Dysregulated Ribosome Biogenesis Is a Targetable Vulnerability in Triple-Negative Breast Cancer: MRPS27 as a Key Mediator of the Stemness-inhibitory Effect of Lovastatin.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

Engineering hyperthermophilic pullulanase to efficiently utilize corn starch for production of maltooligosaccharides and glucose.

Pullulanase is a starch-debranching enzyme that hydrolyzes side chain of starch, oligosaccharides and pullulan. Nevertheless, the limited activities of pullulanases constrain their practical application. Herein, the hyperthermophilic type II pullulanase from Pyrococcus yayanosii CH1 (PulPY2) was evolved by synergistically engineering the substrate-binding pocket and active-site lids. The resulting mutant PulPY2-M2 exhibited 5-fold improvement in catalytic efficiency (kcat/Km) compared to that of PulPY2. PulPY2-M2 was utilized to develop a one-pot reaction system for efficient production of maltooligosaccharides. The maltooligosaccharides conversion rate of PulPY2-M2 reached 96.1%, which was increased by 5.4% compared to that of PulPY2. Furthermore, when employed for glucose production, the glucose productivity of PulPY2-M2 was 25.4% and 43.5% higher than that of PulPY2 and the traditional method, respectively. These significant improvements in maltooligosaccharides and glucose production and the efficient utilization of corn starch demonstrated the potential of the engineered PulPY2-M2 in starch sugar industry.

Enhanced 3-D asynchronous correlation data preprocessing method for Raman spectroscopy of Chinese handmade paper.

We have developed a novel 3D asynchronous correlation method (3D-ACM) designed for the classification and identification of Chinese handmade paper samples using Raman spectra and machine learning. The 3D-ACM approach involves two rounds of tensor product and Hilbert transform operations. In the tensor product process, the outer product of the spectral data from different samples within the same category is computed, establishing inner connections among all samples within that category. The Hilbert transform introduces a 90-degree phase shift, resulting in a true three-dimensional spectral data structure. This expansion significantly increases the number of equivalent frequency points and samples within each category. This enhancement substantially boosts spectral resolution and reveals more hidden information within the spectral data. To maximize the potential of 3D-ACM, we employed six machine learning models: principal component analysis (PCA) with linear regression (LR), support vector machine (SVM) with LR, k-Nearest Neighbors (KNN), random forest (RF), and convolutional neural network (CNN). When applied to the 3D-ACM data preprocessing method, R-squared values of PLS-LR, KNN, RF and CNN supervised models, approached or equaled 1. This indicates exceptional performance comparable to unsupervised models like PCA. 3D-ACM stands as a versatile mathematical technique not confined to spectral data. It also eliminates the necessity for additional experimental setups or external control conditions, distinct from traditional two-dimensional correlation spectroscopy. Moreover, it preserves the original experimental data, setting it apart from conventional data preprocessing methods. This positions 3D-ACM as a promising tool for future material classification and identification in conjunction with machine learning.

Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide.

Cinnamamide and its derivatives are the most common and important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been widely used in large-scale industrial production of nicotinamide and acrylamide, while its catalytic activity is extremely low or inactive for bulky nitrile substrates such as cinnamonitrile. Therefore, beneficial variant ßF37P/L48P/F51N were obtained from PtNHase of Pseudonocardia thermophila JCM3095 by reshaping of substrate access tunnel and binding pocket, which exhibited 14.88-fold improved catalytic efficiency compared to the wild-type PtNHase. Structure analysis, molecular dynamics simulations and dynamical cross-correlation matrix (DCCM) analysis revealed that the introduced mutations enlarged the substrate access tunnel and binding pocket, enhanced overall anti-correlated movements of enzymes, which would promote product release during the dynamic process of catalysis. In a hydration process, the complete conversion of 5 mM cinnamonitrile was achieved by ßF37P/L48P/F51N in a 50 mL reaction, with cinnamamide yield of almost 100 % and productivity of 0.736 g L-1 h-1. The study demonstrates the co-evolution of substrate access tunnel and binding pocket is an effective strategy, and provides a valuable reference for future research. Furthermore, NHases have huge potential for catalyzing bulky nitriles to form corresponding amides in large-scale industrial production.

