Decoding CRISPR-Cas PAM recognition with UniDesign.

The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.

Rapid and Sensitive Detection of Fentanyl and Its Analogs by a Novel Chemiluminescence Immunoassay.

BACKGROUND: Abuse of fentanyl and its analogs is a major contributor to the opioid overdose epidemic in the United States, but detecting and quantifying trace amounts of such drugs remains a challenge without resorting to sophisticated mass spectrometry-based methods. METHODS: A sensitive immunoassay with a sub-picogram limit of detection for fentanyl and a wide range of fentanyl analogs has been developed, using a novel high-affinity antibody fused with NanoLuc, a small-size luciferase that can emit strong and stable luminescence. When used with human urine samples, the assay has a sub-picogram limit of detection for fentanyl, with results fully concordant with LC-MS. RESULTS: When applied to clinical samples, the novel chemiluminescence immunoassay can detect low positive fentanyl missed by routine screening immunoassays, with a limit of detection of 0.8 pg/mL in human urine. When applied to environmental samples, the assay can detect levels as low as 0.25 pg fentanyl per inch2 of environment surface. Assay turnaround time is less than 1 h, with inexpensive equipment and the potential for high-throughput automation or in-field screening. CONCLUSIONS: We have established a novel assay that may have broad applications in clinical, environmental, occupational, and forensic scenarios for detection of trace amounts of fentanyl and its analogs.

Photocatalytic Proton-Coupled Electron Transfer Enabled Radical Cyclization for Isoquinoline-1,3-diones Synthesis.

Radical cyclization has been demonstrated to be an efficient method to access functionalized heterocycles from easily accessible raw materials. Described herein is the development of a photocatalytic proton-coupled electron transfer (PCET) strategy for the synthesis of isoquinoline-1,3-diones using readily prepared naphthalimide (NI)-based organic photocatalysts. The process features free metal-complex photocatalysts, acids, and mild reaction conditions. This mild radical cyclization protocol has a broad substrate scope and can be effectively applied to a variety of medicinally relevant substrates. Furthermore, control experiments were conducted to elucidate the mechanism of this visible light-induced methodology.

A review of 10 patients treated with the masquelet technique and microsurgical technique combined for Gustilo type III open tibial fractures.

BACKGROUND: Open tibial fractures often include severe bone loss and soft tissue defects and requires complex reconstructive operations. However, the optimal treatment is unclear. METHODS: This retrospective study enrolled patients with Gustilo type III open tibial fractures from January 2018 to January 2021 to assess the clinical utility of Masquelet technique together with microsurgical technique as a combined strategy for the treatment of open tibial fractures. The demographics and clinical outcomes including bone union time, infection, nonunion and other complications were recorded for analysis. The bone recovery quality was evaluated by the AOFAS Ankle-Hindfoot Scale score and the Paley criteria. RESULTS: We enrolled 10 patients, the mean age of the patients and length of bone defects were 31.7 years (range, 23-45 years) and 7.5 cm (range, 4.5-10 cm) respectively. Bone union was achieved for all patients, with an average healing time of 12.2 months (range, 11-16 months). Seven patients exhibited a bone healing time of less than 12 months, whereas 3 patients exhibited a bone healing time exceeding 12 months. No significant correlation was found between the length of bone loss and healing time. In addition, no deep infection or nonunion was observed, although 2 patients experienced wound fat liquefaction with exudates and 1 patient presented with a bloated skin flap. The average AOFAS Ankle-Hindfoot Scale score was 80.5 (range, 74-85), and all patients were evaluated as good or exellent based on the Paley criteria. CONCLUSIONS: Our study indicated that the use of the Masquelet technique and the microsurgical technique as a combined strategy is safe and effective for the treatment of Gustilo type III open tibial fractures.

Submucosal Trans-Septal Suturing Technique After a Septal Extension Graft with Porous High-Density Polyethylene: A Technical Report.

