A naturally inspired antibiotic to target multidrug-resistant pathogens.

Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections1,2. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin3. Bacteria-derived antibiotics often appear in nature as collections of similar structures that are encoded by evolutionarily related biosynthetic gene clusters. This structural diversity is, at least in part, expected to be a response to the development of natural resistance, which often mechanistically mimics clinical resistance. Here we propose that a solution to mcr-1-mediated resistance might have evolved among naturally occurring colistin congeners. Bioinformatic analysis of sequenced bacterial genomes identified a biosynthetic gene cluster that was predicted to encode a structurally divergent colistin congener. Chemical synthesis of this structure produced macolacin, which is active against Gram-negative pathogens expressing mcr-1 and intrinsically resistant pathogens with chromosomally encoded phosphoethanolamine transferase genes. These Gram-negative bacteria include extensively drug-resistant Acinetobacter baumannii and intrinsically colistin-resistant Neisseria gonorrhoeae, which, owing to a lack of effective treatment options, are considered among the highest level threat pathogens4. In a mouse neutropenic infection model, a biphenyl analogue of macolacin proved to be effective against extensively drug-resistant A. baumannii with colistin-resistance, thus providing a naturally inspired and easily produced therapeutic lead for overcoming colistin-resistant pathogens.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

Retracted: Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats.

Retracted on the author's request: "We would like to withdraw our manuscript. We restarted the project for further study last year, we found that the results in this study are not solid enough and need to be further explored." Reference: Zong-Qiang Wang, Dian-Hui Xiu, Gui-Feng Liu, Jin-Lan Jiang. Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats. Med Sci Monit 2018; 24: 5779-5787; DOI: 10.12659/MSM.908144.

Metagenome-Guided Analogue Synthesis Yields Improved Gram-Negative-Active Albicidin- and Cystobactamid-Type Antibiotics.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics. Our search for PABA-specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio-informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

Long non-coding RNA XIST binding to let-7c-5p contributes to rheumatoid arthritis through its effects on proliferation and differentiation of osteoblasts via regulation of STAT3.

BACKGROUND: Rheumatoid arthritis (RA), a chronic autoimmune disease, affects around 1% population worldwide, with the life quality of patients severely reduced. In this study, it is intended to explore the role of long non-coding RNA X-inactive specific transcript (lncRNA XIST) in RA and the underlying mechanisms associated with let-7c-5p and signal transducer and activator of transcription 3 (STAT3). METHODS: LncRNA XIST, let-7c-5p, and STAT3 expressions were determined in RA and normal cartilage tissues, and their relationship was analyzed in osteoblasts. The regulatory effects of lncRNA XIST in RA were investigated when XIST expression was upregulated or downregulated in osteoblasts. TNF-α, IL-2, IL-6, alkaline phosphatase (ALP), osteocalcin, TGF-ß1, and IGF1 were measured in vivo in RA rats. RESULTS: LncRNA XIST and STAT3 were expressed at high levels and let-7c-5p expressed at a low level in RA cartilage tissues. LncRNA XIST silencing or let-7c-5p enhancement led to decreased levels of TNF-α, IL-2, and IL-6, suggestive of suppressed inflammatory response, and increased levels of ALP, osteocalcin, TGF-ß1, and IGF-1 as well as reduced damage in cartilage tissues. CONCLUSION: LncRNA XIST downregulation could promote proliferation and differentiation of osteoblasts in RA, serving as a future therapeutic target for RA.

A network meta-analysis on the short-term efficacy and adverse events of different anti-osteoporosis drugs for the treatment of postmenopausal osteoporosis.

A network meta-analysis was conducted to compare the short-term efficacy and adverse events of different drugs for the treatment of postmenopausal osteoporosis (PMO), providing a more effective treatment for PMO. We initially searched through various databases like PubMed, Cochrane Library, and EMBASE from inception till October 2016. All randomized controlled trials (RCTs) of drugs for the treatment of PMO were included for direct and indirect comparison. A combination of direct and indirect evidence of different inhibitors of anti-diabetic drugs for treatment of PMO were considered for calculating the weighted mean difference (WMD) value or odd ratio (OR) value and to draw surface under the cumulative ranking (SUCRA) curves. Twenty-seven RCTs were ultimately incorporated into this network meta-analysis comprising of 48 200 patients suffering from PMO. The network meta-analysis revealed that compared with placebo, alendronate had better efficacy on improving bone mineral density (BMD) at lumbar spine, femoral neck, and total hip. Risedronate and raloxifene had relatively lower incidence of new vertebral fractures. The SUCRA analysis showed that alendronate had better efficacy on improving BMD, risedronate could significantly decrease the incidence of fresh fracture and bazedoxifene was relatively safe. The available evidence suggested that alendronate and risedronate might be the superior choices for the treatment of PMO, while bazedoxifene was a comparatively safer option for patients.

