Category Archives: Cloning

This perennial question came up again in an e-mail from a reader about the abortion debate, so I thought I'd blog again about it. I think the answer is not just "no," but "hell, no"I think it would be an outrageous discrimination against religious believers to have such a constitutional rule, and fortunately nothing in the history or the precedents of the Establishment Clause supports this position.

The argument is this: Isn't it illegitimate for the government to ban abortion, or to ban cloning, or fail to recognize same-sex marriages, when most of the arguments for that position are essentially religious? Isn't that an unconstitutional violation of the separation of church and state, or at least a violation of some democratic norm that people ought not force their religious views on others?

Butmost of the coercive laws that we hotly debate involve the forcing of a majority's views on the minority. That's true of laws protecting endangered species, antislavery laws, antidiscrimination laws, animal cruelty laws, environmental laws, intellectual property lawsor for that matter bans on infanticide, child sexual abuse, or more generally murder, rape, or theft. Some of these laws may be sound on the merits, and others unsound. But the fact that they force one group's views on another doesn't make them violations of the Establishment Clause, regardless of the source of the first group's views.

Likewise, specifically as to abortion, different people have different views about when life begins, or, to be more precise, when the protection against being killed or aborted by one's parent or parents should arise. A few people believe that this line is some point afterbirth. (Indeed, historically, the ancient Romans allowed parents to expose their unwanted children.) Some people believe it's at birth, though that appears to be very much a minority view in America (at least as of 2012).

Some people believe it's at around six months into the pregnancy, the point at which Roe v. Wade held abortion could generally be forbidden. Some people believe it's at viability, the point at which Planned Parenthood v. Casey held abortion could generally be forbidden. Some people believe it's at three months. Some believe it's at conception. Some would draw other lines. But wherever the line is drawnand it must be drawn somewherethat's a legal constraint that forces some people's views on others.

Religious people have moral views just like secular people do, and they'rejust as entitled as secular people to use the political process to enact their views into law. True, religious people's moral views may rest on unproven and probably unprovable metaphysical assumptionsbut the same is generally true as to secular people's moral views.

To say that religious arguments must be excluded from public debate, while equally unprovable secular moral arguments may continue to be made, would be to turn into second-class citizens those people whose basic moral views come from their religion. Neither the Constitution nor sound political morality require this.

In fact, many important political movementsthe antislavery movement, the civil rights movement, and various antiwar movementswere composed in large part of religious people who acted for explicitly religious reasons, and justified their positions using explicitly religious arguments. Would we say that opposition to slavery was illegitimate because it was mostly overtly religious? If not, then we also can't condemn opposition to cloning or abortion or same-sex marriage on these grounds.

But what about the Establishment Clause? Well, the Supreme Court has explicitly held that the Establishment Clause doesn't invalidate laws simply because their supporters backed them for religious reasons. See, e.g.,McGowan v. Maryland,366 U.S. 420, 442 (1961);Bob Jones Univ. v. United States,461 U.S. 574 (1983);Harris v. McRae,448 U.S 297, 319-20 (1980). And for the reasons I mention above, the Court's decisions here were correct.

True, the First Amendment does bar the government from teaching religion, from requiring religious practices such as prayer, and (generally) from singling out conduct for better or worse treatment because it's religiously motivated (e.g., punishing religious animal sacrifices but not secularly motivated animal killing, or giving a sales tax exemption to religious publications but not secular ones). But it doesn't bar the government from implementing religiously-motivated prohibitions on people's conduct, whether as to murder, theft, slavery, civil rights, cloning, or abortion.

Nor do I know of any evidence that the Establishment Clause was generally understood in 1791, in 1868, or any time in between or since as discriminating against religious believers this way. It may be convenient for secularistsand I myself am not religiousto have their moral reasons for lawmaking be permitted, and have their religious rivals' moral reasons declared unconstitutional or otherwise illegitimate. But there's no basis for thinking that the Constitution embodies any such discriminatory rule.

There are lots of good arguments to oppose cloning bans, abortion bans, bans on homosexual conduct, and the like. The supporters of such prohibitions may be wrong on moral or pragmatic or constitutional grounds. But the bans aren't made invalid by the fact that many of their supporters act for religiously influenced moral reasons, as opposed to secularly influenced moral reasons.

(For a different connection between abortion and religion, see my post on abortion and the Free Exercise Clause.)

Originally posted here:

Is It Unconstitutional for Laws to Be Based on Their Supporters' Religiously Founded Moral Beliefs? - Reason

CapVQ329R inhibits swimming motility of E. coli MG1655

The dinucleotide cyclase DncV synthesizes cAMP-GMP to inhibit rdar biofilm formation and motility in the animal commensal strain E. coli ECOR3149. To assess whether the downstream cAMP-GMP receptor, the patatin-like phospholipase CapV, has a function on its own, we expressed CapV in the heterologous host MG1655, an E. coli K-12 derivative which is not known to synthesize cAMP-GMP nor to harbor DncV. Besides wild-type CapV, we overexpressed its variant CapVQ329R, which had been derived by cloning a respective mutated DNA fragment as described in Supplementary Results. To this end, we observed that expression of the variant CapVQ329R caused suppression of swimming motility, whereas overexpression of the wild-type protein CapV did not alter the apparent motility in semi-solid tryptone broth (TB) agar (as described in Supplementary Results; Fig. 1a and Supplementary Figs. 13). TB medium promotes motility of E. coli MG1655 compared to LB medium in the semi-solid agar plate assay50. To further characterize CapVQ329R-induced E. coli MG1655 swimming inhibition, we assessed the production of cell-associated flagella and flagellin upon overexpression of CapVQ329R compared to CapV. Visualization of bacterial cells by transmission electron microscopy (TEM) showed that overexpression of CapVQ329R compared to wild-type CapV dramatically reduced the total number of flagella-producing cells as well as the number of flagella per cell after 6h incubation at 37C (Fig. 1c, d). In agreement, visualization of flagella by Leifson staining upon CapVQ329R overexpression showed cells with intact flagella up to 4h incubation at 37C, but almost no cell with flagella after 6h (Fig. 1c, e). In congruence with the analysis by TEM and light microscopy examination of Leifson staining, we observed inhibition of production of cell-associated extracellular flagellin in a protein gel upon overexpression of CapVQ329R after 6h (Fig. 1f, g). Initial analysis of differential gene expression by qRT-PCR indicated 8% downregulation of expression of the class 1 flagella regulon gene flhD encoding a subunit of the major regulator FlhD4C2, and less than 50% downregulation for the representative genes of class 2 fliA encoding the flagella specific sigma factor and class 3 fliC encoding the subunit of flagella, upon CapVQ329R compared to CapV overexpression. Compared to the vector control, flhD, fliA and fliC were downregulated 28, 83, and 73%, respectively, upon overexpression of CapV. Thus, the expression of CapVQ329R interferes with flagella expression beyond the flagella regulon cascade and can affect, for example, depolymerization or degradation of flagella. Cumulatively, these results indicate that CapVQ329R suppressed swimming motility of MG1655 by post-translationally inhibiting the production of flagellar filaments gradually during the growth phase, while initially production of functional flagellar filaments had been observed.

