Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library.

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.

Dynamic LVEF Decline and Serum NT-proBNP and Uric Acid Levels before Heart Transplantation are Independent Predictors of Adverse Outcomes in Young Adult Patients with Dilated Cardiomyopathy.

Background: The present study investigated the predictors of adverse outcomes in young adult patients with dilated cardiomyopathy (DCM) who underwent heart transplantation (HTx). Methods: Twenty-four young adult patients (aged 18-45 years) with DCM who underwent HTx in our hospital from January 2012 to December 2022 were included in this retrospective analysis. Pre- and post-HTx data were collected for echocardiography, N-terminal pro-brain natriuretic peptide (NT-proBNP), and uric acid (UA). Data collected at the time of DCM diagnosis were designated as baseline data. Post-HTx assessments were conducted at 1 week and 3, 6, 12, and 36 months post-HTx. The primary endpoint was defined as any adverse event, including left ventricular ejection fraction (LVEF) < 50% (n = 3), 50% increase in right or left ventricular diameter (n = 12), or death (n = 2). Patients were categorized into a non-adverse-event group (n = 12) or an adverse-event group (n = 12). Results: Baseline NT-proBNP (p = 0.014) and UA (p = 0.012) were significantly higher in the adverse-event group than in the non-adverse-event group. Baseline NT-proBNP > 7390 pg/mL (relative risk (RR) = 7.412, p = 0.046), UA > 542 µmol/L (RR = 8.838, 95% confidence interval (95% CI) = 1.541-50.694, p = 0.014), and sustained reduction in LVEF ( ≥ 3%) over a 2-year pharmacological treatment prior to HTx (RR = 3.252, p = 0.046) were significantly associated with an increased risk of adverse events post-HTx. Conclusions: In young adult DCM patients post-HTx, heightened baseline levels of NT-proBNP and UA levels and a sustained reduction in LVEF over time prior to undergoing an HTx are significantly associated with an increased risk of adverse events post-HTx. Future studies are needed to observe whether individualized monitoring strategies could reduce the incidence of adverse events following HTx in these patients.

Repurposing plant hormone receptors as chemically-inducible genetic switches for dynamic regulation in yeast.

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.

Three-Component Domino Acylation/Trifluoromethylation of Bicyclic Amidines.

We herein describe a three-component reaction involving bicyclic amidines such as DBN/DBU, acyl fluorides, and TMSCF3 for access to a novel class of N-acyl trifluoromethylated bicyclic aminals. Under mild and operationally simple conditions, bicyclic amidines can undergo difunctionalization (acylation/trifluoromethylation) using readily available reagents. Further Lewis acid-promoted nucleophilic ring-opening can lead to diverse products with quaternary carbon centers containing CF3. The corresponding pentafluoroethylation is also achieved by using TMSC2F5.

acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens.

High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

Saliva microbiome alterations in dental fluorosis population.

Background: We aimed to explore saliva microbiome alterations in dental fluorosis population. Methods: The prevalence of dental fluorosis was examined in 957 college students. Dean's fluorosis index was used to evaluate the dental fluorosis status. Changes in the composition of the salivary microbiome were assessed in a subset of these patients (100 healthy controls, 100 dental fluorosis patients). Results: Dental fluorosis affected 47% of the student sample, and incidence was unrelated to gender. Compared with healthy controls, the microbiota of patients with dental fluorosis exhibited increased diversity, with increased abundance of Treponema lecithinolyticum, Vibrio metschnikovii, Cupriavidus pauculus, Pseudomonas, Pseudomonadaceae, Pseudomonadales, and decreased abundance of Streptococcus mutans, Streptococcus sanguinis, Gemella, and Staphylococcales. Function analyses showed increases in arginine biosynthesis in patients affected by dental fluorosis, together with reductions in amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, and starch and sucrose metabolism. Conclusions: These results suggest that there are striking differences in salivary microbiome between healthy controls and dental fluorosis patients. Dental fluorosis may contribute to periodontitis and systemic lung diseases. There is a need for cohort studies to determine whether altering the salivary microbiota in dental fluorosis patients can alter the development of oral or systemic diseases.

