Rapid spectrophotometric detection for optimized production of landomycins and characterization of their therapeutic potential.

Microbial-derived natural products remain a major source of structurally diverse bioactive compounds and chemical scaffolds that have the potential as new therapeutics to target drug-resistant pathogens and cancers. In particular, genome mining has revealed the vast number of cryptic or low-yield biosynthetic gene clusters in the genus Streptomyces. However, low natural product yields-improvements to which have been hindered by the lack of high throughput methods-have slowed the discovery and development of many potential therapeutics. Here, we describe our efforts to improve yields of landomycins-angucycline family polyketides under investigation as cancer therapeutics-by a genetically modified Streptomyces cyanogenus 136. After simplifying the extraction process from S. cyanogenus cultures, we identified a wavelength at which the major landomycin products are absorbed in culture extracts, which we used to systematically explore culture medium compositions to improve total landomycin titers. Through correlational analysis, we simplified the culture optimization process by identifying an alternative wavelength at which culture supernatants absorb yet is representative of total landomycin titers. Using the subsequently improved sample throughput, we explored landomycin production during the culturing process to further increase landomycin yield and reduce culture time. Testing the antimicrobial activity of the isolated landomycins, we report broad inhibition of Gram-positive bacteria, inhibition of fungi by landomycinone, and broad landomycin resistance by Gram-negative bacteria that is likely mediated by the exclusion of landomycins by the bacterial membrane. Finally, the anticancer activity of the isolated landomycins against A549 lung carcinoma cells agrees with previous reports on other cell lines that glycan chain length correlates with activity. Given the prevalence of natural products produced by Streptomyces, as well as the light-absorbing moieties common to bioactive natural products and their metabolic precursors, our method is relevant to improving the yields of other natural products of interest.

Concurrent use of continuous kidney replacement therapy during extracorporeal membrane oxygenation: what pediatric nephrologists need to know-PCRRT-ICONIC practice points.

Extracorporeal membrane oxygenation (ECMO) provides temporary cardiorespiratory support for neonatal, pediatric, and adult patients when traditional management has failed. This lifesaving therapy has intrinsic risks, including the development of a robust inflammatory response, acute kidney injury (AKI), fluid overload (FO), and blood loss via consumption and coagulopathy. Continuous kidney replacement therapy (CKRT) has been proposed to reduce these side effects by mitigating the host inflammatory response and controlling FO, improving outcomes in patients requiring ECMO. The Pediatric Continuous Renal Replacement Therapy (PCRRT) Workgroup and the International Collaboration of Nephrologists and Intensivists for Critical Care Children (ICONIC) met to highlight current practice standards for ECMO use within the pediatric population. This review discusses ECMO modalities, the pathophysiology of inflammation during an ECMO run, its adverse effects, various anticoagulation strategies, and the technical aspects and outcomes of implementing CKRT during ECMO in neonatal and pediatric populations. Consensus practice points and guidelines are summarized. ECMO should be utilized in patients with severe acute respiratory failure despite the use of conventional treatment modalities. The Extracorporeal Life Support Organization (ELSO) offers guidelines for ECMO initiation and management while maintaining a clinical registry of over 195,000 patients to assess outcomes and complications. Monitoring and preventing fluid overload during ECMO and CKRT are imperative to reduce mortality risk. Clinical evidence, resources, and experience of the nephrologist and healthcare team should guide the selection of ECMO circuit.

Systematic review and network meta-analysis of the risk of Herpes zoster with biological therapies and selective Janus kinase-1 inhibitors in atopic dermatitis.