a, b Flagella-dependent swimming motility of wild-type E. coli MG1655 vector control (VC) and upon overexpression of wild-type CapV, its mutants (red) and mutants of CapVQ329R (blue). In total, 3l of a OD600=5 cell suspension were inoculated into soft agar plates containing 1% tryptone, 0.5% NaCl, and 0.25% agar, and the swimming diameter was measured after 6h at 37C. c Flagella production of a representative E. coli MG1655 VC cell and upon overexpression of CapV, CapVQ329R (Q329R) and CapVQ329R/D197A (D197A) as observed by TEM. d Quantification of the number of flagella per cell upon overexpression of CapV and CapVQ329R after visualization by TEM (c). The number of evaluated cells n=20. Cells were grown in TB medium for 6h at 37C. e Production of surface-associated flagellin of E. coli MG1655 VC, upon overexpression of CapV and CapVQ329R. f Assessment of flagellin subunit FliC expression by colloidal Coomassie staining from E. coli MG1655 culture supernatants after shearing of flagella upon expression of CapV, CapVQ329R, CapVQ329R/D197A and CapVQ329R/S64A. Cells were grown in TB medium at 37C for 6h. g Assessment of flagellin subunit FliC expression over time in E. coli MG1655 overexpressing CapVQ329R. Samples were harvested at different time points in the growth phase for Western blot analysis of FliC. lc, loading control. h Proposed development of filamentation and flagella inhibition of E. coli MG1655 upon CapVQ329R expression over time. Bars represent mean values from three biologically independent replicates with error bars to represent SD. Differences between mean values were assessed by two-tailed Students t test: ns, not significant; *P<0.05, **P<0.01, and ***P<0.001 compared to E. coli MG1655 VC. Vector control VC=pBAD28. pCapV = CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28. S64A=CapV Q329R/S64A cloned in pBAD28. D197A=CapV Q329R/D197A cloned in pBAD28. R27A=CapVQ329R/R27A cloned in pBAD28. G24A/G25A=CapVQ329R/G24A/G25A cloned in pBAD28.

Significantly, upon overexpression of CapVQ329R in E. coli MG1655 TEM and light microscopy demonstrated not only the loss of flagella, but the concomitant development of long thin filamentous cells (Fig. 1c, e). In contrast, E. coli MG1655 cells overexpressing wild-type CapV were only slightly elongated compared to the control (Fig. 1c, e).

Assessment of the temporal development of cell filamentation throughout the growth phase upon induced expression of CapVQ329R by light microscopy after Leifson staining indicated that filamentation did not initiate before 2h after commencement of CapVQ329R expression (Figs. 1e and 2a, b). Subsequently, though the cell length and the frequency of filamentation dramatically increased, whereby after 3h almost all cells displayed as short filaments around 6 times the length of standard rod cells (Fig. 2a, b). After 4h of induction, CapVQ329R expressing E. coli MG1655 cells were on average 25 times longer than control cells. After 6h of induction, CapVQ329R expressing E. coli MG1655 cells were on average more than 50 times longer than the standard rod-shaped cell (Fig. 2, b). Those long cells did not show any movement, while shorter filaments up to approximately 20 times the length of standard E. coli cells, although rare, still showed active swimming motility (Supplementary Movie S1). At the opposite, overexpression of wild-type CapV only slightly increased the cell length compared to E. coli MG1655 control. On note, after 22h induction of CapVQ329R expression by 0.1% L-arabinose, short rod-shaped motile cells dominated again, which suggested filaments to restart cell division after CapVQ329R expression had diminished due to L-arabinose depletion. In line with this hypothesis, induction of CapVQ329R production by 0.2% l-arabinose did not cause reversion to rod-shaped cells nor showed the cells any movement after 22h (Fig. 2a, c and Supplementary Movie S2). Upon transfer of those filamentous cells to fresh TB medium without L-arabinose, however, the emergence of rod-shaped cells was again observed (Fig. 2c). A scheme of this developmental process leading to filamentation with consecutive loss of flagella upon expression of CapVQ329R is displayed in Fig. 1h.

a Light microscopy pictures of cell morphology of E. coli MG1655 VC and upon overexpression of CapV and CapVQ329R (Q329R) in TB medium at different time points at 37C. b Quantification of cell length of E. coli MG1655 VC and upon overexpression of CapV and CapVQ329R in TB medium at different time points. The quantification is based on results from at least three independent experiments with the assessment of 70 cells from each group. c Cell morphology 3h after addition of fresh TB medium to filamentous E. coli MG1655 cells overexpressing CapVQ329R. Arrowheads indicate invaginations at proposed future division sites. d, e, f Assessment of cell length upon overexpression of CapV, CapVQ329R and CapVQ329R derivatives in E. coli MG1655 upon induction with different l-arabinose concentrations. Light microscopy pictures (d), quantification of cell length (e) and protein expression level (lc=loading control) (f) upon induction with 0.01% and 0.1% l-arabinose in TB at 37C for 4h. The quantification is based on results from at least three independent experiments with the assessment of 70 cells from each group. Bar, 5m. Vector control VC=pBAD28. pCapV=CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28. G24A/G25A=CapVQ329R/G24A/G25A cloned in pBAD28. R27A=CapVQ329R/R27A cloned in pBAD28. S64A=CapVQ329R/S64A cloned in pBAD28. D197A=CapVQ329R/D197A cloned in pBAD28.

A positive correlation of the filamentation phenotype of the E. coli MG1655 cells with CapVQ329R production level was demonstrated using increasing L-arabinose concentrations. When incubated for 4h with 0.01% l-arabinose, the low-level CapVQ329R expression created a heterogenous cell population displaying no or restricted cell filamentation (Fig. 2d, e). In contrast upon incubation with 0.1% l-arabinose all cells became filamentous as observed previously. Concomitantly, the CapVQ329R expression level was comparable to the CapV expression level excluding that the observed morphological changes were due to significantly different protein expression levels. As expected, CapV expression was higher upon induction with 0.1% l-arabinose than with 0.01% l-arabinose (Fig. 2f). The 6xHis-tag added to the C-terminus of the protein to detect protein production level did not alter the proficiency of CapVQ329R to induce filamentation (Supplementary Fig. 2g). Cumulatively, filamentation and motility repression are caused by the Q320R substitution in CapV.

Blast search with CapV from E. coli ECOR31 showed that CapV homologs with >60% identity are not only found in individual E. coli and V. cholerae strains, but are widely distributed among gamma-proteobacteria, including Yersinia, Salmonella, Pseudomonas, Shewanella, and Klebsiella species (Supplementary Fig. 4a). Phylogenetic analysis of representative CapV homologs supported classification into four different subgroups (Supplementary Fig. 4b). Alignment of the amino acid sequences of those CapV homologs showed that Q329 is a nearly invariant amino acid even among distantly related CapV proteins (Supplementary Fig. 4a). Q329 is, though, not required for catalytic activity and not part of other characteristic patatin-like phospholipase A2 (PNPLA) consensus motifs and the PNPLA core domain which is restricted to aa 19210 (Prosite) in the 361 aa long protein. In order to clarify the position of R329 in the protein, we generated a structural model of CapV using the closest structural homolog from the PDB database as a template, the lysophospholipase-like protein FabD from Solanum cardiophyllum (PDB: 1oxwC; Fig. 3a) which shows 22% amino acid identity with CapV. According to this model, R329 is located within helix 12, the second last helix of CapVQ329R in the context of the RARGRR329 sequence pointing outward with no obvious change in the overall structure of the monomer or potential oligomer assembly to be observed.