BACKGROUND: Porous high-density polyethylene (pHDPE) is an alternative material for a septal extension graft (SEG) in oriental rhinoplasty when autologous cartilage is limited. Although nasal packing (NP) and trans-septal suturing (TSS) techniques are routine procedures to obviate the dead space after septoplasty, they are associated with certain discomforts and complications. OBJECTIVE: To investigate the application of a submucosal trans-septal suturing (STSS) technique after SEG with pHDPE. METHODS: A prospective study was conducted on 60 female participants who underwent SEG with pHDPE. The participants were randomly divided into the NP group and STSS group. The extra surgical duration of NP and STSS, pain, nasal obstruction, and sleeping disturbance as well as postoperative complications were recorded and compared between groups. RESULTS: No significant difference was found between group NP and group STSS in terms of mean age. The mean extra surgical duration of group STSS was significantly longer than group NP. There were significant higher pains of group NP at 24 hours and 48 hours postoperatively, compared with group STSS. The NP group also experienced significantly more nasal obstruction and sleep disturbance within 48h postoperatively compared to the STSS group. There was one infection in each group, minor bleeding in two NP patients, and one STSS patient. There was no major bleeding, hematoma, graft exposure, or septal perforation in both groups. CONCLUSION: Although STSS needs a longer extra surgical duration than NP, it significantly improves the patient's postoperative comfort with a faster return to normal respiration compared to NP. LEVEL OF EVIDENCE I: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Silver-catalyzed nitrosation and nitration of aromatic amides using NOBF4.

Divergent aromatic ring nitrosation and nitration of aromatic amides are reported using NOBF4 as the electrophile under silver-catalyzed conditions. The reactions proceed efficiently with a wide range of compatible functionalities providing ortho-position nitrosation products, deacylation nitrosation products, and nitration products from different tertiary and secondary aromatic amides.

Gut microbial diversity and function analysis of the final-instar larvae of Protohermes xanthodes (Megaloptera: Corydalidae).

Protohermes xanthodes (Megaloptera, Corydalidae, Protohermes), widespread species in China, is an important insects treated as food and medicine for aquaculture. In this study, the gut microbiota was investigated by 16S rRNA microbial profiling. A total of 600 Amplicon Sequence Variants (ASV) were identified, Proteobacteria and Firmicutes were the main dominant phyla, and 27 genera ran through the entire digestive tract, mainly Hafnia-Obesumbacterium (Proteobacteria), Lactobacillus and Lactococcus (Firmicutes). The PICRUSt2 functional prediction of gut microbiota showed that the foregut abundant pathways related to metabolism, environmental information processing, and genetic information processing. while the midgut had the most abundant metabolic and environmental information processing pathways, including the prominent phosphotransferase system (PTS), propionate metabolism, and ß-lactam resistance. The hindgut had the weakest metabolic function, but its genetic information processing was more abundant than the foregut and midgut. Additionally, 26 strains of bacteria were isolated from the midgut microorganisms, with Firmicutes being the dominant bacteria, and some of the purified bacteria had potential probiotic and anti-pathogen functions. These findings suggest that there are differences in the microorganisms of the different gut floras of the larvae, and each flora has specific metabolic functions. This research could be used to further understand the function of gut microorganisms, explore the co-evolution of P. xanthodes and gut microorganisms, and promote healthy breeding based on gut microorganisms.

Engineered acetaldehyde dehydrogenase for the efficient degradation of acetaldehyde.

