Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation.

Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.

CLDN6 inhibits breast cancer growth and metastasis through SREBP1-mediated RAS palmitoylation.

BACKGROUND: Breast cancer (BC) ranks as the third most fatal malignant tumor worldwide, with a strong reliance on fatty acid metabolism. CLDN6, a candidate BC suppressor gene, was previously identified as a regulator of fatty acid biosynthesis; however, the underlying mechanism remains elusive. In this research, we aim to clarify the specific mechanism through which CLDN6 modulates fatty acid anabolism and its impact on BC growth and metastasis. METHODS: Cell function assays, tumor xenograft mouse models, and lung metastasis mouse models were conducted to evaluate BC growth and metastasis. Human palmitic acid assay, triglyceride assay, Nile red staining, and oil red O staining were employed to investigate fatty acid anabolism. Reverse transcription polymerase chain reaction (RT-PCR), western blot, immunohistochemistry (IHC) assay, nuclear fractionation, immunofluorescence (IF), immunoprecipitation and acyl-biotin exchange (IP-ABE), chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP) were applied to elucidate the underlying molecular mechanism. Moreover, tissue microarrays of BC were analyzed to explore the clinical implications. RESULTS: We identified that CLDN6 inhibited BC growth and metastasis by impeding RAS palmitoylation both in vitro and in vivo. We proposed a unique theory suggesting that CLDN6 suppressed RAS palmitoylation through SREBP1-modulated de novo palmitic acid synthesis. Mechanistically, CLDN6 interacted with MAGI2 to prevent KLF5 from entering the nucleus, thereby restraining SREBF1 transcription. The downregulation of SREBP1 reduced de novo palmitic acid synthesis, hindering RAS palmitoylation and subsequent endosomal sorting complex required for transport (ESCRT)-mediated plasma membrane localization required for RAS oncogenic activation. Besides, targeting inhibition of RAS palmitoylation synergized with CLDN6 to repress BC progression. CONCLUSIONS: Our findings provide compelling evidence that CLDN6 suppresses the palmitic acid-induced RAS palmitoylation through the MAGI2/KLF5/SREBP1 axis, thereby impeding BC malignant progression. These results propose a new insight that monitoring CLDN6 expression alongside targeting inhibition of palmitic acid-mediated palmitoylation could be a viable strategy for treating oncogenic RAS-driven BC.

A Randomized, Evaluator-Blinded, Multi-Centered Study to Compare Injectable Poly-D, L-Lactic Acid Versus Hyaluronic Acid for Nasolabial Fold Augmentation.

BACKGROUND: Injectable poly-D, L-lactic acid (PDLLA), under the brand name of AestheFill (Chaeum Pharma GmbH, Berlin, Germany), is a biocompatible, biodegradable, and biostimulatory product used to correct soft tissue volume loss. Its efficacy and safety have not been fully studied in a large cohort. OBJECTIVES: To evaluate the efficacy and safety of a novel dermal filler injectable poly-D, L-lactic acid. METHODS: This is an evaluator-blinded, multi-centered, randomized controlled trial to compare the efficacy and safety of PDLLA versus hyaluronic acid in the correction of nasolabial fold. Two hundred and sixty patients with moderate to severe nasolabial fold were enrolled and randomized to treatment group (PDLLA) or control group (hyaluronic acid). Each patient received PDLLA or hyaluronic acid injection for nasolabial fold augmentation and followed up for 52 weeks. Wrinkle Severity Rating Scale (WSRS) and Global Aesthetic Improvement Scale (GAIS) were used to evaluate topical nasolabial fold augmentation and overall improvement, respectively. RESULTS: At 24 weeks, 67.6% of patients in the PDLLA group had at least 1-grade improvement in WSRS, compared to 60.9% of patients in the control group with at least 1-grade improvement in WSRS (p<0.05). At each visit, PDLLA group showed more improvement from the baseline in WSRS than the control group. PDLLA was safe and well-tolerated with no severe adverse events. CONCLUSIONS: PDLLA shows non-inferior efficacy in correcting nasolabial fold compared to hyaluronic acid.