a Predicted structural model of the CapV from E. coli ECOR31 shown as ribbon representation. The structural model was built with the I-TASSER server, the result was processed with SWISS-MODEL. The model was based on the coordinates of the 22% identical protein FabD from Solanum cardiophyllum (PDB: 1oxwC). b The graphical representation and schematic indication of the positions of the conserved motifs (indicated by the green bar) and putative active site residues S64 and D197 (marked by red stars) in the PNPLA domain of the 361 aa CapV from E. coli ECOR31 (from L19 to F210). Black arrow, Q329. The graph was assessed by ExPASy_Prosite. c Sequence alignment of CapV from E. coli ECOR31 and selected known phospholipases from other species establishes the conserved motifs of the PNPLA domain, GG-G-x-[K/R]-G, G-x-S-x-G, and DG-[A/G], boxed in black, green, and purple, respectively. Entirely conserved residues are shown in white on a red background. Conserved residues are boxed. Putative catalytic residues of CapV are indicated with filled red triangles. The residues in CapVQ329R mutated to alanine are marked with red asterisks above the sequence. The consensus sequence at the bottom indicates in uppercase letter residues with 100% identity and in lowercase letter residues with higher than 70% conservation. Alignment was performed using CLUSTALW, and the result was processed with ESPript 3.0. Sequence identity as in the Methods section. d 32P-cAMP-GMP-DRaCALA of E. coli cell lysates expressing CapV, CapVQ329R, and CapVQ329R/D197A. VC=pBAD28; pCapV=CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28. D197A=CapVQ329R/D197A cloned in pBAD28. e Assessment of affinity for cAMP-GMP of CapV, CapVQ329R, and CapVQ329R/D197A expressed in E. coli MG1655. 32P-cAMP-GMP was mixed with twofold dilutions of cell extracts starting at a fourfold dilution.

As CapV from V. cholerae46, CapV of E. coli ECOR31 contains a N-terminal canonical PNPLA domain with three main characteristic conserved signature motifs48,51, the phosphate or anion binding motif GG-G-x-[K/R]-G, the esterase box G-x-S-x-G, and the DG-[A/G] motif as part of the catalytic dyad (Fig. 3b, c). The G-x-S-x-G motif includes the conserved nucleophilic serine 64 of the active site characteristic for the phospholipase A2 (PLA2) superfamily48,52.

To investigate if catalysis is required for swimming inhibition and filamentation upon CapVQ329R overexpression, a catalytically inactive S64A variant of the protein (CapVQ329R/S64A) was generated. Compared with CapVQ329R, overexpression of CapVQ329R/S64A equally inhibited swimming motility and induced filamentation (Figs. 1a and 2d, e), demonstrating that the G-x-S-x-G motif of CapVQ329R is not required for the phenotype. However, the substitution of arginine in the GG-G-x-[K/R]-G motif (CapVQ329R/R27A) and aspartic acid of the DG-[A/G] motif (CapVQ329R/D197A) by alanine relieved both the repression of swimming motility and induction of filamentation. Substitution of the two structural glycine residues (CapVQ329R/G24A/G25A) of the GG-G-x-[K/R]-G also partially suppressed swimming motility and induced only a mild filamentous phenotype upon overexpression of the protein (Figs. 1a, b and 2d, e). In summary, the GG-G-x-[K/R]-G and DG-[A/G] motifs of CapVQ329R are required for repression of the swimming phenotype and cell filamentation.

As substitution of the nucleophilic serine 64 did not relieve motility and cell filamentation, we were wondering whether alternative serine residues are involved in the physiological activity of CapVQ329R. To this end, we substituted S33, S113/114, S146, S177, and S206 by alanine residues. Most of these serine residues were selected as they are located close to the catalytic site (Supplementary Fig. 2f). Only serine 206 was required to induce motility inhibition and promote cell filamentation by CapVQ329R (Supplementary Fig. 2f).

Furthermore, we wanted to clarify whether specifically the Q329R mutation is required to induce filamentation. Replacement of Q329 by the other positively charged amino acid lysine still partially repressed the apparent swimming motility and induced filamentation (Fig. 1b), while replacement of Q329 by asparagine retained the wild-type CapV phenotype (Supplementary Fig. 2f, g).

The binding site for cAMP-GMP in CapV has not been identified. The Q329R substitution might alter the binding of cAMP-GMP or other cyclic dinucleotides to CapV. To this end, binding of cAMP-GMP to CapV and CapVQ329R was analyzed by the differential radial capillary action of ligand (DRaCALA) assay. DRaCALA is based on the retention of small molecular compounds at the protein application spot on a nitrocellulose membrane upon binding. The experiment showed that both proteins bound cAMP-GMP with approximately equal affinity while the CapVQ329R/D197A mutant showed diminished binding (Fig. 3d).

Patatin-like phospholipases hydrolyze the sn-2 acyl ester bond of neutral and phospholipids48,53. In order to assess whether overexpression of CapV and CapVQ329R caused significant changes in the lipid profile concomitant with filamentation, we extracted lipids after 4 h of growth extracts to mass spectrometry (Fig. 4). Based on untargeted charged surface hybrid column quadrupole time-of-flight mass spectrometry (CSH-QTOF MS) analysis a total of 326 lipid species were identified (Fig. 4a). Principle component analysis showed distinct classification of samples into groups correlating with the overexpression of the wild-type CapV and CapVQ329R variant protein (Fig. 4b), indicating that the lipid profiles are significantly altered. Subsequently, we applied hierarchal clustering analysis to segregate the samples cumulatively according to overall changes in the individual lipid compounds. Based on the changes in the lipid compounds, the samples can again be classified into distinct groups according to the expressed proteins (Fig. 4f). Lipids known to be most abundant in the E. coli membrane, phosphatidylethanolamines (PE), phosphatidyl-glycerols (PG) and fatty acids (FAs), displayed the highest relative peak intensity (Fig. 4ce and Supplementary Fig. 5). We observed significant changes in the peak intensity of members of phospholipid classes, most abundant membrane components of E. coli such as PEs and PGs, but also of free FAs, lysophospholipids (LPE and LPG), phosphatidylcholines (PCs), ceramides (Cer) and sphingomyelins (SM), although the peak intensity of the latter three classes was at least 100-fold lower (Fig. 4ce and Supplementary Fig. 5). The peak intensity was, however, in none of the cases on the average more than sixfold different. Notably, among the top 50 most significantly altered lipids, PE and PG derivatives with distinct FAs profiles are predominantly represented (Fig. 4f). While CapV overexpression showed downregulation of a restricted group of lipids, a significantly higher number of lipids species were downregulated upon CapVQ329R overexpression. Notably, lipids species were also upregulated upon overexpression of CapV and CapVQ329R suggesting a role of the patatin-like phospholipases in membrane reorganization and/or signaling. Only a few lipid species were distinctively upregulated upon overexpression of CapVQ329R, PE32:2 (16:1 16:1) with two monounsaturated fatty acids and the monounsaturated fatty acid FA18:1. Thus CapV and CapVQ329R might have unique substrate profiles. Whether the observed alterations in the lipid profile are based on distinct residual catalytic activities of the two proteins or indirectly associated with the expression of CapV and CapVQ329R needs to be investigated further. In summary, these results indicate that amino acids in the catalytic motifs are required to induce filamentation and to repress motility. Not or only partially activated CapV and CapVQ329R can alter the lipid profile indicating that CapV is not only a receptor for a second messenger molecule, but might also be involved in alternative second messenger signaling in its nonactivated state.

