pH-Controlled Chemoselective Rapid Azo-Coupling Reaction (CRACR) Enables Global Profiling of Serotonylation Proteome in Cancer Cells.

Serotonylation has been identified as a novel protein posttranslational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM, and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction. Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

pH-Controlled chemoselective rapid azo-coupling reaction (CRACR) enables global profiling of serotonylation proteome in cancer cells.

Serotonylation has been identified as a novel protein post-translational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Chemical proteomic profiling of protein dopaminylation in colorectal cancer cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid residue of H3 ( i.e. , glutamine) and dopamine. In our previous studies, we discovered that the dynamics of this post-translational modification (including installation, removal, and replacement) were regulated by a single enzyme, transglutaminase 2 (TGM2), through reversible transamination. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of TGM2 in colon cancer cells. Due to the enzyme promiscuity of TGM2, non-histone proteins have also been identified as targets of dopaminylation on glutamine residues, however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of modification sites of these dopaminylated proteins were identified, attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that to block the installation of protein dopaminylation may become a promising anti-cancer strategy.

Bioorthogonal labeling and enrichment of histone monoaminylation reveal its accumulation and regulatory function in cancer cell chromatin.

Histone monoaminylation ( i . e ., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

The human microbiome and cancer: a diagnostic and therapeutic perspective.

Recent evidence has shown that the human microbiome is associated with various diseases, including cancer. The salivary microbiome, fecal microbiome, and circulating microbial DNA in blood plasma have all been used experimentally as diagnostic biomarkers for many types of cancer. The microbiomes present within local tissue, other regions, and tumors themselves have been shown to promote and restrict the development and progression of cancer, most often by affecting cancer cells or the host immune system. These microbes have also been shown to impact the efficacy of various cancer therapies, including radiation, chemotherapy, and immunotherapy. Here, we review the research advances focused on how microbes impact these different facets and why they are important to the clinical care of cancer. It is only by better understanding the roles these microbes play in the diagnosis, development, progression, and treatment of cancer, that we will be able to catch and treat cancer early.

Engineering the Substrate Specificity of a P450 Dimerase Enables the Collective Biosynthesis of Heterodimeric Tryptophan-Containing Diketopiperazines.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in NasF5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively.

Production of Heterodimeric Diketopiperazines Employing a Mycobacterium-Based Whole-Cell Biocatalysis System.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. P450-mediated biocatalysis offers a practical avenue to access their structural diversity; however, many of these enzymes are insoluble in Escherichia coli and difficult to operate in Streptomyces. Through validation of the functions of two pairs Mycobacterium smegmatis sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that M. smegmatis is much more efficient, robust, and cleaner in metabolites background than the regularly used E. coli or Streptomyces systems. The M. smegmatis-based system can completely convert 1 g L-1 of cyclodipeptide into HTDKPs within 18 h with minimal background metabolites. On the basis of this efficient system, 12 novel HTDPKs were readily obtained by using two HTDKP-forming P450s (NasbB and NASS1868). Among them, five compounds have neuroprotective properties. Our study significantly expands the bioactive chemical scope of HTDKPs and provides an excellent biocatalysis platform for dealing with problematic enzymes from Actinomycetes.

A Dual Role Reductase from Phytosterols Catabolism Enables the Efficient Production of Valuable Steroid Precursors.

4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.

Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines.

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70-1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.

Exploring the Pathological Role of Collagen in Paravertebral Muscle in the Progression of Idiopathic Scoliosis.

