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Sequestration of carbon by the marine biological pump depends on the processes that alter, remineralize, and preserve particulate organic carbon (POC) during transit to the deep ocean. Here, we present data collected from the Great Calcite Belt, a calcite-rich band across the Southern Ocean surface, to compare the transformation of POC in the euphotic and mesopelagic zones of the water column. The 234Th-derived export fluxes and size-fractionated concentrations of POC, particulate inorganic carbon (PIC), and biogenic silica (BSi) were measured from the upper 1000 m of 27 stations across the Atlantic and Indian sectors of the Great Calcite Belt. POC export out of the euphotic zone was correlated with BSi export. PIC export was not, but did correlate positively with POC flux transfer efficiency. Moreover, regions of high BSi concentrations, which corresponded to regions with proportionally larger particles, exhibited higher attenuation of > 51 μm POC concentrations in the mesopelagic zone. The interplay among POC size partitioning, mineral composition, and POC attenuation suggests a more fundamental driver of POC transfer through both depth regimes in the Great Calcite Belt. In particular, we argue that diatom-rich communities produce large and labile POC aggregates, which not only generate high export fluxes but also drive more remineralization in the mesopelagic zone. We observe the opposite in communities with smaller calcifying phytoplankton, such as coccolithophores. We hypothesize that these differences are influenced by inherent differences in the lability of POC exported by different phytoplankton communities.

We demonstrate a method for estimating absorption and backscattering coefficients by inverting glider-measured profiles of the downwelling irradiance and upwelling radiance. The inversion method was validated against approximately 1,300 profiles of data from 22 glider missions within the Gulf of Maine over a 10 year period. The backscattering coefficient at 532 nm was estimated with a mean absolute error of 21% and bias of 0.01% compared to measured values. We could only quantitatively evaluate the absorption coefficient against the fluorometry data, but found that profiles of fluorescence and absorption were in quantitative agreement. With absorption and backscattering coefficients acting as a basis for studying the biogeochemical parameters of the constituents in the water column, these results show the potential of bio-optical gliders for studying marine ecosystems under varying sky conditions.

In animal behavioral experiments, extended training causes instrumental actions that deliver ingestible substances to lose sensitivity to outcome devaluation and contingency degradation, and to gain direct sensitivity to the current motivational state. These features of habitual control have been attributed to a process that relies on stimulus-response (S-R) associations linking the context to instrumental actions and Pavlovian associations linking the same context to orally-sensed properties of substances attained there. This Pavlovian process was conceived based on the results of irrelevant incentive experiments, but it is not supported by the results of all such experiments. An alternative process is therefore proposed here. In this process, recall of the instrumental action is evoked by an S-R association, but the rate at which this action is then performed is controlled by anticipation of postingestive sensations that have frequently followed it. This anticipation relies on recall of an association linking the action directly to the postingestive sensations. This association is learned during the formation of a chunked action series that begins with the instrumental action and ends with a consummatory response. It enables a prediction of subjective value that is directly influenced by the current motivational state, but is not influenced by devaluation or non-contingent delivery of the substance that has produced the postingestive sensations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Shiga toxin-producing Escherichia coli (STEC) is an enteric foodborne pathogen that can cause mild to severe illness. Here, we report the availability of high-quality whole-genome sequences for 77 STEC strains generated using the PacBio sequencing platform.

Enterotoxigenic Escherichia coli (ETEC) is an important diarrheagenic pathogen. We report here the high-quality whole-genome sequences of 21 ETEC strains isolated from patients in the United States, international diarrheal surveillance studies, and cruise ship outbreaks.

The application of PAT for in-line monitoring of biopharmaceutical manufacturing operations has a central role in developing more robust and consistent processes. Various spectroscopic techniques have been applied for collecting real-time data from cell culture processes. Among these, Raman spectroscopy has been shown to have advantages over other spectroscopic techniques, especially in aqueous culture solutions. Measurements of several process parameters such as glucose, lactate, glutamine, glutamate, ammonium, osmolality and VCD using Raman-based chemometrics models have been reported in literature. The application of Raman spectroscopy, coupled with calibration models for amino acid measurement in cell cultures, has been assessed. The developed models cover four amino acids important for cell growth and production: tyrosine, tryptophan, phenylalanine and methionine. The chemometrics models based on Raman spectroscopy data demonstrate the significant potential for the quantification of tyrosine, tryptophan and phenylalanine. The model for methionine would have to be further refined to improve quantification., (© 2017 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Cerebral oxygen desaturation during cardiac surgery has been associated with adverse perioperative outcomes. Before a large multicenter randomized controlled trial (RCT) on the impact of preventing desaturations on perioperative outcomes, the authors undertook a randomized prospective, parallel-arm, multicenter feasibility RCT to determine whether an intervention algorithm could prevent desaturations., Methods: Eight Canadian sites randomized 201 patients between April 2012 and October 2013. The primary outcome was the success rate of reversing cerebral desaturations below 10% relative to baseline in the intervention group. Anesthesiologists were blinded to the cerebral saturation values in the control group. Intensive care unit personnel were blinded to cerebral saturation values for both groups. Secondary outcomes included the area under the curve of cerebral desaturation load, enrolment rates, and a 30-day follow-up for adverse events., Results: Cerebral desaturations occurred in 71 (70%) of the 102 intervention group patients and 56 (57%) of the 99 control group patients (P = 0.04). Reversal was successful in 69 (97%) of the intervention group patients. The mean cerebral desaturation load (SD) in the operating room was smaller for intervention group patients compared with control group patients (104 [217] %.min vs. 398 [869] %.min, mean difference, -294; 95% CI, -562 to -26; P = 0.03). This was also true in the intensive care unit (P = 0.02). There were no differences in adverse events between the groups., Conclusions: Study sites were successful in reversal of desaturation, patient recruitment, randomization, and follow-up in cardiac surgery, supporting the feasibility of conducting a large multicenter RCT.