Dynamic viral integration patterns actively participate in the progression of BK polyomavirus-associated diseases after renal transplantation.

The classical lytic infection theory along with large T antigen-mediated oncogenesis cannot explain the BK polyomavirus (BKPyV)-associated tumor secondary to BKPyV-associated nephropathy (BKVAN), viremia/DNAemia, and viruria after renal transplantation. This study performed virome capture sequencing and pathological examination on regularly collected urine sediment and peripheral blood samples, and BKVAN and tumor biopsy tissues of 20 patients with BKPyV-associated diseases of different stages. In the early noncancerous stages, well-amplified integration sites were visualized by in situ polymerase chain reaction, simultaneously with BKPyV inclusion bodies and capsid protein expression. The integration intensity, the proportion of microhomology-mediated end-joining integration, and host PARP-1 and POLQ gene expression levels increased with disease progression. Furthermore, multiomics analysis was performed on BKPyV-associated urothelial carcinoma tissues, identifying tandem-like structures of BKPyV integration using long-read genome sequencing. The carcinogenicity of BKPyV integration was proven to disturb host gene expression and increase viral oncoprotein expression. Fallible DNA double-strand break repair pathways were significantly activated in the parenchyma of BKPyV-associated tumors. Olaparib showed an antitumor activity dose-response effect in the tumor organoids without BRCA1/2 genes mutation. In conclusion, the dynamic viral integration patterns actively participate in the progression of BKPyV-associated diseases and thus could be a potential target for disease monitoring and intervention.

N-terminal loops at the tetramer interface of nitrile hydratase act as "hooks" determining resistance to high amide concentrations.

Nitrile hydratase (NHase) has been extensively utilized in industrial acrylamide production. However, the vulnerability to high concentrations of acrylamide limits its further application. Herein, we redesigned the N-terminal loop at the tetramer interface of a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase), and its catalytic activity, resistance to high acrylamide concentrations, and thermostability were improved. Amino acid residues located in the N-terminal loop of the tetramer interface that are responsible for enhancing the resistance to high acrylamide concentrations were identified via static structural analysis and molecular dynamics simulations. A variant library was used to fine-tune the tetramer interface. Variant αL6T exhibited 3.5-fold greater resistance to 50% (v/v) acrylamide, whereas its activity was 1.2-fold higher than that of the wild-type (WT) enzyme, revealing no activity-stability trade-off. Compared to the use of Escherichia coli harboring the WT enzyme, the use of E. coli harboring αL6T increased the acrylamide concentration from 398.1 g/L to 500 g/L. Crystal structure-guided analysis of αL6T and molecular dynamics simulations revealed that increased enzyme surface hydration and the introduction of positive cross-correlation into the N-terminal loop of the tetramer interface caused the two loop regions to hook to each other, thus improving the resistance to high acrylamide concentrations.

Tensor product based 2-D correlation data preprocessing methods for Raman spectroscopy of Chinese handmade paper.

