Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45 000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Design, Synthesis, Bioactive Evaluation, and Molecular Dynamics Simulation of Novel 4H-Pyrano[3,2-c]pyridine Analogues as Potential Sterol 14α-Demethylase (CYP51) Inhibitors.

To discover potential sterol 14α-demethylase (CYP51) inhibitors, thirty-four unreported 4H-pyrano[3,2-c]pyridine derivatives were designed and synthesized. The assay results indicated that most compounds displayed significant fungicidal activity against Sclerotinia sclerotiorum, Colletotrichum lagenarium, Botrytis cinerea, Penicillium digitatum, and Fusarium oxysporum at 16 µg/mL. The half maximal effective concentration (EC50) values of compounds 7a, 7b, and 7f against B. cinerea were 0.326, 0.530, and 0.610, respectively. Namely, they had better antifungal activity than epoxiconazole (EC50 = 0.670 µg/mL). Meanwhile, their half maximal inhibitory concentration (IC50) values against CYP51 were 0.377, 0.611, and 0.748 µg/mL, respectively, representing that they also possessed better inhibitory activities than epoxiconazole (IC50 = 0.802 µg/mL). The fluorescent quenching tests of proteins showed that 7a and 7b had similar quenching patterns to epoxiconazole. The molecular dynamics simulations indicated that the binding free energy of 7a and epoxiconazole to CYP51 was -35.4 and -27.6 kcal/mol, respectively.

Emerging Chemistry for Wide-Temperature Sodium-Ion Batteries.

The shortage of resources such as lithium and cobalt has promoted the development of novel battery systems with low cost, abundance, high performance, and efficient environmental adaptability. Due to the abundance and low cost of sodium, sodium-ion battery chemistry has drawn worldwide attention in energy storage systems. It is widely considered that wide-temperature tolerance sodium-ion batteries (WT-SIBs) can be rapidly developed due to their unique electrochemical and chemical properties. However, WT-SIBs, especially for their electrode materials and electrolyte systems, still face various challenges in harsh-temperature conditions. In this review, we focus on the achievements, failure mechanisms, fundamental chemistry, and scientific challenges of WT-SIBs. The insights of their design principles, current research, and safety issues are presented. Moreover, the possible future research directions on the battery materials for WT-SIBs are deeply discussed. Progress toward a comprehensive understanding of the emerging chemistry for WT-SIBs comprehensively discussed in this review will accelerate the practical applications of wide-temperature tolerance rechargeable batteries.

Design, Synthesis, Antifungal Activity, and Molecular Docking Studies of Novel Chiral Isoxazoline-Benzofuran-Sulfonamide Derivatives.

Succinate dehydrogenase (SDH) is one of the most important molecular targets for the development of novel fungicides. With the emerging problem of resistance in plant fungal pathogens, novel compounds with high fungicidal activity need to be developed, but the study of chiral pesticides for the inhibition of highly destructive plant pathogens has been rarely reported in recent years. Therefore, a series of novel chiral isoxazoline-benzofuran-sulfonamide derivatives were designed to investigate potential novel antifungal molecules. The chiral target compound 3a was cultured as a single crystal and confirmed using X-ray diffraction. All the target compounds were tested for antifungal activity, and compounds 3c, 3i, 3s, and 3r were found to have significant antifungal effects against S. sclerotiorum with EC50 values of 0.42 mg/L, 0.33 mg/L, 0.37 mg/L, and 0.40 mg/L, respectively, which were superior to the commercial fungicide fluopyram (EC50 = 0.47 mg/L). The IC50 value of compound 3i against the SDH of S. sclerotiorum was 0.63 mg/mL, which was further demonstrated by enzyme activity assays. Scanning electron microscopy showed that 3i had a significant inhibitory effect on S. sclerotiorum. In addition, the fluorescence quenching analysis assay indicated that compound 3i had a similar effect with the positive control fluopyram. Molecular docking exhibited that target compounds with chiral configuration had better affinity than racemic configuration, and 3i possessed stronger action than fluopyram, which was in keeping with the in vitro test results. These results would provide a basis and reference for the development of novel chiral fungicides.

