Unpacking the effects of adverse regulatory events: Evidence from pharmaceutical relabeling.

We provide causal evidence that regulation induced product shocks significantly impact aggregate demand and firm performance in pharmaceutical markets. Event study results suggest an average loss between $569 million and $882 million. Affected products lose, on average, $186 million over their remaining effective patent life. This leaves a loss of between $383 million and $696 million attributable to declines in future innovation. Our findings complement research that shows drugs receiving expedited review are more likely to suffer from regulation induced product shocks. Thus, it appears we may be trading off quicker access to drugs today for less innovation tomorrow. Results remain robust to variation across types of relabeling, market sizes, and levels of competition.

Exploring the impact of bulk and substrate physics on hydrolysis rates and biogas yields of anaerobic digesters pretreated with thermal hydrolysis.

This study evaluated the role of bulk and substrate physics on hydrolysis rates and biogas yields in anaerobic digestion (AD) pretreated by thermal hydrolysis (THP). Although THP decreases sludge viscosity, no evidence was found that bulk viscosity impacted the biogas yield or hydrolysis kinetics. In addition, no significant difference between the biogas yields for different total solids concentrations nor floc sizes was detected. However, increased mixing speeds did increase biogas yields. As a result of thermal treatment, the model protein, bovine serum albumin, was harder to degrade in terms of both overall biodegradability and hydrolysis rates when their macrostructures were changed from liquid to gel and to solid structures; the opposite was true for the model polysaccharide, amylopectin. These results demonstrated that hydrolysis in THP-AD systems was impacted mostly by the physical properties of the substrate (gelation) rather than the bulk physical properties within the digester. PRACTITIONER POINTS: Bulk viscosity does not significantly impact hydrolysis efficiency (biogas yield). However, mixing speed impacts hydrolysis beyond biogas holdup effect. Increasing the amount of substrate-microbe collisions through increasing biomass concentration does not have an impact on hydrolysis efficiency or biogas yield. Proteins are harder to degrade when macrostructure changes from liquid to gel/solid as a result of heat treatment. Polysaccharides are easier to degrade when macrostructure changes from liquid to gel/solid as a result of heat treatment. The time required for digesters to reach peak biogas production rates increased with decreasing specific surface available on gel and solid structures.

Impact of anaerobically digested biosolids characteristics and handling conditions on dewatering performance at multiple facilities.

Anaerobically digested biosolids (ABD) characteristics that affect dewatering were assessed at three water resource recovery facilities (WRRF) with different handling practices. Dewatering performance at the three sites corresponded to different levels of soluble chemical oxygen demand (COD), ammonia (NH4 -N), and mono- and divalent cation concentrations in ADB. Capillary suction time (CST) and a modified centrifugal technique were used to determine optimum polymer doses and to assess the impact of handling conditions on dewatering performance. Both techniques indicated that polymer dosing between 15 and 20 kg/dry tonne was optimal for all facilities and that biosolids mixing and pumping did not significantly impact dewaterability. The CST values of anaerobically digested biosolids decreased as temperature increased, but no significant difference was found for either temperature or location of dewatering facilities. Sludge viscosity and rheological properties that vary with temperature appeared to have influenced CST values. Modified centrifugal technique results indicated cake solids were not affected by polymer make-up water or ADB temperature when emulsion polymer was used. This study shows the value of laboratory testing of biosolids under controlled conditions to identify and correct potential problems in full-scale operations. PRACTITIONER POINTS: Capillary suction time and a modified centrifugal technique were used to assess the impact of different process-related and environmental factors on dewatering. Higher concentrations of soluble COD (potentially extracellular polymeric substances - EPS) and low calcium (Ca) in anaerobically digested biosolids align with reduced dewaterability. Cell disruption and break down of floc structures due to storage/mixing and pumping of biosolids did not appear to negatively impact dewatering. Modified centrifugal test results did not provide conclusive evidence of whether dewatering of anaerobically digested biosolids could be significantly impacted by temperature over the range 15-30°C, especially when emulsion polymer is used. This study shows the value of laboratory testing of biosolids under controlled conditions to identify potential problems in the full-scale operations.

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates.