a The number of identified lipid species in CapV and CapVQ329R induced filamentous cells compared to E. coli MG1655 vector control (VC) by untargeted charged surface hybrid column-quadrupole time-of-flight mass spectrometry (CSH-QTOF MS) analysis. For abbreviation of lipid compounds consult Methods. b Principle component analysis of lipid abundance upon overexpression of wild-type CapV and CapVQ329R variant proteins in E. coli MG1655 compared to VC. Of six samples each, outliers have been removed. ce Alternation and relative abundance of PE (c), PG (d), and FA (e) derivatives by untargeted CSH-QTOF MS analysis. Bars represent mean values from five independent replicates with error bars to represent SD. f Heatmap of selected 50 most significantly altered lipid species built based on hierarchical clustering. Each square represents one sample of each group. The color scale presenting the difference of each log2 transformed peak intensity value to the log2 transformed mean for each lipid species and the percentage of each lipid class is indicated on the right of the heatmap. Heatmap analysis was performed on the Tutools platform (https://www.cloudtutu.com), a free online data analysis website. Differences between mean values were assessed by two-tailed Students t test: ns, not significant; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001; black stars in c, d, e and f: compared to MG1655 VC; red stars in f: statistical significance between E. coli MG1655 pCapV and E. coli MG1655 pCapVQ329R. VC=pBAD28. pCapV=CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28.

During cell division, positioning of the FtsZ cytokinetic ring at the site of constriction between nucleoids is coordinated with chromosome replication, nucleoid segregation and cell elongation31,54,55. Impairment of this process leads to cell division arrest and filamentation, which can be induced by DNA damage and nucleoid occlusion15,27. After completion of cell segregation, the nucleoid subsequently becomes more compact56. To determine the effect of CapV and CapVQ329R overexpression during the cell division process, we analyzed FtsZ-ring positioning in an E. coli K-12 MG1655 derivative with a chromosomally encoded FtsZ-GFP fusion protein57 and the position and shape of the nucleoid with DAPI staining. After induction of CapV and CapVQ329R in liquid medium at 37C for 4h, we immediately subjected the cells to fluorescence microscopy on agarose pads to visualize cell shape, septum, and FtsZ-ring formation and nucleoid location. Overexpressing wild-type CapV in the E. coli K-12 MG1655 FtsZ-GFP strain, we observed clearly visible constrictions that corresponded with a correctly positioned FtsZ ring and a single nucleoid in nondividing cells and two fully replicated and/or segregated nucleoids in dividing cells, respectively (Fig. 5a). Of note, nucleoids upon CapV expression appeared slightly more compact than those in the control.

a Phase-contrast and fluorescence images of FtsZ-GFP expressing cells (E. coli MG1655 derivative BS001 harboring vector control (VC), pCapV and pCapVQ329R). Cells were cultured in TB medium at 37C for 4h, stained with DAPI, and assessed immediately under fluorescence microscopy. Large fragments of unsegregated nucleoids are indicated by white arrows. Bar, 3m. b Quantification of the average distance between two adjacent FtsZ rings upon overexpression of CapV and CapVQ329R, refers to Table 1. c Time-lapse analysis of mCherry-MinC expressing cells (E. coli MG1655 derivative PB318 harboring VC, pCapV and pCapVQ329R). A representative elongated cell is displayed. Graphs on the right of fluorescence images display the line profiles of fluorescent signals emanating from the cell. Arbitrary fluorescent units are obtained, analyzed by the Fiji ImageJ 1.8.0 software, and are plotted on the y axis; cell length (in m) is plotted on the x axis. Bar, 3m. VC=pBAD28. pCapV=CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28.

In contrast, upon CapVQ329R-induced cell elongation, most filamentous cells displayed smooth contours with no visible septa (Fig. 5a and Supplementary Fig. 6a, b), suggesting that the block in cell division occurs prior to the development of constriction. However, one or two constrictions were occasionally observed in a few filaments (Supplementary Fig. 6a). We found that most of the CapVQ329R-induced filamentous cells were polynuclear and contained multiple abnormally shaped or positioned nucleoids. Discrete patches of DNA staining could be compact, asymmetrically positioned in the filament or displayed an extended or decondensed shape occupying an extensive part of the filament (Fig. 5a and Supplementary Fig. 6b). We also observed a few filamentous cells containing nucleoids, which were evenly positioned throughout the filament with larger interchromosomal spaces (Supplementary Fig. 6b). Collectively, these observations suggest filamentous cells induced by CapVQ329R overexpression to be (partially) defective in chromosome segregation and nucleoid condensation.

Distinct FtsZ rings were observed in most of the filaments suggesting that the block in cell division occurred after FtsZ-ring formation. Large variations in the distance between two FtsZ rings lead to an unequal number of FtsZ rings in filamentous cells of similar length. CapVQ329R promoted filamentation with an average cell length of 31.2m (n=102), which corresponds to >15 times the standard cell length. The majority of the filamentous cells contained three FtsZ rings with multiple segregated and/or unsegregated nucleoids distributed between the rings. Of note, the distance between two adjacent Z-rings varied dramatically in the population of filamentous cells, with on average fourfold longer spacing in CapVQ329R expressing cells compared to cells expressing CapV and the control (Fig. 5b and Table 1). Equally variable was the number of partitioned nucleoids among filamentous cells.

Abnormal FtsZ expression levels induce cell filamentation58. Comparative immunoblot analysis observed slightly lower FtsZ protein production levels in CapVQ329R and CapV overexpressing cells, while the ftsZ mRNA steady-state level was with 91 and 97% of vector control expression only slightly changed (Supplementary Fig. 6c). Whether this (partial) decrease in FtsZ production levels is responsible for CapVQ329R-induced filamentation needs to be further investigated. In summary, CapVQ329R inhibits cell division at the level of constriction initiation and interferes with nucleoid segregation and condensation.

Previous studies showed that SulA blocks cell division by direct interaction with cell division core component FtsZ during the bacterial SOS response19,32. To investigate whether CapVQ329R induced cell filamentation of E. coli MG1655 is caused by the activation of sulA, we analyzed cell morphology upon CapVQ329R overexpression in a sulA mutant59,60. We found that CapVQ329R induced the same filamentous cell phenotype in the sulA mutant as in wild-type E. coli MG1655 (Supplementary Fig. 6d), suggesting that CapVQ329R-induced cell filamentation is sulA-independent consistent with the observed FtsZ-ring formation.

Multiple factors coordinate the regulation of cell division29. Among them is MinC, which oscillates from pole to pole in order to prevent FtsZ-ring formation at the cellular poles34. Upon overexpression, MinC causes cell filamentation. We investigated the dynamics of MinC mobility by time-lapse fluorescence microscopy (Fig. 5c and Supplementary Movie 3). In rod-shaped wild-type cells, we observed that MinC oscillates between cell poles, moving a large fraction of the total fluorescence signals along the long axis of the cell as described previously61. A complete oscillation cycle lasted about 50s under our experimental conditions. Of note, we found that oscillation of the FtsZ-ring inhibitor MinC is not affected in CapVQ329R-induced filaments (Fig. 5c) (PB318,61). MinC displayed various fluorescence spots ranging from one up to 5 depending on the length of the filament (Fig. 5c and Supplementary Fig. 6e). These fluorescent spots oscillated within multiple invisible cell borders in the filaments suggesting physical restrictions by septa. Within one single filamentous cell, the number of the apparent fluorescence fractions followed thereby a three-step principle: n n1 n (n 2, located at both poles) or n n+1 n (n 1, located within the cell), during an oscillation periodicity of about 50s, which is similar as for rod-shaped control cells and wild-type CapV overexpressing cells (Fig. 5c and Supplementary Fig. 6e). It will be relevant to investigate how long-distance MinC oscillation is maintained in filamentous E. coli cells. In summary, MinC oscillation might remain the effective watchdog for the prevention of FtsZ-ring formation in filamentous cells.

