Recognition of bacteria by inflammasomes.

Inflammasomes are cytosolic multiprotein complexes that assemble in response to a variety of infectious and noxious insults. Inflammasomes play a critical role in the initiation of innate immune responses, primarily by serving as platforms for the activation of inflammatory caspase proteases. One such caspase, CASPASE-1 (CASP1), initiates innate immune responses by cleaving pro-IL-1ß and pro-IL-18, leading to their activation and release. CASP1 and another inflammatory caspase termed CASP11 can also initiate a rapid and inflammatory form of cell death termed pyroptosis. Several distinct inflammasomes have been described, each of which contains a unique sensor protein of the NLR (nucleotide-binding domain, leucine-rich repeat-containing) superfamily or the PYHIN (PYRIN and HIN-200 domain-containing) superfamily. Here we describe the surprisingly diverse mechanisms by which NLR/PYHIN proteins sense bacteria and initiate innate immune responses. We conclude that inflammasomes represent a highly adaptable scaffold ideally suited for detecting and initiating rapid innate responses to diverse and rapidly evolving bacteria.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state.

Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. In kinetoplastid protozoa, including T. brucei, posttranscriptional control mechanisms are the primary means of gene regulation, and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that reportedly interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We validate the DRBD18 interaction with translating ribosomes and the translation initiation factor, eIF3a. We further show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, that is, changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. In DRBD18-depleted cells, a set of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. RNA immunoprecipitation/qRT-PCR indicates that DRBD18 associates with members of both repressed and enhanced cohorts. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.

Plasma from older children in Malawi inhibits Plasmodium falciparum binding in 3D brain microvessels.

A hallmark of cerebral malaria is sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation. Antibodies contribute to malaria immunity, but it remains unclear whether functional antibodies targeting parasite-expressed ligand can block cytoadhesion in the brain. Here, we screened the plasma of older children and young adults in Malawi to characterize the antibody response against the P. falciparum-IE surface and used a bioengineered 3D human brain microvessel model incorporating variable flow dynamics to measure adhesion blocking responses. We found a strong correlation between surface antibody reactivity by flow cytometry and reduced P. falciparum-IE binding in 3D microvessels. Moreover, there was a threshold of surface antibody reactivity necessary to achieve robust inhibitory activity. Our findings provide evidence of the acquisition of adhesion blocking antibodies against cerebral binding variants in people exposed to stable P. falciparum transmission and suggest the quality of the inhibitory response can be influenced by flow dynamics.

Selective Duplex Formation in Mixed Sequence Libraries of Synthetic Polymers.

Recognition-encoded melamine oligomers (REMO) are synthetic polymers that feature an alternating 1,3,5-triazine-piperazine backbone and side-chains equipped with either a phenol or phosphine oxide recognition unit. An automated method for the solid-phase synthesis (SPS) of REMO of any specified sequence has been developed starting from dichlorotriazine monomer building blocks. Complementary homo-oligomers with either six phenols or six phosphine oxides were synthesized and shown to form a stable duplex in nonpolar solvents by NMR denaturation experiments. The duplex was covalently trapped by equipping the ends of the oligomers with an azide and an alkyne group and using a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. The SPS methodology was adapted to synthesize mixed sequence libraries by using a mixture of two different dichlorotriazine building blocks in each coupling cycle of an oligomer synthesis. The resulting libraries contain statistical mixtures of all possible sequences. The self-assembly properties of these libraries were screened by using the CuAAC reaction to trap any duplexes present. In mixed sequence libraries of 6-mers, the trapping experiments showed that only sequence-complementary oligomers formed duplexes at micromolar concentrations in dichloromethane. The automated synthesis approach developed here provides access to large libraries of mixed sequence synthetic polymers, and the covalent trapping experiment provides a convenient tool for screening functional properties of mixtures. The results suggest high-fidelity sequence-selective duplex formation in mixtures of 6-mer sequences of the REMO architecture.

Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

Unique Interactions of the Small Translocases of the Mitochondrial Inner Membrane (Tims) in Trypanosoma brucei.

