Direct Synthesis of C4-Acyl Indoles via C-H Acylation.

The direct synthesis of C4-acyl indoles deprived of C2 and C3 substituents has proven to be challenging, with scarce efficient synthetic routes being reported. Herein, we disclose a highly site-selective palladium-catalyzed C-H acylation for the construction of C4-acyl indoles via a Catellani-Lautens cyclization strategy. In addition, we systematically studied the ortho C-H acylation mechanism of iodoaniline through density functional theory (DFT) calculations and combined experimental results to elucidate the principle of high chemoselectivity brought by triazine benzoate as an acylation reagent.

Deep Learning-Driven Library Design for the De Novo Discovery of Bioactive Thiopeptides.

Broad substrate tolerance of ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic enzymes has allowed numerous strategies for RiPP engineering. However, despite relaxed specificities, exact substrate preferences of RiPP enzymes are often difficult to pinpoint. Thus, when designing combinatorial libraries of RiPP precursors, balancing the compound diversity with the substrate fitness can be challenging. Here, we employed a deep learning model to streamline the design of mRNA display libraries. Using an in vitro reconstituted thiopeptide biosynthesis platform, we performed mRNA display-based profiling of substrate fitness for the biosynthetic pathway involving five enzymes to train an accurate deep learning model. We then utilized the model to design optimal mRNA libraries and demonstrated their utility in affinity selections against IRAK4 kinase and the TLR10 cell surface receptor. The selections led to the discovery of potent thiopeptide ligands against both target proteins (KD up to 1.3 nM for the best compound against IRAK4 and 300 nM for TLR10). The IRAK4-targeting compounds also inhibited the kinase at single-digit µM concentrations in vitro, exhibited efficient internalization into HEK293H cells, and suppressed NF-kB-mediated signaling in cells. Altogether, the developed approach streamlines the discovery of pseudonatural RiPPs with de novo designed biological activities and favorable pharmacological properties.

Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis.

BACKGROUND: In the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China. METHODS: From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed. RESULTS: The isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis. CONCLUSIONS: As a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.

Safety and immunogenicity of heterologous boosting with orally aerosolised or intramuscular Ad5-nCoV vaccine and homologous boosting with inactivated vaccines (BBIBP-CorV or CoronaVac) in children and adolescents: a randomised, open-label, parallel-controlled, non-inferiority, single-centre study.

BACKGROUND: Heterologous booster immunisation with orally administered aerosolised Ad5-nCoV vaccine (AAd5) has been shown to be safe and highly immunogenic in adults. Here, we aimed to assess the safety and immunogenicity of heterologous booster immunisation with orally administered AAd5 in children and adolescents aged 6-17 years who had received two doses of inactivated vaccine (BBIBP-CorV or CoronaVac). METHODS: We did a randomised, open-label, parallel-controlled, non-inferiority study to assess the safety and immunogenicity of heterologous booster immunisation with AAd5 (0·1 mL) or intramuscular Ad5-nCoV vaccine (IMAd5; 0·3 mL) and homologous booster immunisation with inactivated vaccine (BBIBP-CorV or CoronaVac; 0·5 mL) in children (aged 6-12 years) and adolescents (aged 13-17 years) who had received two doses of inactivated vaccine at least 3 months earlier in Hunan, China. Children and adolescents who were previously immunised with two-dose BBIBP-CorV or CoronaVac were recruited for eligibility screening at least 3 months after the second dose. A stratified block method was used for randomisation, and participants were stratified by age and randomly assigned (3:1:1) to receive AAd5, IMAd5, or inactivated vaccine. The study staff and participants were not masked to treatment allocation. Laboratory and statistical staff were masked during the study. In this interim analysis, adverse events within 14 days and geometric mean titre (GMT) of serum neutralising antibodies on day 28 after the booster vaccination, based on the per-protocol population, were used as the primary outcomes. The analysis of non-inferiority was based on comparison using a one-sided 97·5% CI with a non-inferiority margin of 0·67. This study was registered at ClinicalTrials.gov, NCT05330871, and is ongoing. FINDINGS: Between April 17 and May 28, 2022, 436 participants were screened and 360 were enrolled: 220 received AAd5, 70 received IMAd5, and 70 received inactivated vaccine. Within 14 days after booster vaccination, vaccine-related adverse reactions were reported: 35 adverse events (in 13 [12%] of 110 children and 22 [20%] of 110 adolescents) in 220 individuals in the AAd5 group, 35 (in 18 [51%] of 35 children and 17 [49%] of 35 adolescents) in 70 individuals in the IMAd5 group, and 13 (in five [14%] of 35 children and eight [23%] of 35 adolescents) in 70 individuals in the inactivated vaccine group. Solicited adverse reactions were also reported: 34 (13 [12%] of 110 children and 21 [10%] of 110 adolescents) in 220 individuals in the AAd5 group, 34 (17 [49%] of 35 children and 17 [49%] of 35 adolescents) in 70 individuals in the IMAd5 group, and 12 (five [14%] of 35 children and seven [20%] of 35 adolescents) in 70 individuals in the inactivated vaccine group. The GMTs of neutralising antibodies against ancestral SARS-CoV-2 Wuhan-Hu-1 (Pango lineage B) in the AAd5 group were significantly higher than the GMTs in the inactivated vaccine group (adjusted GMT ratio 10·2 [95% CI 8·0-13·1]; p<0·0001). INTERPRETATION: Our study shows that a heterologous booster with AAd5 is safe and highly immunogenic against ancestral SARS-CoV-2 Wuhan-Hu-1 in children and adolescents. FUNDING: National Key R&D Program of China.