A new strategy for grading of Lu'an guapian green tea by combination of differentiated metabolites and hypoglycaemia effect.

Predictable tea grading bears not only scientific merit, but also commercial value. Lu'an guapian green tea (LGGT) is one of the most famous green teas in China. Based on morphology and sensory flavour, LGGT was traditionally graded as first premium (FP), second premium (SP), first grade (FG), second grade (SG), third grade (TG) and summer grade (SuG). The chemical profiles and distinct metabolites distinguishing different grades of LGGT are yet to be defined, neither the grade related health benefits be evaluated. In present study, non-targeted metabolomics combined with chemometrics analysis showed that FP and SP, FG and SG exhibited high similarity, respectively. TG and SuG both exhibited great difference from the other grades. Therefore, LGGT could be regrouped into four grades. Furthermore, eight metabolites were identified and displayed grade related bio-markers of LGGT, which are gallic acid, catechin, gallocatechin, salicylic acid, theasinensin B, theasinensin C, kaempferol 3-(6''-rhanmnosylsoporoside) and l-linalool 3-[xylosyl-(1->6)-glucoside]. Quantitative analysis further confirmed that gallic acid, catechin, gallocatechin and salicylic acid were distinct grade-related metabolites. In vitro and in vivo data showed that methanol-extracts of higher grades LGGT exhibited more potent α-amylase and α-glucosidase inhibitory activity and hypoglyceamia effect than that of lower grades.

Analysis of Differentiated Chemical Components between Zijuan Purple Tea and Yunkang Green Tea by UHPLC-Orbitrap-MS/MS Combined with Chemometrics.

Zijuan tea (Camellia sinensis var. assamica cv. Zijuan) is a unique purple tea. Recently, purple tea has drawn much attention for its special flavor and health benefits. However, the characteristic compounds of purple tea compared with green tea have not been reported yet. The present study employed a non-targeted metabolomics approach based on ultra-high performance liquid chromatography (UHPLC)-Orbitrap-tandem mass spectrometry (MS/MS) for comprehensive analysis of characteristic metabolites between Zijuan purple tea (ZJT) and Yunkang green tea (YKT). Partial least squares-discriminant analysis (PLS-DA) indicated that there are significant differences in chemical profiles between ZJT and YKT. A total of 66 major differential metabolites included catechins, proanthocyanins, flavonol and flavone glycosides, phenolic acids, amino acids and alkaloids were identified in ZJT. Among them, anthocyanins are the most characteristic metabolites. Nine glycosides of anthocyanins and six glycosides of proanthocyanins were found to be significantly higher in ZJT than that in YKT. Subsequently, pathway analysis revealed that ZJT might generate anthocyanins and proanthocyanins through the flavonol and flavone glycosides. Furthermore, quantitative analysis showed absolutely higher concentrations of total anthocyanins in ZJT, which correlated with the metabolomics results. This study presented the comprehensive chemical profiling and the characterized metabolites of ZJT. These results also provided chemical evidence for potential health functions of ZJT.

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two-stage fine chemical production with Kluyveromyces marxianus.

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4 g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

Theaflavin-regulated Imd condensates control Drosophila intestinal homeostasis and aging.

Black tea is the most widely consumed tea drink in the world and has consistently been reported to possess anti-aging benefits. However, whether theaflavins, one type of the characteristic phytochemicals in black tea extracts, are involved in regulating aging and lifespan in consumers remains largely unknown. In this study, we show that theaflavins play a beneficial role in preventing age-onset intestinal leakage and dysbiosis, thus delaying aging in Drosophila. Mechanistically, theaflavins regulate the condensate assembly of Imd to negatively govern the overactivation of Imd signals in fruit fly intestines. In addition, theaflavins prevent DSS-induced colitis in mice, suggesting theaflavins play a role in modulating intestinal integrity. Overall, our study reveals a molecular mechanism by which theaflavins regulate gut homeostasis likely through controlling Imd coalescence.

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus.

BACKGROUND: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. RESULTS: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. CONCLUSIONS: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

Dietary supplementation of Huangshan Maofeng green tea preventing hypertension of older C57BL/6 mice induced by desoxycorticosterone acetate and salt.