The infectious agent for African trypanosomiasis, Trypanosoma brucei, possesses a unique and essential translocase of the mitochondrial inner membrane, known as the TbTIM17 complex. TbTim17 associates with six small TbTims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction patterns of these smaller TbTims with each other and TbTim17 are not clear. Through yeast two-hybrid (Y2H) and co-immunoprecipitation analyses, we demonstrate that all six small TbTims interact with each other. Stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. However, TbTim10 shows weaker associations with TbTim13, which has a stronger connection with TbTim17. Each of the small TbTims also interacts strongly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial for maintaining the steady-state levels of the TbTIM17 complex. Further analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except for TbTim13, is present in ~70 kDa complexes, possibly existing in heterohexameric forms. In contrast, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionates with TbTim17. Altogether, our results demonstrate that, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific to T. brucei.

The potential to manage releases of Bacillus anthracis using bioretention and a high flow media filter: Results of simulated runoff testing with tracer spores Bacillus globigii.

The threat of bioterrorism has spurred research on the decontamination and containment of different agents. Anthrax [causative agent Bacillus anthracis (Ba)] is a disease that can lead to severe infections within human and animals, particularly when inhaled. This research investigated the use of spore-contaminated simulated runoff events into stormwater control measures (SCMs), which are designed to retain and improve the quality of runoff and may have the potential to filter and contain the spores. In this study, the effectiveness of a bioretention cell (BRC) and high flow media filter (HFMF) in Huron, Ohio, were evaluated for removal of Bacillus globigii (Bg) spores (a harmless cognate of Ba). Three 4-8 mm simulated runoff events were created for each SCM using a fire hydrant and Bg spores were injected into the runoff upstream of the SCM inlets. The BRC significantly (p < 0.001) outperformed the HFMF in reducing Bg concentrations and loads, with an average load reduction of 1.9 log (∼99% reduction) compared to 0.4 (∼60% reduction), respectively. A probable critical design factor leading to these differences was the infiltration rate of the media and subsequent retention time within the filters, which was supported by similar disparities in suspended solids reductions. Differences in spore removal may also have been due to particle size distribution of the HFMF, which was more gravelly than the bioretention cell. At 3 and 6 months after the-simulated runoff tests, soil samples taken from both SCMs, yielding detectable Bg spores within the top 15 cm of media, with increased spore concentrations where ponding occurred for longer durations during the tests. This suggests that forebays and areas near inlets may be hotspots for spore cleanup in a real-world bioterrorism incident.

Altered hepatic and intestinal homeostasis in a neonatal murine model of short-term total parenteral nutrition and antibiotics.

Parenteral nutrition (PN) prevents starvation and supports metabolic requirements intravenously when patients are unable to be fed enterally. Clinically, infants are frequently provided PN in intensive care settings along with exposure to antibiotics (ABX) to minimize infection during care. Unfortunately, neonates experience extremely high rates of hepatic complications. Adult rodent and piglet models of PN are well-established but neonatal models capable of leveraging the considerable transgenic potential of the mouse remain underdeveloped. Utilizing our newly established neonatal murine PN mouse model, we administered ABX or controlled drinking water to timed pregnant dams to disrupt the maternal microbiome. We randomized mouse pups to PN or sham surgery controls +/- ABX exposure. ABX or short-term PN decreased liver and brain organ weights, intestinal length, and mucosal architecture (vs. controls). PN significantly elevated evidence of hepatic proinflammatory markers, neutrophils and macrophage counts, bacterial colony-forming units, and evidence of cholestasis risk, which was blocked by ABX. However, ABX uniquely elevated metabolic regulatory genes resulting in accumulation of hepatocyte lipids, triglycerides, and elevated tauro-chenoxycholic acid (TCDCA) in serum. Within the gut, PN elevated the relative abundance of Akkermansia, Enterococcus, and Suterella with decreased Anaerostipes and Lactobacillus compared with controls, whereas ABX enriched Proteobacteria. We conclude that short-term PN elevates hepatic inflammatory stress and risk of cholestasis in early life. Although concurrent ABX exposure protects against hepatic immune activation during PN, the dual exposure modulates metabolism and may contribute toward early steatosis phenotype, sometimes observed in infants unable to wean from PN.NEW & NOTEWORTHY This study successfully established a translationally relevant, murine neonatal parenteral nutrition (PN) model. Short-term PN is sufficient to induce hepatitis-associated cholestasis in a neonatal murine model that can be used to understand disease in early life. The administration of antibiotics during PN protects animals from bacterial translocation and proinflammatory responses but induces unique metabolic shifts that may predispose the liver toward early steatosis.

Machine Learning for Prediction, Classification, and Identification of Immobilized Enzymes for Biocatalysis.