Allantoin induces pruritus by activating MrgprD in chronic kidney disease.

Chronic kidney disease (CKD) is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with CKD, especially in end-stage renal disease. The underlying molecular and neural mechanism of CKD-associated pruritus (CKD-aP) remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons. The calcium influx and action potential reduced significantly in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of phospholipase C, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

Modifiable Risk Factor Score and Fecundability in a Preconception Cohort in Singapore.

Importance: Although multiple modifiable risk factors have been identified for reduced fecundability (defined as lower probability of conception within a menstrual cycle), no scoring system has been established to systematically evaluate fecundability among females who are attempting to conceive. Objective: To examine the association of a risk score based on 6 modifiable factors with fecundability, and to estimate the percentage reduction in incidence of nonconception if all study participants achieved a minimal risk score level. Design, Setting, and Participants: This population-based cohort study obtained data from the S-PRESTO (Singapore Preconception Study of Long-Term Maternal and Child Outcomes) prospective cohort study. Females of reproductive age who were trying to conceive were enrolled from February 2015 to October 2017 and followed for 1 year, ending in November 2018. Data were analyzed from March to May 2022. Exposures: A reduced fecundability risk score was derived by giving participants 1 point for each of the following factors: unhealthy body mass index, unhealthy diet, smoking, alcohol intake, folic acid supplement nonuser, and older maternal age. Total scores ranged from 0 to 6 and were classified into 5 levels: level 1 (score of 0 or 1), level 2 (score of 2), level 3 (score of 3), level 4 (score of 4), and level 5 (score of 5 or 6). Main Outcomes and Measures: Fecundability, measured by time to conception in cycles, was analyzed using discrete-time proportional hazards models with confounder adjustment. Results: A total of 937 females (mean [SD] age, 30.8 [3.8] years) were included, among whom 401 (42.8%) spontaneously conceived within 1 year of attempting conception; the median (IQR) number of cycles before conception was 4 (2-7). Compared with participants with a level 1 risk score, those with level 2, 3, 4, and 5 risk scores had reductions in fecundability of 31% (adjusted fecundability ratio [FR], 0.69; 95% CI, 0.54-0.88), 41% (FR, 0.59; 95% CI, 0.45-0.78), 54% (FR, 0.46; 95% CI, 0.31-0.69) and 77% (FR, 0.23; 95% CI, 0.07-0.73), respectively. Assessment of the population attributable fraction showed that all participants achieving a minimal (level 1) risk level would be associated with a reduction of 34% (95% CI, 30%-39%) in nonconception within a year. Conclusions and Relevance: Results of this study revealed the co-occurrence of multiple modifiable risk factors for lowered fecundability and a substantially higher conception rate among participants with no or minimal risk factors. The risk assessment scoring system proposed is a simple and potentially useful public health tool for mitigating risks and guiding those who are trying to conceive.