Sumoylation of SMAD 4 ameliorates the oxidative stress-induced apoptosis in osteoblasts.

Oxidative stress-induced mitochondrial function and cell apoptosis to osteoblasts, plays a critical role in the pathophysiology of osteoporosis. However, mechanisms underlying such process remain not yet clear. We aims in this study to investigate a possible role of SMAD (the mothers against decapentaplegic homolog 4 (SMAD4) in the oxidative stress-induced apoptosis, in homo sapiens osteoblast hFOB1.19 cells. Results demonstrated that the treatment with more than 100µM H2O2 significantly downregulated the cellular viability, whereas markedly induced apoptosis in hFOB1.19 cells. The SMAD4 was markedly reduced in both mRNA and protein levels in the H2O2 -treated hFOB1.19 cells, along with the reduction of Small ubiquitin-related modifier 1 (SUMO 1) and SUMO 2/3. The immunoprecipitation assay confirmed indicated the interaction between SUMO 1 (or SUMO 2/3) and SMAD4. Moreover, the SMAD4 overexpression markedly ameliorated the H2O2-resulted viability reduction and apoptosis induction in hFOB1.19 cells. Interestingly, such amelioration was blocked by the knockdown of SUMO 2/3. Taken together, we conclued that SMAD4 inhibits the H2O2-induced apoptosis in osteoblast hFOB1.19 cells; such inhibition might depend on the SUMOylation by SUMO 2/3. It implies a promising role of SMAD4 in oxidative stress-promoted damage to osteoblasts.

Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats.

BACKGROUND Surgeons usually transfer the contralateral C7 to the median nerve on the injured side via a nerve graft to recover sensation and movement in a paralyzed hand. The purpose of our study was to determine whether NRG-1 affects the recovery of nerve function in brachial plexus injury after contralateral C7 nerve root transfer in a rat model. MATERIAL AND METHODS An injury model of left brachial plexus and contralateral C7 nerve root transfer was established. Four weeks after the operation, NRG-1 expression was examined by reverse transcription quantitative polymerase chain reaction and Western blot analysis. The diameter rate differences of the healthy limb and affected limb were estimated. The postoperative mass of the left latissimus dorsi, triceps, extensor carpi radialis brevis, and musculus extensor digitorum were examined. The number of nerve fibers and typical area of the affected side were assessed. Postoperative left motor nerve conduction velocity (MNCV) and motor nerve action potential (MNAP) were tested by use of a biological information recording and collecting system. RESULTS Eukaryotic expression plasmid of pcDNA4/myc/A-NRG-1 was successfully constructed, and NRG-1 was overexpressed. Compared with the model group, the NRG-1 group had a lower rate of differences of the limbs; higher mass of left latissimus dorsi, triceps, extensor carpi radialis brevis, and musculus extensor digitorum; more nerve fibers and larger typical area in the affected side, left MNCV, and MNAP; and wider CSA of the left triceps. CONCLUSIONS These results demonstrated that NRG-1 can promote recovery of nerve function in brachial plexus injury after contralateral C7 nerve root transfer in rats.

Expanding the Catalytic Promiscuity of Heparinase†III from Pedobacter heparinus.

Glycosaminoglycans (GAG) lyases are useful biocatalysts for the preparation of oligosaccharides, but their substrate spectra are limited to the same family. Thus, the degradation activity across families of GAG lyases is advantageous and desirable for various applications. In this study, residue Lys130 at the substrate entrance of monomeric heparinase III from Pedobacter heparinus ATCC 13125 was replaced by cysteine, and the resulting mutant K130C showed novel catalytic activity in degrading hyaluronic acid without affecting its native activity toward heparin and heparan sulfate. The broadened catalytic promiscuity by mutant K130C was the result of dimerization through a disulfide bond to expand the substrate binding pocket. This bifunctional enzyme is potentially valuable in the degradation of different types of GAGs.

Enzymatic synthesis of L-norephedrine by coupling recombinant pyruvate decarboxylase and ω-transaminase.

