Relaxing descemetotomy: microscope-integrated OCT-guided technique for acute corneal hydrops.

Introduction: 3 cases are used to illustrate the technique of Descemet membrane (DM) relaxing incisions followed by air descemetopexy for the management of patients with acute corneal hydrops. Patients and Clinical Findings: In each case, anterior-segment optical coherence tomography (OCT) demonstrated taut DM detachments and hydrops was refractory to conservative medical management and intracameral air injection. Diagnosis Intervention and Outcomes: To facilitate the reapproximation of DM and potentiate corneal deturgescence, intraoperative OCT-guided descemetotomy was performed with bent surgical scissors and a bent 30-gauge needle. Subsequent air descemetopexy was successful, and DM reattachment was maintained postoperatively. Corneal edema improved in all patients relatively rapidly postoperatively. Conclusions: Relaxing descemetotomy with air descemetopexy may be useful in cases of acute corneal hydrops with taut DM detachments that are unresponsive to air tamponade alone.

The Diagnostic Accuracy of Transcranial Color-Coded Doppler Ultrasound Technique in Stratifying Intracranial Cerebral Artery Stenoses in Cerebrovascular Disease Patients: A Systematic Review and Meta-Analysis.

The early and accurate stratification of intracranial cerebral artery stenosis (ICAS) is critical to inform treatment management and enhance the prognostic outcomes in patients with cerebrovascular disease (CVD). Digital subtraction angiography (DSA) is an invasive and expensive procedure but is the gold standard for the diagnosis of ICAS. Over recent years, transcranial color-coded Doppler ultrasound (TCCD) has been suggested to be a useful imaging method for accurately diagnosing ICAS. However, the diagnostic accuracy of TCCD in stratifying ICASs among patients with CVD remains unclear. Therefore, this systematic review and meta-analysis aimed at evaluating the diagnostic accuracy of TCCD in the stratification of intracranial steno-occlusions among CVD patients. A total of six databases-Embase, CINAHL, Medline, PubMed, Google Scholar, and Web of Science (core collection)-were searched for studies that assessed the diagnostic accuracy of TCCD in stratifying ICASs. The meta-analysis was performed using Meta-DiSc 1.4. The Quality Assessment of Diagnostic Accuracy Studies tool version 2 (QUADAS-2) assessed the risk of bias. Eighteen studies met all of the eligibility criteria. TCCD exhibited a high pooled diagnostic accuracy in stratifying intracranial steno-occlusions in patients presenting with CVD when compared to DSA as a reference standard (sensitivity = 90%; specificity = 87%; AUC = 97%). Additionally, the ultrasound parameters peak systolic velocity (PSV) and mean flow velocity (MFV) yielded a comparable diagnostic accuracy of "AUC = 0.96". In conclusion, TCCD could be a noble, safe, and accurate alternative imaging technique to DSA that can provide useful diagnostic information in stratifying intracranial steno-occlusions in patients presenting with CVD. TCCD should be considered in clinical cases where access to DSA is limited.

Application of Cas12j for Streptomyces Editing.

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.

Exploring a general multi-pronged activation strategy for natural product discovery in Actinomycetes.

Natural products possess significant therapeutic potential but remain underutilized despite advances in genomics and bioinformatics. While there are approaches to activate and upregulate natural product biosynthesis in both native and heterologous microbial strains, a comprehensive strategy to elicit production of natural products as well as a generalizable and efficient method to interrogate diverse native strains collection, remains lacking. Here, we explore a flexible and robust integrase-mediated multi-pronged activation approach to reliably perturb and globally trigger antibiotics production in actinobacteria. Across 54 actinobacterial strains, our approach yielded 124 distinct activator-strain combinations which consistently outperform wild type. Our approach expands accessible metabolite space by nearly two-fold and increases selected metabolite yields by up to >200-fold, enabling discovery of Gram-negative bioactivity in tetramic acid analogs. We envision these findings as a gateway towards a more streamlined, accelerated, and scalable strategy to unlock the full potential of Nature's chemical repertoire.

Natural Products from Singapore Soil-Derived Streptomycetaceae Family and Evaluation of Their Biological Activities.

Natural products have long been used as a source of antimicrobial agents against various microorganisms. Actinobacteria are a group of bacteria best known to produce a wide variety of bioactive secondary metabolites, including many antimicrobial agents. In this study, four actinobacterial strains found in Singapore terrestrial soil were investigated as potential sources of new antimicrobial compounds. Large-scale cultivation, chemical, and biological investigation led to the isolation of a previously undescribed tetronomycin A (1) that demonstrated inhibitory activities against both Gram-positive bacteria Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) (i.e., MIC90 of 2-4 µM and MBC90 of 9-12 µM), and several known antimicrobial compounds, namely nonactin, monactin, dinactin, 4E-deacetylchromomycin A3, chromomycin A2, soyasaponin II, lysolipin I, tetronomycin, and naphthomevalin. Tetronomycin showed a two- to six-fold increase in antibacterial activity (i.e., MIC90 and MBC90 of 1-2 µM) as compared to tetronomycin A (1), indicating the presence of an oxy-methyl group at the C-27 position is important for antibacterial activity.

Enhancing armeniaspirols production through multi-level engineering of a native Streptomyces producer.

