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text:
Govt Makes Six Airbags Compulsory In Vehicles Carrying Up To 8 Passengers
In a series of tweets, Union Minister for Road Transport and Highways, Nitin Gadkari, said, "In order to enhance the safety of the occupants in motor vehicles carrying up to 8 passengers, I have now approved a draft GSR notification to make a minimum of 6 airbags compulsory."
Published: Friday, 14 January 2022
Last updated: 14 January 2022, 08:49 PM IST
Nitin-Gadkari
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The Centre on Friday approved the draft notification of General Safety Regulations (GSR), making minimum of six airbags compulsory in vehicles carrying up to eight passengers.
"The ministry already mandated the implementation of fitment of the driver airbag w.e.f. July 1, 2019 and front co-passenger airbag w.e.f. January 1, 2022," Gadkari said.
Gadkari said the move is aimed at minimising the impact of frontal and lateral collision to the occupants seated in both front and rear compartments.
"To minimise the impact of frontal and lateral collisions to the occupants seated in both front and rear compartments, it has been decided that 4 additional airbags be mandated in the M1 vehicle category, i.e., two side/side torso airbags and two side curtain/tube airbags covering all outboard passengers. This is a crucial step to make motor vehicles in India safer than ever," Gadkari said.
The minister emphasised that this will ultimately ensure the safety of passengers across all segments, irrespective of the cost/variant of the vehicles.
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National Delhi-Mumbai Expressway To Fetch Rs 1,000 To 1,500 Cr Revenues Every Month, Says Gadkari
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redpajama_set_name: RedPajamaCommonCrawl


Production directly from carbon dioxide by engineered cyanobacteria is one of the promising technologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation.
First, a stoichiometric metabolic model was applied to prediction of cellular metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modified by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic flux distribution predicted by metabolic flux balance analysis (FBA) was used for in silico simulation of gene disruption. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The effect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain.
During 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption.
Direct production of chemicals and fuels from carbon dioxide by cyanobacteria is one of the promising technologies to reduce carbon dioxide emissions and the dependence on fossil fuels [1, 2]. Compared with biological production using heterotrophic microorganisms, biological production using cyanobacteria can avoid various complicated processes for harvesting and decomposition of biomass. The introduction of a synthetic metabolic pathway composed of genes from other organisms into cyanobacteria is a key technology for production of various chemicals directly from carbon dioxide. Some of these valuable chemicals include isobutyraldehyde, isobutanol [3, 4], ethanol , 2,3-butanediol [6, 7], 2-methyl-1-butanol , 1-butanol [9, 10], acetone [11, 12], 3-hydroxybutyrate , ethylene [14, 15], isoprene [16, 17], 1,2-propanediol (1,2-PDO) , and glycerol [19, 20]. In addition, we successfully developed Synechococcus elongatus PCC 7942 to produce 1,3-propanediol (1,3-PDO) by introduction of a synthetic metabolic pathway . 1,3-PDO is a valuable chemical widely used for manufacture of polymers, paint, solvents, and antifreeze agents . In the introduced synthetic metabolic pathway, dihydroxyacetone phosphate (DHAP) in the Calvin cycle is converted to 1,3-PDO via glycerol (Fig. 1). The strain into which the synthetic pathway was introduced was found to produce 3.79 mM (0.29 g/L) 1,3-PDO and 12.6 mM (1.16 g/L) glycerol during 14 days of incubation . Although chemical production directly from carbon dioxide is a key advantage of cyanobacteria compared with other heterotrophic organisms, even the ethanol production by cyanobacteria showing the highest titer (5.5 g/L)  among various chemicals has not been increased over 10 g/L. Therefore, the improvements of productivity are key issues to make production by engineered cyanobacteria practical and cost effective.
In silico simulation is an powerful methodology to improve productivity with low experimental costs. The stoichiometric metabolic model not requiring kinetic parameters of metabolic reactions is comparatively applicable to analysis of whole-cell metabolism. Flux balance analysis (FBA) can predict metabolic flux distribution under constraint conditions . FBA using the genome-scale metabolic model (GSM) has been applied to improvement, for example, of succinic acid , threonine , valine , and lycopene  production by Escherichia coli. For screening of effective candidates for gene manipulation, various strategies have been established for prediction of the effects of a gene knockout, knockdown, and overexpression . Shastri and Morgan  were the first to construct a stoichiometric metabolic model of cyanobacteria composed of the central metabolism and photosystem of Synechocystis sp. PCC 6803. So far, the metabolic models of some cyanobacteria used for bioproduction (Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, Arthrospira platensis, Cyanothece sp. ATCC 51142) have been constructed on the basis of an annotated genome sequence [30–32]. Anfelt et al.  reported that introduction of the phosphoketolase pathway increases cellular acetyl-CoA level and butanol productivity in Synechocystis sp. PCC 6803. FBA was used for validation of this genetic modification to increase theoretical productivity of butanol. Furthermore, some simulations based on FBA have predicted various metabolic modifications (knockout and overexpression) to increase theoretical production by cyanobacteria, for example, of biofuels (1-butanol, 1-octanol, limonene, ethanol) and succinic acid by Synechocystis sp. PCC 6803 [33–35] and biofuels (butanol, ethanol) by Synechococcus sp. PCC 7002 . Nonetheless, there are only a few reports that these predicted strategies for productivity improvement applied experimentally .
In this study, we applied FBA to prediction of metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942 constructed in our previous work . The candidates for the gene knockout to increase 1,3-PDO productivity were estimated by FBA with the minimization of metabolic adjustment (MOMA) algorithm . The positive effect of the gene knockout on 1,3-PDO productivity after screening by in silico simulation was experimentally validated in the 1,3-PDO-producing strain.
All the chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless specified otherwise. Restriction enzymes, phosphatase (New England Biolabs; Ipswich, MA, USA), ligase (Rapid DNA Ligation Kit, Roche; Mannheim, Germany), and DNA polymerase (KOD Plus Neo DNA polymerase, TOYOBO Co., Ltd.; Osaka, Japan) were used for cloning. Oligonucleotides were synthesized by Life Technologies Japan, Ltd. (Tokyo, Japan).
A modified BG11 medium supplemented with 20 mM HEPES–NaOH (pH 7.5) for pH stabilization was used for cultivation of S. elongatus PCC 7942 (Life Technologies Corporation; Carlsbad, CA, USA). Hereafter, this medium is referred to as the BG11 medium. The composition of the BG11 medium used in this study was the same as previously reported . Antibiotics (10 μg/mL kanamycin, 20 µg/mL spectinomycin, and 5 µg/mL chloramphenicol) were added to the BG11 medium when appropriate.
The incubation conditions for 1,3-PDO production were the same as those described previously . Cell density (OD730) was measured using Infinite 200 PRO (TECAN; Männedorf, Switzerland). For determination of dry cell weight (DCW), cells in culture were harvested on the Ominipore™ membrane filter (JHWP04700; Merck Millipore; Darmstadt, Germany) by filtration under reduced pressure. Cell harvest filters were dried at 65 °C for 12 h. The weight differences before and after cell harvesting were determined as DCW in culture.
Plasmids and primers used in this study are listed in Additional file 1: Tables S1 and S2, respectively. To construct a plasmid for gene disruption, the upper and lower regions of a target gene were amplified for homologous recombination by PCR. The primer sets for the upstream region of ndhD1, ndhD2, ndhD3, ndhD4, and ndhF1 were T2711–T2712, T3189–T3190, T2723–T2724, T2719–T2720, and T3208–3209, respectively. The primer sets for the downstream region of ndhD1, ndhD2, ndhD3, ndhD4, and ndhF1 were T3187–T3188, T3191–T3192, T2787–T2726, T2721–2722, and T2713–T2714, respectively. The amplified upstream and downstream regions except for ndhF1 were digested by KpnI–HindIII and HindIII–BamHI, respectively, and ligated into KpnI–BamHI sites of pTA703. Only for ndhF1, the upstream and downstream regions were digested with SalI–HindIII and HindIII–BamHI, respectively, and ligated into SalI–BamHI sites of pTA703. A chloramphenicol resistance cassette was amplified from pZA31-luc (Expressys; Ruelzheim, Germany) by PCR using primers T2128–T2129. The amplified fragment digested with HindIII was ligated into the HindIII site of each plasmid harboring upstream and downstream regions of a target gene, thus creating plasmids pTA1811 (ndhD1 disruption), pTA1812 (ndhD2 disruption), pTA1641 (ndhD3 disruption), pTA1640 (ndhD4 disruption), and pTA1828 (ndhF1 disruption).
