Third-Generation Covalent TMP-Tag for Fast Labeling and Multiplexed Imaging of Cellular Proteins.

Self-labeling protein tags can introduce advanced molecular motifs to specific cellular proteins. Here we introduce the third-generation covalent TMP-tag (TMP-tag3) and showcase its comparison with HaloTag and SNAP-tag. TMP-tag3 is based on a proximity-induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)-acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP-tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18-kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal N-C termini of eDHFR also enable facile insertion into various protein loops. TMP-tag3, HaloTag, and SNAP-tag are orthogonal to each other, collectively forming a toolbox for multiplexed live-cell imaging of cellular proteins under fluorescence nanoscopy.

Red- and Far-Red-Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion.

Zinc biology, featuring intertwining signaling networks and critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red- and far-red-emitting Zn2+ probes with Kd values ranging from 190 nM to 74 µM, which are particularly suitable for real-time monitoring the high concentration of Zn2+ co-released with insulin during vesicular secretory events. Compared to the prototypical rhodamine-based Zn2+ probes, the new class exploits morpholino auxochromes which eliminates phototoxicity during long-term live recording of isolated islets. A Si-rhodamine-based Zn2+ probe with high turn-on ratio (>100), whose synthesis was enabled by a new route featuring late-stage N-alkylation, allowed simultaneous recording of Ca2+ influx, mitochondrial signal, and insulin secretion in isolated mouse islets. The time-lapse multicolor fluorescence movies and their analysis, enabled by red-shifted Zn2+ and other orthogonal physiological probes, highlight the potential impact of biocompatible fluorophores on the fields of islet endocrinology and system biology.

Rh(III)-Catalyzed Redox-Neutral Unsymmetrical C-H Alkylation and Amidation Reactions of N-Phenoxyacetamides.

A Rh(III)-catalyzed unsymmetrical C-H alkylation and amidation of N-phenoxyacetamides with diazo compounds has been developed under mild and redox-neutral conditions, producing dinitrogen as the only byproduct. The reaction represents the first example of one-step, unsymmetrical difunctionalization of two ortho-C-H bonds. Experimental and computational studies support that N-phenoxyacetamides most likely undergo an initial ortho-C-H alkylation with diazo compounds via a Rh(III)-catalyzed C-H activation, and the resulting Rh(III) intermediate subsequently undergoes an intramolecular oxidative addition into the O-N bond to form a Rh(V) nitrenoid species that is protonated and further directed toward electrophilic addition to the second ortho position of the phenyl ring. This work might provide a new direction for unsymmetrical C-H difunctionalization reactions in an efficient manner.

Identification and analysis of glutathione S-transferase gene family in sweet potato reveal divergent GST-mediated networks in aboveground and underground tissues in response to abiotic stresses.

BACKGROUND: Sweet potato, a hexaploid species lacking a reference genome, is one of the most important crops in many developing countries, where abiotic stresses are a primary cause of reduction of crop yield. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in oxidative stress tolerance and cellular detoxification. RESULTS: A total of 42 putative full-length GST genes were identified from two local transcriptome databases and validated by molecular cloning and Sanger sequencing. Sequence and intraspecific phylogenetic analyses revealed extensive differentiation in their coding sequences and divided them into eight subfamilies. Interspecific phylogenetic and comparative analyses indicated that most examined GST paralogs might originate and diverge before the speciation of sweet potato. Results from large-scale RNA-seq and quantitative real-time PCR experiments exhibited extensive variation in gene-expression profiles across different tissues and varieties, which implied strong evolutionary divergence in their gene-expression regulation. Moreover, we performed five manipulated stress experiments and uncovered highly divergent stress-response patterns of sweet potato GST genes in aboveground and underground tissues. CONCLUSIONS: Our study identified a large number of sweet potato GST genes, systematically investigated their evolutionary diversification, and provides new insights into the GST-mediated stress-response mechanisms in this worldwide crop.

Cytogenetic relationships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbitaceae) as inferred from rDNA distribution patterns.