Correlation between plasma glutathione peroxidase 4 and N-acetylneuraminic acid levels with clinical risk stratification and prognosis of patients with acute coronary syndrome.

OBJECTIVES: To investigate the correlation between plasma glutathione peroxidase 4 (GPX4) and N-acetyl-neuraminic acid (Neu5Ac) with clinical risk stratification and outcomes of acute coronary syndrome (ACS) patients. METHODS: Between October 2018 and July 2019, 413 patients that were scheduled for coronary angiography were enrolled in this prospective study at the First Affiliated Hospital of Bengbu Medical College, Bengbu, China. Patients were divided into control and ACS groups. Patients with ACS were divided into 3 risk levels based on their thrombolysis in myocardial infarction risk score. After discharge, ACS patients were followed for the incidence of major adverse cardiac events (MACEs). For the analysis of cumulative endpoint event occurrences, the Kaplan-Meier method was applied. RESULTS: The ACS group had lower plasma GPX4 but higher Neu5Ac levels than the control group. There was a greater increase in plasma Neu5Ac in the high-risk group when compared with the medium-risk and low-risk groups, while GPX4 levels were higher in the low-risk group. The MACEs group had higher plasma Neu5Ac but lower GPX4 levels than the non-MACEs group. The plasma Neu5Ac was an independent risk factor but GPX4 was a protective factor for MACEs. CONCLUSION: Glutathione peroxidase 4 and Neu5Ac levels in plasma can be used to diagnose, stratify risks, and predict long-term outcomes in patients with ACS.

Solid-Phase-Based Synthesis of Lactazole-Like Thiopeptides.

A strategy for the synthesis of de novo discovered lactazole-like thiopeptides is reported. The approach revolves around a convergent and scalable preparation of the central triheterocyclic amino acid and its utilization in Fmoc solid-phase peptide synthesis for modular peptide chain assembly. A technique for preparing C-terminally functionalized thiopeptides for biological studies is also described. The syntheses of 11 TNIK-inhibitor thiopeptides and 6 of their derivatives in multimilligram quantities highlight the practical utility of the developed protocols.

De Novo Discovery of Thiopeptide Pseudo-natural Products Acting as Potent and Selective TNIK Kinase Inhibitors.

Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥1012) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affinity selection against Traf2- and NCK-interacting kinase (TNIK), a protein involved in several cancers, which yielded a plethora of candidate thiopeptides. Of the 11 synthesized compounds, 9 had high affinities for the target kinase (best KD = 1.2 nM) and 10 inhibited its enzymatic activity (best Ki = 3 nM). X-ray structural analysis of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive inhibition exhibited by two of the discovered compounds. The thiopeptides internalized to the cytosol of HEK293H cells as efficiently as the known cell-penetrating peptide Tat (4-6 µM). Accordingly, the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether, our platform enables the exploration of pseudo-natural thiopeptides with favorable pharmacological properties in drug discovery applications.

Association between Vitamin D Deficiency and Levels of Renin and Angiotensin in Essential Hypertension.

Objective: The present study aims to investigate the relationship between vitamin D deficiency and renin-angiotensin-aldosterone levels in patients with essential hypertension. Methods: The present study observed two groups of patients from Urumqi, Xinjiang, China, from April 2017 to March 2018. There were two subject groups: the hypertension group (80 patients with essential hypertension selected by random cluster sampling) and the control group (76 healthy adults). The 25-hydroxyvitamin D (25(OH)D or vitamin D) levels were measured through electrolytes; fasting blood glucose, blood lipids, and other biochemical indicators were detected using immune chemiluminescence; and plasma renin activity and angiotensin II concentrations were detected with radio-immunity. Results: Comparison between the hypertension group and control group showed statistically significant differences in the systolic pressure and levels of 25(OH)D, renin, and triglycerides (P < 0.05). The correlation analysis showed that 25(OH)D was negatively correlated with renin (r = -0.185; P=0.021) and positively correlated with systolic pressure (r = -0.105; P=0.035). There were no statistically significant differences in diastolic pressure, fasting blood glucose, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides between the two groups. Conclusions: The results of the present study show that vitamin D deficiency is common in Urumqi, Xinjiang, China and vitamin D levels are negatively correlated with renin levels. Vitamin D plays an important role in regulating blood pressure by affecting renin levels through the renin-angiotensin-aldosterone system.