The constructed plasmids (pTA1811, 1812, 1641, 1640, and 1828) were used for gene disruption in TA1297 (wild-type strain), TA2984 (1,3-PDO-producing strain), and TA3800 (glycerol-producing strain). TA2984 and TA3800 were constructed in our previous studies [21, 38]. The strains used in this study are listed in Additional file 1: Table S1.
Culture supernatants were centrifuged (20,000×g, 10 min) and filtered using Minisart RC4 (Sartorius; Goettingen, Germany). Concentrations of glycerol and 1,3-PDO in filtered samples were determined using a high-performance liquid chromatograph (LC-20AD, Shimadzu; Kyoto, Japan) equipped with an autosampler (AOC-20; Shimadzu), a SUGAR SP-G guard column (Shodex; Tokyo, Japan), a SUGAR SP0810 column (Shodex), and a refractive index detector (RID-10A; Shimadzu). Operating conditions were as follows: injection volume, 5 µL; mobile phase, MilliQ water; flow rate, 1.0 mL/min in isocratic mode; column temperature, 80 °C.
The stoichiometric metabolic model based on the annotated genome sequence of S. elongatus PCC 7942 published by Knoop  (hereafter called GSM_7942 in this study) served as a basic metabolic model of the wild-type strain in this study. GSM_7942 is composed of 595 genes, 666 reactions, and 533 metabolites. The information on the introduced synthetic metabolic pathway for 1,3-PDO production (8 genes, 6 reactions, and 3 metabolites) was added to GSM_7942, resulting in a metabolic model of TA2984 (hereafter called GSM_7942_PDO in this study). GSM_7942_PDO is composed of 603 genes, 672 reactions, and 536 metabolites. GSM_7942_PDO was used to perform FBA using the COBRA toolbox 2.05  on Matlab (Mathworks Inc., Natick, MA).
This simulation was performed on GSM_7942_PDO with scripts provided in COBRA toolbox 2.05 using Gurobi 6.03 (Gurobi optimization Inc., USA) as a solver.
Chlorophyll concentrations were measured by extraction in methanol . Oxygen consumption (respiration) and evolution (photosynthesis) from whole cells were measured with a Clark-type electrode (OXYT-1, Hansatech Instruments, Norfolk, UK) at a chlorophyll concentration of 10 µg/mL. The temperature of the electrode chamber was maintained at 30°C, and the chamber contents were continuously stirred. Saturated light (1000 μE/m2/s) and growth light (100 μE/m2/s) provided by a digital LED source (PD3-5024-4-EI, CCS, Kyoto, Japan) were used for measurement of photosynthetic activity. Photon flux density was measured with IKS-27 (Koito; Tokyo, Japan). Respiratory activity was similarly measured in complete darkness.
ATP extraction and measurement were basically performed by the method of Wang et al. . The appropriate number of cells at growth phase (corresponding to 1-mL culture at OD730 of 1.0) was collected, and ATP was extracted from pelleted cells by resuspension in a 1% (w/v) trichloroacetic acid solution. After centrifugation at 20,000×g for 10 min at 4 °C, the supernatant was neutralized with 1 M Tris–acetate buffer (pH 7.8). The concentration of ATP in the neutralized sample was determined using the ATP determination kit (A22066, Molecular Probes Inc., Oregon, USA).
Intracellular metabolites of TA2984 (1,3-PDO-producing strain) and TA4058 (ndhF1-null 1,3-PDO-producing strain) were measured by LC-QqQ-MS and GC-Q-MS analyses. Sample preparation and analysis were performed by the method described by Soma and Hanai .
Glycogen extraction was basically performed by the method of Suzuki et al. . Cells at early stationary phase (days 10) were used for glycogen measurement. The extracted glycogen was mixed with glucoamylase (Oriental Yeast Co., Ltd.; Tokyo, Japan) in 2.5 mM Na-acetate (pH 5.0) and incubated at 40 °C for 1 h. After centrifugation, glucose generated from glycogen was measured using BF-7 (Oji Scientific Instruments, Hyogo, Japan) equipping glucose electrode (ED07-0003, Oji Scientific Instruments).
We previously constructed a 1,3-PDO-producing strain, designated as TA2984, by introduction of a synthetic metabolic pathway. A four-step reaction of the introduced pathway converts DHAP, one of the metabolites in the Calvin cycle, into 1,3-PDO via glycerol (Fig. 1). Construction of the stoichiometric metabolic model based on the annotated genome sequence of S. elongatus PCC 7942 was first reported by Triana et al. . In the same year, the comparable scale of a metabolic model of this strain was published by Knoop et al. . We used Knoop's model composed of 595 genes, 666 reactions, and 533 metabolites as a basic metabolic model of the wild-type strain of S. elongatus PCC 7942 (hereafter called GSM_7942 in this study). The additional information on the introduced synthetic metabolic pathway for 1,3-PDO production (8 genes, 6 reactions, and 3 metabolites) was added to GSM_7942, resulting in a metabolic model of the 1,3-PDO-producing strain composed of 603 genes, 672 reactions, and 536 metabolites (hereafter called GSM_7942_PDO in this study).
GSM_7942_PDO was used for prediction of metabolic flux distribution of TA2984 into which the synthetic metabolic pathway for 1,3-PDO was introduced . To predict metabolic flux distribution using a stoichiometric metabolic model, input fluxes to the cell and output fluxes from the cell were needed. For TA2984, the former represents the carbon fixation rate and photon uptake rate, and the latter represents the biomass production rate (corresponding to the specific growth rate) and specific production rates of 1,3-PDO and glycerol. To determine output fluxes, TA2984 was incubated under the same conditions as in our previous report . As a result, TA2984 produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol directly from carbon dioxide during 20 days of incubation (Fig. 2). Although the output fluxes were observed in the experimental data shown in Fig. 2, it was difficult to experimentally determine the input fluxes (carbon fixation rate and photon uptake rate). Therefore, GSM_7942_PDO was used for calculation of the input fluxes capable of satisfying the output fluxes observed experimentally. Because the assumption of a steady state of metabolism is one of the key constraints in FBA , the data from the exponential growth phase (days 2–4) were applied to the prediction. First, the specific growth rate and the specific production rates of 1,3-PDO and glycerol in the exponential growth phase were calculated to be 0.0250/h, 0.0273 mmol/(g DCW)/h, and 0.0515 mmol/(g DCW)/h, respectively. Next, the input fluxes of carbon fixation and photon use were calculated and found to be 1.35 and 19.0 mmol/(g DCW)/h, respectively. These input fluxes were calculated by the method of two-step optimization strategy of Shastri and Morgan  with some modifications. These calculated input fluxes and experimentally obtained output fluxes were used for prediction of the metabolic flux distribution of TA2984 in the exponential growth phase. Under the conditions restricted by input fluxes of carbon fixation and photon use, the maximized specific growth rate corresponded to 0.0290/h without 1,3-PDO and glycerol production. A two-dimensional solution space between the specific growth rate and the specific production rate of 1,3-PDO was then described (Fig. 3a). The experimental data shown in Fig. 2 were plotted in the described solution space.
