Discovery of Lacto-N-biosidases and a Novel N-Acetyllactosaminidase Activity in the CAZy Family GH20: Functional Diversity and Structural Insights.

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both ß1,4- and ß1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.

Classification of myo-connective tissue injuries for severity grading and return to play prediction: A scoping review.

OBJECTIVES: To conduct a systematic literature search to identify currently used classifications of acute non-contact muscle injuries in sporting adults. DESIGNS: Scoping review. METHODS: A systematic literature search from January 1, 2010 to April 19, 2022 of Medline and SPORTDiscus yielded 13,426 articles that were screened for eligibility. Findings from included studies were qualitatively synthesized. Classifications and their grading, as well as outcomes and definitions were extracted. RESULTS: Twenty-four classifications were identified from the 37 included studies, most of which had low evidence study designs. Majority (57 %) of classifications were published after 2009 and were mostly developed for hamstring or other lower limb injuries. The six most cited classifications accounted for 70 % of the reports (BAMIC, modified Peetrons, Munich, Cohen, Chan and MLG-R). Outcome reporting was sparse, making it difficult to draw conclusions. Still, significant relationships between grading and time to return to play were reported for the BAMIC, modified Peetrons, Munich and Cohen classifications. Other classifications either had a very low number of reported associations, reported no associations, reported inconclusive associations, or did not report an assessment of the association. Other outcomes were poorly investigated. CONCLUSIONS: There is no agreed-upon use of muscle classification, and no consensus on definitions and terminology. As a result, reported outcomes and their relationship to severity grading are inconsistent across studies. There is a need to improve the generalizability and applicability of existing classifications and to refine their prognostic value. High-level evidence studies are needed to resolve these inconsistencies.

Anthropogenic legacy of potassium-40 in French large rivers reconstructed from sediment cores.

Environmental imprint of inorganic fertilizer uses was assessed over the last hundred years at the downstream part of large French rivers (Loire, Moselle, Rhine, Rhone, Meuse and Seine rivers) based on Potassium-40 (40K) activity concentration data sets acquired from soil monitoring (1980-2022) and from sediment coes collected from 2020 to 2022 to reconstruct the temporal trajectories of 40K activity concentrations since the beginning of the last century. Cultivated soils were significantly enriched in 40K compared to non-cultivated ones in the 1980s and 1990s when they turned back to the contents of non-cultivated soils during the following decades. In riverine sediments, all the rivers displayed close 40K temporal trajectories with peaking 40K contents in fine grain size sediments in the 1980s. Maximum 40K enrichment factors from this period were related to the proportion of agricultural areas in the river catchment. In the Loire and Moselle rivers, some high 40K contents were associated with sandy sedimentary strata deposited by flood events before the end of the 1950s due to the presence of potassium enriched minerals. The comparison of 40K activity concentration in sediments with potassic fertilizer delivery in France highlighted very similar temporal trajectories giving evidence that the uses of potassic fertilizers imprint the riverine sediments of most French large rivers. Finally, the environmental resilience face to this anthropic pressure was fast as 40K levels decreased immediately after the decreases of the delivery in most of cases.

Polyphenol Oxidase Activity on Guaiacyl and Syringyl Lignin Units.

The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant-based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose-degrading ascomycetes for their capacity to react with G-type and S-type phenolic compounds. All six PPOs catalyze the ortho-hydroxylation of G-type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy-ortho-diphenols. Remarkably, a subset of these PPOs is also active towards S-compounds (syringol, syringic acid, and sinapic acid) resulting in identical methoxy-ortho-diphenols. Assays with 18O2 demonstrate that these PPOs in particular catalyze ortho-hydroxylation and ortho-demethoxylation of S-compounds and generate methanol as a co-product. Notably, oxidative (ortho-)demethoxylation of S-compounds is a novel reaction for PPOs, which we propose occurs via a distinct reaction mechanism compared to aryl-O-demethylases. We further show that addition of a reducing agent can steer the PPO reaction to form methoxy-ortho-diphenols from both G- and S-type substrates rather than reactive quinones that lead to unfavorable polymerization. Application of PPOs opens for new routes to reduce the heterogeneity and methoxylation degree of mixtures of G and S lignin-derived compounds.