Organic waste fractions such as sewage sludge, food waste and manure can be stabilized by anaerobic digestion (AD) to produce renewable energy in the form of biogas. Following AD, the digested solid fraction (digestate) is usually dewatered to reduce the volume before transportation. Post-AD treatments such as the Post-AD thermal hydrolysis process (Post-AD THP) have been developed to improve the dewatering, but the mode of action is not well understood. In this study, samples from 32 commercial full-scale plants were used to assess the impact of Post-AD THP on a broad range of raw materials. Maximum dewatered cake solids after Post-AD THP was predicted by thermogravimetric analysis (TGA). Post-AD THP changed the moisture distribution of the samples by increasing the free water fraction. A consistent improvement in predicted dewatered cake solids was achieved across the 32 samples tested, on average increasing the dry solids concentration by 87%. A full-scale trial showed that dewatering Post-AD THP digestate at 80 °C improved dewatered cake solids above the predictions by TGA at 35 °C. In conclusion, dewatered cake solids were significantly improved by Post-AD THP, reducing the volume of dewatered cake for disposal.

Oxide rupture-induced conductivity in liquid metal nanoparticles by laser and thermal sintering.

Metallic inks with superior conductivity and printability are necessary for high-throughput manufacturing of printed electronics. In particular, gallium-based liquid metal inks have shown great potential in creating soft, flexible and stretchable electronics. Despite their metallic composition, as-printed liquid metal nanoparticle films are non-conductive due to the surrounding metal oxide shells which are primarily Ga2O3, a wide-bandgap semiconductor. Hence, these films require a sintering process to recover their conductivity. For conventional solid metallic nanoparticles, thermal and laser processing are two commonly used sintering methods, and the sintering mechanism is well understood. Nevertheless, laser sintering of liquid metal nanoparticles was only recently demonstrated, and to date, the effect of thermal sintering has rarely been investigated. Here, eutectic gallium-indium nanoparticle films are processed separately by laser or thermal sintering in an ambient environment. Laser and thermally sintered films are compared with respect to electrical conductivity, surface morphology and elemental composition, crystallinity and surface composition. Both methods impart thermal energy to the films and generate thermal stress in the particles, resulting in rupture of the gallium oxide shells and achieving electrical conductivity across the film. For laser sintering, extensive oxide rupture allows liquid metal cores to flow out and coalesce into conductive pathways. For thermal sintering, due to less thermal stress and more oxidation, the oxide shells only rupture locally and extensive phase segregation occurs, leading to non-liquid particle films at room temperature. Electrical conductivity is instead attributed to segregated metal layers and gallium oxide which becomes crystalline and conductive at high temperatures. This comprehensive comparison confirms the necessity of oxidation suppression and significant thermal stress via instantaneous laser irradiation to achieve conductive patterns in liquid form.

Impacts of feed dilution and lower solids retention time on performance of thermal hydrolysis/anaerobic digestion.

The goal of this study was to evaluate using feed dilution/solids retention time (SRT) control to manage potential ammonia inhibition in highly loaded anaerobic digesters after thermal hydrolysis. The study compared three digesters operated at the same target volatile solids (VS) loading rate of 5.5 kg VS/d-m3 , but at different feed concentrations resulting in SRTs of 10, 15, and 18 days. Lowering the feed concentration decreased the digester total ammonia nitrogen concentrations which averaged 1,580, 2,610, and 3,080 mg NH 4 + -N/L for the 10-, 15-, and 18-day digesters. The VS reduction and methane yields were equivalent for the 15- and 18-day digesters and about 4% lower for the 10-day digester. Ammonia inhibition of the 18-day digester occurred early in the study, but the system acclimated over time. Feed dilution reduced the viscosity and the potential for volume expansion due to gas holdup and foaming. PRACTIONER POINTS: Feed dilution reduces digester ammonia concentrations and inhibition potential without sacrificing digester performance at lower SRTs. Feed dilution greatly reduces digester viscosity and associated issues with digester volume expansion due to gas holdup and foaming. Operating at the lower SRT does not impact cake solids after dewatering and substantially decreases polymer demand for conditioning.

Post-anaerobic digestion thermal hydrolysis of sewage sludge and food waste: Effect on methane yields, dewaterability and solids reduction.