The metabolic flux distribution of TA2984 using FBA was applied to the gene disruption simulation based on the MOMA algorithm . This is one of the algorithms used for simulation of sequential gene deletion assuming that the cell adapts to a mutation to minimize the changes of metabolic flux distribution by the gene deletion. The estimated candidates for gene deletion based on this algorithm successfully increased lycopene production in E. coli . Because the objective of our simulation was to screen promising candidates for gene disruption for improvement of 1,3-PDO productivity, we set the specific production rate of 1,3-PDO obtained experimentally [0.0250 mmol/(g DCW)/h] as the lower limit. The metabolic flux distribution corresponding to each of the 595 gene disruptions in GSM_7942_PDO except for the 8 genes of 1,3-PDO producing synthetic metabolic pathway was simulated and the simulated results on 581 strains with single-gene disruption were plotted according to the estimated specific growth rate and estimated specific production rate of 1,3-PDO (Fig. 3b). The simulated results on the 581 strains with single-gene disruption were roughly subdivided into four groups. These four groups are presented as different symbols in Fig. 3b.
The first group (crosses in the figure) was composed of 74 strains that could not satisfy the constraint conditions and lower limit of 1,3-PDO production. The second group (triangles) included 254 strains showing a higher specific production rate of 1,3-PDO, but a null value of the specific growth rate. This may also mean that each gene disrupted in the 254 strains was essential for growth. It was likely that the disruption of any of the 328 genes classified into these two groups was not applicable to improvement of 1,3-PDO productivity. The metabolic flux distributions of the other 267 strains with single-gene disruption could show a certain specific growth rate and higher specific production rate of 1,3-PDO as compared with the experimental data. We screened the 267 candidates for more desirable ones using the product of the specific growth rate and specific production rate as an indicator. Out of the 267 candidates, 231 showed a lower product of the specific growth rate and specific production rate and were classified into the third group (squares).
Finally, 36 genes were classified into the fourth group (circles) as promising candidates to be disrupted for increasing 1,3-PDO productivity (Additional file 1: Table S3). Only 10 patterns of metabolic flux distribution were obtained from 36 strains in the fourth group. First, in GSM_7942, the annotated-gene information was defined by a gene–protein reaction (GPR) association . Under the definition of the GPR association, each disruption of a subunit of a protein complex indicates the same flux distribution. In this study, 22 genes involved in NADPH dehydrogenase-1 (NDH-1) complexes and four genes involved in cytochrome c oxidase were classified into the fourth group. Second, two cases indicating the same metabolic flux distribution were observed in different two-gene disruptions (Synpcc7942_0191 and 1501, and Synpcc7942_2079 and 2080). These genes were annotated as genes encoding an enzyme catalyzing a serial reaction without any blanching pathway. The other six metabolic flux distributions were examined by single-gene disruption. Based on the value of the product of the specific growth rate and specific production, the disruption of 22 genes involved in the NDH-1 complex was found by screening as the most promising candidate for improvement of 1,3-PDO production among the 10 patterns of the fourth group.
Simulated metabolic flux distribution for each strain by gene disruption indicated that the elimination of the NDH-1 complex is the most effective candidate for improvement of 1,3-PDO productivity. The estimated metabolic flux distributions with and without NDH-1 complex elimination were compared (Fig. 4). Each value with an arrow represents the flux [mmol/(g DCW)/h] calculated from experimental data (Fig. 4a) and predicted one of the NDH-1 complex-null strains using the MOMA algorithm (Fig. 4b). In all simulations of gene disruption, the flux of carbon fixation [1.35 mmol/(g DCW)/h] was assumed to not be changed by any gene disruptions. The stoichiometry of ribulose-bisphosphate carboxylase/oxygenase (RubisCO) reaction in GSM_7942 means that 1.97 molecules of 3-phosphoglycerate (3-PG) were produced from 1 molecule of ribulose-1,5-bisphosphate (RuBP), considering photorespiration . Therefore, the flux of 3-phosphoglycerate (3-PG) production corresponded to 2.66 mmol/(g DCW)/h. By means of elimination of the NDH-1 complex, some metabolic fluxes around the 1,3-PDO-producing pathway were predicted to be changed. First, the blanching ratio of the 3-PG-consuming flux into the glycolysis (2-phosphoglycerate; 2-PG) and the Calvin cycle (1,3-bisphosphoglycerate; BPG) was changed. NDH-1 complex elimination increased the flux of fixed carbon into the Calvin cycle. Second, each flux of DHAP production from GAP (glyceraldehyde 3-phosphate) and that of DHAP consumption for S7P (sedoheptulose 7-phosphate) or FBP (fructose-1,6-bisphosphate) synthesis was increased and decreased, respectively, resulting in the increase in the flux into the 1,3-PDO-producing pathway.
NDH-1 complexes in cyanobacteria are involved in several cellular functions (respiration, cyclic electron flow, and carbon dioxide acquisition) and are subdivided into four variants (NDH-1L, L′, MS, and MS′) by function and subunit composition according to reverse-genetic studies . The phenotypic analyses of gene knockout mutants revealed the functional differences among NDH-1 complexes, indicating that NDH-1L and L′ are involved in respiration and cyclic electron flow, whereas NDH-1MS and MS′ participate in carbon dioxide acquisition [48, 49]. Because NdhB is one of the common subunits for all four variants, it was expected that ndhB disruption would be desirable to represent the metabolic flux distribution observed in the gene disruption simulation (Fig. 4b, Additional file 1: Table S3). Nonetheless, all functions of NDH-1 complexes were drastically decreased in the ndhB-null mutant, in particular, this mutant could not grow under ambient air conditions [50–52]. Indeed, ndhB could not be completely disrupted in the wild-type strain and TA2984 (1,3-PDO-producing strain) in our experiment. On the other hand, it was reported that four mutants in which each gene encoding the unique subunit in four NDH-1 complexes is disrupted can grow under photoautotrophic and ambient air conditions . Therefore, we tried to disrupt the genes encoding these unique subunits in TA2984.
In contrast to the strain with ndhB disruption, the gene knockout strains of ndhD1 (Synpcc7942_1976, unique subunit gene for NDH-1L), ndhD2 (Synpcc7942_1439, that for NDH-1L′), ndhD3 (Synpcc7942_2092, that for NDH-MS), and ndhD4 (Synpcc7942_0609, that for NDH-MS′) based on the 1,3-PDO-producing strain (TA2984) were successfully constructed (Additional file 1: Table S1). These four strains were applied to 1,3-PDO production (Additional file 2: Figure S1). All the strains showed a growth defect as compared with TA2984, and the growth defect was more severe in three strains except for TA3672 (ndhD3-null strain; Additional file 2: Figure S1A). Among the four strains, TA4021 (ndhD1-null strain) showed the highest titers of 1,3-PDO (4.04 mM, 0.307 g/L) and glycerol (19.6 mM, 1.81 g/L) during 20 days of incubation (Additional file 2: Figure S1B, C). These titers of 1,3-PDO and glycerol shown by TA4021 were 1.16- and 1.38-fold higher than those of TA2984 (Fig. 2). Table 1 shows output fluxes (specific production rates or specific growth rates) for the wild type and the knockout strains observed in the exponential growth phase (days 2–4) and those calculated by the simulation. This strain revealed a higher specific production rate of 1,3-PDO in the exponential growth phase than TA2984 did (Table 1). The titer of 1,3-PDO produced by TA4022 (ndhD2-null strain) did not increase (Additional file 2: Figure S1B) while a significantly improved production rate of 1,3-PDO was observed in this strain (Table 1). The specific production rate of 1,3-PDO by TA3672 (ndhD3-null strain) was the lowest among the four gene knockout strains and TA2984 (Table 1). On the other hand, TA3671 (ndhD4-null strain) showed the highest production rate of 1,3-PDO (Table 1), but the titer of 1,3-PDO produced by this strain was lower only than the titer of TA2984 (Additional file 2: Figure S1).