Raman-based multivariate calibration models have been developed for real-time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO-based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration., (© 2015 American Institute of Chemical Engineers.)

A simple method for control of lactate accumulation in suspension cultures of Chinese hamster ovary (CHO) cells based on the culture's pH was developed. When glucose levels in culture reach a low level (generally below 1 mM) cells begin to take up lactic acid from the culture medium resulting in a rise in pH. A nutrient feeding method has been optimized which delivers a concentrated glucose solution triggered by rising pH. We have shown that this high-end pH-controlled delivery of glucose can dramatically reduce or eliminate the accumulation of lactate during the growth phase of a fed-batch CHO cell culture at both bench scale and large scale (2,500 L). This method has proven applicable to the majority of CHO cell lines producing monoclonal antibodies and other therapeutic proteins. Using this technology to enhance a 12-day fed-batch process that already incorporated very high initial cell densities and highly concentrated medium and feeds resulted in an approximate doubling of the final titers for eight cell lines. The increase in titer was due to additional cell growth and higher cell specific productivity., (Copyright © 2011 Wiley Periodicals, Inc.)

The Drosophila melanogaster Prat gene encodes amidophosphoribosyltransferase (PRAT; EC 2.4.2.14), which performs the first step in de novo purine nucleotide synthesis. Prat mutations have a recessive lethal phenotype that is found for other genes encoding enzymes in this pathway. The D. melanogaster genome project has revealed a second gene, CG10078 or Prat2, encoding a protein with 76% amino acid sequence identity with Prat. The two genes map to different arms of chromosome 3 and have different intron/exon organizations, as we confirmed by cDNA sequence analysis of Prat2. With the goal to determine the functional significance of this gene duplication, we isolated and sequenced two PRAT-encoding genes from Drosophila virilis. We find that the two D. virilis genes are orthologous to the two D. melanogaster genes in terms of intron/exon organization, amino acid coding sequence, and 5' noncoding sequence. The absence of introns in both DmelPrat and DvirPrat genes suggests that Prat originated from a retrotransposition of Prat2 and that the gene duplication has been preserved in the two species since their divergence approximately 40 million years ago. Analysis of mRNA expression in development shows that maternal expression, detected in adult ovaries and embryos prior to the onset of zygotic transcription, is present for Prat but not Prat2 in both species. Taken together, these findings support the notion that two PRAT-encoding genes have evolved distinct functions in both Drosophila species.

Adenosine, a purine nucleoside found at high levels in solid tumors, is able to suppress the recognition/adhesion and effector phases of killer lymphocyte-mediated tumor cell destruction. Here, we demonstrate that adenosine, at concentrations that are typically present in the extracellular fluid of solid tumors, exerts a profound inhibitory effect on the induction of mouse cytotoxic T cells, without substantially affecting T-cell viability. T-cell proliferation in response to mitogenic anti-CD3 antibody was impaired in the presence of 10 microM adenosine (plus coformycin to inhibit endogenous adenosine deaminase). Antigen-specific T-cell proliferation was similarly inhibited by adenosine. Anti-CD3-activated killer T (AK-T) cells induced in the presence of adenosine exhibited reduced major histocompatibility complex-unrestricted cytotoxicity against P815 mastocytoma cells in JAM and (51)Cr-release assays. Diminished tumoricidal activity correlated with reduced expression of mRNAs coding for granzyme B, perforin, Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), as well as with diminished Nalpha-CBZ-L-lysine thiobenzylester (BLT) esterase activity. Interleukin-2 and interferon-gamma synthesis by AK-T cells was also inhibited by adenosine. AK-T cells express mRNA coding for A(2A), A(2B) and A(3) receptors, but little or no mRNA coding for A(1) receptors. The inhibitory effect of adenosine on AK-T cell proliferation was blocked by an A(3) receptor antagonist (MRS1191) but not by an A(2) receptor antagonist (3,7-dimethyl-1-propargylxanthine [DMPX]). The A(3) receptor agonists (N(6)-2-(4-aminophenyl)ethyladenosine [APNEA] and N(6)-benzyl-5'-N-ethylcarboxamidoadenosine [N(6)-benzyl-NECA]) also inhibited AK-T cell proliferation. Adenosine, therefore, acts through an A(3) receptor to prevent AK-T cell induction. Tumor-associated adenosine may act through the same mechanism to impair the development of tumor-reactive T cells in cancer patients., (Copyright 2002 Wiley-Liss, Inc.)