The paper introduces two new methods, namely the cross correlation method (CCM) and two-dimensional correlation method (TDCM), for preprocessing Raman spectroscopy data for analyzing Chinese handmade paper samples. CCM expands the spectral dimension from 1×N to 1×2N-1 by taking cross-correlation between two spectral data of the same category. TDCM includes two-dimensional synchronous correlation method (TDSCM) and two-dimensional asynchronous correlation method (TDACM), which expand the spectral dimension from 1×N to N×N by taking tensor products between two spectral data and between one spectral data and the Hilbert transformation of the other spectral data of the same category, respectively. The experimental data were preprocessed using baseline removal, CCM, TDSCM, and TDACM methods. Four machine learning models were employed to evaluate the effects of these methods: principal component analysis (PCA) combined with linear regression (LR), support vector machine (SVM) combined with LR, k-Nearest Neighbors (KNN), and random forest (RF). The results show that the R-squared values for the PCA model were nearly 1 for all types of data, indicating high accuracy. However, for SVM-LR, KNN, and RF models, the R-squared values were sorted in the order of raw data, baseline removal data, CCM, TDSCM, and TDACM preprocessed data. The R-squared values of KNN and RF machine learning models for TDACM preprocessed data were approaching 1, indicating that the accuracy of machine learning was significantly improved by nearly 100%. This has led to a remarkable improvement in the accuracy of supervised models such as KNN and RF, bringing them closer to the level of unsupervised models such as PCA.

Enhanced soil function and health by soybean root microbial communities during in situ remediation of Cd-contaminated soil with the application of soil amendments.

The interactions between soil microbiomes at various trophic levels are essential for restoring soil functions. Legumes are considered as "pioneer crops" in degraded or contaminated soils because they can fix nitrogen through symbiotic relationships with rhizobacteria, which promotes soil fertility. However, little is known about the abilities of legumes to contribute to the health of soil contaminated with cadmium (Cd). In this research, we applied a soil amendment (commercial Mg-Ca-Si conditioner, CMC) at two rates (1,500 and 3,000 kg/ha) in a Cd-contaminated soybean field. Bulk and rhizosphere soil samples were collected to assess the amendment-induced effects on four microbial lineages (bacteria, fungi, arbuscular mycorrhizal fungi [AMF], and nematodes) and their functions including Cd stabilization, nutrient cycling, and pathogen control. Compared with the control, both CMC application rates increased the pH and reduced labile Cd fraction in the bulk and rhizosphere soils. Although the total Cd concentrations in the soil were similar, the Cd accumulation in the grains was significantly reduced in treatments of soil amendments. It was observed that the application of CMC can significantly reduce the AMF diversity but increased the diversity of the other three communities. Moreover, the biodiversity within keystone modules (identified by co-occurrence network analysis) played key roles in driving soil multifunctionality. Specifically, key beneficial groups in module 2 such as Aggregicoccus (bacteria), Sordariomycetes (fungi), Glomus (AMF), and Bursaphelenchus (nematode) were strongly associated with soil multifunctionality. By co-culturing bacterial suspensions with the soybean root rot pathogen Fusarium solani in the in vitro assays, we experimentally validated that the application of CMC promoted the suppression of soil bacterial community on pathogens by inhibiting the mycelium growth and spore germination. Also, the bacterial community was more resistant to Cd stress in soils receiving CMC amendment. Our findings provide valuable theoretical references for enhancing soil functions and health via applying a soil amendment (CMC) during Cd-contaminated soil remediation. IMPORTANCE Restoration of microbiome-driven soil functions and health is of great importance during Cd-contaminated soil remediation via soil amendment. Soybean and its symbiotic mutualism can provide abundant nitrogen and phosphorus to relieve the nutrient deficiency of Cd-contaminated soil. This study provides a novel perspective on the potential role of applying a soil amendment (CMC) in enhancing the functions and health of Cd-contaminated soils. Our results showed the distinct differences in soil microbial community responding to amendment-induced changes in edaphic properties. The biodiversity within keystone modules had major contributions to the maintenance of the soil's multifunctionality and health. Additionally, a higher CMC application rate showed more beneficial effects. Collectively, our results enhance our understanding about the effects of applying CMC, together with soybean rotation, to enhance and maintain soil functions and health during the field Cd stabilization process.

Deacetylation of Septin4 by SIRT2 (Silent Mating Type Information Regulation 2 Homolog-2) Mitigates Damaging of Hypertensive Nephropathy.

