Computational Enzyme Design at Zymvol

Directed evolution is the most recognized methodology for enzyme engineering. The main drawback resides in its random nature and in the limited sequence exploration; both require screening of thousands (if not millions) of variants to achieve a target function. Computer-driven approaches can limit laboratorial screening to a few hundred candidates, enabling and accelerating the development of industrial enzymes. In this book chapter, the technology adopted at Zymvol is described. An overview of the current development and future directions in the company is also provided.

KMT5C encodes robust heterochromatin retention and liquid-like behavior using limited sequence features

Cells use multiple strategies to compartmentalize functions through a combination of membrane-bound and membraneless organelles. The latter represent complex assemblies of biomolecules that coalesce into a dense phase through low affinity, multivalent interactions and undergo rapid exchange with the surrounding dilute phase. We describe a liquid-like state for the lysine methyltransferase KMT5C characterized by diffusion within heterochromatin condensates but lacking appreciable nucleoplasmic exchange. Retention was strongly correlated with reduction of condensate surface area, suggesting formation of a liquid droplet with high connectivity. This behavior mapped to a discrete domain whose activity was dependent on multiple short linear motifs. Moreover, it was strikingly resilient to marked phylogenetic differences or targeted changes in intrinsic disorder, charge, sequence, and architecture. Collectively, these findings show that a limited number of sequence features can dominantly encode multivalency, localization, and dynamic behavior within heterochromatin condensates to confer protein retention without progression to a gel or solid.

1301 Early MRI in the Diagnosis of Suspected Scaphoid Fractures – An Audit of a Redesigned Diagnostic Pathway

Introduction MRI is the gold standard for investigation of suspected scaphoid fractures, which can be missed on initial x-rays. This full cycle audit reports the impact of our new patient pathway, which changes repeat x-rays at 2 weeks to urgent limited sequence scaphoid MRI for those with normal initial x-rays, but clinical suspicion of fracture at initial clinic visit. Method A second cycle audited MRI requests for suspected scaphoid fractures at Southmead hospital following implementation of the new pathway in October 2020. We collected wait times from request to scan, and radiologist reports. Results were compared to our first cycle, 6-month time period. Results he results for 24 limited sequence MRIs via our new pathway were compared to 134 full wrist MRIs of the first cycle. Two scans (9.1%) were positive for scaphoid fracture versus 12% in the first cycle. 19 scans (86%) identified alternate pathology including sprain (6), bone bruise (5), non-scaphoid fractures (4) and degenerative change (3). 1 scan (4.5%) was reported as normal. 73% of MRIs were performed within 14 days, compared to 63% in previous cohort. Conclusions Our new pathway using limited sequence MRI identified similar rates of scaphoid fractures. Reduced time to MRI was observed compared to the previous cohort due to shorter scan durations and resulted in earlier diagnosis and fewer outpatient follow-up appointments. Our new pathway has benefits to patient experience and also reduces footfall in hospital, during a time of global Covid-19 pandemic, with no increase in costs.

Melbournevirus-encoded histone doublets are recruited to virus particles and form destabilized nucleosome-like structures

SummaryThe organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of ‘minimalist’ histones in archaea, and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones localize to the cytoplasmic viral factories after virus infection, and ultimately to mature virions. CryoEM structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes, despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of ‘organisms’ that have nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.One Sentence SummarySome large DNA viruses encode fused histone doublets that are targeted to viral factories and assemble into open nucleosome-like structures.

TO STUDY THE ROLE OF LIMITED SEQUENCE MAGNETIC RESONANCE IMAGING IN ASSESSMENT OF CHILDREN WITH HYDROCEPHALUS.

Aim: To evaluate the role of limited sequence MRI (LS MRI) in diagnosing obstructive from nonobstructive hydrocephalus for treatment planning correlating with surgical findings and in follow up cases of shunt treated hydrocephalus to predict the candidate requires revision surgery correlating with final treatment. Materials and Methods: A total of 235 cases were included in the study underwent limited sequence MRI, 121 cases were evaluated for diagnosing obstructive from nonobstructive hydrocephalus out of which 106 cases underwent surgery were correlated with surgical findings and 114 were symptomatic follow up cases evaluated for need of revision surgery. Diagnostic measures such as sensitivity, specificity, PPV, NPV and accuracy were calculated. A p value of <0.05 was considered to be statistically significant. Results: Obstruction was seen in 81 out of the 106 cases who underwent surgery. MRI showed obstruction in 72(88.9%) and no obstruction in 9(11.1%) cases. Out of the 25 cases with no obstruction in surgery, MRI correctly excluded obstruction in 20(80%) cases. MRI misdiagnosed obstruction in 5(20%) cases. No statistically significant difference between the limited sequence MRI and surgery (p value of 0.424 Sensitivity 88.89%, Specificity 80% PPV 93.51%, NPV 68.97% and Accuracy 86.79%). Out of the total 114 follow up cases of hydrocephalus,47 underwent surgery and 67 cases were managed conservatively. MRI criteria predicted surgical candidate in 43(91.5%) and no surgery in 4(8.5%) patients. MRI criteria predicted nonsurgical management in 64 (95.5%) out of the 67 cases and the rest of 3 (4.5%) cases MRI over rated need for surgery. (Sensitivity:91.49% Specificity:95.52% PPV: 93.5% NPV 94.1% Accuracy :93.9%.) Conclusion: LS MRI has good accuracy in detecting an obstruction in paediatric hydrocephalus. In predicting revision surgery for follow-up cases of shunt-treated hydrocephalus, LS MRI has good accuracy.

