Time-Controlled Authentication Strategies for Molecular Information Transfer.

Modern cryptography based on computational complexity theory is mainly constructed with silicon-based circuits. As DNA nanotechnology penetrates the molecular domain, utilizing molecular cryptography for data access protection in the biomolecular domain becomes a unique approach to information security. However, building security devices and strategies with robust security and compatibility is still challenging. Here, this study reports a time-controlled molecular authentication strategy using DNAzyme and DNA strand displacement as the basic framework. A time limit exists for authorization and access, and this spontaneous shutdown design further protects secure access. Multiple hierarchical authentications, temporal Boolean logic authentication, and enzyme authentication strategies are constructed based on DNA networks'good compatibility and programmability. This study gives proof of concept for the detection and protection of bioinformation about single nucleotide variants and miRNA, highlighting their potential in biosensing and security protection.

An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

Exosomes from hypoxic pretreated ADSCs attenuate ultraviolet light-induced skin injury via GLRX5 delivery and ferroptosis inhibition.

Accumulation studies have found that adipose-derived stem cell (ADSC) exosomes have anti-oxidant and anti-inflammatory characteristics. The current study verified their therapeutic potential to elucidate mechanisms of ADSC exosome actions in ultraviolet B (UVB) light-induced skin injury. Exosomes were isolated from ADSCs and hypoxic pretreated ADSCs. Next-generation sequencing (NGS) was applied to characterize differential mRNA expression. A UV-induced mice skin injury model was generated to investigate therapeutic effects regarding the exosomes via immunofluorescence and ELISA analysis. Regulatory mechanisms were illustrated using luciferase report analysis and in vitro experiments. The results demonstrated that exosomes from hypoxic pretreated ADSCs (HExos) inhibited UVB light-induced vascular injury by reversing reactive oxygen species, inflammatory factor expression and excessive collagen degradation. NGS showed that HExos inhibits UV-induced skin damage via GLRX5 delivery, while GLRX5 downregulation inhibited the therapeutic effect of HExos on UV-induced skin damage. GLRX5 upregulation increased the protective Exo effect on UV-induced skin and EPC damage by inhibiting ferroptosis, inflammatory cytokine expression and excessive collagen degradation. Therefore, the data indicate that HExos attenuate UV light-induced skin injury via GLRX5 delivery and ferroptosis inhibition.

Diagnostic and grading criteria for androgenetic alopecia using dermoscopy.

OBJECTIVES: To establish accurate and objective dermoscopic diagnostic criteria and grading standards for males and females with androgenetic alopecia (AGA). METHODS: Twenty patients each with AGA, diffuse alopecia areata, telogen effluvium, and healthy controls were enrolled in the current study. In addition, 60 patients with grades F1/V1, F2/V2, and F3/V3 AGA (20 cases each) were enrolled. The patients underwent dermoscopic examinations. The sensitivity and specificity of the diagnostic criteria were based on the 60 AGA and 60 non-AGA. In addition, 150 patients diagnosed with AGA clinically and by dermoscopy were enrolled to calculate the accuracy of the grading criteria. RESULTS: The diagnostic criteria included primary, secondary, and exclusion criteria. The grading criteria included three indices, which divided the severity of AGA into grades 1, 2, and 3. The sensitivity and specificity of the diagnostic criteria were 98.3% and 96.7% respectively. The accuracy of grade 1, 2, and 3 dermoscopic grading criteria were 96%, 92%, and 100% respectively, with a total accuracy of 96%. LIMITATIONS: To test the diagnostic and grading criteria, more patients need to be collected. CONCLUSIONS: The dermoscopic diagnostic and grading criteria are objective with good accuracy, which could provide a reasonable basis for the early diagnosis, grading treatment, and improved prognosis for AGA.

Combined microfocused ultrasound and delicate pulsed light for facial rejuvenation: A prospective, randomized, and split-face study.