BACKGROUND: Hypertension can lead to podocyte damage and subsequent apoptosis, eventually resulting in glomerulosclerosis. Although alleviating podocyte apoptosis has clinical significance for the treatment of hypertensive nephropathy, an effective therapeutic target has not yet been identified. The function of septin4, a proapoptotic protein and an important marker of organ damage, is regulated by post-translational modification. However, the exact role of septin4 in regulating podocyte apoptosis and its connection to hypertensive renal damage remains unclear. METHODS: We investigated the function and mechanism of septin4 in hypertensive nephropathy to discover a theoretical basis for targeted treatment. Mouse models including Rosa 26 (Gt(ROSA)26Sor)-SIRT2 (silent mating type information regulation 2 homolog-2)-Flag-TG (transgenic) (SIRT2-TG) mice SIRT2-knockout, and septin4-K174Q mutant mice, combined with proteomic and acetyl proteomics analysis, followed by multiple molecular biological methodologies, were used to demonstrate mechanisms of SIRT2-mediated deacetylation of septin4-K174 in hypertensive nephropathy. RESULTS: Using transgenic septin4-K174Q mutant mice treated with the antioxidant Tempol, we found that hyperacetylation of the K174 site of septin4 exacerbates Ang II (angiotensin II)- induced hypertensive renal injury resulting from oxidative stress. Proteomics and Western blotting assays indicated that septin4-K174Q activates the cleaved-PARP1 (poly [ADP-ribose] polymerase family, member 1)-cleaved-caspase3 pathway. In septin4-knockdown human renal podocytes, septin4-K174R, which mimics deacetylation at K174, rescues podocyte apoptosis induced by Ang II. Immunoprecipitation and mass spectrometry analyses identified SIRT2 as a deacetylase that interacts with the septin4 GTPase domain and deacetylates septin4-K174. In Sirt2-deficient mice and SIRT2-knockdown renal podocytes, septin4-K174 remains hyperacetylated and exacerbates hypertensive renal injury. By contrast, in Rosa26-Sirt2-Flag (SIRT2-TG) mice and SIRT2-knockdown renal podocytes reexpressing wild-type SIRT2, septin4-K174 is hypoacetylated and mitigates hypertensive renal injury. CONCLUSIONS: Septin4, when activated through acetylation of K174 (K174Q), promotes hypertensive renal injury. Septin4-K174R, which mimics deacetylation by SIRT2, inhibits the cleaved-PARP1-cleaved-caspase3 pathway. Septin4-K174R acts as a renal protective factor, mitigating Ang II-induced hypertensive renal injury. These findings indicate that septin4-K174 is a potential therapeutic target for the treatment of hypertensive renal injury.

Metagenomic and machine learning-aided identification of biomarkers driving distinctive Cd accumulation features in the root-associated microbiome of two rice cultivars.

Developing low-cadmium (Cd) rice cultivars has emerged as a promising avenue for food safety in Cd-contaminated farmlands. The root-associated microbiomes of rice have been shown to enhance rice growth and alleviate Cd stress. However, the microbial taxon-specific Cd resistance mechanisms underlying different Cd accumulation characteristics between different rice cultivars remain largely unknown. This study compared low-Cd cultivar XS14 and hybrid rice cultivar YY17 for Cd accumulation with five soil amendments. The results showed that XS14 was characterized by more variable community structures and stable co-occurrence networks in the soil-root continuum compared to YY17. The stronger stochastic processes in assembly of the XS14 (~25%) rhizosphere community than that of YY17 (~12%) suggested XS14 may have higher resistance to changes in soil properties. Microbial co-occurrence networks and machine learning models jointly identified keystone indicator microbiota, such as Desulfobacteria in XS14 and Nitrospiraceae in YY17. Meanwhile, genes involved in sulfur cycling and nitrogen cycling were observed among the root-associated microbiome of these two cultivars, respectively. Microbiomes in the rhizosphere and root of XS14 showed a higher diversity in functioning, with the significant enrichment of functional genes related to amino acid and carbohydrate transport and metabolism, and sulfur cycling. Our findings revealed differences and similarities in the microbial communities associated with two rice cultivars, as well as bacterial biomarkers predictive of Cd-accumulation capacity. Thus, we provide new insights into taxon-specific recruitment strategies of two rice cultivars under Cd stress and highlight the utility of biomarkers in offering clues for enhancing crop resilience to Cd stresses in the future.