Acetaldehyde is highly cytotoxic and widely presents in food and the environment. Aldehyde dehydrogenase (ALDH) can degrade acetaldehyde to non-toxic acetic acid, showing potential for acetaldehyde elimination. However, a lack of high-throughput methods for screening efficient variants is a significant obstacle to ALDH design. Here, we established a visualized high-throughput method to screen recombinantly expressed ALDH variants in Bacillus subtilis by fluorescent probes of dual-acceptor cyanine-based in response to NADH, the acetaldehyde degradation product. Molecular docking revealed key amino acids in the binding region of acetaldehyde to ALDH. Combined with saturation mutagenesis and visualization high-throughput methods, a variant ALDHS273N with an activity of 119.82 U·mL-1 was screened. The optimal reaction temperature and pH of ALDHS273N were 60 °C and 9.0, respectively. ALDHS273N showed stability at 30-50 °C and pH 5.0-9.0. The activity of ALDHS273N was increased to 263.52 U∙mL-1 by fermentation optimization, which was 5.58 times that of ALDHWT. The degradation rate of ALDHS273N to 100 mmol L-1 acetaldehyde was 87.34% within 2 h, which was 4.2 times that of the wild enzyme (20.81%). As far as we know, this is the ALDH with the highest activity reported so far, and it is also the first time that ALDH has been used for the efficient degradation of acetaldehyde. Overall, the reported high-throughput screening method and developed mutants represent a significant advance in green bio-elimination technologies of acetaldehyde.

[Advances in animal models of chronic heart failure and its applications in traditional Chinese medicine].

Chronic heart failure(CHF) is a series of clinical syndromes in which various heart diseases progress to their end stage. Its morbidity and mortality are increasing year by year, which seriously threatens people's life and health. The diseases causing CHF are complex and varied, such as coronary heart disease, hypertension, diabetes, cardiomyopathy and so on. It is of great significance to establish animal models of CHF according to different etiologies to explore the pathogenesis of CHF and develop drugs to prevent and treat CHF induced by different diseases. Therefore, based on the classification of the etiology of CHF, this paper summarizes the animal models of CHF widely used in recent 10 years, and the application of these animal models in traditional Chinese medicine(TCM) research, in order to provide ideas and strategies for studying the pathogenesis and treatment of CHF, and provide ideas for TCM modernization research.

Recent developments in the synthesis of nitrogen-containing heterocycles from ß-aminovinyl esters/ketones as CC-N donors.

Nitrogen-containing heterocycles are ubiquitous fragments of numerous natural products, pharmaceuticals, designed bioactive drug candidates and agrochemicals. During the past few decades, these compounds have received considerable attention from the synthetic chemistry community, and great efforts have been focused on the development of concise and efficient methods for the synthesis of these heterocyclic skeletons. In this review, we summarize a diverse range of synthetic methods employing ß-aminovinyl esters(ketones) as key CC-N-synthons to furnish useful bioactive heterocyclic frameworks, such as quinolines, pyridines, pyrazines, pyrroles, indoles, oxazoles, imidazoles, thiazoles, isothiazoles, pyrazoles, triazoles, and azepines, thus offering new opportunities and expanding the toolbox of synthetic chemistry reactions.

Recent developments in the synthesis of the isoquinoline-1,3(2H,4H)-dione by radical cascade reaction.

In recent years, isoquinoline-1,3(2H,4H)-dione compounds have attracted extensive attention from synthetic chemists, with the aim of finding simple, mild, green and efficient synthetic methods. In this review, we summarize the diverse range of synthetic methods employing acryloyl benzamides as key substrates to furnish isoquinoline-1,3-diones using different radical precursors, such as those containing carbon, sulphur, phosphorus, nitrogen, silicon and bromine. This will stimulate the interest of readers to engage in research in this field.

[Trace therapeutic substances of traditional Chinese medicine: great resources of innovative drugs derived from traditional Chinese medicine].

The traditional Chinese medicine(TCM) contains very complex constituents. Besides the major constituents, there are a large number of unclear trace constituents with novel skeletons and potent bioactivities, which have been regarded as one of the important therapeutic substances and the great resources of innovative drugs derived from TCM. The present review highlighted that the development of the trace therapeutic substances of TCM is closely depends on the advanced technologies for their identification, isolation, structure elucidation, and bioactivity evaluation. Additionally, this paper reviewed the novel trace compounds derived from Chinese herbal medicines which have been published in Organic Letters during 2001-2021, and summarized the important licensed drugs originated from the trace therapeutic substances and the discovery and development of trace therapeutic substances of 8 kinds of Chinese herbal medicines. This review provides references for the research and development of TCM therapeutic substances and innovative drugs.