NdhF1 is a unique subunit commonly present in two NDH-1 complexes: NDH-1L and L′ . It was expected that ndhF1 disruption would lead to the additive phenotype of ndhD1 and ndhD2 knockouts. Other studies on gene knockout mutants of ndhD1 and ndhF1 showed that the NDH-1L complex is missing in each mutant [53, 54]. Just as ndhD1-D4, the ndhF1-null 1,3-PDO-producing strain designated as TA4058 was successfully constructed (Additional file 1: Table S1). The growth of TA4058 was more defective than that of TA4021 and TA4022, indicating that the ndhF1 disruption affects cellular function more as compared with each single-gene disruption of ndhD1 or ndhD2 (Fig. 5a and Additional file 2: Figure S1A). During 20 days of incubation, the titers of 1,3-PDO and glycerol produced by TA4058 corresponded to 4.44 mM (0.338 g/L) and 30.3 mM (2.79 g/L), respectively (Fig. 5b, c). Both titers were the highest among the other gene knockout strains and the parental strain for 1,3-PDO production. These results indicated that the disruption of NDH-1 complexes (NDH-1L and L′), which have a function in respiration and cyclic electron flow, positively affected 1,3-PDO and glycerol production. The specific production rate of 1,3-PDO by TA4058 in the exponential growth phase (days 2–4) was significantly enhanced and the highest among all the constructed strains (Table 1). Unfortunately, this value [0.0497 mmol/(g DCW)/h] was inferior to that of simulation of the NDH-1 complex elimination [0.0852 mmol/(g DCW)/h]. Nonetheless, the sum of the specific production rates of 1,3-PDO and glycerol meaning the utilization rate of DHAP for the 1,3-PDO-producing pathway in TA4058 [0.213 mmol/(g DCW)/h] was surprisingly higher than that in simulation [0.162 mmol/(g DCW)/h]. The strain with higher total DHAP utilization showed a lower ratio of 1,3-PDO production to total DHAP utilization, indicating that the later part of the 1,3-PDO-producing pathway converting glycerol to 1,3-PDO is the bottleneck of effective production (Table 1). This result is consistent with findings of our recent study on 1,3-PDO production improvement by promoter exchange in the later part of the 1,3-PDO producing pathway . It is expected that the strategy involving a combination of promoter exchange and NDH-1 complex disruption is a promising method for further improvement of 1,3-PDO productivity.
Previously, we have constructed a glycerol-producing strain (TA3800) into which only the pathway converting DHAP to glycerol was introduced . The effect of ndhF1 disruption was also evaluated in TA3800. As in the 1,3-PDO producing strain, the ndhF1 disruption resulted in a growth defect and improvement of the production titer (Additional file 2: Figure S2). The growth defect caused by the ndhF1 disruption in the 1,3-PDO-producing strain was more significant than that in the wild-type strain and glycerol-producing strain (Additional file 2: Figure S2A–C). The highest titer of glycerol produced by the ndhF1-null glycerol-producing strain designated as TA4059 reached 36.7 mM (3.38 g/L; Additional file 2: Figure S2D). This value was almost equal to the sum of the titers of 1,3-PDO and glycerol (34.7 mM) shown by TA4058 (Fig. 5) and about twofold higher than that of TA3800 (Additional file 2: Figure S2E). Although the reason for the significant growth defect caused by ndhF1 disruption in the 1,3-PDO-producing strain was not clear, glycerol production was successfully stimulated by the ndhF1 disruption, as was 1,3-PDO production. The titer of glycerol produced by TA4059 was the highest among other chemicals (glycerol, 1,3-PDO, and 1,2-PDO) produced from DHAP by engineered cyanobacteria [18–20, 38] and this titer corresponded to high titer compared to other chemicals produced not from DHAP by engineered cyanobacteria, such as ethanol , sucrose , 2,3-butanediol , and lactate . Although various chemicals have been produced by engineered cyanobacteria as summarized in some reviews [1, 2], the research resulting from the titer of product over 3 g/L directly from carbon dioxide seems to be still rare.
The disruption of ndhF1 successfully increased metabolic flux of DHAP into the 1,3-PDO-producing pathway as expected on the basis of the in silico simulation (Table 1) and improved the titers of 1,3-PDO and glycerol production (Fig. 5 and Additional file 2: Figure S2). This result suggests that in silico simulation is an effective method for metabolic improvement of bioproduction in cyanobacteria. To identify the causes of improved productivity, the metabolic changes caused by the ndhF1 disruption were analyzed. It was expected that the ndhF1 disruption affected to respiration and cyclic electron flow . Therefore, at first, the respiration activities of TA4058 (ndhF1-null 1,3-PDO-producing strain) and TA2984 (parental strain) were measured. As the result, TA4058 showed lower respiratory activity in the dark and almost the same photosynthetic activity under saturated light conditions (1000 μE/m2/s) as compared with TA2984 (Additional file 2: Figure S3). This finding was consistent with the phenotype of a ndhF1-null strain of Synechococcus sp. PCC 7002 observed in another study . Under growth light conditions (100 μE/m2/s), the photosynthetic activity of TA4058 was lower than that of TA2984, being consistent with the growth defect of TA4058 (Fig. 5).
The physiological role of cyclic electron flow is known to increase the ATP/NADPH ratio produced by photosynthetic electron flow . So, next, ATP contents in TA4058 and TA2984 were measured. TA4058 showed lower cellular ATP content as compared with TA2984 (Additional file 2: Figure S4). In the respiratory chain, the reducing power (NADH and FADH2) mainly produced by glycolysis and the TCA cycle is used for generating a proton gradient and H2O from O2. The generated proton gradient functions as a driving force of ATP synthase for ATP production. Whole or partial loss of cyclic electron flow and respiratory activity under the influence of the ndhF1 disruption may decrease cellular ATP content.
The intracellular metabolites involved in a reaction with ATP seemed to reflect the ATP deficiency in TA4058. For example, ATP is consumed in reactions producing RuBP and ADP-glucose, and is produced in the reaction producing pyruvate. In ATP-consuming reactions, substrates (ribulose-5-phosphate: Ru5P and glucose-1-phosphate: G1P) and products (RuBP and ADP-glucose) were detected at high and low concentrations, respectively. Conversely, lower concentration of a substrate (phosphoenolpyruvate: PEP) and higher concentration of a product (pyruvate) were observed in an ATP-producing reaction. In particular, the metabolites around the glycogen synthesis pathway seemed to be affected by the ATP deficiency. Fructose-6-phosphate (F6P) is a metabolite of the branching point for glycogen synthesis from the Calvin cycle and is sequentially converted to glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), and ADP-glucose. In this glycogen synthesis pathway, significant accumulation (more than fivefold) was observed for metabolites except for ADP-glucose produced with ATP consumption. This result suggests that glycogen synthesis is repressed in TA4058 caused by ATP deficiency. Because other studies showed that higher accumulation of glycogen is observed in stationary phase [44, 59], the glycogen concentrations of TA2984 and TA4058 were measured at 10 days (Additional file 2: Figure S6). As expected, the intracellular glycogen concentration of TA4058 was about half of that of TA2984 (Additional file 2: Figure S6A).
It is well known that the respiratory activity is strongly related to catabolic metabolism (glycolysis and TCA cycle) in heterotrophic organisms. We hypothesized that the repressed respiratory activity can decrease the metabolic flux in catabolic metabolism generating reducing power in photosynthetic organisms, too. The decreasing of 3-PG-consuming flux into glycolysis by the ndhF1 disruption in the simulation result (Fig. 4) was consistent with the hypothesis. Quantification of intracellular metabolites also supported this hypothesis (Additional file 2: Figure S5). Most of the metabolites in the Calvin cycle showed higher intracellular concentration in TA4058 than in TA2984. On the other hand, the metabolites in the glycolysis and TCA cycle were mostly lower in TA4058 compared with TA2984.
Not only greater flux of 3-PG into the Calvin cycle but also repression of glycogen synthesis may increase the productivity values of 1,3-PDO and glycerol. These results indicate that the productivity improvement was caused by the metabolism adapted to energy imbalance induced by the ndhF1 disruption. Hasunuma et al.  successfully improved the carbon fixation rate in Synechocystis sp. PCC 6803 by overexpression of the flv3 gene involved in NADPH consumption. The strategy of acceleration of NADPH consumption also showed the energy balance to change cellular metabolism of cyanobacteria. In the present study, the metabolism changes intended to adapt to the energy imbalance (ATP deficiency) were applied to chemical production (1,3-PDO and glycerol). Taking into consideration of the points mentioned above, ndhF1 disruption is a good strategy for enhancement of production of other chemicals (for instance, 1,2-PDO and sucrose) from metabolites in the Calvin cycle in cyanobacteria [18, 38, 55].