Construction and application of an efficient dual-base editing platform for Bacillus subtilis evolution employing programmable base conversion.

The functionally evolved bacterial chassis is of great importance to manufacture a group of assorted high value-added chemicals, from small molecules to biologically active macromolecules. However, the current evolution frameworks are less efficienct in generating in vivo genomic diversification because of insufficient tunability, rendering limited evolution spacing for chassis. Here, an engineered genomic diversification platform (CRISPR-ABE8e-CDA-nCas9) leveraging a programmable dual-deaminases base editor was fabricated for rapidly evolving bacterial chassis. The dual-base editor was constructed by reprogramming the CRISPR array, nCas9, and cytidine and adenosine deaminase, enabling single or multiple base conversion at the genomic scale by simultaneous C-to-T and A-to-G conversion in vivo. Employing titration of the Cas-deaminase fusion protein, the platform enabled editing any pre-defined genomic loci with tunable conversion efficiency and editable window, generating a repertoire of mutants with highly diversified genomic sequences. Leveraging the genomic diversification platform, we successfully evolved the nisin-resistant capability of Bacillus subtilis through directed evolution of the subunit of lantibiotic ATP-binding cassette. Therefore, our work provides a portable and programmable genomic diversification platform, which is promising to expedite the fabrication of high-performance and robust bacterial chassis used in the development of biomanufacturing and biopharmaceuticals.

YiaC and CobB regulate lysine lactylation in Escherichia coli.

Lysine lactylation (Kla) has recently been reported to participate in regulating transcription in human cells. However, the characterization, regulatory mechanism and functional consequence of Kla in prokaryotes remain unclear. Here, we report that YiaC functions as a lysine lactylase and that CobB serves as a lysine delactylase in the regulation of metabolism. We demonstrate that YiaC catalyzes the addition of Kla, while CobB erases this PTM both in vitro and intracellularly. Moreover, we show that YdiF can catalyze the formation of a lactyl-coenzyme A, which donates lactyl group for Kla. Quantitative proteomic analysis further reveals 446 endogenous Kla sites targeted by CobB and 79 candidates targeted by YiaC in Escherichia coli (E. coli). Furthermore, we present that Kla can influence the functions of metabolic enzymes. Interestingly, we demonstrate that CobB can specifically modulate the activity of PykF by regulating K382la, promoting glycolysis and bacterial growth. Our study identifies the regulatory enzymes and functional network of Kla and reveals a Kla-mediated molecular mechanism catalyzed by CobB for glycolysis regulation in E. coli.

Diversity-triggered bottom-up trophic interactions impair key soil functions under lindane pollution stress.

A growing amount of evidence suggests that microbial diversity loss may have negative effects on soil ecosystem function. However, less attention has been paid to the determinants of the relationship between community diversity and soil functioning under pollution stress. Here we manipulated microbial diversity to observe how biotic and abiotic factors influenced soil multi-functions (e.g. lindane degradation, soil respiration and nutrient cycling). Results showed that protist community was more sensitive to dilution, pollution stress, and sodium acetate addition than bacterial and fungal community. Acetate addition accelerated the lindane removal. Any declines in microbial diversity reduced the specialized soil processes (NO3-N production, and N2O flux), but increased soil respiration rate. Dilution led to a significant increase in consumers-bacterial and fungi-bacterial interaction as evidenced by co-occurrence network, which possibly played roles in maintaining microbiome stability and resilience. Interestingly, pollution stress and resource availability weaken the relationship between microbial diversity and soil functions through the bottom-up trophic interaction and environmental preference of soil microbiome. Overall, this work provides experimental evidence that loss in microbial diversity, accompanied with changes in trophic interactions mediated biotic and abiotic factors, could have important consequences for specialized soil functioning in farmland ecosystems.