Pd-Catalyzed Tandem Isomerization/Cyclization for the Synthesis of Aromatic Oxazaheterocycles and Pyrido[3,4-b]indoles.

An effient tandem process consisting of palladium-catalyzed double-bond isomerization of long-chain olefins and subsequent intramolecular cyclization promoted by B2(OH)2 for the synthesis of aromatic oxazaheterocycles is disclosed. This strategy can also provide rapid access to pyrido[3,4-b]indoles, trans-2-olefins, and eneamides bearing various functional groups with high regio- and stereoselectivity.

Measuring Software Maintainability with Naïve Bayes Classifier.

Software products in the market are changing due to changes in business processes, technology, or new requirements from the customers. Maintainability of legacy systems has always been an inspiring task for the software companies. In order to determine whether the software requires maintainability by reverse engineering or by forward engineering approach, a system assessment was done from diverse perspectives: quality, business value, type of errors, etc. In this research, the changes required in the existing software components of the legacy system were identified using a supervised learning approach. New interfaces for the software components were redesigned according to the new requirements and/or type of errors. Software maintainability was measured by applying a machine learning technique, i.e., Naïve Bayes classifier. The dataset was designed based on the observations such as component state, successful or error type in the component, line of code of error that exists in the component, component business value, and changes required for the component or not. The results generated by the Waikato Environment for Knowledge Analysis (WEKA) software confirm the effectiveness of the introduced methodology with an accuracy of 97.18%.

CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells.

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.

Retracted: Synthesis of 2-Arylisoindoline Derivatives Catalyzed by Reusable 1,2,4-Triazole Iridium on Mesoporous Silica through a Cascade Borrowing Hydrogen Strategy.

Covalent attachment of a 1,2,4-triazole iridium complex to mesoporous MCM-41 generated a heterogeneous catalyst that was found to be effective in the synthesis of 2-aryl isoindolines, quinolines, cyclic amines, and symmetrical secondary amines through a cascade borrowing hydrogen strategy. Interestingly, the supported heterogeneous iridium catalyst prepared from the 1,2,4-triazole iridium complex and mesoporous MCM-41 exhibited high catalytic activity in the preparation of 2-aryl isoindoline derivatives and symmetrical secondary amines. The catalyst system is highly recyclable for at least five times. Besides the important effect of the triazole, iridium sites grafted on siliceous supports can act as multifunctional catalytic centers and thus greatly enhance the catalytic activity of the catalysts. Furthermore, mechanistic experiments revealed that the reaction is initiated by an initial alcohol dehydrogenation and promoted by an iridium hydride intermediate. Importantly, the direct detection of a diagnostic iridium hydride signal confirmed that the synthesis of 2-aryl isoindolines occurs by a borrowing hydrogen process. This work provides an efficient example of isoindolines synthesis through a borrowing hydrogen strategy.

Multi-Scale Vehicle Detection for Foreground-Background Class Imbalance with Improved YOLOv2.

Vehicle detection is a challenging task in computer vision. In recent years, numerous vehicle detection methods have been proposed. Since the vehicles may have varying sizes in a scene, while the vehicles and the background in a scene may be with imbalanced sizes, the performance of vehicle detection is influenced. To obtain better performance on vehicle detection, a multi-scale vehicle detection method was proposed in this paper by improving YOLOv2. The main contributions of this paper include: (1) a new anchor box generation method Rk-means++ was proposed to enhance the adaptation of varying sizes of vehicles and achieve multi-scale detection; (2) Focal Loss was introduced into YOLOv2 for vehicle detection to reduce the negative influence on training resulting from imbalance between vehicles and background. The experimental results upon the Beijing Institute of Technology (BIT)-Vehicle public dataset demonstrated that the proposed method can obtain better performance on vehicle localization and recognition than that of other existing methods.

Endophytic effects of Aspergillus oryzae on radish (Raphanus sativus) and its herbivore, Plutella xylostella.