BACKGROUND: Paravertebral muscle (PVM) is considered as a contributing factor of idiopathic scoliosis (IS); collagen is crucial for maintaining the mechanical properties of PVM, but only a few researches have described this field. In this study, we observed the muscle stiffness of PVM and the curvature of the spine by adjusting the content of collagen in PVM of rats and explored the role of collagen in the progression of IS. METHODS: 32 female Sprague Dawley rats were randomly divided into four groups: neutralizing antibody (NA) group (group 1), normal control group (group 2), IS group (group 3), and IS with NA group (group 4). TGF-ß1 NA was injected into PVM in group 1 and group 4, while Normal saline in group 2 and group 3. The Cobb angle and muscle stiffness were measured before and after injection; the rats were sacrificed at one week after injection, and performed histological, Western Blot, and qRT-PCR examinations. RESULTS: X-rays showed that scoliosis occurred in group 1 and relieved in group 4. The stiffness of PVM was decreased significantly on the convex side in group 1, while on the concave side in group 4. The expression of TGF-ß1 and COL1 on the concave side in IS rats (group 3) was significantly increased than that in normal rats (group 2), the concentration of COL1 and COL3 in group 3 was significantly higher than that in group 2, and the addition of TGF-ß1 NA significantly downregulated COL1 and COL3 in group 1 and group 4. The concentration of COL1 in convex PVM was negatively related to Cobb angle in group 1 and group 2, and in concave PVM was positively related to Cobb angle in group 3 and group 4. However, no significant correlation was found between COL3 and Cobb angle in group 3 and group 4. CONCLUSIONS: Asymmetric biomechanical characteristics of PVM was an important etiological factor of IS, which was directly correlated with collagen, it could be adjusted by local intramuscular injecting of TGF-ß1 NA, and finally had an effect on the shape of the spine.

Efficient biosynthesis of heterodimeric C3-aryl pyrroloindoline alkaloids.

Many natural products contain the hexahydropyrrolo[2, 3-b]indole (HPI) framework. HPI containing chemicals exhibit various biological activities and distinguishable structural arrangement. This structural complexity renders chemical synthesis very challenging. Here, through investigating the biosynthesis of a naturally occurring C3-aryl HPI, naseseazine C (NAS-C), we identify a P450 enzyme (NascB) and reveal that NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon-carbon bonds with both regio- and stereo-specificity. Surprisingly, the limited freedom is allowed in specificity to generate four types of C3-aryl HPI scaffolds, and two of them were not previously observed. By incorporating NascB into an engineered strain of E. coli, we develop a whole-cell biocatalysis system for efficient production of NAS-C and 30 NAS analogs. Interestingly, we find that some of these analogs exhibit potent neuroprotective properties. Thus, our biocatalytic methodology offers an efficient and simple route to generate difficult HPI framework containing chemicals.

Deciphering Piperidine Formation in Polyketide-Derived Indolizidines Reveals a Thioester Reduction, Transamination, and Unusual Imine Reduction Process.

Piperidine and indolizidine are two basic units of alkaloids that are frequently observed in natural and synthetic compounds. Their biosynthesis in natural products is highly conserved and mostly derived from the incorporation of lysine cyclization products. Through in vitro reconstitution, we herein identified a novel pathway involving a group of polyketide-derived indolizidines, which comprises the processes of tandem two-electron thioester reduction, transamination, and imine reduction to convert acyl carrier protein (ACP)-tethered polyketide chains into the piperidine moieties of their indolizidine scaffolds. The enzymes that catalyze the imine reduction are distinct from previous known imine reductases, which have a fold of acyl-CoA dehydrogenase but do not require flavin for reduction. Our results not only provide a new way for the biosynthesis of the basic units of alkaloids but also show a novel class of imine reductases that may benefit the fields of biocatalysis and biomanufacturing.

A statistical method for characterizing the noise in nonlinearly reconstructed images from undersampled MR data: the POCS example.