L-Norephedrine, a natural plant alkaloid, possesses similar activity as ephedrine and can be used as a vicinal amino alcohol for the asymmetric synthesis of a variety of optically pure compounds, including pharmaceuticals, fine chemicals, and agrochemicals. Because of the existence of two asymmetric centers, efficient synthesis of L-norephedrine has been challenging. In the present study, an R-selective pyruvate decarboxylase from Saccharomyces cerevisiae and an S-selective ω-transaminase from Vibrio fluvialis JS17 were coupled to develop a sequential process for the stereoselective biosynthesis of L-norephedrine. After systematic optimization of the reaction conditions, a green, economic, and practical biocatalytic method to prepare L-norephedrine was established to achieve de and ee values of greater than 99.5 % and a molar yield over 60 %. The present coupling approach can facilitate the development of sequential reactions by various biocatalysts.

Recent advances in the culture-independent discovery of natural products using metagenomic approaches.

Natural products derived from bacterial sources have long been pivotal in the discovery of drug leads. However, the cultivation of only about 1% of bacteria in laboratory settings has left a significant portion of biosynthetic diversity hidden within the genomes of uncultured bacteria. Advances in sequencing technologies now enable the exploration of genetic material from these metagenomes through culture-independent methods. This approach involves extracting genetic sequences from environmental DNA and applying a hybrid methodology that combines functional screening, sequence tag-based homology screening, and bioinformatic-assisted chemical synthesis. Through this process, numerous valuable natural products have been identified and synthesized from previously uncharted metagenomic territories. This paper provides an overview of the recent advancements in the utilization of culture-independent techniques for the discovery of novel biosynthetic gene clusters and bioactive small molecules within metagenomic libraries.

A Family of Antibiotics That Evades Resistance by Binding Polyprenyl Phosphates.

Cilagicin is a Gram-positive active antibiotic that has a dual polyprenyl phosphate binding mechanism that impedes resistance development. Here we bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to search for antibiotics that might similarly avoid resistance development. Synthesis and bioactivity screening of the predicted structures that we identified led to three antibiotics that are active against multidrug-resistant Gram-positive pathogens, two of which, paenilagicin and virgilagicin, did not lead to resistance even after prolonged antibiotic exposure.

Discovery of Paenibacillaceae Family Gram-Negative-Active Cationic Lipopeptide Antibiotics Using Evolution-Guided Chemical Synthesis.

Cationic nonribosomal lipopeptides (CNRLPs) from Paenibacillus spp. have been a rewarding source of Gram-negative-active antibiotics. Here we systematically screened sequenced bacterial genomes for CNRLP biosynthetic gene clusters (BGCs) that we predicted might encode additional Gram-negative-active antibiotics. Total chemical synthesis of the bioinformatically predicted products of seven such BGCs led to our identification of new laterocidine, tridecaptin, and paenibacterin-like antibiotics with potent activity against both multiple-drug-resistant Gram-negative and Gram-positive pathogens.

Bioinformatic prospecting and synthesis of a bifunctional lipopeptide antibiotic that evades resistance.

Emerging resistance to currently used antibiotics is a global public health crisis. Because most of the biosynthetic capacity within the bacterial kingdom has remained silent in previous antibiotic discovery efforts, uncharacterized biosynthetic gene clusters found in bacterial genome-sequencing studies remain an appealing source of antibiotics with distinctive modes of action. Here, we report the discovery of a naturally inspired lipopeptide antibiotic called cilagicin, which we chemically synthesized on the basis of a detailed bioinformatic analysis of the cil biosynthetic gene cluster. Cilagicin's ability to sequester two distinct, indispensable undecaprenyl phosphates used in cell wall biosynthesis, together with the absence of detectable resistance in laboratory tests and among multidrug-resistant clinical isolates, makes it an appealing candidate for combating antibiotic-resistant pathogens.

Lapcin, a potent dual topoisomerase I/II inhibitor discovered by soil metagenome guided total chemical synthesis.

In natural product discovery programs, the power of synthetic chemistry is often leveraged for the total synthesis and diversification of characterized metabolites. The synthesis of structures that are bioinformatically predicted to arise from uncharacterized biosynthetic gene clusters (BGCs) provides a means for synthetic chemistry to enter this process at an early stage. The recent identification of non-ribosomal peptides (NRPs) containing multiple ρ-aminobenzoic acids (PABAs) led us to search soil metagenomes for BGCs that polymerize PABA. Here, we use PABA-specific adenylation-domain sequences to guide the cloning of the lap BGC directly from soil. This BGC was predicted to encode a unique N-acylated PABA and thiazole containing structure. Chemical synthesis of this structure gave lapcin, a dual topoisomerase I/II inhibitor with nM to pM IC50s against diverse cancer cell lines. The discovery of lapcin highlights the power of coupling metagenomics, bioinformatics and total chemical synthesis to unlock the biosynthetic potential contained in even complex uncharacterized BGCs.

