update
http://www.secondbreakfast.net/archives/002875.html
Mike Rentner says:
I'll be whistling and singing all day. That bastard was operating in my area while overseas and he was behind the killing of more than 50 of my battalion. I am SO happy today!


We decided to run at least one archive node per Ethereum blockchain that we work with regularly.
From the word on the Ethereum streets, we knew that an archive node would probably require a couple hundred gigabytes of storage and should be on SSDs and not on spinning rust.
Isn't the latest state (and perhaps the block headers) the only part that needs to be on SSD? The rest of the blockchain and archive are just history and aren't accessed anymore for processing new blocks and transactions. That would reduce the requirement for the SSD to around 10GB. And the rest can be stored on a regular HDD.
Slock.it brings back fond memories. Kind of curious how things are going for them.
Things are going great. We are about 30 people currently and are focusing on IoT and Blockchain Solutions and have done great projects over the last years. Check our website: [slock.it](https://slock.it) for more info.


text:
King's Cross Station
Harry Potter: Warner Bros. Studio Tour from King's Cross
4.6 / 5	1221 Reviews
This Warner Bros. Studio Tour London – The Making of Harry Potter package includes round-trip transportation from King's Cross. You'll have extended time to explore the studios, and be able to see everything from the Hogwarts Express to The Great Hall.
About this ticket
Duration 7 - 7.5 hours
Select participants and date:
Experience pure filmmaking magic at Warner Bros. Studio Tour London – The Making of Harry Potter
Walk through The Great Hall and board the Hogwarts Express
Discover the incredible detail in the props, costumes and special effects
Experience the pure magic of Warner Bros. Studio Tour London with this 7.5-hour package that includes round-trip transportation from London's King's Cross. After your departure from the station, arrive at the studios approximately 2 hours later and enjoy an extended 4-hour visit!
Begin where Harry started, at the Cupboard under the Stairs, before watching an enchanting welcome video and heading into The Great Hall – one of the biggest sets on the site. As you travel through, you'll see an amazing selection of actual costumes, props and special effects pieces, all maintained in pristine condition.
See The Burrow, Hagrid's Hut, the Gryffindor Common Room, Dumbledore's office and much more. Marvel at small details like Dumbledore's Pensieve and larger pieces like the fantastic animatronic door and the Chamber of Secrets.
Have fun riding a broom through London at the Green Screen exhibit and board the Hogwarts Express on Platform 9-3/4. See Privet Drive and the Knight Bus, buy a Butterbeer™ on the backlot and get up close to the prosthetics and animatronics in the special effects department. There is so much to see you will be thankful to have extended time to take it all in!
Round-trip transportation to Warner Bros. Studio by branded bus with free Wi-Fi
Admission ticket to Warner Bros. Studio Tour London
Digital guide (available for rent at the Studio Tour for £4.95)
Harry Potter: Warner Bros. Studio Tour London
Harry Potter Tour: Family Package
• When booking this tour, please choose your "starting time" according to when you would like to board the bus from King's Cross (departure point: King's Cross Station, Pancras Road, Bus Stop T, London). Entrance to the Studio will be around 2 hours later. For example: if you book for 9:30 AM, you will board the bus at 9:30 AM, enter at the studio at 11:30 AM, depart from the studio at 4:00 PM and arrive back in London at approximately 5:30 PM.
• Butterbeer™ may contain nuts and other ingredients that may cause allergies; the tour operator is not responsible for adverse side effects
• During busy periods, alternative transportation may be used
• All vehicles are modern, comfortable, and kept to the highest standards of cleanliness, including a deep clean every day
From £89 per person
See more family activities
Magical London: Harry Potter Guided Walking Tour
GetYourGuide Original
Warner Bros. Studio London: Tour with Coach Transfers
Harry Potter Family Package with Transfers from London
From £336 per group
Harry Potter: Warner Bros. Studio Tour with Transfer
Golden Tours - Gray Line London
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Sightseeing Attraction Tickets
Attractions in London
London Victoria Station