Mini-Review: The Distinct Carbohydrate Active Enzyme Secretome of Rhizopus spp. Represents Fitness for Mycelium Remodeling and Solid-State Plant Food Fermentation.

Rhizopus is a genus of filamentous fungi belonging to the Mucoromycota division. Rhizopus species produce a white, dense mycelium, which is used to create tempeh, a solid-state fermented Asian soybean product, that is gaining renewed attention as a proteinaceous plant food. The profile of carbohydrate-active enzymes (CAZymes) of a fungus or group of fungi, particularly the secretome CAZymes profile, reflects adaptation to different lifestyles and habitats, and has a significant impact on fermentative capacity. This review examines the CAZymes profiles of Rhizopus species focusing on their implication for carbohydrate utilization and solid-state fermentation of plant materials. Through comprehensive genomic assessments and comparisons with other filamentous fungi, we particularly highlight how the unique CAZymes secretome profile is closely correlated with the taxonomy and ecological niches of Rhizopus species. We discuss how the CAZymes secretome capacity of Rhizopus species differs from other fungi and summarize the current state of knowledge regarding the specific CAZymes involved in the modification of carbohydrates in the fungal cell wall and in plant cell walls. We foresee that advanced genomic and proteomic technologies will be used to expand the biotechnology applications of Rhizopus spp.

Identification and characterization of a novel thermostable PL7 alginate lyase from a submarine volcanic metagenomic library.

Seaweed biomass is as an abundant and renewable source of complex polysaccharides, including alginate which has a variety of applications. A sustainable method for exploiting alginate towards the production of valuable oligosaccharides is through enzymatic processing, using alginate lyases. Industrial refinement methods demand robust enzymes. Metagenomic libraries from extreme environments are a new source of unique enzymes with great industrial potential. Herein we report the identification of a new thermostable alginate lyase with only 58 % identity to known sequences, identified by mining a metagenomic library obtained from the hydrothermal vents of the volcano Kolumbo in the Aegean Sea (Kolumbo Alginate Lyase, KAlLy). Sequence analysis and biochemical characterization of KAlLy showed that this new alginate lyase is a Polysaccharide Lyase of family 7 (PL7) enzyme with endo- and exo-action on alginate and poly-mannuronic acid, with high activity at 60°C (56 ± 8 U/mg) and high thermostability (half-life time of 30 h at 50°C). The response surface methodology analysis revealed that the reaction optimum conditions with poly-mannuronic acid as substrate are 44°C, pH of 5.5 with 440 mM NaCl. This novel alginate lyase is a valuable addition to the toolbox of alginate modifying enzymes, due to its diverse sequence and its good thermal stability.

Engineered bacteria that self-assemble "bioglass" polysilicate coatings display enhanced light focusing.

Photonic devices are cutting-edge optical materials that produce narrow, intense beams of light, but their synthesis typically requires toxic, complex methodology. Here we employ a synthetic biology approach to produce environmentally-friendly, living microlenses with tunable structural properties. We engineered Escherichia coli bacteria to display the silica biomineralization enzyme silicatein from aquatic sea sponges. Our silicatein-expressing bacteria can self-assemble a shell of polysilicate "bioglass" around themselves. Remarkably, the polysilicate-encapsulated bacteria can focus light into intense nanojets that are nearly an order of magnitude brighter than unmodified bacteria. Polysilicate-encapsulated bacteria are metabolically active for up to four months, potentially allowing them to sense and respond to stimuli over time. Our data demonstrate that engineered bacterial particles have the potential to revolutionize the development of multiple optical and photonic technologies.

Live to fight another day: The bacterial nucleoid under stress.

The bacterial chromosome is both highly supercoiled and bound by an ensemble of proteins and RNA, causing the DNA to form a compact structure termed the nucleoid. The nucleoid serves to condense, protect, and control access to the bacterial chromosome through a variety of mechanisms that remain incompletely understood. The nucleoid is also a dynamic structure, able to change both in size and composition. The dynamic nature of the bacterial nucleoid is particularly apparent when studying the effects of various stresses on bacteria, which require cells to protect their DNA and alter patterns of transcription. Stresses can lead to large changes in the organization and composition of the nucleoid on timescales as short as a few minutes. Here, we summarize some of the recent advances in our understanding of how stress can alter the organization of bacterial chromosomes.