Multiplexed mobilization and expression of biosynthetic gene clusters.

Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.

Analysis of risk factors related to breast cancer metastasis: a retrospective nested case-control study.

PURPOSE: To explore the laboratory indexes related to breast cancer metastasis, so as to provide scientific basis for the control of breast metastasis. METHODS: A retrospective cohort-based nested case-control study was used to screen 732 breast cancer patients recorded in the First and the Third Hospitals of Jilin University's electronic medical record system between January 2008 through December 2015 without metastasis at admission. Those with subsequent metastasis were classified as the metastasis group and those without metastasis as the control group. The suspected confounders were matched by propensity score matching, then univariate analysis was conducted, and the variables with statistical significance were included in multivariate conditional logistic regression analysis. RESULTS: A total of 86 patients were matched in the transfer group and 315 in the control group, with a total sample size of 401.In univariate analysis, fasting plasma glucose (FPG), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and direct bilirubin (DBIL) in two groups were statistically different (p<0.05), multiple conditional logistic regression showed that FPG (OR=1.335) and ALP (OR=1.016) were factors related to breast cancer metastasis. CONCLUSIONS: For breast cancer patients, the higher FPG and ALP levels may be associated with metastasis. Therefore, daily monitoring and control of these indicators may be helpful for the control of cancer metastasis.

Elucidating the Diversity and Potential Function of Nonribosomal Peptide and Polyketide Biosynthetic Gene Clusters in the Root Microbiome.

Polyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores, and others. Despite their involvement in bacterium-bacterium and bacterium-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics, and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low-similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic data sets revealed higher abundances of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs.IMPORTANCE We identified distinct secondary-metabolite-encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary-metabolite-encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in interbacterial and bacterium-plant interactions.

[Retracted] Diagnostic and prognostic value of contrast­enhanced ultrasound combined with diffusion­weighted magnetic resonance imaging in different subtypes of breast cancer.

Int J Mol Med 42: [Related article:] 105­114, 2018; DOI: 10.3892/ijmm.2018.3591. The authors have requested that their research article entitled 'Diagnostic and prognostic value of contrast­enhanced ultrasound combined with diffusion­weighted magnetic resonance imaging in different subtypes of breast cancer' published in International Journal of Molecular Medicine 42, 105­114, 2018, be retracted. This study was conceived jointly by the research institute of the authors' hospital (Jilin University China­Japan Friendship Hospital) and the Second Affiliated Hospital of Zhengzhou University, and the clinical data were obtained from the two institutes. It is regrettable that the scientific research unit of the Second Affiliated Hospital of Zhengzhou University did not authorize the publication of these results, and the authors have subsequently received an official request from the Second Affiliated Hospital of Zhengzhou University to retract this paper, since the results of their article have infringed the scientific research rights of the third party. The Editor of International Journal of Molecular Medicine agrees that the article should be retracted from the publication in view of the infringement of the scientific rights of the third party. All the named authors agree to this retraction. The authors apologize to the Editor and to the readership of the Journal, and regret any inconvenience this will cause.

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture.

Cis­diammine­dichloro­platinum II­based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non­small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin­treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin­treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20 passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post­cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy­related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15 proteins [glucose­regulated protein, 78 kDa (GRP78), heat shock protein 71, pre­mRNA processing factor 19, polypyrimidine tract binding protein 1, translationally controlled tumor protein, Cathepsin D, Cytochrome c, thioredoxin domain containing 5, MutS homolog (MSH) 6, Annexin A2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor 1A interacting protein, MSH2, protein phosphatase 2A 55 kDa regulatory subunit Bα, Rho glyceraldehyde­3­phosphate­dissociation inhibitor 1 and ANXA4] that were upregulated by >1.5­fold in heavy (H)­ and light (L)­labeled A549R cells. In addition, 16 and 14 proteins were downregulated by >1.5­fold in the H­ and L­labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin­resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin­resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti­apoptosis and/or autophagy promotion.