Substance P Reduces Infarct Size and Mortality After Ischemic Stroke, Possibly Through the M2 Polarization of Microglia/Macrophages and Neuroprotection in the Ischemic Rat Brain.

Substance-P (SP) is an 11 amino acid neuropeptide that is known to stimulate the peripheral mobilization of bone marrow mesenchymal stem cells and M2 polarization in monocytes/macrophages in a variety of acute and chronic tissue injuries. To examine the role of SP in protection and recovery from acute ischemic brain injury, experimental ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAo) in rats for 1 h with subsequent reperfusion. Two injections of SP, immediately and one day post-tMCAo, resulted in approximately threefold lower mortality and 40% less infarct volume than those of saline-treated rats at seven days post-tMCAo. At 4.5 h, SP markedly increased CD11b/c+CD163+/CD 206+ cells in the blood, which were concomitantly decreased in the bone marrow, suggesting that SP preferentially mobilized M2-polarized monocytes. After two days, SP increased the expression of neuroprotective and anti-inflammatory genes in the ischemic brain and induced neuronal survival in the brain penumbra. Additionally, SP markedly increased CD68+CD163+ and CD68+CD206+ M2 microglia/macrophages in the ischemic brain during seven days post-tMCAo. Furthermore, SP preserved the blood‒brain barrier in the ischemic brain, which was confirmed by the abundant levels of SMI71+ brain endothelial cells that colocalized with α-SMA+ pericytes. The beneficial effects of SP on functional recovery and tissue preservation were maintained for six weeks. Collectively, SP treatment in the early phase of ischemic stroke markedly suppressed the destructive inflammatory response and improved the microenvironment for tissue protection and repair.

Labiaplasty in Adolescents: Indications and Treatment Protocol.

BACKGROUND: Adolescents constitute a unique group of labia minora hypertrophy patients, but the necessity and benefits of labiaplasty for adolescents remain controversial. OBJECTIVES: The purpose of this study was to summarize the surgical indications, the details of the treatment procedure, postoperative complications, and therapeutic outcomes of labiaplasty in the adolescent population. METHODS: A retrospective chart review was performed of adolescent patients aged <18 years old who underwent labiaplasty between January 2016 and May 2022. Patient characteristics, surgical method, concomitant procedures, procedure side, operative time, complications, and follow-up data were recorded. RESULTS: A total of 12 patients aged <18 years were included in this study. All procedures were performed for functional reasons. The mean [standard deviation] operative time was 61.75 [20.77] minutes (range, 38-114 minutes). Unilateral labia minora hematoma within 24 hours occurred in 2 of the 12 patients (16.7%) and surgical evacuations were performed immediately. All patients were followed up electronically at 42.33 [16.88] months (range, 14-67 months). Notably, 83.33% (10/12) of patients reported being very satisfied, and 16.67% (2/12) of patients were satisfied. There was no patient dissatisfaction. Preoperative discomfort was completely resolved in 9 patients (75.00%) and significantly improved in 3 patients (25.00%). Furthermore, no patients indicated that symptoms were not improved or made worse. CONCLUSIONS: In the adolescent population, severe hypertrophy of the labia minora and the clitoral hood will cause discomfort, affecting the quality of life and mental health. Therefore, labiaplasty is a safe and effective procedure in adolescents to improve genital appearance and quality of life.

Standardized Photography of Female Genitalia and Anatomical Evaluations.