The simulation result of the metabolic flux distribution of the ndhF1-null and wild type strain (Fig. 4) showed the 3-PG-consuming flux to 2-PG or TCA cycle was decreased by the ndhF1 disruption. This result suggested that the ndhF1-null strain was not suitable for the chemical productions from pyruvate or acetyl-CoA. Yoshikawa et al. , however, showed the ndhF1-null PCC6803 strain with ethanol producing pathway was produced 145% more ethanol (0.132 g/L) from pyruvate than the wild type strain with the same pathway (0.191 g/L). It is difficult to explain clearly the reason why the suggestion based on our simulation result is different from their result, but one possible reason would be the driving force by ATP generation by the reaction from PEP to pyruvate to compensate the ATP deficiency in the ndhF1-null strain.
Recently, Broddrick et al.  constructed GEM of metabolism in PCC7942 composed of 785 genes. This model was constructed by using the information about essential genes revealed by the random barcode transposon site sequencing and physiological data for photoautotrophic metabolism. Using this model or the larger models than that we used, there are possibilities to screen out the other candidate genes for knockout to improve 1,3-PDO productivity.
In this study, the productivity of 1,3-PDO and glycerol in S. elongatus PCC 7942 were successfully improved by gene manipulation based on in silico simulation of gene disruptions. The best candidate for improvement of 1,3-PDO productivity was found to be the elimination of NDH-1 complexes involved in several cellular functions: respiration, cyclic electron flow, and carbon dioxide uptake (Additional file 1: Table S3). Genes encoding NDH-1 complexes were actually disrupted in TA2984 (parental strain for 1,3-PDO production). The effective improvement of productivity was achieved by disruption of the NDH-1 complexes that are related to respiration and cyclic electron flow (Additional file 2: Figure S1). Finally, TA4058 (ndhF1-null 1,3-PDO-producing strain) was found to produce 1.28-fold more 1,3-PDO (4.44 mM, 0.338 g/L) and 2.13-fold more glycerol (30.3 mM, 2.79 g/L) than TA2984 did (Fig. 5). The metabolism adapted to the energy imbalance caused by the ndhF1 disruption may enhance the 3-PG-consuming flux into the Calvin cycle and can be suitable for 1,3-PDO and glycerol production (Additional file 2: Figure S5). This result opens up exciting opportunities for applying in silico simulation to metabolic improvement of cyanobacteria and is suggestive of the feasibility of metabolic changes focused on the energy balance.
YH, SM, and TH designed the experiments. YH and SM carried out all experiments and SM, HH, and FM carried out theoretical analysis and YH drafted the figures and manuscript. TH edited the manuscript. All authors read and approved the final manuscript.
This study was financially supported by the Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST).
The Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST).
12934_2017_824_MOESM1_ESM.docx Additional file 1. Additional tables.
12934_2017_824_MOESM2_ESM.ppt Additional file 2. Additional figures.
Knoop H. Integrative analyse des cyanobakteriellen stoffwechsels. PhD thesis. 2014. https://doi.org/10.18452/17055.


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Jesse Tyler Ferguson Expecting First Child With Justin Mikita
9:24 AM PST 1/23/2020 by Katherine Schaffstall
Charley Gallay/Getty Images
The 'Modern Family' star broke the news on Wednesday's 'Late Late Show.'
Congratulations are in order for Jesse Tyler Ferguson and husband Justin Mikita, who will kick off the new decade as fathers.
The Modern Family star announced the news when he visited The Late Late Show on Wednesday. The actor revealed the news while he and host James Corden were talking about fellow guest Charlie Hunnam's upcoming 40th birthday.
"It's when you finally become an adult, I feel. Like, I need to start getting serious about things," Ferguson said of being in his 40s.
"Actually, this is something I haven't even mentioned to anyone," he continued. "If we could just keep it between the three of us and you all. I'm actually expecting a baby in July with my husband."
Ferguson added that it felt like the right time for him to become a father. "I'm very excited. I'm 44 now. I'm, like, let's get this show going. I mean, it's already, tick tock," he said.
When asked by Corden if he knew his baby's gender, Ferguson responded, "A human."
Ferguson and Mikita became engaged in 2012 after two years of dating and tied the knot in July 2013. This will be the first child for the couple.
The baby will join the family following the conclusion of Modern Family, which Ferguson has starred on for 11 seasons as uptight lawyer Mitchell Pritchett.
Watch Ferguson's announcement below.
Katherine Schaffstall


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text:
MAUDE Adverse Event Report: BD INFUSION THERAPY SYSTEMS INC. S.A. DE C.V. BD SAF-T-INTIMA¿ IV CATHETER SAFETY SYSTEM INTRAVASCULAR CATHETER
BD INFUSION THERAPY SYSTEMS INC. S.A. DE C.V. BD SAF-T-INTIMA¿ IV CATHETER SAFETY SYSTEM INTRAVASCULAR CATHETER Back to Search Results
Catalog Number 383313
Device Problem Leak/Splash (1354)
Patient Problem Chemical Exposure (2570)
Event Type Injury
A device evaluation is anticipated, but has not yet begun. Upon completion of the investigation, a supplemental report will be filed.
It was reported that the bd saf-t-intima¿ iv catheter safety system leaked during use. The protective cap was disconnected after the patient received "pertuzumab" for 15 minutes. The chemotherapy then leaked onto the patient and in the patient's bed from the side adapter. The connection was reportedly protected, the infusion was stopped, and the doctor administered the remainder of the medication. The following information was provided by the initial reporter, translated from (b)(6) to english: "the catheter intima protective cap was disconnected after receiving pertuzumab for 15 minutes with consequent leakage of medication. The connection was protected, infusion was stopped and it was reported to the doctor who released the administration of the rest of the medication. A driver was sent to take new clothes at the patient's house. Patient left well. " information received by email on 8/30/2019: there was exposure of chemotherapy: after the disconnection of the cover, there was a leakage of the drug in the body and bed of the patient. The leakage occurred in side adapter.
BD SAF-T-INTIMA¿ IV CATHETER SAFETY SYSTEM
INTRAVASCULAR CATHETER
BD INFUSION THERAPY SYSTEMS INC. S.A. DE C.V.
periferico luis donaldo
colosio no. 579
brett wilko
9450 south state street
foreign,other,user facility
Other Health Care Professional
1 Patient was Involved in the Event
Device Not Returned to Manufacturer
Patient Treatment Data
Date Received: 09/09/2019 Patient Sequence Number: 1


text:
2 Samuel 21: The Giants of Gath
Marlowe	09. 1-2 Samuel, Bible, Old Testament	2 Samuel, Abishai, Adriel, Aiah, Amorite, Armoni, Barzillai, Benjamin, Bethlehemite, Bethshan, Bible, David, Elhanan, Gath, Gibeon, Gibeonite, Gilboa, Gittite, Gob, Goliath, Hushathite, Ishbibenob, Jaareoregim, Jabeshgilead, Jonathan, Judah, Kish, Meholathite, Mephibosheth, Merab, Michal, Old Testament, Philistine, Rizpah, Saph, Saul, Shimei, Sibbecai, Zela, Zeruiah	1 Comment
The remaining chapters of 2 Samuel are considered a sort of Appendix, relaying various bits and bobs that fit, thematically and chronologically, with the preceding book before the narrative continues in 1 Kings 1.
This chapter in particular appears to take place prior to 2 Sam. 9. The theory goes that Samuel initially ended with 2 Sam. 8, with the material of chapters 9-20 "having been suppressed for a time, though finally restored," according to my study Bible (p.385). Thus, when 2 Sam. 21 was added, it came from different sources and did not fit chronologically with the rest of the book. We'll notice, for example, that at least one story is a repeat (albeit with a surprising change), and a few details seem to come from a different source than what we've been mostly been reading so far.
While the last four chapters of 2 Samuel clearly come from different sources, they do seem to have been arranged with care. My New Bible Commentary notes that "the six sections contained in these four chapters are arranged chiastically: natural disaster, military exploits, poem, poem, military exploits, natural disaster" (p.312).