BACKGROUND: Enzyme immobilization is a beneficial component involved in biocatalytic strategies. Understanding and evaluating the enzyme immobilization system plays an important role in the successful development and implementation of the biocatalysis route. Ensuring the implementation of a successful enzyme immobilization process is vital for realizing a highly functioning and well suited biocatalytic process within pharmaceutical development. AIM: To develop a method which can accurately and objectively identify and classify differences within enzyme immobilization systems, sample preparation methods, and data collection parameters. METHODS: Raman hyperspectral imaging was used to obtain a total of eight spectral data sets from enzyme immobilization samples. Partial least squares discriminant analysis (PLS-DA) was used to classify and identify the samples based on their differences. RESULTS: Several two-class, four-class, and eight-class PLS-DA models were built to classify the different sample data sets. All models reached between 92-100% accuracy after cross-validation and external validation, illustrating great success of the models for identifying differences between the samples. CONCLUSION: Raman hyperspectral imaging with machine learning can be used to investigate, interpret, and classify different data collection parameters, sample preparation methods, and enzyme immobilization supports, providing crucial insight into enzyme immobilization process development.

Exacerbations, treatment patterns, utilization, and costs before and after initiating of benralizumab for the treatment of severe eosinophilic asthma.

OBJECTIVES: The purpose of this study was to examine the number of exacerbations, counts of eosinophils, and asthma-related symptoms 1 year before and after initiating benralizumab for the treatment of severe eosinophilic asthma. METHODS: Patients with prior exacerbations and newly initiating benralizumab were identified in the claims-based Healthcare Integrated Research Database. Claims were used to assess benralizumab treatment patterns, exacerbations, healthcare resource utilization, and other asthma medication used. Among a subset of patients, medical records were abstracted for Asthma Control Test (ACT) scores and asthma symptoms. RESULTS: There were 506 patients meeting inclusion/exclusion criteria for claims-based analyses and 123 for medical-record analyses. The number of patients experiencing exacerbations significantly decreased from baseline to follow-up (40% reduction, McNemar's χ2 = 204.00, p < .001). The mean number of exacerbations also decreased from 3.2 (1.5) to 1.2 (1.4) (paired t = 24.45, p < .001; Cohen's D = 1.09). The effects were larger among patients with eosinophils ≥300 cells/µL. Among patients with an ACT available for baseline and follow-up (n = 47), there was a significant reduction in the number of patients with scores <19 (72% vs. 45%, p < .01). CONCLUSIONS: Treatment with benralizumab resulted in fewer exacerbations, reduced utilization, and improved ACT scores. This study demonstrates that benralizumab is an effective treatment option for patients with severe eosinophilic asthma.

Building an Integrated Data Infrastructure to Examine the Spectrum of Suicide Risk Factors in Philadelphia Medicaid.

While there are many data-driven approaches to identifying individuals at risk of suicide, they tend to focus on clinical risk factors, such as previous psychiatric hospitalizations, and rarely include risk factors that occur in nonclinical settings, such as jails or emergency shelters. A better understanding of system-level encounters by individuals at risk of suicide could help inform suicide prevention efforts. In Philadelphia, we built a community-level data infrastructure that encompassed suicide death records, behavioral health claims, incarceration episodes, emergency housing episodes, and involuntary commitment petitions to examine a broader spectrum of suicide risk factors. Here, we describe the development of the data infrastructure, present key trends in suicide deaths in Philadelphia, and, for the Medicaid-eligible population, determine whether suicide decedents were more likely to interact with the behavioral health, carceral, and housing service systems compared to Medicaid-eligible Philadelphians who did not die by suicide. Between 2003 and 2018, there was an increase in the number of annual suicide deaths among Medicaid-eligible individuals, in part due to changes in Medicaid eligibility. There were disproportionately more suicide deaths among Black and Hispanic individuals who were Medicaid-eligible, who were younger on average, compared to suicide decedents who were never Medicaid-eligible. However, when we accounted for the racial and ethnic composition of the Medicaid population at large, we found that White individuals were four times as likely to die by suicide, while Asian, Black, Hispanic, and individuals of other races were less likely to die by suicide. Overall, 58% of individuals who were Medicaid-eligible and died by suicide had at least one Medicaid-funded behavioral health claim, 10% had at least one emergency housing episode, 25% had at least one incarceration episode, and 22% had at least one involuntary commitment. By developing a data infrastructure that can incorporate a broader spectrum of risk factors for suicide, we demonstrate how communities can harness administrative data to inform suicide prevention efforts. Our findings point to the need for suicide prevention in nonclinical settings such as jails and emergency shelters, and demonstrate important trends in suicide deaths in the Medicaid population.