Introduction: Atopic dermatitis (AD) patients have an increased risk of herpes zoster (HZ). The relationship of dupilumab, tralokinumab, upadacitinib, and abrocitinib to HZ incidence in AD patients remains unclear. Aim: To evaluate and compare the incidence and risk of HZ among patients with moderate to severe atopic dermatitis treated with advanced systemic therapies. Material and methods: Systematic searches were conducted in Ovid Medline and Embase. The primary outcome was incidence of HZ in patients with moderate to severe AD receiving placebo or the aforementioned treatments. A frequentist random-effects NMA was conducted with odds ratio. Results: Our search identified 16 trials comprising 10,689 patients. Upadacitinib was associated with a dose-dependent increase in the incidence of HZ compared to placebo (OR = 2.55 [1.09, 5.95] and (OR = 4.29 [1.89, 9.74], respectively) and compared to various dupilumab doses (OR = 4.48 [1.29, 15.57], 3.61 [1.28, 10.18] and 7.54 [2.21, 25.68], 6.09 [2.24, 16.52], respectively). Upadacitinib 30 mg was associated with a higher incidence of HZ when compared to upadacitinib 15 mg (OR = 1.68 [1.19, 2.38]). Abrocitinib 200 mg was associated with a higher increase in HZ compared to placebo (OR = 3.34 [1.34, 8.31]). According to SUCRA ranks, both JAK-1 inhibitors had a higher cumulative incidence of HZ compared to dupilumab. Conclusions: JAK-1 inhibitors are associated with a significantly higher incidence of HZ compared to dupilumab and placebo. Our results suggest that recombinant HZ vaccination should be highly considered for all adult patients prior to starting oral JAK-1 inhibitors.

Regulation of water flow in the ocular lens: new roles for aquaporins.

The ocular lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. The lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the transparency and refractive properties of the lens. This flow of water generates a substantial hydrostatic pressure gradient which is regulated by a dual feedback system that uses the mechanosensitive channels TRPV1 and TRPV4 to sense decreases and increases, respectively, in the pressure gradient. This regulation of water flow (pressure) and hence overall lens water content, sets the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Here we focus on the roles played by the aquaporin family of water channels in mediating lens water fluxes, with a specific focus on AQP5 as a regulated water channel in the lens. We show that in addition to regulating the activity of ion transporters, which generate local osmotic gradients that drive lens water flow, the TRPV1/4-mediated dual feedback system also modulates the membrane trafficking of AQP5 in the anterior influx pathway and equatorial efflux zone of the lens. Since both lens pressure and AQP5-mediated water permeability ( P H 2 O ${P_{{{\mathrm{H}}_{\mathrm{2}}}{\mathrm{O}}}}$ ) can be altered by changes in the tension applied to the lens surface via modulating ciliary muscle contraction we propose extrinsic modulation of lens water flow as a potential mechanism to alter the refractive properties of the lens to ensure light remains focused on the retina throughout life.

Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

Flow cytometric evaluation of yeast-bacterial cell-cell interactions.

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichia coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high-throughput discovery of bacterial surface-binding molecules.

Challenges and unique considerations of adolescents with chronic kidney disease.

Chronic kidney disease (CKD) is an increasingly prevalent disease that burdens patients of all ages significantly. While much data have been gathered on adult and paediatric populations, there is a lack of data on the effects of CKD on young adults and adolescents. The aim of this review was to look at the prevalence and comorbidities of this patient population, considering the rapid biological growth and development during this time frame. We present this review to illustrate the need for further research into this special patient group.

Modulation of Membrane Trafficking of AQP5 in the Lens in Response to Changes in Zonular Tension Is Mediated by the Mechanosensitive Channel TRPV1.

In mice, the contraction of the ciliary muscle via the administration of pilocarpine reduces the zonular tension applied to the lens and activates the TRPV1-mediated arm of a dual feedback system that regulates the lens' hydrostatic pressure gradient. In the rat lens, this pilocarpine-induced reduction in zonular tension also causes the water channel AQP5 to be removed from the membranes of fiber cells located in the anterior influx and equatorial efflux zones. Here, we determined whether this pilocarpine-induced membrane trafficking of AQP5 is also regulated by the activation of TRPV1. Using microelectrode-based methods to measure surface pressure, we found that pilocarpine also increased pressure in the rat lenses via the activation of TRPV1, while pilocarpine-induced removal of AQP5 from the membrane observed using immunolabelling was abolished by pre-incubation of the lenses with a TRPV1 inhibitor. In contrast, mimicking the actions of pilocarpine by blocking TRPV4 and then activating TRPV1 resulted in sustained increase in pressure and the removal of AQP5 from the anterior influx and equatorial efflux zones. These results show that the removal of AQP5 in response to a decrease in zonular tension is mediated by TRPV1 and suggest that regional changes to PH2O contribute to lens hydrostatic pressure gradient regulation.

A Silk-Based Platform to Stabilize Phenylalanine Ammonia-lyase for Orally Administered Enzyme Replacement Therapy.

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ∼1 in 15 000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 °C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, which will enable oral administration of PAL to treat PKU.