Post-anaerobic digestion (PAD) treatment technologies have been suggested for anaerobic digestion (AD) to improve process efficiency and assure hygenization of organic waste. Because AD reduces the amount of organic waste, PAD can be applied to a much smaller volume of waste compared to pre-digestion treatment, thereby improving efficiency. In this study, dewatered digestate cakes from two different AD plants were thermally hydrolyzed and dewatered, and the liquid fraction was recirculated to a semi-continuous AD reactor. The thermal hydrolysis was more efficient in relation to methane yields and extent of dewaterability for the cake from a plant treating waste activated sludge, than the cake from a plant treating source separated food waste (SSFW). Temperatures above 165 °C yielded the best results. Post-treatment improved volumetric methane yields by 7% and the COD-reduction increased from 68% to 74% in a mesophilic (37 °C) semi-continuous system despite lowering the solid retention time (from 17 to 14 days) compared to a conventional system with pre-treatment of feed substrates at 70 °C. Results from thermogravimetric analysis showed an expected increase in maximum TS content of dewatered digestate cake from 34% up to 46% for the SSFW digestate cake, and from 17% up to 43% in the sludge digestate cake, after the PAD thermal hydrolysis process (PAD-THP). The increased dewatering alone accounts for a reduction in wet mass of cake leaving the plant of 60% in the case of sludge digestate cake. Additionaly, the increased VS-reduction will contribute to further reduce the mass of wet cake.

Pretreatment of a primary and secondary sludge blend at different thermal hydrolysis temperatures: Impacts on anaerobic digestion, dewatering and filtrate characteristics.

A study was performed to evaluate the effect of thermal hydrolysis pretreatment (THP) temperature on subsequent digestion performance and operation, as well as downstream parameters such as dewatering and cake quality. A blend of primary and secondary solids from the Blue Plains treatment plant in Washington, DC was dewatered to about 16% total solids (TS), and thermally hydrolyzed at five different temperatures 130, 140, 150, 160, 170 °C. The thermally hydrolyzed solids were then fed to five separate, 10 L laboratory digesters using the same feed concentration, 10.5% TS and a solids retention time (SRT) of 15 days. The digesters were operated over a six month period to achieve steady state conditions. The higher thermal hydrolysis temperatures generally improved the solids reduction and methane yields by about 5-6% over the temperature range. The increased temperature reduced viscosity of the solids and increased the cake solids after dewatering. The dissolved organic nitrogen and UV absorbance generally increased at the higher THP temperatures. Overall, operating at a higher temperature improved performance with a tradeoff of higher dissolved organic nitrogen and UV adsorbing materials in the return liquor.

Bench-to-bench bottlenecks in translation.

Transfer of biopharmaceutical inventions between firms has bottlenecks in upstream development that suggest a role for repurposing mechanisms well before the clinical trials stage.

Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43-mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

Do alternate bacterial indicators and pathogens increase after centrifuge dewatering of anaerobically digested biosolids?

The objectives of this research were to evaluate the potential for sudden increase and/or regrowth of alternative bacteria as either indicators or pathogens after dewatering of thermophilic and mesophilically digested biosolids. The results showed that, in general, for thermophilic processes, even when a statistically significant (p < 0.05) sudden increase and regrowth occurred for fecal coliforms, Escherichia coli, and Enterococci, it did not occur for Salmonella or Aeromonas. For the mesophilic process evaluated, sudden increase did not occur, but regrowth occurred for fecal coliforms, E. coli, Enterococci, and Salmonella. The results have implications for Class A and B biosolids regulations, as both fecal coliform and Salmonella are part of the regulatory limits. The results also suggest that the public health risks are minimal, as a result of the potential sudden increase and regrowth that may occur.

The effect of digestion and dewatering on sudden increases and regrowth of indicator bacteria after dewatering.

Several investigators have reported higher densities of indicator bacteria after dewatering of anaerobically digested biosolids. The increases appear to occur at two points in the biosolids process: the first, referred to as "sudden increase", occurs immediately after dewatering; the second, "regrowth", occurs during storage over longer periods. The objectives of this study were to examine the effect of digestion and dewatering processes on sudden increase and regrowth of fecal coliform and E. coli. Samples were collected from five thermophilic and five mesophilic digestion processes, with either centrifuge or belt filter press dewatering. Sudden increase typically was observed in the thermophilic processes with centrifuge dewatering and was not observed in the mesophilic processes with either centrifuge or belt filter press dewatering. Regrowth was observed in both thermophilic and mesophilic processes with centrifuge dewatering but not belt filter press dewatering.

Effect of feeding patterns on the performance of activated sludge systems.

Excessive amounts of monovalent cations are known to cause deterioration in settling and dewatering properties of activated sludge. In this study, variations in the feeding pattern to a sequencing batch reactor (SBR) were evaluated to determine if the feed pattern could influence effluent quality and sludge characteristics under high monovalent cation concentrations. Data showed that deflocculation caused by high concentrations of sodium could be mitigated by using a feed cycle where the influent to the SBR was provided over a period of 1 minute. In contrast, when the feed was provided over 4 hours, deterioration in settling and effluent water quality was observed, as reflected by an increase in effluent suspended solids, effluent chemical oxygen demand, and capillary suction time.