Prospective Multicenter Comparison of Open and Robotic Radical Prostatectomy: The PROST-QA/RP2 Consortium.

PURPOSE: Our goal was to evaluate the comparative effectiveness of robot-assisted laparoscopic prostatectomy (RALP) and open radical prostatectomy (ORP) in a multicenter study. MATERIALS AND METHODS: We evaluated men with localized prostate cancer at 11 high-volume academic medical centers in the United States from the PROST-QA (2003-2006) and the PROST-QA/RP2 cohorts (2010-2013) with a pre-specified goal of comparing RALP (549) and ORP (545). We measured longitudinal patient-reported health-related quality of life (HRQOL) at pre-treatment and at 2, 6, 12, and 24 months, and pathological and perioperative outcomes/complications. RESULTS: Demographics, cancer characteristics, and margin status were similar between surgical approaches. ORP subjects were more likely to undergo lymphadenectomy (89% vs 47%; p <0.01) and nerve sparing (94% vs 89%; p <0.01). RALP vs ORP subjects experienced less mean intraoperative blood loss (192 vs 805 mL; p <0.01), shorter mean hospital stay (1.6 vs 2.1 days; p <0.01), and fewer blood transfusions (1% vs 4%; p <0.01), wound infections (2% vs 4%; p=0.02), other infections (1% vs 4%; p <0.01), deep venous thromboses (0.5% vs 2%; p=0.04), and bladder neck contractures requiring dilation (1.6% vs 8.3%; p <0.01). RALP subjects reported less pain (p=0.04), less activity interference (p <0.01) and higher incision satisfaction (p <0.01). Surgical approach (RALP vs ORP) was not a significant predictor of longitudinal HRQOL change in any HRQOL domain. CONCLUSIONS: In high-volume academic centers, RALP and ORP patients may expect similar long-term HRQOL outcomes. Overall, RALP patients have less pain, shorter hospital stays, and fewer post-surgical complications such as blood transfusions, infections, deep venous thromboses, and bladder neck contractures.

Turbulence strength C n2 estimation from video using physics-based deep learning.

Images captured from a long distance suffer from dynamic image distortion due to turbulent flow of air cells with random temperatures, and thus refractive indices. This phenomenon, known as image dancing, is commonly characterized by its refractive-index structure constant C n2 as a measure of the turbulence strength. For many applications such as atmospheric forecast model, long-range/astronomy imaging, and aviation safety, optical communication technology, C n2 estimation is critical for accurately sensing the turbulent environment. Previous methods for C n2 estimation include estimation from meteorological data (temperature, relative humidity, wind shear, etc.) for single-point measurements, two-ended pathlength measurements from optical scintillometer for path-averaged C n2, and more recently estimating C n2 from passive video cameras for low cost and hardware complexity. In this paper, we present a comparative analysis of classical image gradient methods for C n2 estimation and modern deep learning-based methods leveraging convolutional neural networks. To enable this, we collect a dataset of video capture along with reference scintillometer measurements for ground truth, and we release this unique dataset to the scientific community. We observe that deep learning methods can achieve higher accuracy when trained on similar data, but suffer from generalization errors to other, unseen imagery as compared to classical methods. To overcome this trade-off, we present a novel physics-based network architecture that combines learned convolutional layers with a differentiable image gradient method that maintains high accuracy while being generalizable across image datasets.

Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Tracking spatial regimes as an early warning for a species of conservation concern.

In this era of global environmental change and rapid regime shifts, managing core areas that species require to survive and persist is a grand challenge for conservation. Wildlife monitoring data are often limited or local in scale. The emerging ability to map and track spatial regimes (i.e., the spatial manifestation of state transitions) using advanced geospatial vegetation data has the potential to provide earlier warnings of habitat loss because many species of conservation concern strongly avoid spatial regime boundaries. Using 23 yr of data for the lek locations of Greater Prairie-Chicken (Tympanuchus cupido; GPC) in a remnant grassland ecosystem, we demonstrate how mapping changes in the boundaries between grassland and woodland spatial regimes provide a spatially explicit early warning signal for habitat loss for an iconic and vulnerable grassland-obligate known to be highly sensitive to woody plant encroachment. We tested whether a newly proposed metric for the quantification of spatial regimes captured well-known responses of GPC to woody plant expansion into grasslands. Resource selection functions showed that the grass:woody spatial regime boundary strength explained the probability of 80% of relative lek occurrence, and GPC strongly avoided grass:woody spatial regime boundaries at broad scales. Both findings are consistent with well-known expectations derived from GPC ecology. These results provide strong evidence for vegetation-derived delineations of spatial regimes to serve as generalized signals of early warning for state transitions that have major consequences to biodiversity conservation. Mapping spatial regime boundaries over time provided interpretable early warnings of habitat loss. Woody plant regimes displaced grassland regimes starting from the edges of the study area and constricting inward. Correspondingly, the relative probability of lek occurrence constricted in space. Similarly, the temporal trajectory of spatial regime boundary strength increased over time and moved closer to the observed limit of GPC lek site usage relative to grass:woody boundary strength. These novel spatial metrics allow managers to rapidly screen for early warning signals of spatial regime shifts and adapt management practices to defend and grow habitat cores at broad scales.