BACKGROUND: Comparative mapping of 5S and 45S rDNA by fluorescent in situ hybridization (FISH) technique is an excellent tool to determine cytogenetic relationships among closely related species. RESULTS: In this study, the number and position of 5S and 45S rDNA loci in all Citrullus species and subspecies were determined. The cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus, C. colocynthis and C. naudinianus (or Acanthosicyos naudinianus) had two 45S rDNA loci and one 5S rDNA locus which was located syntenic to one of the 45S rDNA loci. C. ecirrhosus and C. lanatus subsp. lanatus had one 45S rDNA locus and two 5S rDNA loci, each located on a different chromosome. C. rehmii had one 5S and one 45S rDNA locus positioned on different chromosomes. The distribution of 5S and 45S rDNA in several species belonging to other genera in Benincaseae tribe was also investigated. The distribution pattern of rDNAs showed a great difference among these species. CONCLUSIONS: The present study confirmed evolutionary closeness among cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus and C. colocynthis. Our result also supported that C. lanatus subsp. lanatus was not a wild form of the cultivated watermelon instead was a separate crop species. In addition, present cytogenetic analysis suggested that A. naudinianus was more closely related to Cucumis than to Citrullus or Acanthosicyos, but with a unique position and may be a link bridge between the Citrullus and the Cucumis.

Morphine preconditioning confers cardioprotection in doxorubicin-induced failing rat hearts via ERK/GSK-3ß pathway independent of PI3K/Akt.

Preconditioning against myocardial ischemia-reperfusion (I/R) injury can be suppressed in some pathological conditions. This study was designed to investigate whether morphine preconditioning (MPC) exerts cardioprotection in doxorubicin (DOX)-induced heart failure in rats and the mechanisms involved. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt), extracellular signal-regulated kinase (ERK) and glycogen synthase kinase (GSK)-3ß pathways were examined. Normal and DOX-induced failing rat hearts were subjected to I/R injury using a Langendorff perfusion system with or without MPC or ischemic preconditioning (IPC). The PI3K inhibitor (wortmannin) or ERK inhibitor (PD98059) was infused before MPC. In normal hearts, both MPC and IPC significantly reduced infarct size and the rise in lactate dehydrogenase (LDH) level caused by I/R injury. Pretreatment with wortmannin or PD98059 abrogated the protective effects of MPC and suppressed the phosphorylation of Akt, ERK and GSK-3ß. In failing rat hearts, however, MPC retained its cardioprotection while IPC did not. This protective effect was abolished by PD98059 but not wortmannin. MPC increased the level of p-ERK rather than p-Akt. The phosphorylation of GSK-3ß induced by MPC was reversed by PD98059 only. IPC did not elevate the expression of p-ERK, p-Akt and p-GSK-3ß in failing rat hearts. We conclude that MPC is cardioprotective in rats with DOX-induced heart failure while IPC is not. The effect of MPC appears to be mediated via the ERK/GSK-3ß pathway independent of PI3K/Akt.

Iridium(III)-catalyzed C-7 selective C-H alkynylation of indolines at room temperature.

An iridium-catalyzed direct C-7 selective C-H alkynylation of indolines at room temperature, for the first time, has been developed via C-H bond activation. Furthermore, the first example of direct C-H alkynylation of carbazoles at the C1 position is also achieved. More importantly, the resulting product can be readily transformed into C7-alkynylated indoles, further widening the C-7 derivatization of indoles and highlighting the synthetic utility of this methodology.

Redox-neutral rhodium-catalyzed C-H functionalization of arylamine N-oxides with diazo compounds: primary C(sp(3))-H/C(sp(2))-H activation and oxygen-atom transfer.

An unprecedented rhodium(III)-catalyzed regioselective redox-neutral annulation reaction of 1-naphthylamine N-oxides with diazo compounds was developed to afford various biologically important 1H-benzo[g]indolines. This coupling reaction proceeds under mild reaction conditions and does not require external oxidants. The only by-products are dinitrogen and water. More significantly, this reaction represents the first example of dual functiaonalization of unactivated a primary C(sp(3) )H bond and C(sp(2) )H bond with diazocarbonyl compounds. DFT calculations revealed that an intermediate iminium is most likely involved in the catalytic cycle. Moreover, a rhodium(III)-catalyzed coupling of readily available tertiary aniline N-oxides with α-diazomalonates was also developed under external oxidant-free conditions to access various aminomandelic acid derivatives by an O-atom-transfer reaction.