Anaerobically digested biosolids odor generation and pathogen indicator regrowth after dewatering.

The objective of this research was to investigate whether a preferential stimulation of microorganisms in anaerobically digested biosolids can occur after dewatering and if it can lead to pathogen indicator regrowth and odor generation upon storage. Laboratory incubation simulating biosolids storage indicates that both odorant generation, based on total volatile organic sulfur compound concentrations (TVOSCs) and pathogen indicator regrowth, based on fecal coliform densities follow similar formation and reduction patterns. The formation and reduction patterns of both odor compounds and fecal coliforms imply that groups of microorganism are induced if shearing disturbance is imposed during dewatering, but a secondary stabilization can be achieved soon after 1-2 weeks of storage. The occurrence of the induction is likely the microbial response to substrate release and environmental changes, such as oxygen, resulting from centrifuge shearing. The new conditions favor the growth of fecal coliforms and odor producing bacteria, and therefore, results in the observed fecal coliforms regrowth and odor accumulation during subsequent storage. However, when both substrate and oxygen deplete, a secondary stabilization can be achieved, and both odor and fecal coliforms density will drop.

Regulation of human cytidine triphosphate synthetase 2 by phosphorylation.

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K(m) and IC(50) values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [(32)P]H(3)PO(4) demonstrated that Ser(568) and Ser(571) were two major phosphorylation sites, and additional studies demonstrated that Ser(568) was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser(568) (S568A) but not Ser(571) significantly increased hCTPS2 activity, demonstrating that Ser(568) is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V(max) of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser(568) as a major site of CTPS2 regulation by phosphorylation.

Role of protein, amino acids, and enzyme activity on odor production from anaerobically digested and dewatered biosolids.

The main objective of this research was to test the hypothesis that bioavailable protein and, more specifically, the sulfur-containing amino acids within the protein, can be degraded by proteolytic enzymes to produce odor-causing compounds--mainly volatile sulfur compounds (VSCs)--during biosolids storage. To achieve these objectives, samples of digester effluent and cake solids were collected at 11 different wastewater treatment plants in North America, and the samples were analyzed for protein and amino acid content and general protein-degrading enzyme activity. At the same time, cake samples were stored using headspace bottles, the concentration of VSCs were measured using gas chromatography, and olfactometry measurements were made by a trained odor panel. The results showed that the bound cake protein content and methionine content was well-correlated with VSC production and the detection threshold measured by the odor panel.

Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3.

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.

Reactivation and growth of non-culturable indicator bacteria in anaerobically digested biosolids after centrifuge dewatering.

Recent literature has reported that high concentrations of indicator bacteria such as fecal coliforms (FCs) were measured in anaerobically digested sludges immediately after dewatering even though low concentrations were measured prior to dewatering. This research hypothesized that the indicator bacteria can enter a non-culturable state during digestion, and are reactivated during centrifuge dewatering. Reactivation is defined as restoration of culturability. To examine this hypothesis, a quantitative polymerase chain reaction (qPCR) method was developed to enumerate Escherichia coli, a member of the FC group, during different phases of digestion and dewatering. For thermophilic digestion, the density of E. coli measured by qPCR could be five orders of magnitude greater than the density measured by standard culturing methods (SCMs), which is indicative of non-culturable bacteria. For mesophilic digestion, qPCR enumerated up to about one order of magnitude more E. coli than the SCMs. After centrifuge dewatering, the non-culturable organisms could be reactivated such that they are enumerated by SCMs, and the conditions in the cake allowed rapid growth of FCs and E. coli during cake storage.

Generation pattern of sulfur containing gases from anaerobically digested sludge cakes.

Eleven dewatered sludge cakes collected from anaerobic digesters at different treatment plants were evaluated for the amount, type, and pattern of odorous gas production. All but one of the sludge cakes were from mesophilic anaerobic digesters. One was from a thermophilic digester. The pattern and quantities of sulfur gases were found to be unique for each of the samples with regard to the products produced, magnitude, and subsequent decline. The main odor-causing chemicals were volatile sulfur compounds, which included hydrogen sulfide, methanethiol, and dimethyl sulfide. Volatile sulfur compound production peaked in 3 to 8 days and then declined. The decline was a result of conversion of organic sulfur compounds to sulfide. In one side-by-side test, a high-solids centrifuge cake generated more odorous compounds than the low-solids centrifuge cake. The data show that anaerobic digestion does not eliminate the odor potential of anaerobically digested dewatered cakes.