Literature Review of the Efficacy of High-Volume Hemofiltration in Critically Ill Pediatric Patients.

BACKGROUND: Pediatric sepsis is a significant public health issue. This condition is exacerbated by rising serum creatinine and inflammatory cytokines that lead to deleterious effects upon the body. The current standard of care involves the use of continuous kidney replacement therapy to remove harmful cytokines until the body returns to homeostasis. In order to promote faster clearance and reduced stay in the ICU, high-volume hemofiltration (HVHF) has shown promise. However, there is a paucity of studies to fully elucidate its benefits. METHODS: A literature search was done using PubMed/ MEDLINE and Embase. The literature was reviewed by two independent reviewers, who independently assessed the quality of randomized controlled trials by using the Cochrane risk of bias tool for RCTs and Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized controlled trials. Data were combined from studies with a similar design. RESULTS: The primary endpoint of all-cause mortality was found to be reduced by 40% across all of the pooled studies. For secondary endpoints, significant reductions of serum creatinine were found. Additionally, duration of ICU stays and treatment course was found to be significantly shorter in HVHF patients than the current standard of care. The rate of adverse effects was analyzed, and there was no difference in the proportion of patients developing hypokalemia, hyperkalemia, hypernatremia, or hyponatremia. The proportion of patients developing hyperglycemia was higher in patients undergoing HVHF, whereas the proportions of patients developing bleeding were significantly less in patients undergoing HVHF. One study reported a total number of adverse events between the two groups which were significantly lesser in patients undergoing HVHF. CONCLUSION: HVHF shows promise as a modality to treat pediatric patients with sepsis. In order to confirm the benefits of this modality, future studies need significantly more patients for analysis.

Early detection of SARS-CoV-2 and other infections in solid organ transplant recipients and household members using wearable devices.

The increasing global prevalence of SARS-CoV-2 and the resulting COVID-19 disease pandemic pose significant concerns for clinical management of solid organ transplant recipients (SOTR). Wearable devices that can measure physiologic changes in biometrics including heart rate, heart rate variability, body temperature, respiratory, activity (such as steps taken per day) and sleep patterns, and blood oxygen saturation show utility for the early detection of infection before clinical presentation of symptoms. Recent algorithms developed using preliminary wearable datasets show that SARS-CoV-2 is detectable before clinical symptoms in >80% of adults. Early detection of SARS-CoV-2, influenza, and other pathogens in SOTR, and their household members, could facilitate early interventions such as self-isolation and early clinical management of relevant infection(s). Ongoing studies testing the utility of wearable devices such as smartwatches for early detection of SARS-CoV-2 and other infections in the general population are reviewed here, along with the practical challenges to implementing these processes at scale in pediatric and adult SOTR, and their household members. The resources and logistics, including transplant-specific analyses pipelines to account for confounders such as polypharmacy and comorbidities, required in studies of pediatric and adult SOTR for the robust early detection of SARS-CoV-2, and other infections are also reviewed.

Synthetic Biology Approaches to Engineer Probiotics and Members of the Human Microbiota for Biomedical Applications.

An increasing number of studies have strongly correlated the composition of the human microbiota with many human health conditions and, in several cases, have shown that manipulating the microbiota directly affects health. These insights have generated significant interest in engineering indigenous microbiota community members and nonresident probiotic bacteria as biotic diagnostics and therapeutics that can probe and improve human health. In this review, we discuss recent advances in synthetic biology to engineer commensal and probiotic lactic acid bacteria, bifidobacteria, and Bacteroides for these purposes, and we provide our perspective on the future potential of these technologies.

Establishing synthesis pathway-host compatibility via enzyme solubility.

Current pathway synthesis tools identify possible pathways that can be added to a host to produce the desired target molecule through the exploration of abstract metabolic and reaction network space. However, not many of these tools explore gene-level information required to physically realize the identified synthesis pathways, and none explore enzyme-host compatibility. Developing tools that address this disconnect between abstract reactions/metabolic design space and physical genetic sequence design space will enable expedited experimental efforts that avoid exploring unprofitable synthesis pathways. This work describes a workflow, termed Probabilistic Pathway Assembly with Solubility Confidence Scores (ProPASS), which links synthesis pathway construction with the exploration of the physical design space as imposed by the availability of enzymes with predicted characterized activities within the host. Predicted protein solubility propensity scores are used as a confidence level to quantify the compatibility of each pathway enzyme with the host Escherichia coli (E. coli). This study also presents a database, termed Protein Solubility Database (ProSol DB), which provides solubility confidence scores in E. coli for 240,016 characterized enzymes obtained from UniProtKB/Swiss-Prot. The utility of ProPASS is demonstrated by generating genetic implementations of heterologous synthesis pathways in E. coli that target several commercially useful biomolecules.