Theoretical Investigation on Dialumenes toward Dihydrogen Activation: Mechanism and Ligand Effect.

Herein, we report a computation study based on the density functional theory calculations to understand the mechanism and ligand effect of the base-stabilized dialumenes toward dihydrogen activation. Among all of the examined modes of dihydrogen activation using the base-stabilized dialumene, we found that the concerted 1,2-hydrogenation of the Al═Al double bond is kinetically more preferable. The concerted 1,2-hydrogenation of the Al═Al double bond adopts an electron-transfer model with certain asynchrony. That is, the initial electron donation from the H-H σ bonding orbital to the empty 3p orbital of the Al1 center is followed by the backdonation from the lone pair electron of the Al2 center to the H-H σ antibonding orbital. Combined with the energy decomposition analysis on the transition states of the concerted 1,2-hydrogenation of the Al═Al double bond and the topographic steric mapping analysis on the free dialumenes, we ascribe the higher reactivity of the aryl-substituted dialumene over the silyl-substituted analogue in dihydrogen activation to the stronger electron-withdrawing effect of the aryl group, which not only increases the flexibility of the Al═Al double bond but also enhances the Lewis acidity of the Al═Al core. Consequently, the aryl-substituted dialumene fragment suffers less geometric deformation, and the orbital interactions between the dialumene and dihydrogen moieties are more attractive during the 1,2-hydrogenation process. Moreover, our calculations also predict that the Al═Al double bond has a good tolerance with the stronger electron-withdrawing group (-CF3) and the weaker σ-donating N-heterocyclic carbene (NHC) analogue (e.g., triazol carbene and NHSi). The reactivity of the dialumene in dihydrogen activation can be further improved by introducing these groups as the supporting ligand and the stabilizing base on the Al═Al core, respectively.

[Changes in mild cognitive impairment and sociodemographic disparity among adults aged 55 years and above in 4 provinces of China].

OBJECTIVE: To investigate the changes in mild cognitive impairment(MCI) and sociodemographic disparity among adults aged 55 years and above in 4 provinces of China. METHODS: A total of 4687 adults aged 55 years and above from Community-based Cohort Study on Nervous System Disease who did not have Alzheimer's disease, participated in both rounds of the survey, and had complete baseline sociodemographic data and two rounds of data on cognitive function were selected. Generalized estimation equations were used to analyse the effect of sociodemographic factors on MCI. RESULTS: The detection rates of MCI in adults aged 55 years and above without Alzheimer's disease in 4 provinces of China in 2018 and 2020 were 48.56% and 42.56% respectively. MCI occurred in 30.11% of those with normal cognition(NC) at baseline, and 44.24% of those with MCI at baseline reverted to NC. The risk of MCI increased and the likelihood of MCI reversion decreased with increasing age and decreasing per capita monthly household income. In the baseline NC population, the risk of MCI in the junior high school and above group was 35% lower than that in the illiterate group(RR=0.65, 95%CI 0.53-0.80), the risk of MCI was lower in those living in rural areas(RR=0.56, 95%CI 0.49-0.65), and the risk of MCI was 1.17 times(95%CI 1.03-1.32) higher in those with a history of chronic diseases than in those without it. In the baseline MCI population, the likelihood of MCI reversion increased with education, the likelihood of MCI reversion was 1.04 times higher for workers than for non-workers(95%CI 1.00-1.08). CONCLUSION: The incidence and reversal rates of MCI were high in adults aged ≥55 years in four provinces of China. Advanced age, low education and low income level are risk factors for cognitive dysfunction.