CapV has been identified as a patatin-like phospholipase that causes growth retardation upon activation by cAMP-GMP in V. cholerae El Tor46. In E. coli MG1655, though overexpression of CapV of E. coli ECOR31 wild-type did not affect cell division during the entire growth phase. Though no effect was observed during the first 3h of cell growth in liquid culture, overexpression of CapVQ329R induced a mild decrease in optical density after 6h (Fig. 6a). As extensive filamentation might not permit a direct correlation between OD and cell number, we tested cell viability by spotting E. coli MG1655 cells on agar plates for counting of viable individual cells. Compared to the control and cells expressing CapV, ~50% of the colonies were recovered from E. coli MG1655 cell cultures expressing CapVQ329R (Fig. 6b). Consistently, cells stained with the nucleic acid stain SYTO 9 for viability and propidium iodide (PI) for cell death showed that CapVQ329R production induced approximately 50% E. coli MG1655 cells to stain selectively with PI after 6h, while no DNA staining with PI was observed after 3h (Fig. 6c and Supplementary Fig. 7a, b). Of note, cells in some filaments took up both dyes seemingly live and dead (Supplementary Fig. 7). Interestingly, the uptake of SYTO 9 into individual cells varied widely in particular in CapV expressing cells, which showed no decrease in cell viability. These data indicate a highly heterogeneous nucleic acid content in individual cells. Thus, expression of not or only partially activated CapV and CapVQ329R might differentially affect membrane permeability and cell viability.

a Growth curves of E. coli MG1655 upon CapV and CapVQ329R overexpression induced by 0.1% l-arabinose in TB at 37C. Each data point represents the meanSD of six biological replicates. tb = TB medium. b Colony-spotting assay on agar plates. Cells were grown at 37C and harvested at different time points. Cell viability determined by spotting serial dilutions (100106) on LB plates to assess colony-forming units. c Quantification of Live/Dead staining of E. coli MG1655 cells after 3h and 6h in TB medium at 37C. n=1200. VC=pBAD28. pCapV=CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28.

Filamentation has been shown to be affected by environmental conditions26. We found that filamentation was particularly pronounced during cell growth in TB medium (1% tryptone, 0.5% NaCl), while it was restricted when cultured in LB medium (1% tryptone, 1% NaCl, 0.5% yeast extract) (Fig. 7a). Supplementation of TB medium with 0.5% yeast extract (YE) repressed the filamentous phenotype dramatically, while 5% restored the rod shape of all cells. Supplementation with 0.5% NaCl did not affect filamentation.

a Light microscopy pictures of E. coli MG1655 cell morphology and, b quantification of cell length in LB and TB medium supplemented with 0.5% NaCl, 0.5% YE, 5% YE, and VB6 (pyridoxine, 5mg/ml), respectively. The quantification is based on results from at least three independent experiments with the assessment of 70 cells from each group. Bar, 5m. VC=pBAD28. Q329R=CapVQ329R cloned in pBAD28.

Yeast extract is the water-soluble portion of autolyzed yeast cells used to prepare microbiological culture media62. As a nutrient source, it provides nitrogen, amino acids, peptides, carbohydrates, vitamin B complex, and other components that promote microbial growth63. In particular, yeast extract contains B vitamins, water-soluble precursors of structurally unrelated enzyme cofactors including thiamine (B1), riboflavin (B2), nicotinamide (B3), pantothenate (B5), pyridoxine (B6), biotin (B7), folic acid (B9), and cobalamin (B12). To identify the component(s) in YE that contribute(s) to the repression of cell filamentation upon CapVQ329R overexpression, we supplemented TB medium with these individual B vitamins. While supplementation with thiamine, riboflavin, nicotinamide, pantothenate, biotin, folic acid, and cobalamin showed no effect, supplementation with the vitamin B6 precursor pyridoxine at 5mg/ml decreased the length of filaments by 50% (Fig. 7a, b and Supplementary Fig. 8). It will be relevant to investigate the molecular mechanism of pyridoxine to inhibit CapVQ329R-induced cell filamentation. Further, the functionality of interconvertible pyridoxal and pyridoxamine vitamin B6 complex compounds and their 5 phosphate biologically active counterparts can be investigated individually and in combination. In summary, pyridoxine is one component in LB medium, which is effectively inhibiting cell filamentation upon overexpression of CapVQ329R.

To determine if the effect of CapVQ329R is restricted to E. coli K-12 MG1655, we overexpressed CapVQ329R in commensal and UPEC E. coli strains64,65 and the gastrointestinal pathogen S. typhimurium UMR1. The NCBI database (accessed latest December 15, 2021) indicates CapV homologs to be encoded predominantly by human fecal E. coli strains and strains derived from animals and animal and plant products. UPEC E. coli can develop extended filamentation upon host cell escape20. In all cases, CapVQ329R overexpression inhibited apparent swimming motility (Supplementary Fig. 9a, b).

Furthermore, CapVQ329R induced different degrees of cell filamentation in the E. coli strains and S. typhimurium UMR1 (Supplementary Fig. 9c). CapVQ329R production induced mild cell filamentation in commensal E. coli Fec32 and Fec89, moderate cell filamentation was observed in commensal Fec67 and extensive filamentation occurred in ECOR31, UPEC CFT073, and S. typhimurium UMR1. A heterogeneous population of moderately filamented cells as well as individual rod-shaped cells was observed in the UPEC strain E. coli No. 12 and the commensal E. coli strain Tob1 upon CapVQ329R overexpression (Supplementary Fig. 9c). In summary, our results showed that the phenotypes observed upon CapVQ329R expression were not restricted to E. coli MG1655, but were common to genetically unrelated commensal and UPEC E. coli strains and S. typhimurium, indicating a general effect of CapVQ329R on bacterial cell morphology, regulation of flagella-mediated swimming motility and biofilm formation.

We were subsequently wondering, whether CapVQ329R affects phenotypes other than cell filamentation and flagella expression. To this end, we investigated the colony morphotype on agar plates. A significant number of E. coli isolates display a rdar biofilm morphotype on Congo red agar plates characterized by the expression of the extracellular matrix components amyloid curli and the exopolysaccharide cellulose64.