A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells.

Advances in recombinant DNA manufacturing technology have now made possible the production of a highly purified and active recombinant factor IX (rFIX) product. Recombinant factor IX was developed by (1) stable insertion of the genes for both factor IX and PACE-SOL (a truncated, soluble serine protease needed to enhance the capacity of cells to remove the amino-terminal propeptide from rFIX) into Chinese hamster ovary cells; (2) selection of a cell line that was capable of expressing high amounts of active rFIX while growing in bioreactors containing a completely defined culture medium that does not contain blood or plasma products; and (3) inclusion of four independent chromatography steps, none of which require monoclonal antibodies. Furthermore, rFIX has been extensively tested to demonstrate similarity to plasma-derived factor IX and has been shown to be a consistent, high-purity product. For example, a high-specific-activity product (276+/-23 IU/mg) has been consistently produced throughout 65 consecutive batches from five consecutive manufacturing campaigns. Thus, rFIX offers a consistent and high-purity source of factor IX treatment for patients with hemophilia B.

In a batch-refeed continuous process involving a recombinant Chinese hamster ovary cell line, a brief upset was occasionally observed during which cell growth halted and cell viability dropped. This was found to be associated with depletion of insulin from the medium early during the affected passage. Insulin depletion was found to be primarily the result of insulin degrading activity released by the cells during the preceding passage.

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

The attractiveness of fermentor cultures of DIOSCOREA DELTOIDEA Wall. (Dioscoreaceae) as a source of diosgenin has been greatly improved by switching away from the traditional product recovery method that has been used in all previous studies. By using a known but little-used hydrolysis method involving 2N H (2)SO (4) in 70% isopropanol rather than using 2N aqueous HCl, diosgenin was found to be a growth-associated product instead of a non-growth-associated product as was formerly thought. This is an important improvement from a biotechnological standpoint because it means that diosgenin can be obtained directly from growth-phase tissue and that a non-growth phase is unnecessary. The reason that switching hydrolysis methods has this impact is that the non-traditional method gives high diosgenin yields from a broader group of steroidal glycosides. During the non-growth phase, steroidal glycosides were found to spontaneously change from furostanol saponins to spirostanol saponins. Whereas the nontraditional hydrolysis method gives high diosgenin yields from both types, the traditional method gives high yields only from the latter type.

A fundamentally new method is described for the separation of the dimeric indole alkaloids vincristine and vinblastine from monomeric indole alkaloid impurities. This method uses an RP-18 high-performance liquid chromatography column with a methanol-water mobile phase containing an inorganic acid and an unusually low concentration of inorganic buffer. By keeping the buffer concentration low, the elution of all indole alkaloids is retarded, but the dimeric ones are retarded more than the monomeric ones. A theoretical model developed to explain this behavior postulates that the anions of the buffer solubilize the protonated indole alkaloids by pairing with them. Lowering the buffer concentration reduces the availability of pairing ions and thus decreases the mobile phase affinity of protonated alkaloids, particularly those having a 2+ charge. A similar approach may be applicable in other situations where ionogenic organic compounds having a particular valence must be separated from related compounds having different valences, or from non-ionogenic compounds.

A 141, stirred-tank bioreactor was used to investigate the effect of 7% glucose solutions on CATHARANTHUS ROSEUS suspension cultures. Measurement of oxygen uptake rate indicated that alkaloid accumulation occurred primarily during a 20-day transition period between growth-oriented metabolism and maintenance-oriented metabolism. Exposure of the cells to light during this period stimulated catharanthine accumulation and triggered a switch from ajmalicine accumulation to serpentine accumulation. In addition, it suppressed the secretion of both ajmalicine and serpentine; without light nearly 80% of the ajmalicine and serpentine was found in the medium, whereas with light less than 20% was secreted. Alkaloid accumulation was found to be adversely affected by increasing the volume of inoculum culture transferred to a given volume of fresh glucose solution, apparently due to the entry of 2,4-D with the inoculum.