Accurate Models of Substrate Preferences of Post-Translational Modification Enzymes from a Combination of mRNA Display and Deep Learning.

Promiscuous post-translational modification (PTM) enzymes often display nonobvious substrate preferences by acting on diverse yet well-defined sets of peptides and/or proteins. Understanding of substrate fitness landscapes for PTM enzymes is important in many areas of contemporary science, including natural product biosynthesis, molecular biology, and biotechnology. Here, we report an integrated platform for accurate profiling of substrate preferences for PTM enzymes. The platform features (i) a combination of mRNA display with next-generation sequencing as an ultrahigh throughput technique for data acquisition and (ii) deep learning for data analysis. The high accuracy (>0.99 in each of two studies) of the resulting deep learning models enables comprehensive analysis of enzymatic substrate preferences. The models can quantify fitness across sequence space, map modification sites, and identify important amino acids in the substrate. To benchmark the platform, we performed profiling of a Ser dehydratase (LazBF) and a Cys/Ser cyclodehydratase (LazDEF), two enzymes from the lactazole biosynthesis pathway. In both studies, our results point to complex enzymatic preferences, which, particularly for LazBF, cannot be reduced to a set of simple rules. The ability of the constructed models to dissect such complexity suggests that the developed platform can facilitate a wider study of PTM enzymes.

Unique bacterial communities associated with components of an artificial aquarium ecosystem and their possible contributions to nutrient cycling in this microecosystem.

In order to better understand the bacterial distribution characteristics in a whole microecosystem, the bacterial communities in different components of an artificial aquarium (i.e., plants, fishes, sand and water) were characterized using high throughput sequencing of bacterial 16S rRNA genes. Across all samples, 2873 operational taxonomic units were identified and assigned to 771 genera in 36 phyla. In a principle coordinate analysis, samples clustered according to their origin, indicating that bacterial communities from the same component were most similar. Further taxonomic analysis revealed that most dominant genera, even those with the similar functions, were biased to one component: Nitrospira and Rhodobacter were mainly abundant in plant samples; Rhodococcus, Serratia, Ralstonia, Sphingobacterium and Pseudomonas were most common in sand samples; Cetobacterium and Aeromonas dominated fish samples; and Flavobacterium, Alpinimonas and Limnobacter were especially common in water samples. Functional predictions performed by PICRUSt and the dominant genera exhibited that bacteria detected in each component could participate in all nutrient cycles in the aquarium. However, those involved in carbon and nitrogen cycling were most common in plant and fish samples, while phosphate metabolism-related pathways were more abundant in sand and water samples. Moreover, the aquarium plants, in association with their bacterial communities might be the most important component in the aquarium, as indicated by their highest bacterial richness and diversity. This study adds to our understanding on the differences in the microbiome of different components and their possible contributions to nutrient cycling in a self-sustaining aquarium.

One-year outcome after transcatheter aortic valve replacement for aortic regurgitation: A single-center study.

BACKGROUND: Presently, there are limited reports in the literature on the postoperative (midterm) clinical outcome for pure aortic regurgitation (AR) following transcatheter aortic valve replacement (TAVR). METHODS: Between March 2014 and June 2019, a total of 134 high-risk patients with pure, symptomatic severe AR patients were enrolled in the current study. The outcome was assessed according to the Valve Academic Research Consortium-2 criteria. Procedural results, clinical outcomes, and the patients' hemodynamics for a period of 1 year were analyzed. RESULTS: The patient mean was 73.1 ± 6.4 years and 25.4% were female. The average Society of Thoracic Surgeons score was 9.8 ± 5.3%. Procedural success was 97.1% (130/134), and the device success rate was 96.3% (129/134). Five cases were converted to open surgery, while two patients underwent valvular reinterventions (surgical aortic valve replacement for thrombosis and increasing paravalvular regurgitation). The mean aortic valve gradient was 10.2 ± 4.1 mmHg, while the moderate and severe AR was 1.6% at 1 year. Paravalvular regurgitation was none/trivial in 79.8% and mild in 18.5%. The 1-year all-cause mortality rate was 7.4%. At 1 year, the stroke incidence rate was 2.2%, and a pacemaker was implanted in 8.9% of the enrolled patients. CONCLUSIONS: In high-risk patients undergoing transapical TAVR for AR, the use of the J-Valve is safe, and effective TAVR should be considered as a reasonable option for high-risk patients with pure AR.