"Toolbox" construction of an extremophilic nitrile hydratase from Streptomyces thermoautotrophicus for the promising industrial production of various amides.

Nitrile hydratase (NHase; EC 4.2.1.84) is widely used to synthesize the corresponding amides from nitriles, which is the most successful green biocatalyst. However, the limited acceptability of substrates and instability under harsh reaction conditions have hindered its widespread industrial application. Here, a gene encoding an extremophilic NHase from Streptomyces thermoautotrophicus (S.t NHase) was successfully overexpressed in Escherichia coli. The enzyme exhibited excellent thermostability, retaining >50 % of residual activity after heat treatment at 65 °C for 252 min. To further improve the catalytic performance of S.t NHase, semi-rational engineering of its substrate access tunnel was performed. A mutant ßL48D showed a specific activity of 566.18 ± 18.86 U/mg towards 3-cyanopyridine, which was 7.7 times higher than its parent enzyme (73.80 ± 5.76 U/mg). Molecular dynamics simulation showed that the introduction of aspartic acid into ßLeu48 resulted in a larger and more frequent opening of the substrate access tunnel entrance. On this basis, a "toolbox" containing various mutants on the substrate access tunnel was further established, whose catalytic activity towards various nitrile substrates was extensively improved, showing great potential for efficient synthesis of multiple high-value amides.

Insight into the broadened substrate scope of nitrile hydratase by static and dynamic structure analysis.

The narrow substrate scope limits the wide industrial application of enzymes. Here, we successfully broadened the substrate scope of a nitrile hydratase (NHase) through mutation of two tunnel entrance residues based on rational tunnel calculation. Two variants, with increased specific activity, especially toward bulky substrates, were obtained. Crystal structure analysis revealed that the mutations led to the expansion of the tunnel entrance, which might be conducive to substrate entry. More importantly, molecular dynamics simulations illustrated that the mutations introduced anti-correlated movements to the regions around the substrate tunnel and the active site, which would promote substrate access during the dynamic process of catalysis. Additionally, mutations on the corresponding tunnel entrance residues on other NHases also enhanced their activity toward bulky substrates. These results not only revealed that residues located at the enzyme surface were a key factor in enzyme catalytic performance, but also provided dynamic evidence for insight into enzyme substrate scope broadening.

Modulating the pH profile of the pullulanase from Pyrococcus yayanosii CH1 by synergistically engineering the active center and surface.

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5-5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (PulPY2) was successfully shifted from 6.4 to 5 with a 2-fold increase in the specific activity based on synergistic engineering of the active center and surface. Synergistic engineering was performed by introducing histidine within 6 Å of the active sites, and by enhancing negative charges on the enzymatic surface. Two single-site mutants of PulPY2-Q13H and PulPY2-I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant PulPY2-Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to PulPY2. These results not only obtained an applicable pullulanase for industrial application, but also provided a strategy for shifting the optimal pH of the enzyme based on synergistic engineering of the active center and surface.

Ectopic expression of a combination of 5 genes detects high risk forms of T-cell acute lymphoblastic leukemia.

BACKGROUND: T cell acute lymphoblastic leukemia (T-ALL) defines a group of hematological malignancies with heterogeneous aggressiveness and highly variable outcome, making therapeutic decisions a challenging task. We tried to discover new predictive model for T-ALL before treatment by using a specific pipeline designed to discover aberrantly active gene. RESULTS: The expression of 18 genes was significantly associated with shorter survival, including ACTRT2, GOT1L1, SPATA45, TOPAZ1 and ZPBP (5-GEC), which were used as a basis to design a prognostic classifier for T-ALL patients. The molecular characterization of the 5-GEC positive T-ALL unveiled specific characteristics inherent to the most aggressive T leukemic cells, including a drastic shut-down of genes located on the mitochondrial genome and an upregulation of histone genes, the latter characterizing high risk forms in adult patients. These cases fail to respond to the induction treatment, since 5-GEC either predicted positive minimal residual disease (MRD) or a short-term relapse in MRD negative patients. CONCLUSION: Overall, our investigations led to the discovery of a homogenous group of leukemic cells with profound alterations of their biology. It also resulted in an accurate predictive tool that could significantly improve the management of T-ALL patients.