BACKGROUND: Standardized photographic recording and anatomic evaluation are crucial to refined and comprehensive preoperative design and enhanced aesthetic effect of female genital cosmetic surgery. OBJECTIVES: The authors aim to propose a standard photographic scheme and physical examination form for the anatomical assessment of patients undergoing female genital surgery. METHODS: The scheme containing 2 positions (standing and lithotomy positions) and 11 views (1 frontal and 2 oblique views from standing position; 6 frontal views with labia minora open and closed, pulled to the opposite side, clitoral hood pushed up, posterior fourchette stretched; 2 oblique views from lithotomy position) (2P11V) is applied to record pre- and postoperative appearance of the vulva. The evaluation form is utilized to record characteristics of different anatomical subunits during photography. RESULTS: Two hundred forty-five patients who underwent female genital surgery were enrolled in the research from October 2018 to October 2022. All the patients received preoperative and postoperative 2P11V photography with about 5-minutes' shooting time. Various anatomical variations containing hypertrophy and prolapse of mons pubis, redundant types of labia minora and clitoral hood, incremental exposure of clitoral glans, hypo- to hypertrophy of labia majora, disappearance of interlabial groove, hypertrophy of posterior fourchette, and relation of subunits were accurately documented. CONCLUSIONS: 2P11V photographic scheme displays the isolated features of each organ and proportion relation among different parts of vulva. The standard photographic record and physical examination form offer detailed anatomical structure to surgeons and facilitate surgeons to carry out an accurate surgical design, which deserve to be promoted and applied.

Risk factors associated with keloid infections: A five-year retrospective study.

Keloid infections reduce patient-reported quality of life greatly. Characteristics and risk factors of keloid infections have not been thoroughly studied. So, a retrospective cohort study was conducted focusing on the potential risk factors, microbiologic cultures and histological findings. Keloid patients consulting for surgical interventions were included in this study. Data were collected from their electronic medical records. 564 patients were recruited with the keloid infection rate being 22.4%. For adult patients, age above 40 years (OR, 2.84; P = .000), disease duration of 12 years or more (OR, 3.03; P = .000), the number of keloids over 3 (OR, 1.59; P = .050) and the presence of family history (OR, 1.91; P = .027) were significantly associated with keloid infections. Suppurative keloids were located mostly in thorax (61.79%). For the under-age subgroup(n = 25), family history was frequently seen in patients with infections. Microbiologic cultures revealed a mixed spectrum of bacteria including Staphylococcus (25%), Actinomyces (30%) and Prevotella (10%). The rate of epidermoid cysts was 19.7% in histological examination. Age > 40 years, disease duration ≥12 years, the number of keloids >3 and the presence of family history are risk factors for keloid infections.

Deep Learning Assisted Diagnosis of Musculoskeletal Tumors Based on Contrast-Enhanced Magnetic Resonance Imaging.

BACKGROUND: Misdiagnosis of malignant musculoskeletal tumors may lead to the delay of intervention, resulting in amputation or death. PURPOSE: To improve the diagnostic efficacy of musculoskeletal tumors by developing deep learning (DL) models based on contrast-enhanced magnetic resonance imaging and to quantify the improvement in diagnostic performance obtained by using these models. STUDY TYPE: Retrospective. POPULATION: Three hundreds and four musculoskeletal tumors, including 212 malignant and 92 benign lesions, were randomized into the training (n = 180), validation (n = 62) and testing cohort (n = 62). FIELD STRENGTH/SEQUENCE: A 3 T/T1 -weighted (T1 -w), T2 -weighted (T2 -w), diffusion-weighted imaging (DWI), and contrast-enhanced T1-weighted (CET1 -w) images. ASSESSMENT: Three DL models based, respectively, on the sagittal, coronal, and axial MR images were constructed to predict the malignancy of tumors. Blinded to the prediction results, a group of specialists made independent initial diagnoses for each patient by reading all image sequences. One month after the initial diagnoses, the same group of doctors made another round of diagnoses knowing the malignancy of each tumor predicted by the three models. The reference standard was the pathological diagnosis of malignancy. STATISTICAL TESTS: Sensitivity, specificity, and accuracy (all with 95% confidential intervals [CI]) corresponding to each diagnostic test were computed. Chi-square tests were used to assess the differences in those parameters with and without DL models. A P value < 0.05 was considered statistically significant. RESULTS: The developed models significantly improved the diagnostic sensitivities of two oncologists by 0.15 (95% CI: 0.06-0.24) and 0.36 (95% CI: 0.24-0.28), one radiologist by 0.12 (95% CI: 0.04-0.20), and three of the four orthopedists, respectively, by 0.12 (95% CI: 0.04-0.20), 0.29 (95% CI: 0.18-0.40), and 0.23 (95% CI: 0.13-0.33), without impairing any of their diagnostic specificities (all P > 0.128). DATA CONCLUSION: The DL models developed can significantly improve the performance of doctors with different training and experience in diagnosing musculoskeletal tumors. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 2.