Senile hypertension affects the life quality of aged population. Dietary intervention plays a pivotal role in the prevention of hypertension. There are few reports concerning the effects and mechanisms of green tea supplementation preventing age related hypertension. The current study investigated the effect and mechanism of dietary supplement of Huangshan Maofeng green tea (HSMF) on prevention of hypertension induced by deoxycorticosterone acetate (DOCA) and salt in old C57BL/6 mice. Our results showed that HSMF dose-dependently prevented the increase of systolic blood pressure and diastolic blood pressure induced by DOCA plus salt (DS) at 51-week-old mice. And HSMF significantly reduced the agonists' stimulated contraction of mesenteric arteries isolated from the old mice. The expression of vasoconstrictor genes and inflammatory cytokines in aorta were suppressed observably by HSMF supplementation compared with DS group. The protein expression of PKCα in the aorta was dose-dependently decreased by HSMF compared to DS group. The phosphorylation level of MYPT1, CPI-17and MLC20 was also restrained by HSMF in the aorta. Furthermore, HSMF protected kidney by maintaining integrity of glomeruli and tubules and remarkably decreased the NGAL level in plasma. HSMF also suppressed the kidney inflammation by decreasing inflammatory cytokines expression and the macrophage infiltration. Our results proved that dietary supplement of HSMF remarkably improved the vascular functions and protected kidney injury, and thus prevented hypertension induced by DS in older C57BL/6 mice. Our data indicated that the dietary supplement of HSMF may potentially be used as a food additive for preventing hypertension for aged people.

Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 â€‹°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, P SSA3 and P ADH1 , increased expression above 40 â€‹°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 â€‹°C. Cultures at 41 â€‹°C exhibited a similar TAL biosynthesis capability, while at 30 â€‹°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.

The effect of CYP2C19 genotype-guided antiplatelet therapy on outcomes of selective percutaneous coronary intervention patients: an observational study.

Aim: To observe if personalized antiplatelet therapy according to the CYP2C19 phenotype can improve the outcomes of patients receiving selective percutaneous coronary intervention (PCI). Methods: In this observational study, 677 Chinese patients undergoing selective PCI were divided into gene group (n = 369) and conventional group (n = 308), and given antiplatelet therapy according to the CYP2C19 genotype or clinical features, respectively. Incidence of MACE (death, non-fatal myocardial infarction, and unplanned repeat revascularization) and bleeding was compared between the two groups after 18 months. Results: Diabetes, heart dysfunction and SYNTAX score (>15), but not routinely CYP2C19 genotype test-guided antiplatelet therapy, were associated with MACE. The incidence of bleeding showed no difference. Conclusion:CYP2C19 phenotype-guided antiplatelet therapy may have no influence on the outcomes of selective PCI patients. Clinical features-guided antiplatelet therapy may be reasonable.

Accurate quantification of creatinine in serum by coupling a measurement standard to extractive electrospray ionization mass spectrometry.

Ambient ionization (AI) techniques have been widely used in chemistry, medicine, material science, environmental science, forensic science. AI takes advantage of direct desorption/ionization of chemicals in raw samples under ambient environmental conditions with minimal or no sample preparation. However, its quantitative accuracy is restricted by matrix effects during the ionization process. To improve the quantitative accuracy of AI, a matrix reference material, which is a particular form of measurement standard, was coupled to an AI technique in this study. Consequently the analyte concentration in a complex matrix can be easily quantified with high accuracy. As a demonstration, this novel method was applied for the accurate quantification of creatinine in serum by using extractive electrospray ionization (EESI) mass spectrometry. Over the concentration range investigated (0.166 ~ 1.617 µg/mL), a calibration curve was obtained with a satisfactory linearity (R(2) = 0.994), and acceptable relative standard deviations (RSD) of 4.6 ~ 8.0% (n = 6). Finally, the creatinine concentration value of a serum sample was determined to be 36.18 ± 1.08 µg/mL, which is in excellent agreement with the certified value of 35.16 ± 0.39 µg/mL.