Congruent with the pronounced temperature-dependent effect of CapVQ329R, we investigated the effect of CapVQ329R in strains that displayed the rdar morphotype at 37C. We choose the UPEC strain No. 12, which expresses a semi-constitutive csgD-dependent rdar morphotype65. While overexpression of wild-type CapV had no major effect compared to the No. 12 control, overexpression of CapVQ329R dramatically disrupted the rdar morphotype (Fig. 8a). Observations of colonies by scanning electron microscopy and TEM demonstrated that CapVQ329R induced even more extensive cell filamentation on agar plates compared to liquid culture, without affecting cell arrangement (Fig. 8b). While nonfilamented cells produced a pronounced extracellular matrix, the filaments produced only little or no matrix (Fig. 8b).

a Rdar morphotype in wild-type E. coli No. 12 vector control (VC) and upon overexpression of CapV and its mutant CapVQ329R. Cells were grown on a salt-free LB agar plate for 24h at 37C. b Scanning electron microscopy of plate-grown colonies. Colony morphotypes grown on a salt-free LB agar plate were fixed after 24h of growth at 37C. c CsgD production upon overexpression of CapV and CapVQ329R in E. coli strain No. 12 compared to VC. Only colony morphotypes from the same plate and signals from the same western blot are compared. d LC-MS/MS quantification of in vivo amounts of c-di-GMP, cAMP, and cAMP-GMP upon overexpression of CapV and CapVQ329R in E. coli strain No. 12 compared to VC. Data are displayed as absolute amounts referred to the original cell suspension. VC=pBAD28. pCapV=CapV cloned in pBAD28; Q329R=CapVQ329R cloned in pBAD28.

In conjunction with rdar morphotype downregulation, its major transcriptional activator CsgD was downregulated (Fig. 8c), demonstrating that CapVQ329R expression affects the central regulatory hub for rdar biofilm formation in strain No. 12. Furthermore, CapVQ329R overexpression equally downregulated the rdar morphotype in the commensal E. coli strains ECOR31, Fec67, Fec89, and Tob1 (Supplementary Fig. 9d). Downregulation of CsgD expression upon expression of CapVQ329R was exemplarily observed for strain ECOR31 (Supplementary Fig. 9e).

We then investigated the effect of CapVQ329R overexpression in commensal E. coli MG1655 and UPEC CFT073 which display a smooth and white morphotype when grown at 37C, indicative for the lack of rdar biofilm expression under these experimental conditions. After 48h of growth, though, a distinctly structured brown colony morphology with dye uptake was displayed upon CapVQ329R, but not upon CapV overexpression (Supplementary Fig. 9d). In the same line, S. typhimurium strain UMR1 displayed a smooth and white morphotype at 37C66, with its colony to develop a similar brown morphotype after 48h of CapVQ329R overexpression (Supplementary Fig. 9d). Again, this colony morphotype seem to be distinct from the rdar biofilm in coloration with no or below detection limit production of the biofilm regulator CsgD in E. coli MG1655 (Supplementary Fig. 9). Semi-constitutive rdar morphotype expressing ECOR31 and No. 12 displaying reduced morphology and coloration retained low CsgD levels upon CapVQ329R production (Fig. 8c and Supplementary Fig. 9c). Thus, CapVQ329R production affects biofilm-associated colony morphotype formation in a complex way in different E. coli strains.

In Enterobacteriaceae, cyclic di-GMP is a ubiquitous bacterial second messenger, which stimulates the development of the rdar biofilm morphotype via csgD expression40,67,68. We investigated the cyclic (di) nucleotide levels exemplarily in the UPEC strain E. coli No. 12. Along with inhibition of csgD expression, the in vivo cyclic di-GMP level was concomitantly decreased upon CapVQ329R overexpression (Fig. 8d). Besides cyclic di-GMP, both cAMP and cAMP-GMP regulate E. coli biofilm formation43,69. Consistent with a downregulated rdar biofilm phenotype, a reduction in cAMP and an increase in cAMP-GMP levels were observed upon CapVQ329R overexpression (Fig. 8d). Thereby, CapVQ329R might, for example, inhibit a diguanylate cyclase or promote phosphodiesterase activity to downregulate the cyclic di-GMP level. Taken together, these results indicate that CapVQ329R inhibits rdar biofilm formation potentially through regulation of the intracellular level of various cyclic (di)-nucleotide signals.

Recently, the patatin-like phospholipase CapV and the dinucleotide cyclase DncV have been shown to take part in bacterial antiphage defense70. Since CapVQ329R showed a physiological function independent of DncV, we wondered whether CapVQ329R still contributes to antiphage defense. While overexpression of wild-type CapV had no effect, interestingly, an approximately tenfold higher plaque formation was observed upon CapVQ329R overexpression compared to the control when MG1655 cells were infected with P1 phage (Supplementary Fig. 10a), indicating that CapVQ329R enhances susceptibility to bacteriophage P1 infection.

We also observed that overexpression of CapVQ329R, but not CapV renders the laboratory strain E. coli MG1655, the commensal strain ECOR31, and UPEC No. 12 more susceptible or sensitive against the beta-lactam antibiotic cephalexin (Supplementary Fig. 10b). A systematic investigation of altered susceptibility to various antibiotic classes awaits to be performed.

We were also wondering whether CapVQ329R-induced cell elongation and concomitant physiological changes impair the interaction of E. coli with host cells. To this end, we exposed the bladder epithelial cell line T24 to UPEC E. coli strain No. 12 overexpressing CapV and CapVQ329R. CapVQ329R expressing E. coli strain No. 12 associated significantly less with the T24 bladder epithelial cell line than the control and CapV expressing cells. Although a trend to lower induction of mRNA steady-state levels of genes coding for the pro-inflammatory markers IL-1 and CXCL-8 was observed for T24 cells exposed to CapVQ329R expressing bacteria, the obtained results were not statistically significant (Supplementary Fig. 10c). Thus, in summary, CapVQ329R production causes substantial physiological alterations in E. coli MG1655.

Read more:

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution | npj Biofilms and...

Biochemistry Professor John Browse and anthropology Professor Tim Kohler were elected to the National Academy of Sciences in recognition of their achievements in original research.

Browse and Kohler are among just 150 new members announced on May3. First established by U.S.Congress and President Abraham Lincoln in 1863, the National Academy of Sciences is a nonprofit society of scholars charged with providing independent, objectiveadvice about science and technology to the nation.

Browse is a pioneer and leader in plant biology. His research focuses on investigating the biosynthesis of membrane and seed-storage lipids in plants, using Arabidopsis, or thale cress, a model organism often used to understand plant biology. His work has improved understanding of plant defenses and helped bioengineer plants with higher amounts of useful chemicals, such as increased levels of heart-healthy monounsaturated fatty acids. He is also well known for identifying and cloning the desaturase gene in Arabidopsis, responsible for the synthesis of polyunsaturated fatty acids in plants.

Kohler studies the social dynamics of prehistoric cultures, specializing in the U.S. Southwest. His research explores the relationships among demography, violence, wealth inequality, social evolution, and climate variability. While his work has improved methods of understanding the past, his findings have echoes into the present which was recently recognized when the United Nations named Kohler as a lead author on a recent Intergovernmental Panel on Climate Change report. Kohlers current projects include the SKOPE project to make interpreted paleoenvironmental data widely accessible, and another National Science Foundation-funded project to generate and analyze measures of wealth inequality in societies around the world over the last 10,000years.

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Two WSU faculty named to National Academy of Sciences WSU Insider - WSU News

Season 10, Episode 6, "Saturday Morning Fun Pit," is one of those episodes that play with the typical series formula. Taking chance with episodes like this can be a hit or miss for sitcoms. Sadly, it arguably misses in this case.

The episode sees Richard Nixon enjoying Saturday morning cartoons that feature the "Futurama" gang. The three segments parody various cartoons from different eras. The first is "Bendee Boo and the Mystery Crew." The segment sees them investigate the connection between George Takei's kabuki theater and a cloning lab being used by the Harlem Globetrotters to train for their next game (by going against five clones of NBA legend Larry Bird). It is revealed that Takei was trying to halt the game to bring attention to his theater.