In situ real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need. PAT involves the collection of in situ data, thereby providing real time information about the monitored process as well as increasing understanding of that process. Here, three separate PAT tools - optical particle imaging, in situ particle analysis, and Raman spectroscopy - were used to monitor two key steps involved in the formation of a stable emulsion product, emulsification and homogenization, as well as perform a stability assessment. The obtained results provided new insights-particle size decreases during emulsification and homogenization, and molecular changes do not occur during either the emulsification or homogenization steps. Further, the stability assessment indicated that the coarse emulsion product obtained from the emulsification step is stable over the course of 24 hours when mixed. To the best of our knowledge, this is the first report of an analytical methodology for in situ, real time analysis of emulsification and homogenization processes for vaccine adjuvants. Using our proposed analytical methodology, an improved understanding of emulsion-based vaccine adjuvants can now be achieved, ultimately impacting the ability to develop and deliver successful pharmaceuticals.

Medication use among patients with Crohn's disease or ulcerative colitis before and after the initiation of advanced therapy.

BACKGROUND: Although various treatments help reduce abdominal pain, real-world pain medication utilization among patients with Crohn's disease (CD) or ulcerative colitis (UC) receiving advanced therapies is poorly understood. The aim is to understand the utilization of pain medication 12 months before and after the initiation of advanced therapies among patients with newly diagnosed CD or UC. METHODS: This retrospective, observational cohort study used administrative medical and pharmacy claims data of patients with CD or UC from HealthCore Integrated Research Database (HIRD®). The data from patients with use of pain medication over 12 months follow-up (after the initiation date of advanced therapies) were collected and analyzed. Differences in the use of pain medication 12 months before and after the initiation of advanced therapies were assessed using McNemar's and Wilcoxon signed-rank test. RESULTS: Prior to initiating advanced therapies, 23.1% of patients with CD (N = 540) received nonsteroidal anti-inflammatory drugs (NSAIDs), 78.1% glucocorticoids, 49.4% opioids, and 29.3% neuromodulators; similarly, 20.9% of patients with UC (N = 373) received NSAIDs, 91.4% glucocorticoids, 40.8% opioids, and 29.5% neuromodulators. After receiving advanced therapies for 12 months, patients reported a reduction in the use of steroids (78.1% vs. 58.9%, P < 0.001 in CD; 91.4% vs. 74.3%, P < 0.001 in UC), opioids (49.4% vs. 41.5%, P = 0.004 in CD; 40.8% vs. 36.5%, P = 0.194 in UC), and NSAIDs (23.1% vs. 15.0%, P < 0.001 in CD; 20.9% vs. 15.8%, P = 0.035 in UC), while the use of neuromodulators significantly increased (29.3% vs. 33.7%, P = 0.007 in CD; 29.5% vs. 35.7%; P = 0.006 in UC). CONCLUSIONS: The use of pain medications such as NSAIDs, glucocorticoids, opioids, and neuromodulators was common among patients with CD or UC. These results highlight that patients with CD or UC continued to receive pain medications even after initiating advanced therapies.

Novel IM-associated protein Tim54 plays a role in the mitochondrial import of internal signal-containing proteins in Trypanosoma brucei.

BACKGROUND: The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus-encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS). Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex. RESULTS: TbTim54, a trypanosome-specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited the in vitro mitochondrial import and assembly of the internal signal-containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N-terminal signal-containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate-specificity of TbTim54 for internal-signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N-terminal domain of TbTim17. CONCLUSION: TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal-containing IM proteins in T. brucei. SIGNIFICANCE: We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 in T. brucei.