Primary Gouty Tophi Involving the Eyelid.

A 51-year-old male with no known history of gout was referred by his optometrist for bilateral lower eyelid cysts near the puncta. The lesions were not painful but were cosmetically concerning and excision was desired. Utilizing local anesthesia, the lesions were excised and sent to pathology for review. Pathology noted lesions to be "gouty tophus." Given that our patient had no history of gout, this is the first case report of gouty tophus on the eyelid being the initial manifestation of gout.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases.

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Improving Equity in Deep Learning Medical Applications with the Gerchberg-Saxton Algorithm.

Deep learning (DL) has gained prominence in healthcare for its ability to facilitate early diagnosis, treatment identification with associated prognosis, and varying patient outcome predictions. However, because of highly variable medical practices and unsystematic data collection approaches, DL can unfortunately exacerbate biases and distort estimates. For example, the presence of sampling bias poses a significant challenge to the efficacy and generalizability of any statistical model. Even with DL approaches, selection bias can lead to inconsistent, suboptimal, or inaccurate model results, especially for underrepresented populations. Therefore, without addressing bias, wider implementation of DL approaches can potentially cause unintended harm. In this paper, we studied a novel method for bias reduction that leverages the frequency domain transformation via the Gerchberg-Saxton and corresponding impact on the outcome from a racio-ethnic bias perspective.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Bioinformatics-based identification of GH12 endoxyloglucanases in citrus-pathogenic Penicillium spp.

Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6-5.3 and 35-37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite -4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity.

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5-12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Rapid Expansion of the Healing Emotional Lives of Peers Program During COVID-19: A Second Victim Peer Support Program for Healthcare Professionals.

OBJECTIVES: In 2018, the Healing Emotional Lives of Peers (HELP) Program was implemented at Mayo Clinic Rochester to guide healthcare professionals (HCPs) after a second victim experience, such as adverse patient events or medical errors. The HELP program was expanded to all HCPs in response to the anticipated stressors of the COVID-19 pandemic. This article aims to describe the rapid expansion of the peer support program and evaluate the effectiveness of peer support provided to affected colleagues (ACs). METHODS: Quantitative data collected from workshop evaluations, activations, and associated metrics ( TPS Self-Assessment , Encounter Form , and AC Self-Assessment ) were summarized through standard descriptive statistics using SAS version 9.4 software. Open-ended responses were qualitatively analyzed for iterative themes about the HELP program and associated workshops. RESULTS: Between April 2020 and December 2021, 22 virtual workshops to train peer supporters were conducted with 827 attendees. Of these, 464 employees completed the workshop evaluation. A total of 94.2% rated the workshop as excellent or very good. Participants perceived the workshop to be highly effective and felt more prepared to support ACs. Between May 2020 and December 2021, 247 activations were submitted through the HELP Program's intranet Web site and peer support was requested for 649 employees. Of the 268 TPS Self-Assessments , 226 (84.3%) felt that they provided helpful support to an AC. One hundred ACs evaluated support received, with 93% being "extremely" or "very satisfied." Affected colleagues appreciated having a TPS provide judgment-free support. CONCLUSIONS: The HELP Program promotes a culture of safety by helping HCPs process traumatic events. To effectively meet the needs of patients, healthcare organizations need to prioritize the well-being of their employees through interpersonal support.

Glucuronan lyases from family PL7 use a Tyr/Tyr syn ß-elimination catalytic mechanism for glucuronan breakdown.

TpPL7A and TpPL7B, members of CAZy family PL7, act as ß-glucuronan lyases. TpPL7A diverges by lacking the catalytic histidine, identified as the Brønsted base in PL7 alginate lyases. Our research, including TpPL7A's crystal structure, and mutagenesis studies, reveals a shared syn-ß-elimination mechanism with a single tyrosine serving as both base and acid catalyst. This mechanism may extend to subfamily PL7_4 glucuronan lyases.