The second segment, birthed from a riot where the mob complains cartoons aren't educational, is titled "Purpleberry Pond." It sees the gang as adorable characters that live in a happy purple land. When an orange character named Lord Loquat arrives, they learn the value of not judging others. They also preach healthy eating, despite every variety of cereal they advertise being loaded with sugar.

The final segment, "G.I. Zapp," is another one that the mob demands be changed for being too violent. The segment sees the crew, led by Zapp Brannigan, engage in a violent battle that Nixon attempts to edit live with a special device in his office. He changes the characters from mercenaries to "patriotic peacekeepers" and alters dialogue such as"We're gonna blow them straight to HeCHURCH!" Ultimately, he gives up and cuts to a PSA.While all in good fun, the episode has gone on to be an infamous one amongst fans for one big reason in particular.

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The Futurama Episode That Wouldn't Make Sense To Modern Fans - Looper

This Conversational Artificial Intelligence and Voice Cloning Market report studies the diverse and growth picture of the Conversational Artificial Intelligence and Voice Cloning industry. It is an executive summary of the Conversational Artificial Intelligence and Voice Cloning industry highlighting its major trends, other findings, and recommendations. The report studies the Conversational Artificial Intelligence and Voice Cloning industry by highlighting the individual companies, investors, producers, distributors, and providers of raw materials. The report details the risks, opportunities, mature segments, and emerging segments in the market. The report forecasts the revenue growth of the global Conversational Artificial Intelligence and Voice Cloning market at regional, global, and country level and analyzes the latest trends in the industry in the every sub-segment in the industry.

Key Players in the Conversational Artificial Intelligence and Voice Cloning market:

, Acapela Group, Alt Inc., Amazon (AWS), AmplifyReach, Aristech GmbH, Artificial Solutions, AT&T;, Avaamo, Baidu, CandyVoice, Cepstral, CereProc, Clinc, Cognigy, Conversica, Creative Virtual, Descript Inc., Exceed.ai, exClone, Facebook, FIS, Google, Haptik, IBM, Inbenta, Interactions, iSpeech, Kasisto (KAI), Kata.ai, Kore.ai, LumenVox, Lyrebird, Microsoft, Mindsay, Nuance Communications, Oracle, Pypestream, Quosphere, Rasa, ReadSpeaker (rSpeak), Resemble AI, Rulai, Saarthi.ai, SAP, Smartbox Assistive Technology, Solvvy, SoundHound, VivoTek, VocaliD, Voctro Labs, Voicery , ,

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The yearly production volumes, amount of products and goods consumed annually, the market of Conversational Artificial Intelligence and Voice Cloning industry across the globe is studied in the report. Moreover, the diverse consumer base of the Conversational Artificial Intelligence and Voice Cloning industry, investors, previous data, and financial value of the market is determined in the report. The diversity of demand for the products and services available in the market and the robust nature of the Conversational Artificial Intelligence and Voice Cloning industry that is enabling market players for investing in the market are studied in the report. The government-developed policies and Conversational Artificial Intelligence and Voice Cloning industry standards are detailed in the study. The policies developed by the governments, international agencies, and policy-makers that have reduced the barriers and increased access to the market are elaborated in the study.

Conversational Artificial Intelligence and Voice Cloning Market Types:

Type I,Type II,Type III

Conversational Artificial Intelligence and Voice Cloning Market Applications:

Application I,Application II,Application III

The Goals of the Conversational Artificial Intelligence and Voice Cloning Industry Report Are To:

Report the demand dynamics, changing profit dynamics, market and distribution trends, new product innovations and technology interventions, and new business strategies.

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To highlight the risks, opportunities, mature segments, and emerging segments in the market.

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The report studies the global Conversational Artificial Intelligence and Voice Cloning industry covering the growth, challenges and digital disruption in the industry.

The growth prospects of the Conversational Artificial Intelligence and Voice Cloning industry are studied in the report.

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The present situation of all the sectors in the global Conversational Artificial Intelligence and Voice Cloning market is detailed here.

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Conversational Artificial Intelligence and Voice Cloning Market Key Players Change the View of the Global Face of Industry by 2028: , Acapela Group,...

"People ask me, 'Why is it so expensive?' and I tell them because there are so many complicated steps involved in the whole process," says Rodriguez. "It's definitely an emotional reason for pet clients. They want to be able to carry on that strong emotional bond that they have with the pet."

The industry has since expanded elsewhere in the globe. Sooam Biotech in South Korea offer dog cloning services, as well as Sinogene in China.

However, many scientists remain uncomfortable about the whole premise. Lovell-Badge argues that there is "no justification" for pet cloning as while the resulting animals will be genetically identical, they will not have the same behavioural characteristics and personalities as all creatures are a product of both genes and their environment.

"People really want their pet that knows them and knows certain tricks and so forth," says George Church, professor of genetics at Harvard Medical School. "In that sense, it's a little bit taking advantage of people's grief."

Reviving extinct species

In the years that followed Dolly's cloning, the central question was whether scientists would ever extend the technology to humans, and the many moral and ethical issues that would invoke.

But while a human embryo was successfully cloned in 2013, the process of creating an entire human being has never been attempted because of the likely public outcry. Chinese scientists did clone the first primates in January 2018, long-tailed macques Zhong Zhong and Hua Hua, but there are currently no suggestions that this work will continue into further primate species.

Instead, most funding is being devoted to using cloning to resurrect animals on the verge of extinction. Efforts are underway to clone both the giant panda and the northern white rhino a species for which there are just two animals left on the planet while in the last two years, ViaGen have cloned the black footed ferret and Przewalski's horse, both of which are endangered.

Church is leading the most ambitious project, a quest to revive the woolly mammoth, a species that last lived some 4,000 years ago. His de-extinction company Colossal has already raised 11m ($14.5m) in funding to support the idea, which will involve creating an elephant-mammoth hybrid through taking skin cells from Asian elephants and using cloning technology to reprogram them with mammoth DNA.

The rest is here:

The people cloning their pets - BBC.com

The flashing blue lights of an unmarked police car burst into the cabin of my SUV just seconds after Id eased off the motorway and into the service station for a welcome pit stop.

But it wasnt unexpected, I had an inkling it could and probably would happen.

And for the past few miles Id been watching the distinctive headlights of the grey BMW through my rear-view mirror always keeping a steady pace and distance behind.

So as the car drew alongside next to the Shell filling station, just off the Southbound A1 (M), I wound down the window and looked across at the officer in the passenger seat eyeing me up,

South Wales? I ventured? He nodded.

Thankfully, I was not the guilty party but an innocent victim. As PCs Dan Stoppard and Gav Pearson of North Yorkshire police were happy to confirm.

The full story began to emerge as we compared notes at Barnsdale Bar South services near Pontefract.

My car had been cloned and I was the latest victim of a rising tide of automotive identity theft.

So-called car-cloning is on the increase as criminals steal the identities of cars in a variety of scams that leave thousands of innocent motorists with parking tickets and speeding fines for which theyre not responsible.

But in the worst cases, consumers are tricked into unwittingly buying stolen cars with a false number plate and identity and left thousands of pounds out of pocket when the vehicle is impounded and returned to its rightful owner.

For me, it all began with a phone call from Nissan while I was in the North-East visiting the firms Sunderland plant last week. Id driven up in a new Qashqai, which had been built there.

