Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26-His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

Multiomics endotyping of preterm infants with bronchopulmonary dysplasia and pulmonary hypertension-A pilot study.

Pulmonary hypertension associated with bronchopulmonary dysplasia is a severe complication of preterm birth resulting in high mortality of up to 50% within the first 2 years of life. There is a direct relationship between bronchopulmonary dysplasia severity and incidence of associated pulmonary hypertension. However, it is challenging to clinically characterize severe bronchopulmonary dysplasia with and without pulmonary hypertension and there is need for better understanding of the two entities. Our main objective is to identify markers to help understand biological processes and characterize infants with pulmonary hypertension associated with bronchopulmonary dysplasia using tracheal aspirates. We conducted an unbiased multiomic analysis of tracheal aspirates via microRNA (miRNA) polymerase chain reaction arrays, RNA sequencing, and mass spectrometry proteomics in preterm infants with severe bronchopulmonary dysplasia with and without pulmonary hypertension (n = 46). Our pilot study analysis revealed 12 miRNAs (hsa-miR-29a, has-miR-542-3p, has-miR-624, has-miR-183, hsa-miR-501-3p, hsa-miR-101, hsa-miR-3131, hsa-miR-3683, hsa-miR-3193, hsa-miR-3672, hsa-miR-3128, and hsa-miR-1287), 6 transcripts (IL6, RPL35P5, HSD3B7, RNA5SP215, OR2A1-AS1, and RNVU1-19), and 5 proteins (CAPS, AAT, KRT5, SFTPB, and LGALS3BP) with significant differential expression in preterm infants with severe lung disease with pulmonary hypertension when compared with infants with severe lung disease but no pulmonary hypertension. Pathway analysis of the integrated multiomic expression signatures revealed NFkB, VEGF, SERPINA1, IL6, and ERK1/2 as target molecules and cellular development, cellular growth and proliferation, and cellular movement as key affected molecular functions. Our multiomic analysis of tracheal aspirates revealed a comprehensive thumbprint of miRNAs, mRNAs, and proteins that could help endotype infants with severe lung disease and pulmonary hypertension.

Balancing Trade-Offs Imposed by Growth Media and Mass Spectrometry for Bacterial Exometabolomics.

The bacterial exometabolome consists of a vast array of specialized metabolites, many of which are only produced in response to specific environmental stimuli. For this reason, it is desirable to control the extracellular environment with a defined growth medium composed of pure ingredients. However, complex (undefined) media are expected to support the robust growth of a greater variety of microorganisms than defined media. Here, we investigate the trade-offs inherent to a range of complex and defined solid media for the growth of soil microorganisms, production of specialized metabolites, and detection of these compounds using direct infusion mass spectrometry. We find that complex media support growth of more soil microorganisms, as well as allowing for the detection of more previously discovered natural products as a fraction of total m/z features detected in each sample. However, the use of complex media often caused mass spectrometer injection failures and poor-quality mass spectra, which in some cases resulted in over a quarter of samples being removed from analysis. Defined media, while more limiting in growth, generated higher quality spectra and yielded more m/z features after background subtraction. These results inform future exometabolomic experiments requiring a medium that supports the robust growth of many soil microorganisms. IMPORTANCE Bacteria are capable of producing and secreting a rich diversity of specialized metabolites. Yet, much of their exometabolome remains hidden due to challenges associated with eliciting specialized metabolite production, labor-intensive sample preparation, and time-consuming analysis techniques. Using our versatile three-dimensional (3D)-printed culturing platform, SubTap, we demonstrate that rapid exometabolomic data collection from a diverse set of environmental bacteria is feasible. We optimized our platform by surveying Streptomyces isolated from soil on a variety of media types to assess viability, degree of specialized metabolite production, and compatibility with downstream LESA-DIMS analysis. Ultimately, this will enable data-rich experimentation, allowing for a better understanding of bacterial exometabolomes.

Sample-efficient identification of high-dimensional antibiotic synergy with a normalized diagonal sampling design.

Antibiotic resistance is an important public health problem. One potential solution is the development of synergistic antibiotic combinations, in which the combination is more effective than the component drugs. However, experimental progress in this direction is severely limited by the number of samples required to exhaustively test for synergy, which grows exponentially with the number of drugs combined. We introduce a new metric for antibiotic synergy, motivated by the popular Fractional Inhibitory Concentration Index and the Highest Single Agent model. We also propose a new experimental design that samples along all appropriately normalized diagonals in concentration space, and prove that this design identifies all synergies among a set of drugs while only sampling a small fraction of the possible combinations. We applied our method to screen two- through eight-way combinations of eight antibiotics at 10 concentrations each, which requires sampling only 2,560 unique combinations of antibiotic concentrations.

Early Salivary miRNA Expression in Extreme Low Gestational Age Newborns.