High glucose intake exacerbates experimental autoimmune prostatitis through mitochondrial reactive oxygen species-dependent TGF-ß activation-mediated Th17 differentiation.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common inflammatory immune disease of the urogenital system. High glucose intake is considered to be a potential promoter of autoimmune diseases. However, the influence of high glucose intake on CP/CPPS is unknown. This research aimed to explore the influences of high glucose intake on experimental autoimmune prostatitis (EAP), a valid animal model of CP/CPPS, and the underlying mechanism. NOD mice received 20% glucose water or normal water treatment during EAP induction. EAP severity and Th17 cell responses were evaluated. Then, we explored the effects of an IL-17A neutralizing antibody, an inhibitor of TGF-ß, the reactive oxygen species (ROS) inhibitor NAC, and the mitochondrial ROS (mtROS) antioxidant MitoQ on glucose-fed EAP mice. The results demonstrated that high glucose intake aggravated EAP severity and promoted Th17 cell generation, which could be ameliorated by the neutralization of IL-17A. In vitro experiments showed that high dextrose concentrations promoted Th17 cell differentiation through mtROS-dependent TGF-ß activation. Treatment with TGF-ß blockade, NAC, or MitoQ suppressed Th17 cell generation both in vivo and in vitro, resulting in the amelioration of EAP manifestations caused by high glucose intake. This study revealed that high glucose intake exacerbates EAP through mtROS-dependent TGF-ß activation-mediated Th17 differentiation. Our results may provide insights into the molecular mechanisms underlying the detrimental effects of an environmental factor, such as high glucose intake, on CP/CPPS.

Protective effect of Tetrandrine on optic nerve by inhibiting glial activation through NF-κB pathway.

Introduction: This study aimed to explore the effect and molecular mechanism of Tetrandrine (Tet) onlipopolysaccharide (LPS)-induceduveitis andoptic nerve injury in vivo and in vitro. Methods: Uveitis was induced by LPS injected into the hindlimb foot pad of Wistar rats and was intervened by retroeyeball injection of Tet (100 nM, 1 µM or 10 µM).The anterior segment inflammation was observed by slit lamp. Tunelassay was used to detect the survival state of ganglion cells and nuclear layers of inner and outer. The detection of characteristic markers in different activation states of glial cells were performed by qualitative and quantitative test of immunofluorescence and western blotting. Also, western blotting was used to detect the expression of inflammatory factors in retina and the activation of nuclear factor kappa B (NF-κB) signal pathway. Meanwhile, routine blood test and function of liver and renal were performed. Results: The ciliary hyperemia was obvious, and the iris vessels were dilated and tortuous in rats with LPS-induced uveitis. Tet-pretreated obviously elieved these symptoms. In addition, the dilation and hyperemia in Tet group were alleviated compared with LPS group, and the inflammatory scores in Tetgroup were significantly lower than those of LPS group. TUNEL Staining showed that the number ofretinal ganglion cell (RGCs) in Tetgroup was slightly less than that in normal group, but significantly more than that in LPS group, and the cells arranged orderly. Besides, the number of apoptotic cells was significantly less than that in LPS group. Tet reduced LPS-activated gliocyte in a dose-dependent manner. Tumour necrosis factor alpha (TNF-α), interleukin (IL)-1ß, interferon gamma (γ-IFN) and IL-2 in retina were increased by LPS but decreased significantly viaTet-pretreatment. Moreover, LPS activate NF-κB signal pathway, while Tet efficiently inhibited this effect.Furthermore, injection of Tet did not damage theroutineblood, liver and kidney. Conclusions: Retrobulbar injection of Tet significantly alleviatedLPS-induced uveitisand optic nerve injuryof rats by activating gliocyte and NF-κB signaling pathway.

The predictive value of peripheral blood CD4 cells ATP concentration for immune-related adverse events in advanced non-small cell lung cancer patients.