Towards creating an extended metabolic model (EMM) for E. coli using enzyme promiscuity prediction and metabolomics data.

BACKGROUND: Metabolic models are indispensable in guiding cellular engineering and in advancing our understanding of systems biology. As not all enzymatic activities are fully known and/or annotated, metabolic models remain incomplete, resulting in suboptimal computational analysis and leading to unexpected experimental results. We posit that one major source of unaccounted metabolism is promiscuous enzymatic activity. It is now well-accepted that most, if not all, enzymes are promiscuous-i.e., they transform substrates other than their primary substrate. However, there have been no systematic analyses of genome-scale metabolic models to predict putative reactions and/or metabolites that arise from enzyme promiscuity. RESULTS: Our workflow utilizes PROXIMAL-a tool that uses reactant-product transformation patterns from the KEGG database-to predict putative structural modifications due to promiscuous enzymes. Using iML1515 as a model system, we first utilized a computational workflow, referred to as Extended Metabolite Model Annotation (EMMA), to predict promiscuous reactions catalyzed, and metabolites produced, by natively encoded enzymes in Escherichia coli. We predict hundreds of new metabolites that can be used to augment iML1515. We then validated our method by comparing predicted metabolites with the Escherichia coli Metabolome Database (ECMDB). CONCLUSIONS: We utilized EMMA to augment the iML1515 metabolic model to more fully reflect cellular metabolic activity. This workflow uses enzyme promiscuity as basis to predict hundreds of reactions and metabolites that may exist in E. coli but may have not been documented in iML1515 or other databases. We provide detailed analysis of 23 predicted reactions and 16 associated metabolites. Interestingly, nine of these metabolites, which are in ECMDB, have not previously been documented in any other E. coli databases. Four of the predicted reactions provide putative transformations parallel to those already in iML1515. We suggest adding predicted metabolites and reactions to iML1515 to create an extended metabolic model (EMM) for E. coli.

Computational prediction of functional abortive RNA in E. coli.

Failure by RNA polymerase to break contacts with promoter DNA results in release of bound RNA and re-initiation of transcription. These abortive RNAs were assumed to be non-functional but have recently been shown to affect termination in bacteriophage T7. Little is known about the functional role of these RNA in other genetic models. Using a computational approach, we investigated whether abortive RNA could exert function in E. coli. Fragments generated from 3780 transcription units were used as query sequences within their respective transcription units to search for possible binding sites. Sites that fell within known regulatory features were then ranked based upon the free energy of hybridization to the abortive. We further hypothesize about mechanisms of regulatory action for a select number of likely matches. Future experimental validation of these putative abortive-mRNA pairs may confirm our findings and promote exploration of functional abortive RNAs (faRNAs) in natural and synthetic systems.

Co-utilization of hexoses by a microconsortium of sugar-specific E. coli strains.

Escherichia coli is an important commercial species used for production of biofuels, biopolymers, organic acids, sugar alcohols, and natural compounds. Processed biomass and agroindustrial byproducts serve as low-cost nutrient sources and contain a variety of hexoses available for bioconversion. However, metabolism of hexose mixtures by E. coli is inefficient due to carbon catabolite repression (CCR), where the transport and catabolic activity of one or more carbon sources is repressed and/or inhibited by the transport and catabolism of another carbon source. In this work, we developed a microconsortium of different E. coli strains, each engineered to preferentially catabolize a different hexose-glucose, galactose, or mannose. We modified the specificity and preference of carbon source using a combination of rational strain design and adaptive evolution. The modifications ultimately resulted in strains that preferentially catabolized their specified sugar. Finally, comparative analysis in galactose- and mannose-rich sugar mixtures revealed that the consortium grew faster and to higher cell densities compared to the wild-type strain. Biotechnol. Bioeng. 2017;114: 2309-2318. © 2017 Wiley Periodicals, Inc.