Background: MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression playing a key role in organogenesis. MiRNAs are studied in tracheal aspirates (TA) of preterm infants. However; this is difficult to obtain in infants who are not intubated. This study examines early salivary miRNA expression as non-invasive early biomarkers in extremely low gestational age newborns (ELGANs). Methods: Saliva was collected using DNA-genotek swabs, miRNAs were analyzed using RNA seq and RT PCR arrays. Salivary miRNA expression was compared to TA using RNA seq at 3 days of age, and longitudinal changes at 28 days of age were analyzed using RT PCR arrays in ELGANs. Results: Approximately 822 ng of RNA was extracted from saliva of 7 ELGANs; Of the 757 miRNAs isolated, 161 miRNAs had significant correlation in saliva and TA at 3 days of age (r = 0.97). Longitudinal miRNA analysis showed 29 miRNAs downregulated and 394 miRNAs upregulated at 28 days compared to 3 days of age (adjusted p < 0.1). Bioinformatic analysis (Ingenuity Pathway Analysis) of differentially expressed miRNAs identified organismal injury and abnormalities and cellular development as the top physiological system development and cellular function. Conclusion: Salivary miRNA expression are source for early biomarkers of underlying pathophysiology in ELGANs.

A Pilot Trial Using Telemedicine in Radiation Oncology: The Future of Health Care Is Virtual.

Background: Social determinants of health directly affect cancer survival. Driven by advances in technology and recent demands due to COVID-19, telemedicine has the ability to improve patient access to care, lower health care costs, and increase workflow efficiency. The role of telemedicine in radiation oncology is not established. Materials and Methods: We conducted an IRB-approved pilot trial using a telehealth platform for the first post-radiation visit in patients with any cancer diagnosis. The primary endpoint was feasibility of using telehealth defined by completion of five telehealth visits per month in a single department. Secondary endpoints included the ability to assess patients appropriately, patient and physician satisfaction. Physicians were surveyed again during the pandemic to determine whether viewpoints changed. Results: Between May 27, 2016 and August 1, 2018, 37 patients were enrolled in the Telehealth in Post-operative Radiation Therapy (TelePORT) trial, with 24 evaluable patients who completed their scheduled telehealth visit. On average, 1.4 patients were accrued per month. All patients were satisfied with their care, had enough time with their physician and 85.7% believed the telehealth communication was excellent. All physicians were able to accurately assess the patient's symptoms via telehealth, whereas 82.3% felt they could accurately assess treatment-related toxicity. Physicians assessing skin toxicity from breast radiation were those who did not feel they were able to assess toxicity. Discussion and Conclusions: Both health care providers and patients have identified telemedicine as a suitable platform for radiation oncology visits. Although there are limitations, telemedicine has significant potential for increasing access of cancer care delivery in radiation oncology.

MicroRNA Signatures Associated with Bronchopulmonary Dysplasia Severity in Tracheal Aspirates of Preterm Infants.

Bronchopulmonary dysplasia (BPD) is a form of chronic lung disease that develops in neonates as a consequence of preterm birth, arrested fetal lung development, and inflammation. The incidence of BPD remains on the rise as a result of increasing survival of extremely preterm infants. Severe BPD contributes to significant health care costs and is associated with prolonged hospitalizations, respiratory infections, and neurodevelopmental deficits. In this study, we aimed to detect novel biomarkers of BPD severity. We collected tracheal aspirates (TAs) from preterm babies with mild/moderate (n = 8) and severe (n = 17) BPD, and we profiled the expression of 1048 miRNAs using a PCR array. Associations with biological pathways were determined with the Ingenuity Pathway Analysis (IPA) software. We found 31 miRNAs differentially expressed between the two disease groups (2-fold change, false discovery rate (FDR) < 0.05). Of these, 4 miRNAs displayed significantly higher expression levels, and 27 miRNAs had significantly lower expression levels in the severe BPD group when compared to the mild/moderate BPD group. IPA identified cell signaling and inflammation pathways associated with miRNA signatures. We conclude that TAs of extremely premature infants contain miRNA signatures associated with severe BPD. These may serve as potential biomarkers of disease severity in infants with BPD.

Tracheal aspirate transcriptomic and miRNA signatures of extreme premature birth with bronchopulmonary dysplasia.

OBJECTIVE: Extreme preterm infants are a growing population in neonatal intensive care units who carry a high mortality and morbidity. Multiple factors play a role in preterm birth, resulting in major impact on organogenesis leading to complications including bronchopulmonary dysplasia (BPD). The goal of this study was to identify biomarker signatures associated with prematurity and BPD. STUDY DESIGN: We analyzed miRNA and mRNA profiles in tracheal aspirates (TAs) from 55 infants receiving invasive mechanical ventilation. Twenty-eight infants were extremely preterm and diagnosed with BPD, and 27 were term babies receiving invasive mechanical ventilation for elective procedures. RESULT: We found 22 miRNAs and 33 genes differentially expressed (FDR < 0.05) in TAs of extreme preterm infants with BPD vs. term babies without BPD. Pathway analysis showed associations with inflammatory response, cellular growth/proliferation, and tissue development. CONCLUSIONS: Specific mRNA-miRNA signatures in TAs may serve as biomarkers for BPD pathogenesis, a consequence of extreme prematurity.