There was a famine in Israel for three years in a row. The people are suffering and, finally, David calls on God. One might wonder why he let the famine get into its third year before doing this, but I suppose it just takes that long before a palace starts to feel the pinch.
Of course, God shows a bit of his own weird sense of time, because he claims to have sent the famine as punishment for Saul killing the Gibeonites (a story not recorded in our text). Israel had sworn not to kill them (Jos. 9:3-27, albeit through trickery), but Saul had done so anyway "in his zeal" (2 Sam. 21:2). We've had hints of this zeal in, for example, the story of the witch of Endor (1 Sam. 28:9). This paints a picture of a Saul who was very concerned with establishing a Yawehist Israelite homogeneity, compared to David's liberal use of Philistines and other non-Israelites in his personal guard.
Why Israel should be punished now for Saul's actions is left unexplained. A cynic might wonder if perhaps David wanted to find a reason for the famine that he could bring back to his people, but didn't want it to be anything that was his fault (particularly if we're placing this story fairly early on in his rule). In fact, isn't it convenient that the famine is a punishment against his deposed predecessor? Doesn't that just every so nicely discourage any lingering support for Saul?
David goes to the Gibeonites and asks them what can be done to appease them. It seems that God's retributive justice was not initiated by himself, but rather by a Gibeonite curse that either took this long to come into effect, or they've been biding their time until the responsible party is dead and his dynasty collapsed.
The Gibeonites claim that they do not want to be repaid in blood or gold, except that they do actually want seven of Saul's sons to be hanged on the mountain of God at Gibeon – which sounds an awful lot like the blood vengeance they claimed not to be asking for. This only avoids being a contradiction if a) the number seven is a symbolic one, replacing the one-to-one killing of a blood vengeance, or b) the nature of the killing is ritually/legally different from a blood vengeance. In other words, if this is meant to be a human sacrifice to God rather than a tribal justice matter.
David agrees to their terms, though we get a clunky, clearly added later note that he spares Mephibosheth because of his oath to Jonathan. Instead of Mephibosheth, he chooses Armoni and Mephibosheth (a case of name recycling, at one end or another) – the sons of Saul and his concubine Rizpah. It seems that some of Saul's survived him, though 1 Sam. 31 implied that they all died with him at the battle of Gilboa.
For the other five, he got the five sons of Michal, Saul's daughter, and Adriel, son of Barzillai the Meholathite. Obviously an issue because it is Merab who married Adriel in 1 Sam. 18:19. Also a problem because we were told in 2 Sam. 6:23 that Michal died childless.
Some theories have been proposed to fix the discrepancy; for example, that Merab's sons were given to Michal to bring up. Others, such as my RSV, simply change the name to Michal to "fix" the error. According to Paul Davidson of Is That In The Bible?, there are some problems with this bandaid:
(1) We have already shown that the mention of Merab marrying Adriel in 1Sam 18 is a separate tradition and a later addition to 1 Samuel. It is difficult to assume "Merab" is the correct reading once we realize that the earlier reference to Merab's marriage – the very passage scholars would like to harmonize 2Sam 21 with – is a later insertion. (2) The LXX confirms the reading of "Michal" in 2Sam 21:8, which means that if there was such an error, it was very widespread, and it happened before the LXX was produced. (3) Josephus, Pseudo-Jerome, and rabbinic sources confirm the reading of "Michal" and propose harmonizations. (4) Targum Jonathan appears to have been based on a vorlage that reads "Michal", and it solves the problem by asserting that Michal simply raised the children on behalf of Merab.
The record is clearly a bit dodgy, however you cut it.
These seven sons and grandsons of Saul are hanged and God is appeased (despite the excuse that God is appeased because the Gibeonites withdraw their curse, this still smells rather strongly of human sacrifice).
So the Gibeonites are happy, but poor Rizpah isn't. She camps out at the spot where her two sons are left hanging and keeps all the carrion eaters away until the rain comes (it being the sign that the drought-induced famine would soon be over). From context cues, it seems that the bodies were left hanging the entire summer, from late April or May until the Autumn.
Rizpah, by Peter Graham, c.1850
When David hears of Rizpah's vigil, we're told that he fetched Saul and Jonathan's bones from Jabesh Gilead and buries them along with the bones of the men sacrificed by the Gibeonites in Zela, the tomb of Saul's father. It is after the funeral that God finally relented and the famine was over.
The text seems to want to tell us that Rizpah's grief convinced David to bury Saul and Jonathan's bones, yet he expressed more than enough grief himself to do it way back in 2 Sam. 1. It makes it rather difficult to believe that it had never occurred to David before now to give them a proper burial – particularly Jonathan, whom he claimed to love so much.
It's difficult not to see the political motivations behind David's decision to bury them now. It could be that he needed this big show of love for Saul and Saul's dynasty to avoid repercussions from Saul's remaining supporters. Or perhaps it was an attempt to show that he didn't give in to the Gibeonites' demands too readily.
It could also be to smooth over the fact that David had allowed the men's bodies to hang, exposed to the elements, for what could be as long as six months – a huge insult, as well as a clear violation of the law (Deut. 21:23).
In fact, the entire Gibeonite desire for revenge (particularly its timing) looks awfully suspicious. A cynic might wonder if David used a natural disaster as an excuse to get rid of a bunch of Saul's descendents and thereby solidify his own hold to power.
Philistine Aggression
The Philistines are at it again! In this chapter, we hear of four Philistine champions, all descended from giants, and the Israelite heroes who defeated them.
There's Ishbibenob, whose spear weighed as much as three hundred shekels of bronze. With a new sword in hand, he comes after David, but Abishai steps in (again) and kills the threat. After this, David's men forbid him from coming out to fight with them, "lest you quench the lamp of Israel" (2 Sam. 21:17). If I were to venture a guess, I'd say it was known that David did not participate in his own military campaigns. Some people, like the author of this passage, tried to excuse his absence. Others, like the author of 2 Samuel 11, clearly did not approve.
The next champion is Saph, dispatched by Sibbecai the Hushathite.
The third might be a little familiar: Goliath the Gittite, once again armed with a spear like a weaver's beam (2 Sam. 21:19; 1 Sam. 17:7). This time, however, he is defeated by Elhanan, son of Jaareoregim. According to Kenneth C. Davis, "the King James translators of 1611 tried to cover up the discrepancy by inserting the words "brother of" before the second mention of Goliath, but older texts don't bear that version out" (Don't Know Much About the Bible, p.177).
Paul Davidson has a discussion of the episode on Is That In The Bible? that I recommend, but here's an excerpt:
It is commonly thought by scholars that this was the original Goliath legend, for various reasons. In the earliest folktales, it was the champion Elhanan who slew Goliath when Israel was threatened by an ancient race of giants. Elhanan, Abishai, and Jonathan were all members of the Shalishim (the "Thirty"), a group of elite warriors who are listed in 2Sam 23. (Sibbecai is also included in the parallel list in 1 Chr 11:10–47.) Later on, as the figure of David the warrior king became more important to Jews and the other characters more obscure, the story of Goliath was retold with David as the hero instead.
The last Philistine champion is unnamed, but we're told that he had six fingers on each hand and six toes on each foot, and was slain by Jonathan, the son of Shimei and David's nephew.


Pet lovers who enjoy traveling!
Hi everyone! I'm Amanda, and my husband is Rick. We are entertainers who have done everything from balloon twisting and stilt walking to stunts such as sword fighting and scuba diving. I'm between contracts right now and looking for something to do. My husband is a full time math tutor.
Although we ADORE cats and dogs, we travel too much to have one of our own right now so please let us live vicariously through your pet family!
We love our home here in Florida where we can visit the beach, go on cruises occasionally, and see beautiful sunsets while reading on the porch.
I'm new to this website but we've been house sitting, babysitting and pet sitting for years. We have experience with large and small breeds. I paid my way through school as a lifeguard and then an EMT so I have a cool head under pressure and don't panic should an emergency arise.
