Accelerated discovery and miniaturization of novel single-stranded cytidine deaminases.

Cytidine base editors (CBEs) hold significant potential in genetic disease treatment and in breeding superior traits into animals. However, their large protein sizes limit their delivery by adeno-associated virus (AAV), given its packing capacity of <4.7 kb. To overcome this, we employed a web-based fast generic discovery (WFG) strategy, identifying several small ssDNA deaminases (Sdds) and constructing multiple Sdd-CBE 1.0 versions. SflSdd-CBE 1.0 demonstrated high C-to-T editing efficiency, comparable to AncBE4max, while SviSdd-CBE 1.0 exhibited moderate C-to-T editing efficiency with a narrow editing window (C3 to C5). Utilizing AlphaFold2, we devised a one-step miniaturization strategy, reducing the size of Sdds while preserving their efficiency. Notably, we administered AAV8 expressing PCSK9 targeted sgRNA and SflSdd-CBEs (nSaCas9) 2.0 into mice, leading to gene-editing events (with editing efficiency up to 15%) and reduced serum cholesterol levels, underscoring the potential of Sdds in gene therapy. These findings offer new single-stranded editing tools for the treatment of rare genetic diseases.

Genetically modified pigs with CD163 point mutation are resistant to HP-PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is a globally prevalent contagious disease caused by the positive-strand RNA PRRS virus (PRRSV), resulting in substantial economic losses in the swine industry. Modifying the CD163 SRCR5 domain, either through deletion or substitution, can eff1ectively confer resistance to PRRSV infection in pigs. However, large fragment modifications in pigs inevitably raise concerns about potential adverse effects on growth performance. Reducing the impact of genetic modifications on normal physiological functions is a promising direction for developing PRRSV-resistant pigs. In the current study, we identified a specific functional amino acid in CD163 that influences PRRSV proliferation. Viral infection experiments conducted on Marc145 and PK-15 CD163 cells illustrated that the mE535G or corresponding pE529G mutations markedly inhibited highly pathogenic PRRSV (HP-PRRSV) proliferation by preventing viral binding and entry. Furthermore, individual viral challenge tests revealed that pigs with the E529G mutation had viral loads two orders of magnitude lower than wild-type (WT) pigs, confirming effective resistance to HP-PRRSV. Examination of the physiological indicators and scavenger function of CD163 verified no significant differences between the WT and E529G pigs. These findings suggest that E529G pigs can be used for breeding PRRSV-resistant pigs, providing novel insights into controlling future PRRSV outbreaks.

Effects of anthropogenic subsidy and glyphosate on macroinvertebrates in streams.

Streams and surrounding terrestrial ecosystems are closely linked by numerous resource subsidies including anthropogenic subsidies which are increasingly entering streams due to intensive human activities. Also, streams are threatened by stressors such as glyphosate-the most widely used herbicide worldwide. However, the ecological consequences of anthropogenic subsidies and glyphosate on freshwaters are not fully understood. Here, we deployed leaf litter (Cinnamomum camphora) bags containing neither, either, or both treatments of anthropogenic carrion subsidy (chicken meat) and glyphosate (coated in agar) in four streams, which had different land use (i.e., forest, village, and suburban) in Huangshan, Anhui Province, China. We aimed to investigate the individual and combined effects of anthropogenic carrion subsidy and glyphosate on macroinvertebrates in streams and whether these effects differ with land use change. Macroinvertebrate communities significantly differed among streams: biodiversity index and total taxon richness were highest in village streams and lowest in suburban stream. Overall effects of carrion subsidy and glyphosate on macroinvertebrates were not significant. However, several taxa were affected in one or more streams by the individual or combined effects of carrion subsidy and glyphosate, indicating the importance of local community structure and physical habitats in driving the response of macroinvertebrates to carrion subsidy and glyphosate. Collectively, these results imply that the effects of carrion subsidy and glyphosate on macroinvertebrates are site-specific, and future studies should cover more streams and last longer time to better understand the ecological mechanisms driving such pattern.

Impacts of pollution, sex, and tide on the time allocations to behaviours of Uca arcuata in mangroves.