OBJECTIVES: Public's interest in noninvasive skin rejuvenation treatments continues to grow. The advantage of combination therapy lies in that it can target different aspects of skin rejuvenation. This study aimed to assess the efficacy and safety of microfocused ultrasound (MFU) combined with delicate pulsed light (DPL) for facial rejuvenation. METHODS: Twenty-one patients with facial relaxation were enrolled. All patients received whole-face MFU treatment, and one side of the face was randomly assigned to receive DPL. MFU treatment was performed at Months 0 and 3, while DPL treatment was performed at Months 1, 2, 4, and 5. The length and angle of the nasolabial fold and perioral wrinkles, melanin index (MI), erythema index (EI), transepidermal water loss (TEWL), and follow-up time were recorded at Months 0, 3, and 6. Side effects were recorded during treatment and each follow-up visit. RESULTS: Twenty patients successfully completed the study. At the sixth month, the average length of perioral wrinkles and nasolabial folds on the combined side decreased by 11.5% (pwithin < 0.001) and 6.5% (pwithin = 0.011), while 8.3% (pwithin = 0.012) and 3.8% (pwithin = 0.02) on the MFU side. Compared with MFU treatment alone, the combined treatment also showed significant improvements in nasolabial fold angle (from 28.8 ± 3.4° to 32.7 ± 5.0°) and perioral wrinkle angle (from 39.3 ± 5.0° to 43.7 ± 5.1°). In addition, the combined side had greater benefits than the MFU side in improving MI, EI, TEWL, and skin elasticity (pbetween < 0.05). Except for one patient who withdrew due to increased skin sensitivity after MFU treatment, other subjects did not experience permanent or serious side effects. CONCLUSIONS: The combination of MFU and DPL for facial rejuvenation treatment is safe and effective. The combined treatment has better efficacy in skin firmness, and improving skin tone.

Treatment effect of ultra-pulse dynamic CO2 laser and comedone extractor in dense comedones: a prospective, randomized, split-face, evaluator-blind, controlled clinical trial.

Clearance of comedone is challenging in the treatment of acne, as it is very likely to develop into inflammatory lesions. However, there is lack of effective treatments for dense comedones. Comedone extractor has been widely employed by dermatologists, but the effect is temporary and may cause irritation. CO2 laser is a potential method for dense comedones, but the efficacy and safety need to be explored. In this single-center, randomized, single-blind, self-controlled study, the faces of patients with dense comedones were randomly assigned into two sides receiving either ultra-pulse dynamic CO2 laser or comedone extraction at an interval of 2 weeks for 4 sessions. After 4 treatments, the average comedone reduction rate of the CO2 laser was 64.49%, which was higher than that by the extractor (46.36%) (P < .001). 79.16% of the patients reached over 50% reduction by CO2 laser, while only 37.5% on extractor treated side reached 50% clearance. Texture index, porphyrin index, red zone, erythema index, and transepidermal water loss decreased after both treatments, and CO2 laser showed more improvement. There was no difference in hydration index and melanin index between the two treatments. No permanent or severe side effects were observed on both sides. The CO2 laser showed higher comedone clearance with lower pain scores than the comedone extractor.

Combining superpulse dynamic CO2 laser and supramolecular salicylic acid in the treatment of dense comedones with higher clearance in a shorter time: A prospective, randomized, split-face clinical trial.

OBJECTIVES: Dense comedones are common in patients with acne vulgaris, and promoting treatment can prevent the progression of acne lesions. However, the efficacy-time conflict makes the treatment challenging and the medication options are limited by the side effects. MATERIALS AND METHODS: Thirty-five patients with symmetrical dense comedones were enrolled and the two sides of the face were randomly assigned to receive 30% supramolecular salicylic acid (SSA) combined with CO2 laser or CO2 laser monotherapy at an interval of 2 weeks for six treatment sessions. Comedones count, porphyrin index (PI), texture index (TI), melanin index, erythema index, hydration index (HI), transepidermal water loss (TEWL), and side effects were recorded at each visit till the 12th week. RESULTS: Thirty-one patients completed the study. Comedones on the combined-SSA side were reduced more after six treatments, that the mean reduction rate of the combined-SSA side was 85.76%, and that of the CO2 laser-treated side was 62.32% (Pbetween < 0.001). Combining SSA also showed a better effect on reducing PI and TI than CO2 laser singly (Pbetween < 0.001). TEWL and HI between the two sides showed no significant differences after treatments. No permanent or severe side effects were observed on both side. CONCLUSIONS: The treatment combined CO2 laser with 30% SSA dealt with the efficacy-time conflict while significantly reducing comedones and improving skin texture in 12 weeks and no serious adverse reactions occurred. LIMITATIONS: It is a single-center study and the number of subjects was small.

1927nm fractional thulium fiber laser combined with 30% salicylic acid for the treatment of acne and acne scars: A prospective, randomized, and split-face study.