MAIN CONCLUSION: We provide evidence that Aspergillus oryzae not only acts as an endophyte in Raphanus sativus, but also works as a plant growth promoter and provides some protection against its herbivore, Plutella xylostella affecting its feeding rate, mortality and fitness parameters, thereby contributing to the pest population suppression. Seed inoculation of radish seeds with the fungus Aspergillus oryzae allowed its establishment as an endophyte promoting plant growth and negatively affecting fitness parameters of its major herbivore, diamondback moth, Plutella xylostella. Endophytic fungi may contribute to the growth of their host plants and enhance resistance to herbivores and diseases. We evaluated the effect of A. oryzae (Ahlburg) E. Cohn as an endophyte in radish (Raphanus sativus L.) on growth and development of the plants themselves and their major herbivore, the diamondback moth P. xylostella (L). A. oryzae colonization rates in leaves were significantly higher than in roots and stems, with a rate of 80% in leaves, 40% in stems and 20% in roots 1 week after seed inoculation. Colonization gradually decreased in the various plant tissues, and disappeared completely in roots, stems and leaves within 2, 5 and 7 weeks, respectively. A. oryzae did not affect seed germination; however, it promoted radish growth with endophytic plants attaining average heights of 116 mm compared to 99.6 mm in the controls at the third week post-inoculation. The P. xylostella fitness parameters, consumption, larval and pupal weights, and feeding on the endophytic plants were significantly lower than the controls, while larval mortality was significantly higher. Larvae fed on endophytic plants consumed 0.46 mg less leaf matter in the first week post seed inoculation and weighed 0.83 mg less as mature 4th instars than controls. We have demonstrated that A. oryzae can establish as an endophyte in R. sativus through seed inoculation providing some plant growth promotion and protection against its herbivore by increasing its mortality and negatively affecting its fitness parameters, suggesting that adopting seed treatments with A. oryzae may be beneficial in the commercial cultivation of radish.

Mn-Catalyzed 1,6-conjugate addition/aromatization of para-quinone methides.

A series of ferrocenyl triazole ligands have been synthesized and characterized, which proved to be effective for the Mn-catalyzed 1,6-conjugate addition/aromatization of para-quinone methides with good to high yields under mild conditions. This protocol provided an efficient and practical route to the synthetically interesting functionalized quinones, methines and their analogues.

Characterization and expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

BACKGROUND: Serine protease inhibitors (SPIs) have been found in all living organisms and play significant roles in digestion, development and innate immunity. In this study, we present a genome-wide identification and expression profiling of SPI genes in the diamondback moth, Plutella xylostella (L.), a major pest of cruciferous crops with global distribution and broad resistance to different types of insecticides. RESULTS: A total of 61 potential SPI genes were identified in the P. xylostella genome, and these SPIs were classified into serpins, canonical inhibitors, and alpha-2-macroglobulins based on their modes of action. Sequence alignments showed that amino acid residues in the hinge region of known inhibitory serpins from other insect species were conserved in most P. xylostella serpins, suggesting that these P. xylostella serpins may be functionally active. Phylogenetic analysis confirmed that P. xylostella inhibitory serpins were clustered with known inhibitory serpins from six other insect species. More interestingly, nine serpins were highly similar to the orthologues in Manduca sexta which have been demonstrated to participate in regulating the prophenoloxidase activation cascade, an important innate immune response in insects. Of the 61 P.xylostella SPI genes, 33 were canonical SPIs containing seven types of inhibitor domains, including Kunitz, Kazal, TIL, amfpi, Antistasin, WAP and Pacifastin. Moreover, some SPIs contained additional non-inhibitor domains, including spondin_N, reeler, and other modules, which may be involved in protein-protein interactions. Gene expression profiling showed gene-differential, stage- and sex-specific expression patterns of SPIs, suggesting that SPIs may be involved in multiple physiological processes in P. xylostella. CONCLUSIONS: This is the most comprehensive investigation so far on SPI genes in P. xylostella. The characterized features and expression patterns of P. xylostella SPIs indicate that the SPI family genes may be involved in innate immunity of this species. Our findings provide valuable information for uncovering further biological roles of SPI genes in P. xylostella.