The projection-onto-convex-sets (POCS) algorithm is a powerful tool for reconstructing high-resolution images from undersampled k-space data. It is a nonlinear iterative method that attempts to estimate values for missing data. The convergence of the algorithm and its other deterministic properties are well established, but relatively little is known about how noise in the source data influences noise in the final reconstructed image. In this paper, we present an experimental treatment of the statistical properties in POCS and investigate 12 stochastic models for its noise distribution beside its nonlinear point spread functions. Statistical results show that as the ratio of the missing k-space data increases, the noise distribution in POCS images is no longer Rayleigh as with conventional linear Fourier reconstruction. Instead, the probability density function for the noise is well approximated by a lognormal distribution. For small missing data ratios, however, the noise remains Rayleigh distributed. Preliminary results show that in the presence of noise, POCS images are often dominated by POCS-enhanced noise rather than POCS-induced artifacts. Implicit in this work is the presentation of a general statistical method that can be used to assess the noise properties in other nonlinear reconstruction algorithms.

[Efficacy of acupuncture in treatment of cancer pain: a systematic review].

BACKGROUND: Although acupuncture is a well-established treatment for cancer pain and its effects have been widely reported in recent two decades, there is still controversy over whether its efficacy is better than placebo. OBJECTIVE: To evaluate the efficacy of acupuncture therapy on cancer pain. SEARCH STRATEGY: Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 3, 2008), EMBASE, PubMed, ScienceDirect database, Current Controlled Trials, Chongqin VIP database and CNKI database were searched, and the search date ended in June 2008. The authors also hand-searched six Chinese Journals related to the question. INCLUSION CRITERIA: All randomized controlled trials (RCTs) comparing acupuncture therapy with placebo, Western drugs, Chinese herbal medicines, or comparing acupuncture therapy plus drug treatment with drug treatment. DATA EXTRACTION AND ANALYSIS: Two separate evaluators assessed the quality of the included reports and extracted the useful information. Disagreements were resolved through discussion. Meta-analysis of the included trials was done with RevMan 5.0, and qualitative analysis was employed when meta-analysis was not appropriate. RESULTS: Seven published RCTs with a total of 634 patients met the inclusion criteria, and the quality of one of the included trials was high. Due to flaws in design and reporting, meta-analysis was precluded, and only qualitative analysis was done on the majority of the reports. The high-quality trial showed that auricular acupuncture therapy was significantly superior to placebo in pain alleviation. The other six low-quality trials with non-placebo showed that acupuncture therapy had some positive effects. CONCLUSION: Acupuncture is effective for pain relief. However, the poor quality of the majority of the trials reduces the reliability of the conclusion. More high-quality RCTs are needed to verify the effects.

Mechanisms of treatment of cancer pain with a topical Chinese herbal formula in rats.

BACKGROUND: Pain has a substantial impact on patients' activities and overall quality of life, but current conventional drugs have debilitating side effects, including gastrointestinal disorders. Thus there is a pressing need for new therapies with fewer side effects to alleviate cancer pain. We recently developed a topical herbal formula Xiaotan Tongluo analgesic gel (XTTL gel) based on the principles of traditional Chinese herbalism, and we have received positive feedback from bone cancer pain patients. The aim of this study was to determine the analgesic effects and explore the mechanisms of XTTL gel in a rat model of bone cancer pain. METHODS: The rat model of bone cancer pain was established by inoculating Walker-256 rat carcinoma cells directly into the right tibial medullary cavity of Wistar rats. The rats were randomly assigned to three groups (n = 10 per group): (1) sham bone cancer control (sham group): vehicle (PBS) inoculation without carcinoma cells plus topical administration of blank gel; (2) Sham treatment control (vehicle group): Walker-256 cell inoculation plus topical administration of blank gel; (3) XTTL gel treatment (treatment group): Walker-256 cell inoculation plus topical administration of XTTL gel. XTTL gel treatments were applied daily for 7 days starting on day 14 following inoculation. Outcomes were assessed 21 days after inoculation by mechanical allodynia, histological staining, and by measuring concentrations of type I collagen carboxy-terminal telopeptide (ICTP) and bone-specific alkaline phosphatase (BAP) in serum. RESULTS: Fourteen days after cancer cell incubation, significant mechanical allodynia in the ipsilateral hind paw and tumor growth in proximal end of the tibia were observed in the vehicle and treatment groups but not in the sham group. At day 21, mechanical withdrawal thresholds in treatment group rats were significantly higher ((4.8557 +/- 0.8336) g) compared with those of the vehicle group ((1.8630 +/- 1.4369) g, P < 0.05). ICTP and BAP levels increased significantly in vehicle group rats ((101.5176+/- 11.0694) U/L and (370.7838 +/- 12.8273) U/L, respectively) compared with those of the sham group ((11.7553 +/- 1.1885) U/L and (185.7338 +/- 3.6761) U/L, respectively; P < 0.05). XTTL gel decreased the level of blood serum ICTP ((41.8998 +/- 6.4970) U/L, P < 0.05) but had little effect on blood serum BAP ((365.5338 +/- 18.5361) U/L, P > 0.05). CONCLUSION: Topical use of XTTL gel may have an analgesic effect on bone cancer pain, an effect mediated by lowering of ICTP levels and inhibiting bone resorption.