OBJECTIVES: Patients with acne usually develops acne scars subsequently, early intervention of scars is crucial in acne management. 1927nm fractional thulium fiber laser (TFL) is effective in scars improvement and chemical peels with 30% supramolecular salicylic acid (SSA) can be applied for the treatment of acne. The purpose of this study is to evaluate and compare the efficacy and safety of TFL monotherapy versus the concomitant application of TFL and 30% SSA on acne and acne scars. MATERIALS AND METHODS: Thirty-three patients with acne and acne scars were enrolled, and two sides of the face were randomly divided to receive either TFL and SSA chemical peeling or TFL. Four sessions of TFL treatments were applied with 4-week intervals for both sides, SSA combined treatment side received eight SSA chemical peels with 2-week intervals additionally. GAGS, ECCA score, the number of acne lesions, melanin index (MI) and erythema index (EI), transepidermal water loss (TEWL), and side effects were recorded at Weeks 0, 4, 8, 12, and 18. Satisfaction of patients was recorded on both sides at the end of the study. RESULTS: Thirty patients completed the study. Both control group (TFL monotherapy) and SSA group (TFL combined with SSA chemical peeling) significantly improved GAGS and ECCA score. SSA group showed higher efficacy in terms of GAGS and ECCA score, acne lesion count, TEWL, MI, EI, and satisfaction than control group. All the side effects were temporary and tolerable, no adverse effects were observed. CONCLUSIONS: Both TFL and the TFL combined with 30% SSA chemical peeling are safe and effective for the treatment and prevention of acne and acne scars, though the combined group has higher efficacy.

Prevalence and characteristics of accessory mandibular canals in an eastern Chinese population by cone beam computed tomography.

PURPOSE: This retrospective study aimed to determine the prevalence and morphological characteristics of accessory mandibular canals (AMCs) in an eastern Chinese population to provide clinical guidance for reducing intraoperative and postoperative complications. METHODS: Cone beam computed tomography (CBCT) scans of 300 Chinese patients were used to identify AMCs according to a modification of Naitoh's classification. The length of the branch (L0) and the upper and lower angles between the branch and mandibular canal were measured on sagittal images. Additionally, the branches were divided into narrow or wide types by calculating the ratio of the branch diameter to the main canal diameter. The location of the bifurcation point was characterized by measuring its distance to the buccal wall of the mandible (L1), lingual wall of the mandible (L2) and alveolar ridge (L3). RESULTS: The prevalence rate of AMCs was 40.7% (95% CI: 35.1-46.3), and the most common type was the retromolar canal, followed by the forward canal, dental canal, trifid mandibular canals (TMCs) or others, inferior canal and buccolingual canal. Twenty-one cases of multiple branches with unusual patterns were observed in the study. The average values of L0, L1, L2 and L3 were 15.05 ± 0.63 mm, 5.79 ± 0.14 mm, 4.40 ± 0.18 mm and 14.61 ± 0.31 mm, respectively. The mean upper angle and lower angle were 141.59° ± 2.44° and 50.64° ± 2.57°, respectively. Approximately 20.8% of the branches were defined as wide type, and no statistical significance was found between different types. CONCLUSION: AMCs are not rare anatomic variations of the mandibular canal in the eastern Chinese population; thus, CBCT examination is highly recommended for precise evaluation before surgeries involving the mandibles.

Reactivity Differences Enable ROS for Selective Ablation of Bacteria.

An effective strategy to engineer selective photodynamic agents to surmount bacterial-infected diseases, especially Gram-positive bacteria remains a great challenge. Herein, we developed two examples of compounds for a proof-of-concept study where reactive differences in reactive oxygen species (ROS) can induce selective ablation of Gram-positive bacteria. Sulfur-replaced phenoxazinium (NBS-N) mainly generates a superoxide anion radical capable of selectively killing Gram-positive bacteria, while selenium-substituted phenoxazinium (NBSe-N) has a higher generation of singlet oxygen that can kill both Gram-positive and Gram-negative bacteria. This difference was further evidenced by bacterial fluorescence imaging and morphological changes. Moreover, NBS-N can also successfully heal the Gram-positive bacteria-infected wounds in mice. We believe that such reactive differences may pave a general way to design selective photodynamic agents for ablating Gram-positive bacteria-infected diseases.

Heat Shock Repressor HspR Directly Controls Avermectin Production, Morphological Development, and H2O2 Stress Response in Streptomyces avermitilis.

The heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces, which accounts for ∼70% of commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H2O2 stress responses in the industrially important species Streptomyces avermitilis. HspR directly activated ave structural genes (aveA1 and aveA2) and H2O2 stress-related genes (katA1, catR, katA3, oxyR, ahpC, and ahpD), whereas it directly repressed heat shock genes (HSGs) (the dnaK1-grpE1-dnaJ1-hspR operon, clpB1p, clpB2p, and lonAp) and developmental genes (wblB, ssgY, and ftsH). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to corepress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs (htpG, hsp18_1, and hsp18_2) different from those of HspR (clpB1p, clpB2p, and lonAp). A consensus HspR-binding site, 5'-TTGANBBNNHNNNDSTSHN-3', was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in the coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H2O2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors but not as activators. The results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP cross talks with HspR at dnaK1p to coregulate the heat shock response (HSR), but it also has its own specific target heat shock genes (HSGs). The novel role of PhoP in the HSR further demonstrates the importance of this regulator in Streptomyces. Overexpression of hspR strongly enhanced avermectin production in Streptomyces avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP and facilitate methods for antibiotic overproduction in Streptomyces species.

