Generalized biomolecular modeling and design with RoseTTAFold All-Atom.

Deep-learning methods have revolutionized protein structure prediction and design but are presently limited to protein-only systems. We describe RoseTTAFold All-Atom (RFAA), which combines a residue-based representation of amino acids and DNA bases with an atomic representation of all other groups to model assemblies that contain proteins, nucleic acids, small molecules, metals, and covalent modifications, given their sequences and chemical structures. By fine-tuning on denoising tasks, we developed RFdiffusion All-Atom (RFdiffusionAA), which builds protein structures around small molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we designed and experimentally validated, through crystallography and binding measurements, proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and the light-harvesting molecule bilin.

A comparative analytical study on reagent-fused silica gel plate and wax painted paper-based microfluidic device for serological testing.

The detection and identification of body fluids at a crime scene shed light on the events which might have occurred and the people involved in the crime; the techniques used being lengthy, makes the on-scene detection a tough process to carry out and handle. This study aims to develop an on-spot detection method for serological testing using silica gel encapsulation technique and a modified microfluidic paper-based analytical device (µPADs). Kastle-Meyer reagent was incorporated into the µPAD and Silica Gel plate and was subsequently validated for the detection of blood. This study revealed that the µPAD was a better option. The emerging technique of µPADs allows a cost-effective and simple method of detection for body fluids. The fibrous network of the paper is manipulated to fabricate a guided channel for the fluid flow. The guided channels in the modified µPADs were fabricated by patterning a hydrophobic barrier out of wax. The analyte of interest for this study is blood but the principle can be modified to include other biological fluids.

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Structural Mechanism of Regioselectivity in an Unusual Bacterial Acyl-CoA Dehydrogenase.

Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an γ,δ-ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. While TcsD is homologous to canonical α,ß-ACADs, it acts regioselectively at the γ,δ-position and only on α,ß-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the α,ß position of substrates. From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative γ,δ-ACADs that belong to diverse natural product biosynthetic gene clusters. These predictions are supported by the demonstration that a phylogenetically distant homologue of TcsD also regioselectively oxidizes α,ß-unsaturated substrates. This work exemplifies a powerful approach to understand unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.

Robust Characterization of Two Distinct Glutarate Sensing Transcription Factors of Pseudomonas putida l-Lysine Metabolism.

A significant bottleneck in synthetic biology involves screening large genetically encoded libraries for desirable phenotypes such as chemical production. However, transcription factor-based biosensors can be leveraged to screen thousands of genetic designs for optimal chemical production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida. The genomic contexts of csiR homologues were analyzed, and their DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro. CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added, confirming that it acts as a genetic repressor. Both transcription factors and cognate promoters were then cloned into broad host range vectors to create two glutarate biosensors. Their respective sensing performance features were characterized, and more sensitive derivatives of the GcdR biosensor were created by manipulating the expression of the transcription factor. Sensor vectors were then reintroduced into P. putida and evaluated for their ability to respond to glutarate and various lysine metabolites. Additionally, we developed a novel mathematical approach to describe the usable range of detection for genetically encoded biosensors, which may be broadly useful in future efforts to better characterize biosensor performance.

Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism.

Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research.IMPORTANCEP. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.

E-O technique is superior to E-C technique in manikins during single person bag mask ventilation performed by novices.

Conventional E-C technique of mask holding is unreliable during single person bag mask ventilation (BMV) due mainly to leak around the mask and inexperience of the persons. In this manikin study, conventional E-C technique was compared with E-O technique during single person BMV both with experienced (n = 50) and novice (n = 50) volunteers. The E-O technique involved encircling the mask neck with the web between thumb and index finger while the other digits provided chin lift. Two independent observers recorded the chest expansion as 1 (nil), 2 (minimal), 3 (moderate) and 4 (good). For analysis ideal and average chest expansion were clubbed as acceptable. E-C technique in experienced volunteers showed acceptable results in 49 (31 + 18) occasions, while with novices acceptable is 39 (17 + 22). With E-O technique, expansion was acceptable in 47 (38 + 9) experienced volunteers, and acceptable in 46 (32 + 14) novices. (P = 0.003). In cross over analysis for experienced volunteers, similar chest expansion was obtained on 30 occasions with both techniques, E-C better than E-O on 8 and E-O better than E-C on 12 occasions. Novices had comparable results on 17 occasions, E-C better than E-O on 8 and E-O better than E-C on 25 occasions (P = 0.016). The conventionally taught E-C technique of single person BMV provides acceptable chest expansion on most occasions with experienced operators than novices. Novices should use E-O technique as the first choice for single person BMV. Both techniques may be used interchangeably when one fails.

Anesthetic management of congenital epulis in neonate.

The most common cause of difficult intubation in pediatrics is due to congenital anomalies of airway. We report a case of neonate with congenital epulis (CE) who presented with a difficult airway. A 7-day old neonate weighted 3.2 kg with a large mass occupying the oral cavity that was diagnosed as congenital epulis was scheduled for excision biopsy. The mass was large, mobile, and moving in and out with no clear pedicle. An intravenous line was inserted and secured. The airway was then assessed while the patient was awake and an assistant displaced the mass and a laryngoscope was placed to visualize the larynx easily. After preoxygenation, inhalation induction of anesthesia was accomplished using sevoflurane in oxygen. Endotracheal intubation was performed with conventional laryngoscopy. The baby made uneventful recovery after the surgical procedure. In conclusion, epulis presents a real challenge to anesthesiologists. It can be excised either under local or general anesthesia, depending on the size of its pedicle. If done under general anesthesia, assessment of the airway is mandatory for better airway management and safe endotracheal intubation.