Had I by any chance been to South Wales as well? came the question from Nissan.

The answer was no. And Nissan knew this, but needed to confirm because a near identical blue Nissan Qashqai, with exactly the same (but fake) number plate as mine, had been twice clocked speeding and twice been involved in high-speed pursuits with police in South Wales.

How could that be?

Usually, criminals will wander around car showrooms looking for a model that resembles one they have stolen, and put copies of those legitimate plates on to their stolen example.

But my case was slightly different. My car was part of the Nissan press fleet made available for journalists to road test.

The vehicle had already been extensively photographed with the images appearing in motoring magazines and online.

The suspicion was that a similar-looking car had been stolen and some fake number plates made up matching those of the car I was driving. So the legitimate number plate on my car was now in the police system and would flag up if caught on camera.

In my case, the suspect number plate was spotted on the A1 by an ANPR number-plate recognition camera and flagged as being of interest. Apparently, a number of police cars were sent in pursuit, but roadworks meant it fell to the unmarked BMW to pull me in.

Police cars themselves have an array of all-round cameras whose digitised images link via computer to the DVLA, insurance and MOT records, highlighting vehicles that may be untaxed, unroadworthy, uninsured or suspected of being used in criminal or anti-social activity.

Nissan had already told the police that my car was probably not the one theyd been looking for in Wales. The officers too had been tipped via their alert system that my car was probably the honest one.

The two officers who pulled me in said there tended to be an increase in cloning in March and September when the new six-monthly plates come out as thats when a lot of number plates are made and often discarded.

To help distinguish my legitimate car from the imposter clone, the officers made a subtle tweak to my plates which would signal to other police that this was the real deal.

Motoring groups say Im far from alone and that this is a growing problem. The RAC said: Complaints about the trade in cloned registration number plates are on the rise.

Criminals armed with duplicate plates are better equipped to dodge ANPR cameras while also duping police officers into pursuing the wrong suspects.

It added: Cloning is a very real problem that could not only leave you thousands of pounds out of pocket, but also see you wrongly accused of a serious crime. Criminals either attempt to sell the cloned vehicle or use it to carry out further crimes.

If the cloned car is caught breaking the law, or involved in unlawful activity, these offences will often be attributed to the owner of the car that has been cloned.

Some unscrupulous drivers are using green number plates on diesel and petrol cars to avoid paying green penalties when driving older, polluting vehicles in Londons ULEZ or other Clean Air Zones.

And some number plate suppliers are selling plates without verifying the cars original documents which makes it easier for criminals to get their hands on illegal number plates.

Funnily enough, personalised number plates have a lower chance of being cloned even though they might appear more simple. Keeping photos of your car can help as differences such as dents, scrapes or modifications will distinguish your car from the one committing the crime.

The AA said: Cloning is done either to mask the true identity of a stolen vehicle or, more basically, to avoid parking tickets, speeding fines, congestion charges and the like the ticket would be sent to the keeper of the vehicle from which the identity was stolen.

But if you are unfortunate enough to buy a cloned vehicle, then you will lose both the car and any money paid for it, so its important to check out carefully any car youre thinking of buying and to walk away if you suspect anything may be wrong.

The AA also cautioned to avoid paying cash: Dont pay less than 70 per cent of the market value of the vehicle. If its too good to be true, it may well be.

The dog is mans best friend, as the saying goes.

Yet millions of drivers could be breaking the law and putting the life of their favourite pet in peril while driving.

This could even lead to fines of up to 5,000, motoring experts have warned.

Research by road safety charity IAM RoadSmart reveals that more than four out of ten (42 per cent) of dog owners put their pet in an unsafe place while on the road.

Of this group, more than a third said they left their pet unrestrained in the car: either in the back seat, the passenger seat, the footwell or loose in the boot.

And although 1 in 12 left their dog restrained on the passenger seat which is legal but not best practice the report said: This could lead to a pet being killed or badly injured in a collision if the airbag is activated, even at low speeds.

With around 36 million licence holders in the UK and approximately 12 million dogs, these survey figures represent a huge amount of risk taking on our roads.

The charity points out that the Highway Code states dogs must be suitably restrained when travelling in a car so they cannot distract the driver or cause injury to either themselves or the motorist.

Also, insurers are unlikely to pay out for an accident in the event of driving without proper control, leaving the motorist with a sizeable repair bill.

Link:

Catching the car clones! Automotive identity theft' is on the rise - msnNOW

Show more information on RDC...

As with Rapid Delta Restore (RDR) the concept of RDR has been something that has been thought about for quite some time here at Macrium Software. We wanted to build a clone solution that would effectively and rapidly copy only the differences between the source and target file systems. The advantage of this is obvious, RDC offers similar a performance increase as an Incremental disk image offers over a Full image and enables regular clones to be a viable and fast DR solution.

How does it work?

The NTFS file system resident on the clone source is compared with file system on the target disk. The two file systems are first verified that they originated from the same format command and then the target NTFS file system structures are analyzed for differences. All the NTFS file system structures are copied to the target disk and any that do not exist or have been modified on the target disk cause the data records for each NTFS file or object to be copied as well. The result is an 'Incremental' clone applying only file system changes detected between the source and the target.

Note: RDC works with NTFS file systems only. All other file systems will perform a full clone

Note: RDC is not available when shrinking partitions during a clone.

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Cloning a disk - KnowledgeBase - Macrium Reflect

MarketsandResearch.biz just released a market research Global Voice Cloning Market that looks at the sensitive data discovery industry from a variety of angles. Its objective is to determine the markets size and growth potential in a variety of categories, including components, organisation size, deployment style, application, vertical, and geographic regions. It includes company profiles, critical insights about product and business offerings, recent developments, and significant market strategies, as well as a thorough competition analysis of the markets top competitors.

The report would provide revenue figures for the overall sensitive data discovery market and its sub segments that are the closest approximations to Voice Cloning market market leaders/new entrants in this industry.

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All aspects of the sensitive data discovery business are covered in the Voice Cloning market study. Its objective is to determine the markets size and growth potential in a variety of categories, including components, organisation size, deployment style, application, vertical, and geographic regions. It includes company profiles, critical insights about product and business offerings, recent developments, and significant market strategies, as well as a thorough competition analysis of the markets top competitors.

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Contact UsMark StoneHead of Business DevelopmentPhone: 1-201-465-4211Email: sales@marketsandresearch.biz

Original post:

Global Voice Cloning Market 2022 Industry Insights and Key Players like IBM, Google, Lyrebird, Nuance Communications, Baidu The Bite - The Bite

Sign in to GitHub.com and GitHub Desktop before you start to clone.

On GitHub.com, navigate to the main page of the repository.

Above the list of files, click Code.

Click Open with GitHub Desktop to clone and open the repository with GitHub Desktop.

Click Choose... and, using the Finder window, navigate to a local path where you want to clone the repository.

Note: If the repository is configured to use LFS, you will be prompted to initialize Git LFS.

Click Clone.

Sign in to GitHub.com and GitHub Desktop before you start to clone.

On GitHub.com, navigate to the main page of the repository.

Above the list of files, click Code.

Click Open with GitHub Desktop to clone and open the repository with GitHub Desktop.

Click Choose... and, using Windows Explorer, navigate to a local path where you want to clone the repository.

Note: If the repository is configured to use LFS, you will be prompted to initialize Git LFS.

Click Clone.

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Cloning a repository from GitHub to GitHub Desktop ...