12-Lead ECG arrhythmia classification using cascaded convolutional neural network and expert feature.

Owing to widely available digital ECG data and recent advances in deep learning techniques, automatic ECG arrhythmia classification based on deep neural network has gained growing attention. However, existing neural networks are mainly validated on single­lead ECG, not involving the correlation and difference between multiple leads, while multiple leads ECG provides more complete description of the cardiac activity in different directions. This paper proposes a 12­lead ECG arrhythmia classification method using a cascaded convolutional neural network (CCNN) and expert features. The one-dimensional (1-D) CNN is firstly designed to extract features from each single­lead signal. Subsequently, considering the temporal correlation and spatial variability between multiple leads, features are cascaded as input to two-dimensional (2-D) densely connected ResNet blocks to classify the arrhythmia. Furthermore, features based on expert knowledge are extracted and a random forest is applied to get a classification probability. Results from both CCNN and expert features are combined using the stacking technique as the final classification result. The method has been validated against the first China ECG Intelligence Challenge, obtaining a final score of 86.5% for classifying 12­lead ECG data with multiple labels into 9 categories.

Multifunctional Antimicrobial Biometallohydrogels Based on Amino Acid Coordinated Self-Assembly.

There is a real need for new antibiotics against self-evolving bacteria. One option is to use biofriendly broad-spectrum and mechanically tunable antimicrobial hydrogels that can combat multidrug-resistant microbes. Whilst appealing, there are currently limited options. Herein, broad-spectrum antimicrobial biometallohydrogels based on the self-assembly and local mineralization of Ag+ -coordinated Fmoc-amino acids are reported. Such biometallohydrogels have the advantages of localized delivery and sustained release, reduced drug dosage and toxicity yet improved bioavailability, prolonged drug effect, and tunable mechanical strength. Furthermore, they can directly interact with the cell walls and membrane, resulting in the detachment of the plasma membrane and leakage of the cytoplasm. This leads to cell death, triggering a significant antibacterial effect against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria in cells and mice. This study paves the way for developing a multifunctional integration platform based on simple biomolecules coordinated self-assembly toward a broad range of biomedical applications.

Self-Assembled Bio-Organometallic Nanocatalysts for Highly Enantioselective Direct Aldol Reactions.

Supramolecular nanocatalysts were designed for asymmetric reactions through the self-assembly process of a bio-organometallic molecule, ferrocene-l-prolinamide (Fc-CO-NH-P). Fc-CO-NH-P could self-assemble into versatile nanostructures in water, including nanospheres, nanosheets, nanoflowers, and pieces. In particular, the self-assembled nanoflowers exhibited a superior specific surface area, high stability, and delicate three-dimensional (3D) chiral catalytic active sites. The nanoflowers could serve as heterogeneous catalysts with an excellent catalytic performance toward direct aldol reactions in aqueous solution, achieving both high yield (>99%) and stereoselectivity (anti/syn = 97:3, ee% >99%). This study proposed a significant strategy to fabricate supramolecular chiral catalysts, serving as a favorable template for designing new asymmetric catalysts.

Porphyrin/Ionic-Liquid Co-assembly Polymorphism Controlled by Liquid-Liquid Phase Separation.

Understanding and controlling multicomponent co-assembly is of primary importance in different fields, such as materials fabrication, pharmaceutical polymorphism, and supramolecular polymerization, but these aspects have been a long-standing challenge. Herein, we discover that liquid-liquid phase separation (LLPS) into ion-cluster-rich and ion-cluster-poor liquid phases is the first step prior to co-assembly nucleation based on a model system of water-soluble porphyrin and ionic liquids. The LLPS-formed droplets serve as the nucleation precursors, which determine the resulting structures and properties of co-assemblies. Co-assembly polymorphism and tunable supramolecular phase transition behaviors can be achieved by regulating the intermolecular interactions at the LLPS stage. These findings elucidate the key role of LLPS in multicomponent co-assembly evolution and enable it to be an effective strategy to control co-assembly polymorphism as well as supramolecular phase transitions.

Supramolecular Phthalocyanine Assemblies for Improved Photoacoustic Imaging and Photothermal Therapy.