Discovery of the ATPase Activity of a Cobalt-Type Nitrile Hydratase Activator and Its Promoting Effect on Enzyme Maturation.

An activator protein and a metal ion are two factors known to be indispensable for the maturation of nitrile hydratase (NHase). Here, the third key factor, adenosine triphosphate (ATP), was identified to play an important role in the activation of Co-type NHase. Free phosphate measurements revealed that the Co-type activator protein can hydrolyze ATP/GTP with appreciable performance and that such catalytic performance is related to NHase activity. Computational analysis and site-directed mutagenesis identified several potential hot spot residues involved in the binding of ATP to Co-type activator protein, and an E60A/W61A/D62A/I139A/T141A combinatorial variant reduced the ATPase activity to 18% of its original level. Further NHase activation studies using the combinatorial variant demonstrated that although the ATPase activity of the Co-type activator protein correlated with NHase activity, a low ATP concentration of 0.5 mmol/L was optimal for NHase activation, with higher ATP concentrations potentially inhibiting NHase activity.

Abnormal global alternative RNA splicing in COVID-19 patients.

Viral infections can alter host transcriptomes by manipulating host splicing machinery. Despite intensive transcriptomic studies on SARS-CoV-2, a systematic analysis of alternative splicing (AS) in severe COVID-19 patients remains largely elusive. Here we integrated proteomic and transcriptomic sequencing data to study AS changes in COVID-19 patients. We discovered that RNA splicing is among the major down-regulated proteomic signatures in COVID-19 patients. The transcriptome analysis showed that SARS-CoV-2 infection induces widespread dysregulation of transcript usage and expression, affecting blood coagulation, neutrophil activation, and cytokine production. Notably, CD74 and LRRFIP1 had increased skipping of an exon in COVID-19 patients that disrupts a functional domain, which correlated with reduced antiviral immunity. Furthermore, the dysregulation of transcripts was strongly correlated with clinical severity of COVID-19, and splice-variants may contribute to unexpected therapeutic activity. In summary, our data highlight that a better understanding of the AS landscape may aid in COVID-19 diagnosis and therapy.

Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor.

Transcription factor (TF)-based biosensors are expected to serve as powerful tools for the high-throughput screening of biocatalytic systems; however, most of them respond to ligands in a narrow concentration range, which limits their application. In this study, we constructed a heterogenous niacin biosensor using the repressive TF BsNadR and its target promoters from Bacillus subtilis. The fine-tunable output of the niacin biosensor was expanded to a wide range of niacin concentrations (0-50 mM) through desensitization engineering, which was suitable for the accurate identification of differences in enzyme activity. Structural mechanism analysis indicated that weakening the affinity of BsNadR with the ligand niacin and with DNA alters its regulatory properties. Based on the desensitized niacin biosensor, a high-throughput in vivo screening platform was developed for evolving nitrile metabolism-related enzymes. The evolved nitrilase, amidase, and nitrile hydratase with 6.6-, 2.1-, and 21.3-fold improvements in activity were achieved, respectively. In addition, these mutants also exhibited elevated activity toward other cognate substrates, indicating the broad applicability of the screening platform. This study not only provided a universal high-throughput screening platform for different nitrile metabolism-related enzymes but also demonstrated the advantages of repressive biosensors and the vital role of desensitization engineering of the TF in the development of high-throughput screening platforms for enzymes.