OBJECTIVE: Lung cancer with the highest incidence and mortality in the world. Immune checkpoint inhibitors (ICIs), can bring long-term survival benefits to patients, but also can bring immune-related adverse events (irAEs) in some patients during therapy. Therefore, the aim of this study was to investigate the predictive effect of peripheral blood WBC, NLR, sATPCD4 and nATPCD4 on irAEs in advanced non-small cell lung cancer (NSCLC). METHODS: Clinical data of 112 patients with advanced NSCLC who were treated with PD -1/PD -L1 inhibitor in the Fifth Affiliated Hospital of Guangzhou Medical University from December 15, 2019 to April 30, 2023 were retrospectively analyzed. These patients were divided into the irAEs group (n = 27) and non-irAEs group (n = 85). The clinical data of the two groups were compared. Receiver operating characteristic (ROC) curves were drawn to determine the threshold value of baseline peripheral blood parameters to predict the occurrence of irAEs. Multivariate logistic regression analysis was used to explore the relationship between peripheral blood markers and the incidence of irAEs. RESULTS: The patient characteristics have no significant difference between irAEs and non-irAEs group. But the baseline peripheral blood WBC, sATPCD4 and nATPCD4 of patients in the irAEs group were higher than those in the non-irAEs group (p < 0.05), and the NLR in irAEs group was similar to in the non-irAEs group (p = 0.639).Univariate analysis showed that high WBC, sATPCD4 and nATPCD4 may the risk factors for the occurrence of irAEs (p < 0.05). Multivariate logistic regression analysis showed that high sATPCD4 and nATPCD4 were independent risk factors for the occurrence of irAEs (p < 0.05). The best critical values of WBC, sATPCD4 and nATPCD4 before treatment for predicting the occurrence of irAEs were 8.165 × 109cells/L (AUC = 0.705) ,484.5 ng/mL (AUC = 0.777), and 156 ng/mL (AUC = 0.840), respectively. CONCLUSIONS: sATPCD4 and nATPCD4 were independent risk factors for the occurrence of irAEs in advanced NSCLC patients. This discovery provides a new method to predict the occurrence of irAEs in patients. Based on the prediction results, corresponding treatment measures can be taken to reduce the incidence of adverse events.

Injectable hydrogels embedded with chitosan nanoparticles coated with hyaluronic acid for sequential release of dual drugs.

An effective treatment for some disease, such as the model disease acute retinal necrosis (ARN), requires a combination of different drugs which should be administered at a certain interval. The precise sequential and long-term drug release are the critical questions. In this work, the as-prepared chitosan nanoparticles (CS-NPs) coated with hyaluronic acid (HA) were embedded in the aldehyde ß-cyclodextrin (ACD)/aminated hyaluronic acid (NHA) hydrogels to synthesize injectable hydrogels loaded with dual drugs named DEX-CS-NPs/GCV-Gel and HA-DEX-CS-NPs/GCV-Gel. In the first 24 h and 48 h, the releases of DEX from DEX-CS-NPs/GCV-Gel were 128.5 % and 82.8 % faster than those from HA-DEX-CS-NPs/GCV-Gel, respectively. There was no DEX released from HA-DEX-CS-NPs/GCV-Gel at the first 7 h, which has never been reported before, although some hydrogel systems loaded with different drugs release different drugs simultaneously at different rate which have been well studied. This is a good start of a precise sequence release. The composite hydrogels possessed appropriate rheology, gel time, degradation performance, and ideal cytocompatibility. The injectable hydrogel loaded with dual drugs presenting a precise sequential and long-term release has great potential in the treatment of diseases requiring combinations of drugs being released sequentially at different treating stages.

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway.

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments in vitro and in vivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.

Soil microorganisms and methane emissions in response to short-term warming field incubation in Svalbard.

Introduction: Global warming is caused by greenhouse gases (GHGs). It has been found that the release of methane (CH4) from Arctic permafrost, soil, ocean, and sediment is closely related to microbial composition and soil factors resulting from warming over several months or years. However, it is unclear for how long continuous warming due to global warming affects the microbial composition and GHG release from soils along Arctic glacial meltwater rivers. Methods: In this study, the soil upstream of the glacial meltwater river (GR) and the estuary (GR-0) in Svalbard, with strong soil heterogeneity, was subjected to short-term field incubation at 2°C (in situ temperature), 10°C, and 20°C. The incubation was carried out under anoxic conditions and lasted for few days. Bacterial composition and CH4 production potential were determined based on high-throughput sequencing and physiochemical property measurements. Results: Our results showed no significant differences in bacterial 16S rRNA gene copy number, bacterial composition, and methanogenic potential, as measured by mcrA gene copy number and CH4 concentration, during a 7- and 13-day warming field incubation with increasing temperatures, respectively. The CH4 concentration at the GR site was higher than that at the GR-0 site, while the mcrA gene was lower at the GR site than that at the GR-0 site. Discussion: Based on the warming field incubation, our results indicate that short-term warming, which is measured in days, affects soil microbial composition and CH4 concentration less than the spatial scale, highlighting the importance of warming time in influencing CH4 release from soil. In summary, our research implied that microbial composition and CH4 emissions in soil warming do not increase in the first several days, but site specificity is more important. However, emissions will gradually increase first and then decrease as warming time increases over the long term. These results are important for understanding and exploring the GHG emission fluxes of high-latitude ecosystems under global warming.

