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1. Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single-Stranded RNA

   https://doi.org/10.1021/acs.est.1c01255  

   Zhang, Ke; Hodge, Joseph; Chatterjee, Anamika; Moon, Tae Seok; Parker, Kimberly M. (May 2021, Environmental science technology)

   null (Ed.)

   Phosphodiester bonds in the backbones of double-stranded (ds)RNA and single-stranded (ss)RNA are known to undergo alkaline hydrolysis. Consequently, dsRNA agents used in emerging RNA interference (RNAi) products have been assumed to exhibit low chemical persistence in solutions. However, the impact of the duplex structure of dsRNA on alkaline hydrolysis has not yet been evaluated. In this study, we demonstrated that dsRNA undergoes orders-of-magnitude slower alkaline hydrolysis than ssRNA. Furthermore, we observed that dsRNA remains intact for multiple months at neutral pH, challenging the assumption that dsRNA is chemically unstable. In systems enabling both enzymatic degradation and alkaline hydrolysis of dsRNA, we found that increasing pH effectively attenuated enzymatic degradation without inducing alkaline hydrolysis that was observed for ssRNA. Overall, our findings demonstrated, for the first time, that key degradation pathways of dsRNA significantly differ from those of ssRNA. Consideration of the unique properties of dsRNA will enable greater control of dsRNA stability during the application of emerging RNAi technology and more accurate assessment of its fate in environmental and biological systems, as well as provide insights into broader application areas including dsRNA isolation, detection and inactivation of dsRNA viruses, and prebiotic molecular evolution. 

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2. Distinct actors drive different mechanisms of biopolymer processing in polar marine coastal sediments

   https://doi.org/10.1111/1462-2920.16687  

   Knittel, Katrin; Miksch, Sebastian; Moncada, Chyrene; Silva‐Solar, Sebastian; Moye, Jannika; Amann, Rudolf; Arnosti, Carol (August 2024, Environmental Microbiology)

   Abstract Heterotrophic bacteria in the ocean initiate biopolymer degradation using extracellular enzymes that yield low molecular weight hydrolysis products in the environment, or by using a selfish uptake mechanism that retains the hydrolysate for the enzyme‐producing cell. The mechanism used affects the availability of hydrolysis products to other bacteria, and thus also potentially the composition and activity of the community. In marine systems, these two mechanisms of substrate processing have been studied in the water column, but to date, have not been investigated in sediments. In surface sediments from an Arctic fjord of Svalbard, we investigated mechanisms of biopolymer hydrolysis using four polysaccharides and mucin, a glycoprotein. Extracellular hydrolysis of all biopolymers was rapid. Moreover, rapid degradation of mucin suggests that it may be a key substrate for benthic microbes. Although selfish uptake is common in ocean waters, only a small fraction (0.5%–2%) of microbes adhering to sediments used this mechanism. Selfish uptake was carried out primarily byPlanctomycetotaandVerrucomicrobiota. The overall dominance of extracellular hydrolysis in sediments, however, suggests that the bulk of biopolymer processing is carried out by a benthic community relying on the sharing of enzymatic capabilities and scavenging of public goods. 

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3. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass

   https://doi.org/10.1021/acssuschemeng.3c06266  

   DeChellis, Antonio; Nemmaru, Bhargava; Sammond, Deanne; Douglass, Jenna; Patil, Nivedita; Reste, Olivia; Chundawat, Shishir_P S (March 2024, ACS Sustainable Chemistry & Engineering)

   Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates. 

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4. Effect of Mechanical Loading on PLGA Biodegradation

   Samanta, Devleena; Koithan, John A; Muliana, Anastasia H; Pharr, Matt (June 2025, Polymer degradation and stability)

   Poly(lactic-co-glycolic) acid (PLGA) has been widely implemented in tissue engineering and drug delivery systems, stemming from its biocompatibility, controllable biodegradation, non-toxicity, non-immunogenicity, and tunable mechanical properties. PLGA exhibits a broad range of degradation times and modes, which can be finely tuned by adjusting various parameters, namely by altering the ratio of lactide and glycolide units, molecular weight, end group functionality, specimen geometry, processing temperature, and chemistry of the surrounding medium. To tailor the degradation profile, the in vitro profile should closely reflect the in vivo profile; however, the effects of mechanical loading coupled with hydrolysis on PLGA biodegradation are typically overlooked. To this end, this study investigates the combined effects of mechanical loading and hydrolysis at 37ºC on the changes in the chemical and physical properties of PLGA as it degrades with time. We found that after several days of combined loading and hydrolysis at 37ºC PLGA significantly creeps, whereas non-loaded (but hydrolyzed) specimens only slightly elongated after relatively long-term hydrolysis (~60 days). Despite this observation and perhaps counterintuitively, the hydrolyzed non-loaded samples exhibited faster degradation than hydrolyzed loaded samples. Additionally, our studies indicated the presence of bulk erosion in hydrolyzed non-loaded samples and surface erosion in hydrolyzed loaded samples. We also observed (only) physical ageing in control samples (loaded and non-loaded samples that were not immersed in PBS but exposed to 37 °C). Based on these observations, we discuss potential underlying mechanisms for the observed differences in the biodegradation behavior of PLGA specimens with and without mechanical loading. 

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5. Degradable Plastics Are Vulnerable to Cracks

   https://doi.org/10.1016/j.eng.2021.02.009  

   Yang, Xuxu; Steck, Jason; Yang, Jiawei; Wang, Yecheng; Suo, Zhigang (May 2021, Engineering)

   A plastic may degrade in response to a trigger. The kinetics of degradation have long been characterized by the loss of weight and strength over time. These methods of gross characterization, however, are misleading when plastic degrades heterogeneously. Here, we study heterogeneous degradation in an extreme form: the growth of a crack under the combined action of chemistry and mechanics. An applied load opens the crack, exposes the crack front to chemical attack, and causes the crack to outrun gross degradation. We studied the crack growth in polylactic acid (PLA), a polyester in which ester bonds break by hydrolysis. We cut a crack in a PLA film using scissors, tore it using an apparatus, and recorded the crack growth using a camera through a microscope. In our testing range, the crack velocity was insensitive to load but was sensitive to humidity and pH. These findings will aid the development of degradable plastics for healthcare and sustainability. 

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