Relative perfusion index: An objective, quantitative and noninvasive method for evaluating the severity of keloids.

BACKGROUND: Keloids are the result of abnormal wound healing, and they differ from the normal skin of the patient in the level of blood perfusion and the degrees of inflammation, hypoxia, regeneration of vessels, and expression of sensory receptors. However, there is no objective assessment method to accurately characterize the severity of keloids. OBJECTIVES: The purpose of this study was to evaluate the perfusion levels of keloids and the expression levels of various internal cytokines, including hypoxia-induced factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), interleukin-17 (IL-17), HT2A receptor subtype (5-HT2A R), and H1R, in keloids and nonadjacent normal skin and to propose a laser speckle contrast imaging (LSCI)-based relative perfusion index (RPI), through which keloids can be divided into five grades to objectively characterize their severity. METHODS: This population-based cross-sectional study included 70 untreated keloid patients who each had only one keloid on the chest. LSCI was used to measure the area of each patient's keloid ( K area ${K}_{\mathrm{area}}$ ) and the perfusion level of each patient's keloid ( K perfusion ${K}_{\mathrm{perfusion}}$ ) and normal skin ( N perfusion ${N}_{\mathrm{perfusion}}$ ). The Vancouver Scar Scale (VSS) and Visual Analog Scale (VAS) for pain and pruritus were also used to assess each keloid. Immunohistochemistry and Western blot were used to detect the expression levels of various internal cytokines in keloids and normal skin. We compared the perfusion and expression levels of intrinsic cytokines between keloids and normal skin. We established the RPI to grade the severity of keloids and applied different methods to test the utility of the RPI. RESULTS: The mean perfusion level of keloids was significantly higher than that of normal skin (p < 0.001). The expression levels of HIF-1α, VEGF, IL-17, 5-HT2A R, and H1R in keloids were significantly higher than those in normal skin (p < 0.05). RPI was defined as: [ ( K perfusion - N perfusion ) × 0.03 + K area × 0.001 ] . $[({K}_{\mathrm{perfusion}}-{N}_{\mathrm{perfusion}})\times 0.03+{K}_{\mathrm{area}}\times 0.001].$ The severity of keloids could be divided into five grades based on RPI. The RPI had a higher correlation with the pain-VAS, pruritus-VAS, and the expression levels of internal cytokines in keloids than blood perfusion levels and the VSS. T-SNE (t-distributed stochastic neighbor embedding) was also used to verify the clinical discriminatory abilities of this RPI model. CONCLUSIONS: The proposed RPI based on LSCI showed the highest accuracy, unlike the VSS and assessment of perfusion, and can be utilized as a reliable, objective, quantitative, and noninvasive tool to evaluate the severity of keloids.

Corrigendum to "Adipose-Derived Stromal Cells Attenuate Adipose Inflammation in Obesity through Adipocyte Browning and Polarization of M2 Macrophages".

[This corrects the article DOI: 10.1155/2019/1731540.].

The Effect of Yes-Associated Protein on the Interaction Between the MEK/Extracellular Signal-Regulated Kinase and Hippo Pathways in Osteoblasts Co-Cultured With Fibroblast Growth Factor Receptor 2-Mutated Dura Cells.