Tracking of dietary patterns between pregnancy and 6 years post-pregnancy in a multiethnic Asian cohort: the Growing Up in Singapore Towards healthy Outcomes (GUSTO) study.

PURPOSE: Few studies have described adherence to dietary patterns over time in women of childbearing age. This study aims to describe, examine the stability and changes in dietary patterns between pregnancy and 6 years post-pregnancy and the sociodemographic and lifestyle factors influencing the adherence over time. METHODS: During pregnancy and at 6 years post-pregnancy, 24-h recalls and food frequency questionnaires were collected, respectively, from 709 women. Data on sociodemographic and lifestyle factors were collected via questionnaires. Dietary patterns were identified using principal component analysis and stability assessed using Pearson's correlation coefficients (r) and Cohen's weighted kappa (κ). Associations with sociodemographic characteristics were assessed by multiple logistic regression. RESULTS: The 'Fruits, Vegetables and Legumes' (FVL) and 'Seafood, Noodle, Soup' (SNS) patterns were identified at both time points, with low correlation for the dietary pattern z scores (r 0.2 and 0.3, respectively) and modest agreement in tertile assignment, suggesting poor stability. An 'unhealthy' pattern was only observed at 6 years post-pregnancy. Women who showed increased adherence to FVL pattern had higher educational attainment and exhibited healthy lifestyle behaviours. Women who had gestational diabetes during pregnancy were less likely to decrease adherence to FVL pattern over time. Women who adhered more closely to the 'unhealthy' pattern at 6 years post-pregnancy tended to be younger, of Malay ethnicity, had lower socioeconomic status, were less physically active and had additional pregnancies. CONCLUSIONS: Dietary habits of women became less healthy during the transition from pregnancy to 6 years post-pregnancy. However, results should be interpreted with caution due to the different dietary assessment tools used at the two time points.

Prepregnancy adherence to plant-based diet indices and exploratory dietary patterns in relation to fecundability.

BACKGROUND: Modest associations have been reported between specific food groups or nutrients and fecundability [measured by time to pregnancy (TTP)]. Examining overall diets provides a more holistic approach towards understanding their associations with fecundability. It is not known whether plant-based diets indices or exploratory dietary patterns are associated with fecundability. OBJECTIVES: We examine the associations between adherence to 1) plant-based diet indices; and 2) exploratory dietary patterns and fecundability among women planning pregnancy. METHODS: Data were analyzed from the Singapore Preconception Study of Long-Term Maternal and Child Outcomes (S-PRESTO) study. Prepregnancy diet was assessed using a semi-quantitative FFQ from which the overall, healthful, and unhealthful plant-based diet indices (oPDI, hPDI, and uPDI, respectively) were calculated. Exploratory dietary patterns were derived using factor analysis based on 44 predefined food groups. Participants were categorized into quintiles based on their dietary pattern scores. TTP (expressed in menstrual cycles) was ascertained within a year from the prepregnancy dietary assessment. Discrete-time proportional hazard models, adjusted for confounders, were used to estimate fecundability ratios (FRs) and 95% CIs, with FR > 1 indicating a shorter TTP. RESULTS: Among 805 women, 383 pregnancies were confirmed by ultrasound scans. Compared with women in the lowest quintile, those in the highest quintile of the uPDI had reduced fecundability (FR of Q5 compared with Q1, 0.65; 95% CI, 0.46-0.91; P trend, 0.009). Conversely, greater adherence to the hPDI was associated with increased fecundability (1.46; 95% CI, 1.02-2.07; P trend, 0.036). The oPDI was not associated with fecundability. Among the 3 exploratory dietary patterns, only greater adherence to the Fast Food and Sweetened Beverages (FFSB) pattern was associated with reduced fecundability (0.61; 95% CI, 0.40-0.91; P trend, 0.018). CONCLUSIONS: Greater adherence to the uPDI or the FFSB dietary pattern was associated with reduced fecundability among Asian women. Greater adherence to the hPDI may be beneficial for fecundability, though this requires confirmation by future studies.