Amanda was amazing. She deserves way more then the 5 stars allowed in each category. Amanda is extremely easy to communicate with and treated our fur babies as if they were her own. She truly went above and beyond. My neighbor even texted me while we were away and commented about how happy our dog looked and how good Amanda was with him. Amanda made us feel extremely comfortable with the entire process and we hope she will come back again!!
Amanda was awesome taking care of my 3 pups! She was very accommodating working around my leave and return schedule. She sent pictures everyday which was great, usually funny ones. Amanda is very friendly and laid back and took care of everything I needed her to while I was away. I would definitely recommend her to sit your house and pets!
I've known recommended for many years and they have been nothing but responsible and reliable the entire time I have known them. Whoever has them watch their house would not only be privileged but would find their house in a better condition than how they left it.
Barkley loves Amanda! My dog is a 12 year old min-pin and when I say her name, he runs to the door. Amanda has sat for Barkley over the past 3 years. She has so much love and patience for him, which makes me feel worry-free when I have to travel on business trips or go on vacation. Sometimes I have her come over, if he needs an extra walk and I'm too busy. I recommend Amanda for house and pet sitting.


This fantastic directory provides explanation and examples of: parts of speech, punctuation, types of sentences, sentence features and structures. Perfect for KS2 S.P.A.G. teaching and revision, plus EAL to enable effective communication!


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AgTech Garage leaders.
Why a global consulting firm acquired an open innovation hub for agtech in Brazil
Louisa Burwood-Taylor
PwC, the global management consultancy, accounting and professional services business, acquired AgTech Garage, an open innovation hub for agtech in Brazil last month.
According to a press release announcing the deal, "the acquisition reinforces the importance of Brazilian agribusiness in the global scenario and reaffirms PwC's focus on the sector."
To find out more about how the deal came about and what it means for Brazil's agtech sector, we caught up with AgTech Garage CEO José Tomé (JT).
AFN: Why did PwC buy AgTech Garage? Does it add totally new capabilities to the firm, or will it be an extension of work already being done at the firm?
JT: The AgTech Garage has created an open innovation community management model that is new and unique. We created a strong brand, well positioned and protagonist in the ecosystem with high potential of scale. Agribusiness is strategic for PwC, and Brazil is where the Global Center of Excellence in this industry is located.
PwC and AgTech Garage add new capabilities. Together, we will offer complete solutions to players in this sector, with unprecedented support from strategy to execution, with a lot of innovation and technology.
AFN: How will the work of AgTech Garage change? Will you still interact with startups in the same way?
JT: The way AgTech Garage interacts with startups does not change with the arrival of PwC. We will, in fact, generate even more value and provide new services for the community of entrepreneurs. As startups scale up, we can also support them on several fronts, such as strategy, financing, mergers and acquisitions, processes, access to capital markets, technology, taxes, ESG, among others. Nothing changes in our relationship with startups, corporates, or any other agent in the ecosystem. We will be more comprehensive and more solid, providing confidence to our partners and a new look at innovation.
AFN: Please describe how AgTech Garage was created and why? What was the journey like from then to now?
JT: AgTech Garage was founded in 2017 by three entrepreneurs in Piracicaba (SP), a region that is an innovation area for agribusiness in Brazil. Our intention has always been to encourage open innovation in the country and, through it, increase the competitiveness of entrepreneurs and large companies. Especially in the last two years, we have had very fast growth, becoming one of the main innovation hubs worldwide.
Nowadays, AgTech Garage has a network of more than 80 large partner companies, more than 1,000 connected startups (including almost 15% from outside Brazil), and a team of more than 50 people. Even though we are an Agribusiness hub, our network comprises companies from more than nine sectors of the economy that interact in some way with Agribusiness. Given all this, we began to analyze how to accelerate our growth, and the acquisition by PwC was natural. PwC has been part of our community for over 2 years and the conversations that resulted in this movement, to unite skills and capabilities, were an evolution of this relationship.
AFN: What is next for Brazil's agtech ecosystem in 2023 and beyond?
JT: The ecosystem, of which we are a part, is not only of agtechs. It is an agribusiness ecosystem that attracts traditional agtechs and agfintechs and food techs. In addition, it attracts edtechs, because we have to transform education in agribusiness. So it is a more complex ecosystem, where different sectors and technologies converge to transform agribusiness. I believe all the techs will be in agribusiness, and we will work hard on this in 2023. And our community is not only after agtechs. It demands the solutions of fintechs, edtechs, logitechs, climate techs… all of this is agribusiness.
On the investment side, we are already noticing the diversification of the venture capital industry. We follow the structuring of many corporate venture capitals (CVCs), for example, which come with a much more strategic look than a financial one. Many other investor profiles are also consolidating, such as large producer groups that invest directly in startups, groups of specialized executive angels and there is also crowdfunding. The moment is for diversification.
Regarding VC exits, some may happen in 2023, very much along the lines of the early exits. I think we will also see corporations making more acquisitions and generating early exits. We are heading toward a very dynamic scenario.
consulting, corporates, latam, latin america
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northampton college lrc
Quick Links. Date: Description: This is a book and journal collection supporting the Drama, Dance and Music courses at the University College Northampton. It was awesome! Learning Support Manager: Mrs B Oliver: Learning Mentor: Mrs T Squires: LSU Administrative Assistant: Ms L Giacobbe: LSAs: Mrs V Angel. MyWestmoreland Portal Last modified: Wednesday, 23 September 2020, 3:32 PM. LRC Ground Floor Summer Opening Hours Have a wonderful summer everyone! Sponne School. Learning Resource Centre (LRC) LRC Manager: Mrs R Brooking: Learning Support Unit. The University of Northampton is a higher education establishment based in Northampton, which offers students undergraduate and postgraduate courses and university degrees. Our participation in VA's VetSuccess on Campus Program began with a partnership between Rutgers, Middlesex County College, and the United States Department of Veterans Affairs (VA) with the purpose of providing a supportive, on-campus resource to help you make a smooth transition to college life. Our Church Street Campus redevelopment began in 2019 following funding from SEMLEP's Local Growth Fund and opened to students in September 2020. The opinion of the court was delivered by: Stengel, J. Northampton Community College July 7, 2010 RONALD HENEGHAN, PLAINTIFF v. NORTHAMPTON COMMUNITY COLLEGE, ET AL, DEFENDANTS. Posted: 23 March 2016 A regional skills competition designed to seek out some of the best engineering talents was hosted at West Nottinghamshire College last week. 276 connections. Before coming to college, I'd always aspired to be… The VR experience is one I will not forget in a hurry. Other facilities. The University of Northampton. Open to the public. play video. You can study in Avenue Campus's own library, which includes the Humanities course collection, and a collection of 4,000 films in many modern languages for viewing on-site. Northampton Early College (NCEC) Second Floor -200's. Articles returned will match all of the requested criteria. ORDER. View Rebekah Groves' full profile. It really put into practice the theory that we have… Find out more about Kingston University London's Library and Learning Services, including information on our learning resources centres, including their opening hours. City of Westminster College entered a new and exciting phase in its 100-plus year history in January 2011, with the opening of the new Paddington Green Campus. ... before deciding whether it is something of benefit to the College in the long term? Social workers support and protect society's most vulnerable people. LRC Research Resources. The centre is equipped with up to date and industry standard electronic, automation and robotic equipment suitable for learners studying this course. Student Zone Campus Info & Contacts Careers, HE & Employability Counselling Exams Office Finance Go Higher West Yorkshire LRC NCLT Baccalaureate Study Resources Progress Tutors Safeguarding, Well-Being & Support Student Executive Study Support Work Experience View Elaine Galer's profile on LinkedIn, the world's largest professional community. Wrenn Remembrance Day 2020 Elaine has 2 jobs listed on their profile. If you're hungry before or after studying, try the Avenue cafe, which provides hot and cold meals and snacks. The guide to the Harvard system of referencing (4th ed.) Engineering students prepare for success. Build an advanced search. ; Subject and database guides Learning Resources, University College Northampton, Tresham College, part of The Bedford College Group, is committed to providing quality post-16 skills training in Wellingborough. Please click on the links below for further help on using the Harvard Referencing system. Join Facebook to connect with Abington Northampton and others you may know. Halifax Community College 100 College Drive P.O. City, University of London is an independent member institution of the University of London. LRC Library / Harvard Referencing ... Harvard Referencing is the