Rhodium(III)- and iridium(III)-catalyzed C7 alkylation of indolines with diazo compounds.

A Rh(III)-catalyzed procedure for the C7-selective C-H alkylation of various indolines with α-diazo compounds at room temperature is reported. The advantages of this process are: 1) simple, mild, and pH-neutral reaction conditions, 2) broad substrate scope, 3) complete regioselectivity, 4) no need for an external oxidant, and 5) N2 as the sole byproduct. Furthermore, alkylation and bis-alkylation of carbazoles at the C1 and C8 positions have also been developed. More significantly, for the first time, a successful Ir(III)-catalyzed intermolecular insertion of arene C-H bonds into α-diazo compounds is reported.

Inflammatory bowel disease and rheumatoid arthritis share a common genetic structure.

Background: The comorbidity rate of inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) is high; nevertheless, the reasons behind this high rate remain unclear. Their similar genetic makeup probably contributes to this comorbidity. Methods: Based on data obtained from the genome-wide association study of IBD and RA, we first assessed an overall genetic association by performing the linkage disequilibrium score regression (LDSC) analysis. Further, a local correlation analysis was performed by estimating the heritability in summary statistics. Next, the causality between the two diseases was analyzed by two-sample Mendelian randomization (MR). A genetic overlap was analyzed by the conditional/conjoint false discovery rate (cond/conjFDR) method.LDSC with specific expression of gene analysis was performed to identify related tissues between the two diseases. Finally, GWAS multi-trait analysis (MTAG) was also carried out. Results: IBD and RA are correlated at the genomic level, both overall and locally. The MR results suggested that IBD induced RA. We identified 20 shared loci between IBD and RA on the basis of a conjFDR of <0.01. Additionally, we identified two tissues, namely spleen and small intestine terminal ileum, which were commonly associated with both IBD and RA. Conclusion: Herein, we proved the presence of a polygenic overlap between the genetic makeup of IBD and RA and provided new insights into the genetic architecture and mechanisms underlying the high comorbidity between these two diseases.

Readily releasable ß cells with tight Ca2+-exocytosis coupling dictate biphasic glucose-stimulated insulin secretion.

Biphasic glucose-stimulated insulin secretion (GSIS) is essential for blood glucose regulation, but a mechanistic model incorporating the recently identified islet ß cell heterogeneity remains elusive. Here, we show that insulin secretion is spatially and dynamically heterogeneous across the islet. Using a zinc-based fluorophore with spinning-disc confocal microscopy, we reveal that approximately 40% of islet cells, which we call readily releasable ß cells (RRßs), are responsible for 80% of insulin exocytosis events. Although glucose up to 18.2 mM fully mobilized RRßs to release insulin synchronously (first phase), even higher glucose concentrations enhanced the sustained secretion from these cells (second phase). Release-incompetent ß cells show similarities to RRßs in glucose-evoked Ca2+ transients but exhibit Ca2+-exocytosis coupling deficiency. A decreased number of RRßs and their altered secretory ability are associated with impaired GSIS progression in ob/ob mice. Our data reveal functional heterogeneity at the level of exocytosis among ß cells and identify RRßs as a subpopulation of ß cells that make a disproportionally large contribution to biphasic GSIS from mouse islets.

Survival Predictors of Resectable Gallbladder Carcinoma: An Analysis of the Surveillance, Epidemiology, and End Results Database.