Reducing Intrathecal Baclofen Related Infections: Service Evaluation and Best Practice Guidelines.

OBJECTIVES: Intrathecal baclofen (ITB) pumps are an effective treatment for spasticity; however infection rates have been reported in 3-26% of patients in the literature. The multidisciplinary ITB service has been established at The National Hospital for Neurology and Neurosurgery, UCLH, Queen Square, London for over 20 years. Our study was designed to clarify the rate of infection in our ITB patient cohort and secondly, to formulate and implement best practice guidelines and to determine prospectively, whether they effectively reduced infection rates. METHODS: Clinical record review of all patients receiving ITB pre-intervention; January 2013-May 2015, and following practice changes; June 2016-June 2018. RESULTS: Four of 118 patients receiving ITB during the first time period (3.4%, annual incidence rate of infection 1.4%) developed an ITB-related infection (three following ITB pump replacement surgery, one after initial implant). Infections were associated with 4.2% of ITB-related surgical procedures. Three of four pumps required explantation. Following change in practice (pre-operative chlorhexidine skin wash and intraoperative vancomycin wash of the fibrous pocket of the replacement site), only one of 160 ITB patients developed infection (pump not explanted) in the second time period (0.6%, annual incidence rate 0.3%). The infection rate related to ITB surgical procedures was 1.1%. In cases of ITB pump replacement, the infection rate was reduced to 3.3% from 17.6%. CONCLUSIONS: This study suggests that a straightforward change in clinical practice may lower infection rates in patients undergoing ITB therapy.

A de novo enzyme catalyzes a life-sustaining reaction in Escherichia coli.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Here we describe Syn-F4, the first de novo protein that meets both criteria. Purified Syn-F4 hydrolyzes the siderophore ferric enterobactin, and expression of Syn-F4 allows an inviable strain of Escherichia coli to grow in iron-limited medium. These findings demonstrate that entirely new sequences can provide life-sustaining enzymatic functions in living organisms.

Transcriptional, posttranscriptional, and posttranslational regulation of SHOOT MERISTEMLESS gene expression in Arabidopsis determines gene function in the shoot apex.

The activity of SHOOT MERISTEMLESS (STM) is required for the functioning of the shoot apical meristem (SAM). STM is expressed in the SAM but is down-regulated at the site of leaf initiation. STM is also required for the formation of compound leaves. However, how the activity of STM is regulated at the transcriptional, posttranscriptional, and posttranslational levels is poorly understood. We previously found two conserved noncoding sequences in the promoters of STM-like genes across angiosperms, the K-box and the RB-box. Here, we characterize the function of the RB-box in Arabidopsis (Arabidopsis thaliana). The RB-box, along with the K-box, regulates the expression of STM in leaf sinuses, which are areas on the leaf blade with meristematic potential. The RB-box also contributes to restrict STM expression to the SAM. We identified FAR1-RELATED SEQUENCES-RELATED FACTOR1 (FRF1) as a binding factor to the RB-box region. FRF1 is an uncharacterized member of a subfamily of four truncated proteins related to the FAR1-RELATED SEQUENCES factors. Internal deletion analysis of the STM promoter identified a region required to repress the expression of STM in hypocotyls. Expression of STM in leaf primordia under the control of the JAGGED promoter produced plants with partially undifferentiated leaves. We further found that the ELK domain has a role in the posttranslational regulation of STM by affecting the nuclear localization of STM.

Aminopyridinecarboxamide-based inhibitors: Structure-activity relationship.

Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1beta stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with improved cellular activity.

Assessment of outcomes and parental effect on Quality-of-Life endpoints in the management of atopic dermatitis.

The objective of this study was to assess the consequences of atopic dermatitis/ eczema on two areas: (1) the quality of life of parents/caregivers and (2) resource utilization from two large group practices. Data from 414 patients with atopic dermatitis, aged two to 12 years, were collected between January 2001 and December 2003. Parents/caregivers completed the Parent's Index of Quality of Life-Atopic Dermatitis (PIQoL-AD). One-way analysis of variance and analysis of covariance models determined statistical significance. Pairwise significance testing was performed to determine statistical differences (P < .05). Mean patient age was 6.7 years and 55% of patients were males; mild and moderate atopic dermatitis was present in 82% and 13% of patients, respectively. Mean PIQoL-AD scores worsened (5.9 +/- 5.4 vs. 3.0 +/- 3.6, P < .001) for caregivers whose child had disease flares versus those without disease flares. Patients with atopic dermatitis incurred an additional 1.8 unscheduled visits annually at a cost of $93.54 per patient. It was determined that atopic dermatitis may have considerable quality-of-life and financial consequences to both family and community.

Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides.

A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.

IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2.

NF-kappaB is sequestered in the cytoplasm by the inhibitory IkappaB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IkappaBs leading to their degradation that results in NF-kappaB activation. IKK-1 and IKK-2 are two direct IkappaB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IkappaBalpha peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.

Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer.

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.