An integrative protocol for one-step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data.

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a "one-step PCR approach" has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00182-1.

BLCA prognostic model creation and validation based on immune gene-metabolic gene combination.

BACKGROUND: Bladder cancer (BLCA) is a prevalent urinary system malignancy. Understanding the interplay of immunological and metabolic genes in BLCA is crucial for prognosis and treatment. METHODS: Immune/metabolism genes were extracted, their expression profiles analyzed. NMF clustering found prognostic genes. Immunocyte infiltration and tumor microenvironment were examined. Risk prognostic signature using Cox/LASSO methods was developed. Immunological Microenvironment and functional enrichment analysis explored. Immunotherapy response and somatic mutations evaluated. RT-qPCR validated gene expression. RESULTS: We investigated these genes in 614 BLCA samples, identifying relevant prognostic genes. We developed a predictive feature and signature comprising 7 genes (POLE2, AHNAK, SHMT2, NR2F1, TFRC, OAS1, CHKB). This immune and metabolism-related gene (IMRG) signature showed superior predictive performance across multiple datasets and was independent of clinical indicators. Immunotherapy response and immune cell infiltration correlated with the risk score. Functional enrichment analysis revealed distinct biological pathways between low- and high-risk groups. The signature demonstrated higher prediction accuracy than other signatures. qRT-PCR confirmed differential gene expression and immunotherapy response. CONCLUSIONS: The model in our work is a novel assessment tool to measure immunotherapy's effectiveness and anticipate BLCA patients' prognosis, offering new avenues for immunological biomarkers and targeted treatments.

Advances in diagnosis and prediction for aggression of pure solid T1 lung cancer.

A growing number of early-stage lung cancers presenting as malignant pulmonary nodules have been diagnosed because of the increased adoption of low-dose spiral computed tomography. But pure solid T1 lung cancer with ≤3 cm in the greatest dimension is not always at an early stage, despite its small size. This type of cancer can be highly aggressive and is associated with pathological involvement, metastasis, postoperative relapse, and even death. However, it is easily misdiagnosed or delay diagnosed in clinics and thus poses a serious threat to human health. The percentage of nodal or extrathoracic metastases has been reported to be >20% in T1 lung cancer. As such, understanding and identifying the aggressive characteristics of pure solid T1 lung cancer is crucial for prevention, diagnosis, and therapeutic strategies, and beneficial to improving the prognosis. With the widespread of lung cancer screening, these highly invasive pure solid T1 lung cancer will become the main advanced lung cancer in future. However, there is limited information regarding precision medicine on how to identify these "early-stage" aggressive lung cancers. To provide clinicians with new insights into early recognition and intervention of the highly invasive pure solid T1 lung cancer, this review summarizes its clinical characteristics, imaging, pathology, gene alterations, immune microenvironment, multi-omics, and current techniques for diagnosis and prediction.

Multiple Trajectories of Cereal Consumption and Their Associations with Type 2 Diabetes in Chinese Adults - China, 1997-2018.

What is already known about this topic?: Consuming refined grains, specifically white rice, elevates the risk of developing type 2 diabetes (T2D). Conversely, incorporating whole grains into the diet is linked to a reduced risk. What is added by this report?: This study employed a novel multi-trajectory modeling technique to account for the intercorrelations among various cereal consumption patterns. Four distinct multi-trajectory groups of cereal intake, identified from 1997 to 2018 within the Chinese population, were associated with varying levels of T2D risk. What are the implications for public health practice?: This research investigates the implications of evolving cereal consumption patterns on T2D in nondiabetic adults. This study delineates unique trajectories linked with cereal intake patterns, thereby providing a robust foundation for policymakers to craft initiatives to prevent T2D among adults in China.