BACKGROUND: To analyze population-level data for resectable gallbladder carcinoma (GBC) according to the eighth edition of the American Joint Committee on Cancer staging system. METHODS: We queried the Surveillance, Epidemiology, and End Results database to identify all patients aged 18 years or older with T1-3 M0 GBC diagnosed between 2004 and 2015. Multivariate cox hazard regression analysis was used to identify prognostic factors of cancer-specific survival (CSS). RESULTS: Of the 1601 eligible patients, 1310 (81.8%) underwent cholecystectomy only and 291 (18.2%) underwent an en bloc resection. Overall, 219 (13.7%) patients were in stage I, 400 (25%) were in stage II, 260 (16.2%) were in stage IIIA, 653 (40.8%) were in stage IIIB, and 69 (4.3%) were in stage IVB. The 5-year survival rates for patients were 82.7% for stage I, 73.4% for stage II, 31.9% for stage IIIA, 24.1% for stage IIIB, and 10% for stage IVB. Multivariate cox analysis indicated that predictors of decreased CSS included age at diagnosis >65 years, tumor size >3.2 cm, adenocarcinoma, increasing tumor spread, and lymph node involvement. Besides, chemotherapy and radiation were predictors of increased CSS. CONCLUSIONS: Older age, increasing tumor size, adenocarcinoma, and advanced tumor/node stage were associated with a poorer prognosis after resection for GBC. Furthermore, patients with resectable GBC can benefit from adjuvant therapy.

Genetically predicted levels of circulating cytokines and the risk of six immune skin diseases: a two-sample Mendelian randomization study.

Background: Circulating cytokines play a crucial role in the onset and progression of immune skin diseases. However, the causal relationships and the direction of causal effects require further investigation. Methods: Two-sample Mendelian randomization (MR) analyses were conducted to assess the causal relationships between 41 circulating cytokines and six immune skin diseases including alopecia areata, chloasma, hidradenitis suppurativa (HS), lichen planus (LP), seborrheic dermatitis, and urticaria, using summary statistics from genome-wide association studies. Reverse MR analyses was performed to test for the reverse causation. Pleiotropy and heterogeneity tests were conducted to assess the robustness of the findings. Results: Twelve unique cytokines showed a suggestive causal relationship with the risk of six immune skin diseases. Among them, the causal effects between 9 unique cytokines and immune skin diseases have strong statistical power. Additionally, the concentrations of six cytokines might be influenced by LP and urticaria. After Bonferroni correction, the following associations remained significant: the causal effect of beta-nerve growth factor on HS (odds ratio [OR] = 1.634, 95% confidence interval [CI] = 1.226-2.177, p = 7.97e-04), interleukin (IL)-6 on LP (OR = 0.615, 95% CI = 0.481-0.786, p = 1.04e-04), IL-4 on LP (OR = 1.099. 95% CI = 1.020-1.184, p = 1.26e-02), and IL-2 on urticaria (OR = 0.712, 95% CI = 0.531-0.955, p = 2.33e-02). Conclusion: This study provides novel perspectives on the relationship between circulating cytokines and immune skin diseases, potentially providing valuable insights into their etiology, diagnostic approaches, and treatment.

Rhodium(III)-Catalyzed Selective C-H Acetoxylation and Hydroxylation Reactions.

An efficient Cp*Rh(III)-catalyzed, chelation-assisted C(sp2)-H acetoxylation and hydroxylation reaction has been developed for the first time. The reaction proceeds under mild conditions and allows for selective preparation of C-H acetoxylation and hydroxylation products, thus providing a good complement to previous C-H oxygenation reactions and expanding the field of Cp*Rh(III)-catalyzed C-H functionalizations.

Rhodium(iii)-catalyzed alkylation of primary C(sp(3))-H bonds with α-diazocarbonyl compounds.

Rh(iii)-catalyzed intermolecular chelation-assisted insertion of carbenes derived from α-diazocarbonyl compounds into non-acidic primary sp(3) C-H bonds, for the first time, is reported under mild reaction conditions, thus affording a good complement to previous metal-carbenoid-induced primary C(sp(3))-H insertion reactions. We believe that this method will open up a new avenue for primary sp(3) C-H functionalization with α-diazocarbonyl compounds.

Rh(III)- or Ir(III)-catalyzed ynone synthesis from aldehydes via chelation-assisted C-H bond activation.

A Rh(III)- or Ir(III)-catalyzed direct aldehyde C-H alkynylation was developed for the first time as a simple and practical method for the synthesis of ynones. This catalytic reaction proceeds under mild reaction conditions and tolerates a variety of synthetically important functional groups (e.g., chloro, bromo, aldehyde), thus providing a good complement to previous methods.