Study on analgesic effect of traditional Chinese medicine.

Chinese medicine has been used in treating pain for a long time. Much progress has been made in studies on the mechanism of the analgesic effect of Chinese medicine in animal experiments. It is found that the analgesic action may be related to the following actions: (1) Reducing the secretion of peripheral algogenic substances and inducing the secretion of pain-sensitive substances; (2) Alleviating the accumulation of local algogenic substances; (3) Increasing the release of endogenous analgesic substances; (4) Regulating c-fos gene and increasing the secretion of such substances in the central nervous system, etc. In this paper, the experimental methods and analgesic effect of Chinese medicines are reviewed.

Rapid passive MR catheter visualization for endovascular therapy using nonsymmetric truncated k-space sampling strategies.

Passive catheter tracking guidance by MRI is a promising approach for endovascular therapy that may have several clinical advantages over the more frequently employed active MR approaches. However, real-time MR passive tracking is problematic because it is difficult to have an image update rate >1 Hz and preserve adequate spatial and image contrast resolution. One solution for improving real-time temporal performance is the use of nonsymmetric truncated k-space sampling strategies, which acquire only a fraction of the data in both the readout and phase-encoding directions. This article investigated these acquisition strategies in combination with using (a) multicycle projection dephaser (mcPD) gradients for background suppression and (b) the projection-onto-convex sets (POCS) algorithm to reconstruct the images. The use of mcPD gradients allowed the data sampling strategies to exploit the k-space energy structure of the catheter, and POCS allowed reconstruction of high-quality MR images that were suitable for real-time passive catheter tracking and demonstrated improved geometric representations of catheter width and tip position compared to zero filling. The use of nonsymmetric truncated k-space reduced the total acquisition time.

MR image reconstruction of sparsely sampled 3D k-space data by projection-onto-convex sets.

In many rapid three-dimensional (3D) magnetic resonance (MR) imaging applications, such as when following a contrast bolus in the vasculature using a moving table technique, the desired k-space data cannot be fully acquired due to scan time limitations. One solution to this problem is to sparsely sample the data space. Typically, the central zone of k-space is fully sampled, but the peripheral zone is partially sampled. We have experimentally evaluated the application of the projection-onto-convex sets (POCS) and zero-filling (ZF) algorithms for the reconstruction of sparsely sampled 3D k-space data. Both a subjective assessment (by direct image visualization) and an objective analysis [using standard image quality parameters such as global and local performance error and signal-to-noise ratio (SNR)] were employed. Compared to ZF, the POCS algorithm was found to be a powerful and robust method for reconstructing images from sparsely sampled 3D k-space data, a practical strategy for greatly reducing scan time. The POCS algorithm reconstructed a faithful representation of the true image and improved image quality with regard to global and local performance error, with respect to the ZF images. SNR, however, was superior to ZF only when more than 20% of the data were sparsely sampled. POCS-based methods show potential for reconstructing fast 3D MR images obtained by sparse sampling.