BACKGROUND: C342Y (Cys342Tyr) point mutation of FGFR2 (fibroblast growth factor receptor 2) is closely associated with the pathogenesis of Crouzon syndrome. The dura mater plays an important role in mediating the closure of cranial sutures. However, the underlying mechanisms of these pathological processes have been rarely investigated. in this study, the authors analyzed the effects of dura cells with FGFR2 mutations on the biological function of osteoblasts. METHODS: Dura cells and cranial osteoblasts from C57BL/6 mice were extracted and cultured. C342Y-FGFR2 mutant constructs were established via lentivirus and applied to infect dura cells. A co-cultured trans-well system with dura cells and osteoblasts was established. Three experimental groups were set up: oste group, Oste + Dura-vector group, and Oste + Dura-C342Y group. The expression levels of key factors in MEK (Mitogen-activated protein kinase kinase, MAPKK)/extracellular signal-regulated kinase (ERK) and Hippo pathway were detected by western blot and RT-qPCR (Real Time Quantitative PCR). Finally, a rescue experiment was carried out with small interference RUA. RESULTS: The proliferation level of osteoblasts in Oste + Dura- C342Y group was significantly up-regulated. Our studies indicated that the activation of MEK/ERK pathway in Oste + Dura-C342Y group could inhibit the Hippo pathway, lead to down-regulation of large tumor suppressor 1 and promote the activation and nuclear localization of yes-associated protein, and the results of rescue experiments showed a reverse expression trend, further confirming the effects of C342Y-FGFR2 mutation in dura cells on osteoblasts and its potential mechanism. CONCLUSIONS: This study suggested that the C342Y-FGFR2 mutation in dura cells could promote osteoblastic proliferation, and shown the crosstalk between MEK/ERK and Hippo pathways. As the regulatory machinery center, yes-associated protein might play a bridging role in these pathways, and might influence the pathogenesis of craniosynostosis by activating downstream transcriptional factors.

Fused Lateral Clitoral Hood and Labia Minora: New Classification Based on Anatomic Variation of the Clitoral Hood-Labia Minora Complex and Simple Surgical Management.

BACKGROUND: Existing classifications of the clitoral hood-labia minora complex (CLC) have neglected its integrity and anatomic variation, resulting in failure to optimize approaches tailored to individuals. OBJECTIVES: The aim of this study was to present a new classification system for comprehensive evaluation of variations of the CLC and to introduce a simple surgical approach for the fused type. METHODS: Anatomic variations of the CLC were classified into 3 types: isolated labia minora or lateral clitoral hood hypertrophy (Type 1); conventional combined hypertrophy (Type 2); and fused lateral clitoral hood and labia minora (Type 3). A modified procedure for the fused type was performed in 4 steps: the anterior border of labia minora was defined first, then the hypertrophic lateral clitoral hood and labia minora were each removed separately, and finally the junction region was trimmed. Satisfaction questionnaires were administered during follow-ups. RESULTS: Among all 301 patients (602 sides), Type 2 was the most common variation (285 sides, 47.3%). Type 3 variations in 67 patients (105 sides, 17.5%) were identified, and 77.6% of these patients answered the questionnaires 3 months after surgery. For patients with type 3 variations, the satisfaction rate in the 4-step excision group was 91.7%, which was significantly higher than that in the wedge excision group (56.3%) (P = 0.01). The complication rate of the 4-step excision was 2.5%. CONCLUSIONS: Preoperative evaluation based on the new classification facilitated recognition of variations of the CLC, especially of the fused type. The 4-step excision is a simple, effective, and safe approach to treat the fused variation with high satisfaction.

Heterogeneous Features of Keloids Assessed by Laser Speckle Contrast Imaging: A Cross-Sectional Study.