Fiddler crabs (Uca) are ecosystem engineers in coastal ecosystems. Many anthropogenic and natural factors can affect the time allocated to various behaviours in Uca. However, the behaviour of U. arcuata, a widely distributed fiddler crab in Asia, has not been studied in mainland China. Here, we used binoculars to record the time budget of ten behaviours of U. arcuata to investigate the potential effects of sex, tides, and pollution on these behaviours. We found that the crabs spent 42.3%, 27.0%, and 10.6% of their time on feeding, feeding while walking, and stationary respectively. The crabs spent <1.5% of their time on copulation and grooming. The total foraging time (feeding + feeding while walking) did not differ among the three polluted sites. However, crabs spent more time on feeding but less time on feeding while walking. The feeding rate and probability of burrowing and grooming decreased while the possibility of locomotion and stay in burrow increased with increasing nutrient concentration. Females spent 13.9% more time on feeding and fed 54.9% faster than males. Males had a higher tendency to grooming and combat while they were less likely to walk than females. Regarding to the influence of tide, fiddler crabs fed 11.2% faster at ebb tides than at flood tides, and they were more likely to walk and stay in burrows at flood tides than at ebb tides. Our results indicated that nutrient pollution had stronger impacts on the behaviours of crabs than sex and tide. In polluted mangroves, increasing nutrient concentration reduced the quantity of sediment processed by fiddler crabs due to their smaller feeding area, slower feeding rate, and reduced frequency of burrowing activities. These results imply that mitigating nutrient pollution in mangroves may benefit the restoration and management of coastal ecosystems through the enhanced engineering functions of fiddler crabs.

Combined effects of water temperature, grazing snails and terrestrial herbivores on leaf decomposition in urban streams.

The decomposition of organic matter in freshwaters, such as leaf litter, can affect global nutrient (e.g., carbon) cycling. This process can be influenced by fast urbanization through increased water temperature, reduced aquatic diversity and changed leaf litter quality traits. In this study, we performed a mesocosm experiment to explore the individual and combined effects of warming (8°C higher and ambient), the presence versus absence of grazing snails (Parafossarulus striatulus), and intraspecific difference of leaf litter quality (intact versus > 40% area of Liriodendron chinense leaves grazed by terrestrial insects) on litter decomposition in urban streams. Litter decomposition rates ranged from 0.019 d-1 to 0.058 d-1 with an average decomposition rate of 0.032 ± 0.002 d-1. All the three factors had significant effects on litter decomposition rate. Warming and the presence of snails accelerated litter decomposition rates by 60% and 35% respectively. Litter decomposition rates of leaves damaged by terrestrial insects were 5% slower than that of intact leaves, because litter quality of terrestrial insect-damaged leaves was lower (i.e., higher specific leaf weight) than intact leaves. For treatments with snails, warming stimulated microbial and snail mediated litter decomposition rates by 35% and 167%, respectively. All combinations of treatments showed additive effects on litter decomposition except for the interaction between warming and snails which showed positive synergistic effects. In addition, neither temperature nor litter quality affected snail growth rate. These results imply that higher water temperature and the presence of abundant snails in urban streams greatly enhanced litter decomposition. Moreover, the effect of pest outbreaks, which resulted in lower litter quality, can cascade to aquatic ecosystems by retarding microbe-mediated litter decomposition. When these factors co-occurred, warming could synergistically interact with snails to speed up the depletion of organic matter, while the effect of leaf quality on litter decomposition may be diminished at high water temperature. These effects could further influence stream food webs and nutrient cycling.

A wearable iontophoresis enables dual-responsive transdermal delivery for atopic dermatitis treatment.

Atopic dermatitis is a chronic, inflammation skin disease that remains a major public health challenge. The current drug-loading hydrogel dressings offer numerous benefits with enhanced loading capacity and a moist-rich environment. However, their development is still limited by the accessibility of a suitable driven source outside the clinical environment for precise control over transdermal delivery kinetics. Here, we prepare a sulfonated poly(3,4-ethylenedioxythiophene) (PEDOT) polyelectrolyte hydrogel drug reservoir that responds to different stimuli-both endogenous cue (body temperature) and exogenous cue (electrical stimulation), for wearable on-demand transdermal delivery with enhanced efficacy. Functioned as both the drug reservoir and cathode in a Zn battery-powered iontophoresis patch, this dual-responsive hydrogel achieves high drug release efficiency (68.4 %) at 37 °C. Evaluation in hairless mouse skin demonstrates the efficacy of this technology by facilitating transdermal transport of 12.2 µg cm-2 dexamethasone phosphate when discharged with a 103 Ω external resistor for 3 h. The Zn battery-driven iontophoresis results in an effective treatment of atopic dermatitis, displaying reductions in epidermal thickness, mast cell infiltration inhibition, and a decrease in IgE levels. This work provides a new treatment modality for chronic epidermal diseases that require precise drug delivery in a non-invasive way.