BACKGROUND: Nature has provided unique molecular scaffolds for applications including therapeutics, agriculture, and food. Due to differences in ecological environments and laboratory conditions, engineering is often necessary to uncover and utilize the chemical diversity. Although we can efficiently activate and mine these often complex 3D molecules, sufficient production of target molecules for further engineering and application remain a considerable bottleneck. An example of these bioactive scaffolds is armeniaspirols, which are potent polyketide antibiotics against gram-positive pathogens and multi-resistance gram-negative Helicobacter pylori. Here, we examine the upregulation of armeniaspirols in an alternative Streptomyces producer, Streptomyces sp. A793. RESULTS: Through an incidental observation of enhanced yields with the removal of a competing polyketide cluster, we observed seven-fold improvement in armeniaspirol production. To further investigate the improvement of armeniaspirol production, we examine upregulation of armeniaspirols through engineering of biosynthetic pathways and primary metabolism; including perturbation of genes in biosynthetic gene clusters and regulation of triacylglycerols pool. CONCLUSION: With either overexpression of extender unit pathway or late-stage N-methylation, or the deletion of a competing polyketide cluster, we can achieve seven-fold to forty nine-fold upregulation of armeniaspirol production. The most significant upregulation was achieved by expression of heterologous fatty acyl-CoA synthase, where we observed not only a ninety seven-fold increase in production yields compared to wild type, but also an increase in the diversity of observed armeniaspirol intermediates and analogs.

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces hosts for natural product discovery.

With the advent of rapid automated in silico identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species, Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as alternative NP hosts. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.

Antibacterial Spirotetronate Polyketides from an Actinomadura sp. Strain A30804.

Large scale cultivation and chemical investigation of an extract obtained from Actimonadura sp. resulted in the identification of six previously undescribed spirotetronates (pyrrolosporin B and decatromicins C-G; 7-12), along with six known congeners, namely decatromicins A-B (1-2), BE-45722B-D (3-5), and pyrrolosporin A (6). The chemical structures of compounds 1-12 were characterized via comparison with previously reported data and analysis of 1D/2D NMR and MS data. The structures of all new compounds were highly related to the spirotetronate type compounds, decatromicin and pyrrolosporin, with variations in the substituents on the pyrrole and aglycone moieties. All compounds were evaluated for antibacterial activity against the Gram-negative bacteria, Acinetobacter baumannii and Gram-positive bacteria, Staphylococcus aureus and were investigated for their cytotoxicity against the human cancer cell line A549. Of these, decatromicin B (2), BE-45722B (3), and pyrrolosporin B (7) exhibited potent antibacterial activities against both Gram-positive (MIC90 between 1-3 µM) and Gram-negative bacteria (MIC90 values ranging from 12-36 µM) with weak or no cytotoxic activity against A549 cells.

Antibacterial Thiopeptide GE2270-Congeners from Nonomuraea jiangxiensis.

Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopeptides (1−9) were obtained from a Nonomuraea jiangxiensis isolated from a terrestrial soil sample collected in Singapore. Even though some of these compounds were previously synthesized or isolated from engineered strains, herein we report the unprecedented isolation of these thiopeptides from a native Nonomuraea jiangxiensis. A comparison with the literature and a detailed analysis of the NMR and HRMS of compounds 1−9 was conducted to assign their chemical structures. The structures of all new compounds were highly related to the thiopeptide antibiotics GE2270, with variations in the substituents on the thiazole and amino acid moieties. Thiopeptides 1−9 exhibited a potent antimicrobial activity against the Gram-positive bacteria, Staphylococcus aureus with MIC90 values ranging from 2 µM to 11 µM. In addition, all compounds were investigated for their cytotoxicity against the human cancer cell line A549, none of the compounds were cytotoxic.

In-Silico Identified New Natural Sortase A Inhibitors Disrupt S. aureus Biofilm Formation.

Sortase A (SrtA) is a membrane-associated enzyme that anchors surface-exposed proteins to the cell wall envelope of Gram-positive bacteria such as Staphylococcus aureus. As SrtA is essential for Gram-positive bacterial pathogenesis but dispensable for microbial growth or viability, SrtA is considered a favorable target for the enhancement of novel anti-infective drugs that aim to interfere with key bacterial virulence mechanisms, such as biofilm formation, without developing drug resistance. Here, we used virtual screening to search an in-house natural compound library and identified two natural compounds, N1287 (Skyrin) and N2576 ((4,5-dichloro-1H-pyrrol-2-yl)-[2,4-dihydroxy-3-(4-methyl-pentyl)-phenyl]-methanone) that inhibited the enzymatic activity of SrtA. These compounds also significantly reduced the growth of S. aureus but possessed moderate mammalian toxicity. Furthermore, S. aureus strains treated with these compounds exhibited reduction in adherence to host fibrinogen, as well as biofilm formation. Hence, these compounds may represent an anti-infective therapy without the side effects of antibiotics.

Identification and engineering of 32 membered antifungal macrolactone notonesomycins.

Notonesomycin A is a 32-membered bioactive glycosylated macrolactone known to be produced by Streptomyces aminophilus subsp. notonesogenes 647-AV1 and S. aminophilus DSM 40186. In a high throughput antifungal screening campaign, we identified an alternative notonesomycin A producing strain, Streptomyces sp. A793, and its biosynthetic gene cluster. From this strain, we further characterized a new more potent antifungal non-sulfated analogue, named notonesomycin B. Through CRISPR-Cas9 engineering of the biosynthetic gene cluster, we were able to increase the production yield of notonesomycin B by up to 18-fold as well as generate a strain that exclusively produces this analogue.

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

BACKGROUND: Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. RESULTS: Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. CONCLUSIONS: We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.

Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity.

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K(+) ions and only one in the presence of Na(+) ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K(+) but not Na(+). Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na(+) solution, which comprises a novel core arrangement distinct from the known topologies. This structure complements the previously elucidated basket-type human telomeric G-quadruplex to serve as reference structures in Na(+)-containing environment. These structures, together with the coexistence of other conformations in Na(+) solution as observed by nuclear magnetic resonance spectroscopy, establish the polymorphic nature of human telomeric repeats beyond the influence of K(+) ions.