Sequence of Trypanosoma cruzi reference strain SC43 nuclear genome and kinetoplast maxicircle confirms a strong genetic structure among closely related parasite discrete typing units

Chagas disease is a zoonotic, parasitic, vector-borne neglected tropical disease that affects the lives of over 6 million people throughout the Americas. Trypanosoma cruzi, the causative agent, presents extensive genetic diversity. Here we report the genome sequence of reference strain SC43cl1, a hybrid strain belonging to the TcV discrete typing unit (DTU). The assembled diploid genome was 79 Mbp in size, divided into 1236 contigs with an average coverage reaching 180×. There was extensive synteny of SC43cl1 genome with closely related TcV and TcVI genomes, with limited sequence rearrangements. TcVI genomes included several expansions not present in TcV strains. Comparative analysis of both nuclear and kinetoplast sequences clearly separated TcV from TcVI strains, which strongly supports the current DTU classification.

First phylogenetic analysis of Malian SARS-CoV-2 sequences provide molecular insights into the genomic diversity of the Sahel region

We are currently facing a pandemic of COVID-19, caused by a spillover from an animal-originating coronavirus to humans occuring in the Wuhan region, China, in December 2019. From China the virus has spread to 188 countries and regions worldwide, reaching the Sahel region on the 2nd of March 2020. Since whole genome sequencing (WGS) data is very crucial to understand the spreading dynamics of the ongoing pandemic, but only limited sequence data is available from the Sahel region to date, we have focused our efforts on generating the first Malian sequencing data available. Screening of 217 Malian patient samples for the presence of SARS-CoV-2 resulted in 38 positive isolates from which 21 whole genome sequences were generated. Our analysis shows that both, the early A (19B) and the fast evolving B (20A/C) clade, are present in Mali indicating multiple and independent introductions of the SARS-CoV-2 to the Sahel region.

Bile Facilitates Human Norovirus Interactions with Diverse Histoblood Group Antigens, Compensating for Capsid Microvariation Observed in 2016–2017 GII.2 Strains

Human norovirus (HuNoV) is the leading cause of global infectious acute gastroenteritis, causing ~20% of reported diarrheal episodes. Typically, GII.4 strains cause 50–70% of yearly outbreaks, and pandemic waves of disease approximately every 2–7 years due to rapid evolution. Importantly, GII.4 dominance is occasionally challenged by the sudden emergence of other GII strains, most recently by GII.2 strains which peaked in 2016–2017, dramatically increasing from 1% to 20% of total HuNoV outbreaks. To determine if viral capsid evolution may account for the sudden rise in GII.2 outbreaks, Virus Like Particles (VLPs) of two 2016–2017 GII.2 strains were compared by antigenic and histo blood group antigen (HBGA) binding profiles to the prototypic 1976 GII.2 Snow Mountain Virus (SMV) strain. Despite >50 years of GII.2 strain persistence in human populations, limited sequence diversity and antigenic differences were identified between strains. However, capsid microvariation did affect HBGA binding patterns, with contemporary strains demonstrating decreased avidity for type A saliva. Furthermore, bile salts increased GII.2 VLP avidity for HBGAs, but did not alter antigenicity. These data indicate that large changes in antigenicity or receptor binding are unlikely to explain GII.2 emergence, in contrast to the pandemic GII.4 strains, and indicate that host factors such as waning or remodeling of serum or mucosal immunity likely contributed to the surge in GII.2 prevalence.

Short, Rich, and Powerful: a New Family of Arginine-Rich Small Proteins Have Outsized Impact in Agrobacterium tumefaciens

ABSTRACT Due to minute size and limited sequence complexity, small proteins can be challenging to identify but are emerging as important regulators of diverse processes in bacteria. In this issue of the Journal of Bacteriology, Kraus and coworkers (A. Kraus, M. Weskamp, J. Zierles, M. Balzer, et al., J Bacteriol 202:e00309-20, 2020, https://doi.org/10.1128/JB.00309-20) report a comprehensive analysis of a fascinating subfamily of arginine-rich small proteins in Agrobacterium tumefaciens, conserved among Alphaproteobacteria. Their findings reveal that these small proteins are under complex regulation and have a disproportionately large impact on metabolism and behavior.