format used by Havering College. "Ambition and Excellence with Care for All " Remote Learning. LRC Advisor Luton, United Kingdom Photography. Twitter; Facebook; Linkedin; Youtube; Instagram; This website contains a wide variety of images relating to the University's campus and activities. Grant Campus (631) 851-6700 Admissions (631) 451-4000 Sayville Center: School of Nursing (631) 851-6972 The vehicle mounted meter ensures that the taxi price is neither higher nor lower than mandated. Office Administration Department (215) Business Administration Department (202) Mathematics Department; Medical Office Administration; Learning Resouce Center (LRC) Learning Resouce Center (LRC) Electronic Resource Center (ERC) Center for Academic Success; Student Center Building (300 Building) This later merged to become Liverpool Polytechnic. Licensed taxi operators are bound by these transport fees for rides in the core area of Northampton. The Bedford College group has a newly built Advanced Engineering Centre located at the Caudwell Street campus. Smith College - Lewis Global Studies Center, Northampton, MA. Allison Fitzpatrick Vice President, Monroe Campus at Northampton Community College Belmar, New Jersey 472 connections This inspirational Schmidt Hammer Lassen-designed building was constructed on Paddington Green to replace a previously purpose-built block constructed in 1967. Student & Employee Email. I came to Lakes College in 2010 to study on the Access to Higher Education course. It's free! Fancy an evening out or a new hair cut? Brackley Road Towcester Northamptonshire NN12 6DJ. Please see the Bedford College Group LRC statement for HE learners policy. A post shared by North Kent College (@northkent_college) on May 2, 2017 at 2:02pm PDT. Suffolk County Community College Selden • Brentwood • Riverhead Long Island, New York Ammerman Campus (631) 451-4110 Eastern Campus (631) 548-2500 Michael J. Important Links for Westmoreland Students & Staff. Your Choice Your Career Your Future. Northampton fixed the taxi fare in a regional regulation. 1,019 likes. Telephone: 01327 350284 You can also find out how to purchase membership and our libraries. Spreadsheet of resources from Northampton University Some are specific to Northampton, others are already included in the list above. Social Work courses. Making our college a COVID-safe environment for staff and students. Or looking for child care for your little one? Our Facilities to hire. Central Bedfordshire College. See the complete profile on LinkedIn and discover Elaine's connections and jobs at similar companies. The Incuba. Oct 11, 2017 - Human Biology by Mike Boyle, 9780007267514, available at Book Depository with free delivery worldwide. If you're looking to work with those in need, from children and the elderly, to individuals or families, our social work and related courses have much to offer. Drawer 809 Weldon, NC 27890 252-536-2551 252-536-4144 252-536-6351 Useful online referencing tools. Last week the Learning Resources Centre (LRC) team attended the Council for Learning Resources in Colleges (CoLRiC) roadshow on Information Literacy at Northampton College.As well as having a tour of the Beacon award winning Library we took part in some practical sessions on referencing material.. Back in the day of the LRC team referencing was easy – books, journals and newspapers. We're Mind, the mental health charity. View the profiles of people named Abington Northampton. Engages students in international studies and cultivates an understanding of the global context and implications of their work. Established by Royal Charter in 1836, the University of London consists of 18 independent member institutions with outstanding global reputations and several prestigious central academic bodies and activities. Liverpool John Moores University (abbreviated LJMU) is a public research university in the city of Liverpool, England.The university can trace its origins to the Liverpool Mechanics' School of Arts, established in 1823. We're here to make sure no one has to face a mental health problem alone. College Store. COVID-19 Updates: For advice and updates for staff, students, and applicants who may have concerns about the coronavirus outbreak please visit our Situation Updates page. ... Liberation Referencing from University of Northampton. Library. The health, safety and wellbeing of our staff and students is our number one priority and we have been working hard to make the College a safe environment during these unprecedented times.For the latest communication and guidance, please visit the following page.
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Q: Android antivirus I am new in computing. I have read that android is based on Linux OS.
And Linux rather any distro of UNIX is virus free.
Then my question is that what is the need of anti virus s/w's in Android phones??
Please correct if I am carrying wrong knowledge.
Thanks in advance.

A: You may be asking yourself, what if I have a cell phone virus and what is it anyway? You know you keep a lot of precious, valuable data on your phone, and when you hear in the news that mobile threats are on the rise, it's easy to lose sight of the context behind the numbers and worry that you've gotten a dreaded mobile phone virus that's going to steal your personal info and eat your children. Hopefully we can clarify things by addressing some of the questions that we hear most about so-called Android "viruses."
Is there really such a thing as an Android virus?
Historically carried over from the old PC world, a "virus" is a program that replicates itself by attaching to another program. Hackers often used this method to spread their nefarious work, and virus became a popular term to refer to all types of malicious software (malware) on computers. In the case of smartphones, to date we have not seen malware that replicate itself like a PC virus can, and specifically on Android this does not exist, so technically there are no Android viruses. However, there are many other types of Android malware. Most people think of any malicious software as a virus, even though it is technically inaccurate.
So what is Android malware?
Malware, short for malicious software, is software designed to secretly control a device, steal private information or money from the device's owner. Malware has been used to steal passwords and account numbers from mobile phones, put false charges on user accounts and even track a user's location and activity without their knowledge. Learn about some of the most notable malware Lookout has blocked in Resources Top Threats.
How would I get malware on my phone?
Through Lookout's research for the State of Mobile Security 2012, we've found that user behavior and geography greatly influence your risk of encountering malware. The safest bet is to stick with downloading well-known apps from well-known apps from reputable markets like Google Play in addition to having a security app. Fraudsters make it their job to disguise malware as innocent-looking mobile apps on app stores and websites. So if you're thinking that it's a good idea to download a just-published, supposedly free version of Angry Birds you found on a random Chinese app store, it's probably not. Once installed, these apps may appear to work just as described, but they are can be busy with additional secret tasks. Some apps start out clean, but are given malicious capabilities after a seemingly routine software update.
And conscientious app downloading won't always minimize your risk. Sneaky, drive-by-download sites can download a potentially malicious app file without any user intervention. Safe Browsing in Lookout Premium for Android will block web-based threats like that, but even so, you also shouldn't install random downloads from your download manager that you didn't expect to find there.
How can I protect myself and my mobile device?
It's pretty simple to minimize the risk of encountering malware, and we've got 5 simple mobile security tips right here. The top two ways to protect yourself are to download a mobile security app like Lookout to catch those pesky "phone viruses" and to be judicious about what apps you download and were you download them from. Lookout will scour your phone or tablet for any existing malware, and also examine every new app you download to ensure it is safe. But even before you let Lookout scan your newly downloaded app, you should only download apps from sites you trust, check the ratings and read reviews to make sure they're widely used and respected.
So, should you worry about getting a phone virus? Nope, because they technically don't exist. (If they ever do crop up, Lookout will weed them out.) And should you worry about the more accurately termed malware? Well, with a little bit of awareness and Lookout on your phone and by your side, you can keep malware and other mobile threats at bay.

redpajama_set_name: RedPajamaStackExchange


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Adventure Puzzles & Escape Rooms
Bungee/Base Jumping
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Extreme Competitions
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Zip-Lining
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Bear, DE, United States
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East Hanover, NJ, United States
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3rd St, San Francisco, CA, United States
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Arlington, TX, United States
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Steuart St, San Francisco, CA, United States
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Kennesaw, GA, United States
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Brooklyn, NY, United States
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Building this site was a good opportunity to try out Zurb Foundation for the first time. This is how I got it to play nicely with CakePHP and LiveReload.
If Compass works fine on the command line, but LiveReload throws errors you may need this solution, which configures LiveReload to use the gems that are installed with Foundation.
This means that variables can be written to override Foundation's variables.
<project_name>.scss is imported after the Foundation component files at the end of app.scss and includes plain css and and scss.