text:
> Home > UCLA > Library Special Collections, Charles E. Young Research Library
∞ https://oac.cdlib.org/findaid/ark:/13030/c87d2xg1/
L.A. Watts Times newspapers and ephemera
Contact UCLA::Library Special Collections, Charles E. Young Research Library
Restrictions on Use and Reproduction
Provenance/Source of Acquisition
Organization and Arrangement
UCLA Catalog Record ID
Title: L.A. Watts Times newspapers and ephemera
Collection number: 2122
Contributing Institution: UCLA Library Special Collections
Physical Description: 13.8 linear ft. (33 flat boxes)
Abstract: The Watts Times was founded in 1965 following the Watts Riots. In 1976, Charles Cook purchased the publication and changed the name to L.A. Watts Times. The collection consists of original L.A. Watts Times newspapers, a small amount of awards, and a very small amount of ephemera.
Language of Materials: Materials are in English.
Physical Location: Stored off-site at SRLF. Advance notice is required for access to the collection. Please contact the UCLA Library Special Collections Reference Desk for paging information.
Creator: L.A.Watts Times.
COLLECTION STORED OFF-SITE AT SRLF: Open for research. Advance notice required for access. Contact the UCLA Library Special Collections Reference Desk for paging information.
Property rights to the physical object belong to the UCLA Library Special Collections. Literary rights, including copyright, are retained by the creators and their heirs. It is the responsibility of the researcher to determine who holds the copyright and pursue the copyright owner or his or her heir for permission to publish where The UC Regents do not hold the copyright.
[Identification of item], L.A. Watts Times Newspapers and Ephemera (Collection 2122). Library Special Collections, Charles E. Young Research Library, UCLA.
Danny Bakewell, Chairman of Bakewell Media, 2012.
The Watts Times was founded in 1965 following the Watts Riots and eventually became the premiere African American publication in Los Angeles. In 1976, the newspaper was purchased by Charles Cook, an African American activist and former leader of the Congress of Racial Equality. As an effort to include news about Los Angeles' entire African American community, Cook changed the publication's name to L.A. Watts Times. Cook was credited for transforming the L.A. Watts Times from being a tabloid circulated in Watts to becoming a financially stable weekly publication distributed in Los Angeles, Long Beach, Carson, Compton, Inglewood, Gardena, Santa Monica and West Hollywood.
After Charles Cook's death in 1998, his daughter Melanie Polk became publisher of L.A. Watts Times. In June 2010, Polk sold the publication to Danny Bakewell, publisher of Los Angeles Sentinel and owner of Bakewell Media
The collection consists of issues of original L.A. Watts Times newspapers along with a small amount of awards and ephemera. The bulk of the newspapers are loose publications; a small portion are hard bound together by year. Many of the awards, honors, and commendations are from the California State Government and City of Los Angeles and they acknowledge the L.A. Watts Times as well as publishers Charles and Beverly Cook and Melanie Polk.
The collection is not arranged.
UCLA Catalog Record ID: 7397589
African Americans--California--Los Angeles--Newspapers
Watts (Los Angeles, Calif.)--Newspapers.
Container List


Dense collagen implants were developed which can be easily manufactured by extrusion at room temperature without the need of organic solvents. The physicochemical properties (matrix surface pattern, apparent matrix density, melting temperatures and swelling behavior) of the collagen materials and matrices were investigated. Furthermore, the diffusion coefficients of water inside the collagen devices (5.76*E-02cm²/h) and of various FITC dextrans in solution (e.g. FITC dextran 70: 2.4*E-03cm²/h) were determined by PFG-NMR and FCS, respectively. The developed collagen devices were used to investigate the enzymatic collagen matrix degradation and the release of higher molecular weight drugs, e.g. proteins. Several processes, i.e. diffusion, swelling and erosion, contribute to the overall release profile from collagen devices. Since it was desired to obtain a delivery system which controls release mainly by erosion, insoluble collagen type I materials were used to enhance the resistance against enzymatic attack. Besides this, collagen was physically or chemically cross-linked in some experiments to further restrict collagen digestion and drug delivery. It was shown that model compounds like BSA or FITC dextran 20, 70 and 150, respectively, could be incorporated and that their delivery could be controlled by the used collagen matrix material, e.g. animal source or cross-linking degree, the matrix dimensions (length or diameter of the extrudates), the molecular weight of the incorporated model compound and the drug load. The in vitro release of FITC dextrans and BSA was investigated and delivery of 80% model drug was in the range between 7h and 5d. Comparsion of the in vitro and the in vivo release (monitored in adult domestic pigs) of BSA was made by ESR. Similar results were obtained and it was shown that the mechanism of release changed from mainly diffusion towards erosion control by increasing the degree of matrix cross-linking. The degradation of insoluble collagen type I by bacterial collagenase was studied in detail to gain further insights into the enzymatic hydrolysis of collagen. In contrast to a simple Michaelis-Menten kinetic, adsorption of collagenase onto the substrate surface plays an important role. Based on the obtained in vitro results a mathematical model was developed to describe drug release from collagen matrices undergoing enzymatic degradation. Equations for the collagen degradation and the drug release were implemented, adsorption and diffusion phenomena were incorporated and a mixture of experimentally determined and fitted parameters was used to feed the model. Good correlation between experimental and simulated data was found. Histological evaluations demonstrated that the developed minirods showed good biocompatibility, with only minor inflammation reactions and normal tissue remodeling. This emphasized the assumption that collagen extrudates could be used in vivo without surgical removal after drug depletion.