Phototheranostic nanoplatforms are of particular interest for cancer diagnosis and imaging-guided therapy. Herein, we develop a supramolecular approach to fabricate a nanostructured phototheranostic agent through the direct self-assembly of two water-soluble phthalocyanine derivatives, PcS4 and PcN4. The nature of the molecular recognition between PcS4 and PcN4 facilitates the formation of nanostructure (PcS4-PcN4) and consequently enables the fabrication of PcS4-PcN4 with completely quenched fluorescence and reduced singlet oxygen generation, leading to the high photoacoustic and photothermal activity of PcS4-PcN4. In vivo evaluations suggest that PcS4-PcN4 could not only efficiently visualize a tumor with high contrast through whole-body photoacoustic imaging but also enable excellent photothermal therapy for cancer.

The variant at TGFBRAP1 is significantly associated with type 2 diabetes mellitus and affects diabetes-related miRNA expression.

While the transforming growth factor-ß1 (TGF-ß1) regulates the growth and proliferation of pancreatic ß-cells, its receptors trigger the activation of Smad network and subsequently induce the insulin resistance. A case-control was conducted to evaluate the associations of the polymorphisms of TGF-ß1 receptor-associated protein 1 (TGFBRAP1) and TGF-ß1 receptor 2 (TGFBR2) with type 2 diabetes mellitus (T2DM), and its genetic effects on diabetes-related miRNA expression. miRNA microarray chip was used to screen T2DM-related miRNA and 15 differential expressed miRNAs were further validated in 75 T2DM and 75 normal glucose tolerance (NGT). The variation of rs2241797 (T/C) at TGFBRAP1 showed significant association with T2DM in case-control study, and the OR (95% CI) of dominant model for cumulative effects was 1.204 (1.060-1.370), Bonferroni corrected P < 0.05. Significant differences in the fast glucose and HOMA-ß indices were observed amongst the genotypes of rs2241797. The expression of has-miR-30b-5p and has-miR-93-5p was linearly increased across TT, TC, and CC genotypes of rs2241797 in NGT, Ptrend values were 0.024 and 0.016, respectively. Our findings suggest that genetic polymorphisms of TGFBRAP1 may contribute to the genetic susceptibility of T2DM by mediating diabetes-related miRNA expression.

Inconsistency of Species Tree Methods under Gene Flow.

Coalescent-based methods are now broadly used to infer evolutionary relationships between groups of organisms under the assumption that incomplete lineage sorting (ILS) is the only source of gene tree discordance. Many of these methods are known to consistently estimate the species tree when all their assumptions are met. Nonetheless, little work has been done to test the robustness of such methods to violations of their assumptions. Here, we study the performance of two of the most efficient coalescent-based methods, ASTRAL and NJst, in the presence of gene flow. Gene flow violates the assumption that ILS is the sole source of gene tree conflict. We find anomalous gene trees on three-taxon rooted trees and on four-taxon unrooted trees. These anomalous trees do not exist under ILS only, but appear because of gene flow. Our simulations show that species tree methods (and concatenation) may reconstruct the wrong evolutionary history, even from a very large number of well-reconstructed gene trees. In other words, species tree methods can be inconsistent under gene flow. Our results underline the need for methods like PhyloNet, to account simultaneously for ILS and gene flow in a unified framework. Although much slower, PhyloNet had better accuracy and remained consistent at high levels of gene flow.

Risk assessment of ammonia bunkering operations: Perspectives on different release scales.

Ammonia is an alternative marine fuel to reduce greenhouse gas emissions. Conducting studies on ammonia bunkering risk is essential as ammonia is toxic and corrosive to humans and the environment. This study aims to assess the ammonia bunkering operational risk from the perspectives of small, medium and large release scales. Scaling releases from small to medium results in more changes in cloud footprints at lower gas concentrations. Conversely, transitioning from medium to large releases causes more changes in cloud footprints at higher gas concentrations and lethality footprints with higher values. Moreover, this study performs a sensitivity analysis on ammonia bunkering supply, release, and meteorological factors. Wind speed is the most significant factor in small and medium releases, while hose diameter is the most significant factor in large releases. Under the given inputs, a 50% change in wind speed can have up to 100% change in the 1100 ppm maximum cloud footprint for small releases and a 663% change for medium releases. Similarly, a 50% change in hose diameter can result in a 1689% change in the 1100 ppm maximum cloud footprint for large releases. The research provides valuable insights into analysing ammonia bunkering operational risk considering different risk assessment criteria.

Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 µg/mL - 4.36-fold higher than WT value (48.9 µg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 µg/mL. Overexpression of fatty acid ß-oxidation pathway genes in the strain further increased B1a titer to 452.8 µg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 µg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.