BACKGROUND AND OBJECTIVES: Keloids are described as benign dermal fibroproliferative lesions, and vascularization may play a significant role in their pathogenesis. In this study, laser speckle contrast imaging (LSCI) was used to assess perfusion within keloids and surrounding skin, and perfusion of keloids at different stages was compared. STUDY DESIGN/MATERIALS AND METHODS: A total of 59 patients with 110 untreated keloids on the anterior chest were enrolled in this study. Different keloid stages (progressive, stable, and regressive) were defined according to patients' descriptions of whether keloids became larger, stable, or smaller during the previous year. Vancouver Scar Scale (VSS) was assessed by a plastic surgeon, and patient reports on pain and itching were documented. LSCI was used to evaluate blood perfusion of keloids (K), skin adjacent to keloids (A), and nonadjacent skin (N). The mean perfusion of these regions was determined, and ratios (K/N, A/N) were calculated. RESULTS: A heterogeneous perfusion map was observed among the keloid groups, as well as within each keloid. A positive correlation was found between keloid perfusion and VSS. There were 62 (56.4%) keloids in the progressive stage, 33 (30.0%) keloids in the stable stage, and 15 (13.6%) keloids in the regressive stage. The mean K/N ratios in the progressive, stable, and regressive stages were 2.3 ± 0.5, 1.8 ± 0.3, and 1.5 ± 0.5, respectively. The mean A/N ratios were 1.2 ± 0.4, 1.2 ± 0.2, and 1.0 ± 0.5, respectively. Within each keloid, significantly higher perfusion was noted in the keloid and adjacent skin compared with nonadjacent skin. CONCLUSION: These results indicate that LSCI is a promising technique for evaluating keloid blood perfusion and distinguishing heterogeneous keloids. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Briarenols W-Z: Chlorine-Containing Polyoxygenated Briaranes from Octocoral Briareum stechei (Kükenthal, 1908).

Briareum stechei is proven to be a rich source of 3,8-cyclized cembranoids (briarane) with a bicyclo[8.4.0] carbon core. In the present study, four previously unreported briaranes, briarenols W-Z (1-4), along with solenolide A (5), briarenolide M (6), briaexcavatolide F (7), and brianolide (8), were isolated and characterized through spectroscopic analysis, and the absolute configuration of 8 was corroborated by a single-crystal x-ray diffraction analysis. Briaranes 2 and 5 were found to induce significant inflammatory activity in lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophage cells by enhancing the expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins.

Adipose-Derived Stem Cells Improve the Aging Skin of Nude Mice by Promoting Angiogenesis and Reducing Local Tissue Water.

BACKGROUND: Adipose-derived stem cells (ASCs) are considered promising cells for skin rejuvenation. However, whether the angiogenetic effect of ASCs plays an important role in the treatment of aging skin and its influence on skin tissue remain elusive. OBJECTIVES: The aim of this study was to evaluate the effect of ASCs on angiogenesis and local tissue water (LTW) in the aging skin of nude mice. METHODS: Twelve nude mice were randomly divided into a UVB-induced photoaging group and a natural aging group. After the mouse model had been established, ASCs and phosphate-buffered saline (PBS) were then each injected into different sides of the dorsal skin of the mice. Blood perfusion and LTW content were measured. After 7 weeks, mice were killed, and skin samples were collected to measure the thickness of the dermis, the density of the capillaries, and the expression of angiogenic growth factors. RESULTS: ASC therapy significantly increased the thickness of the dermis, the number of capillaries, and the expression of some angiogenic growth factors (vascular endothelial growth factor, insulin-like growth factor 1, and epidermal growth factor). At 7 weeks after injection, blood perfusion was significantly higher on the side injected with ASCs than on the side injected with PBS. LTW content was increased in the PBS-injected side, but the ASC-injected side showed no significant changes over time. CONCLUSIONS: ASCs increased dermal thickness, promoted angiogenesis, and reduced LTW content in the skin of photoaging mice, providing a potential clinical therapy for skin rejuvenation.

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin.

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.

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.

miR-625-5p/PKM2 negatively regulates melanoma glycolysis state.

PKM2 plays an important role in cancer glycolysis, however, the link of PKM2 and microRNAs (miRNAs) in melanoma is still unclear. The study will investigate the role of miRNAs in regulating PKM2 mediated melanoma cell glycolysis. We found that high PKM2 expression in melanoma tissues and cell lines was positively associated with glycolysis. Further study indicated that miR-625-5p regulated PKM2 expression on mRNA and protein levels in melanoma cells. There was a negative relationship between miR-625-5p and PKM2 expression in the clinical melanoma samples. These findings provide an evidence that miR-625-5p/PKM2 plays a role in melanoma cell glucose metabolism.

CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters.

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes.

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high-efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 Cas9 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology-directed repair-mediated knock-in and deletion. In strains where spCas9 was nonfunctional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production, and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.