Genomics evolution of Jingmen viruses associated with ticks and vertebrates.

Jingmen virus (JMV) associated with ticks and vertebrates have been found to be related to human disease. We obtained the genome of a Jingmen tick virus (JMTV) strain from Rhipicephalus microplus in Guizhou province and compared the genomes of seven JMV species associated with ticks and vertebrates to understand the evolutionary relationships. The topology of the phylogenetic tree of segment 1 and segment 3 is similar, and segment 2 and segment 4 formed two different topologies, with the main differences being between Alongshan virus (ALSV), Takachi virus, Yanggou tick virus and Pteropus lylei jingmen virus (PLJV), and the possibility of genetic reassortment among these viruses. Moreover, we detected recombination within JMTV and between PLJV and ALSV. The genetic reassortment and recombination that occurs during cross-species transmission of these JMV associated with ticks and vertebrates not only complicates their evolutionary relationships, but also raises the risk of these viruses to humans.

Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Role of SLC5A2 polymorphisms and effects of genetic polymorphism on sodium glucose cotransporter 2 inhibitorsinhibitor response.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease characterized by hyperglycaemia. T2DM is a highly heterogeneous polygenic disease. Due to genetic variation, variations in lifestyle and other environmental exposures, there are certain variations in the phenotype of T2DM patients. Sodium glucose cotransporter 2 (SGLT2) inhibitors are novel hypoglycaemic agents that increase urinary glucose excretion by inhibiting glucose reabsorption in the proximal tubules of the kidney. For glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors, studies have confirmed a variety of gene variants that may modify their effects. For SGLT2 inhibitors, research has focused on the SLC5A2 gene encoding SGLT2 and UGT1A9 gene polymorphisms affecting SGLT2 inhibitor metabolism. The SLC5A2 polymorphism rs9934336 have been associated with decreased HbA1c during the oral glucose tolerance test. Common variants of the SLC5A2 gene are related to blood glucose and insulin concentrations, but not glucagon concentrations. SLC5A2 rs9934336 and rs3116150 are related to a lower risk of heart failure. SGLT2 inhibitor exposure of UGT1A9*3 carriers is commonly higher than that of noncarriers, while these effects commonly have no obvious clinical significance on SGLT2 inhibitor pharmacokinetics. In terms of efficacy, general SLC5A2 variants show no significant effect on the response to the SGLT2 inhibitor empagliflozin. At present, research on the relationship between genetic polymorphisms and the efficacy of SGLT2 inhibitors is limited. The main purpose of this review is to elucidate the general effects of SGLT2 polymorphisms and the association between polymorphisms and the treatment response to SGLT2 inhibitors.

Molecular engineering and bioimaging applications of C2-alkenyl indole dyes with tunable emission wavelengths covering visible to NIR light.

As an important member of dyes, small-molecule fluorescent dyes show indispensable value in biomedical fields. Although various molecular dyes have been developed, full-color dyes covering blue to red region derived from a single chromophore are still in urgent demand. In this work, a series of dyes based on C2-alkenyl indole skeleton were synthesized, namely AI dyes, and their photophysical properties, cytotoxicity, and imaging capacity were verified to be satisfactory. Particularly, the maximal emission wavelengths of these dyes could cover a wide range from visible to NIR light with large Stokes shifts. Besides, the optical and structural discrepancies between the C2- and C3- alkenyl AI dyes were discussed in detail, and the theoretical calculations were conducted to provide insights on such structure-activity relationship. Finally, as a proof-of-concept, a fluorescent probe AI-Py-B capable of imaging endogenous ONOO- was presented, demonstrating the bioimaging potentials of these alkenyl indole dyes. This work is anticipated to open up new possibilities for developing dye engineering and bio-applications of natural indole framework.