Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

Potential Antifungal Targets for Aspergillus sp. from the Calcineurin and Heat Shock Protein Pathways.

Aspergillus species, especially A. fumigatus, and to a lesser extent others (A. flavus, A. niger, A. terreus), although rarely pathogenic to healthy humans, can be very aggressive to immunocompromised patients (they are opportunistic pathogens). Although survival rates for such infections have improved in recent decades following the introduction of azole derivatives, they remain a clinical challenge. The fact that current antifungals act as fungistatic rather than fungicide, that they have limited safety, and that resistance is becoming increasingly common make the need for new, more effective, and safer therapies to become more acute. Over the last decades, knowledge about the molecular biology of A. fumigatus and other Aspergillus species, and particularly of calcineurin, Hsp90, and their signaling pathway proteins, has progressed remarkably. Although calcineurin has attracted much interest, its adverse effects, particularly its immunosuppressive effects, make it less attractive than it might at first appear. The situation is not very different for Hsp90. Other proteins from their signaling pathways, such as protein kinases phosphorylating the four SPRR serine residues, CrzA, rcnA, pmcA-pmcC (particularly pmcC), rfeF, BAR adapter protein(s), the phkB histidine kinase, sskB MAP kinase kinase, zfpA, htfA, ctfA, SwoH (nucleoside diphosphate kinase), CchA, MidA, FKBP12, the K27 lysine position from Hsp90, PkcA, MpkA, RlmA, brlA, abaA, wetA, other heat shock proteins (Hsp70, Hsp40, Hsp12) currently appear promising and deserve further investigation as potential targets for antifungal drug development.

Tumor selective uptake of drug-nanodiamond complexes improves therapeutic outcome in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.

Fungal Pigments: Carotenoids, Riboflavin, and Polyketides with Diverse Applications.

Natural pigments and colorants have seen a substantial increase in use over the last few decades due to their eco-friendly and safe properties. Currently, customer preferences for more natural products are driving the substitution of natural pigments for synthetic colorants. Filamentous fungi, particularly ascomycetous fungi (Monascus, Fusarium, Penicillium, and Aspergillus), have been shown to produce secondary metabolites containing a wide variety of pigments, including ß-carotene, melanins, azaphilones, quinones, flavins, ankaflavin, monascin, anthraquinone, and naphthoquinone. These pigments produce a variety of colors and tints, including yellow, orange, red, green, purple, brown, and blue. Additionally, these pigments have a broad spectrum of pharmacological activities, including immunomodulatory, anticancer, antioxidant, antibacterial, and antiproliferative activities. This review provides an in-depth overview of fungi gathered from diverse sources and lists several probable fungi capable of producing a variety of color hues. The second section discusses how to classify coloring compounds according to their chemical structure, characteristics, biosynthetic processes, application, and present state. Once again, we investigate the possibility of employing fungal polyketide pigments as food coloring, as well as the toxicity and carcinogenicity of particular pigments. This review explores how advanced technologies such as metabolic engineering and nanotechnology can be employed to overcome obstacles associated with the manufacture of mycotoxin-free, food-grade fungal pigments.

Recent advances in probiotication of fruit and vegetable juices.

Probiotics are live bacteria beneficial to health when consumed adequately. Health professionals now recommend probiotics on regular diets due to their positive effects on human health. The probiotics that are usually consumed from the market through food products are mostly dairy-based. Fruit and vegetables are gaining popularity as preferred matrices for probiotic carriers to the human body, owing to their high cholesterol content and the lactose intolerance of dairy products. On the other hand, fruits and vegetable juices are rich in nutrient content such as vitamins, minerals, and antioxidants and do not contain a starter culture that can compete with the nutrients. The probiotication of fruit and vegetable juices (apple, carrot, citrus fruit, pome-granate, watermelon, tomato, and pineapple) are performing as efficient probiotic bacteria carriers. This review covers the previous works that highlighted the variety of probiotic fruit and vegetable juices as well as the viability of each probiotic in various products after proper fermentation and storage. In addition, physicochemical and sensory changes that occurred during the processing and storage period have been discussed. Furthermore, strategies (microencapsulation, adding prebiotics, antioxidant addition, maintaining optimum pH, temperature, adaptation with resistance, and good packaging) to improve the stability of probiotic bacteria are outlined, as it is difficult to maintain the stability of probiotic bacteria during storage. Finally, the manuscript discusses the effect of probiotic fruit and vegetable juices on human health.

Human skin specific long noncoding RNA HOXC13-AS regulates epidermal differentiation by interfering with Golgi-ER retrograde transport.

After a skin injury, keratinocytes switch from a state of homeostasis to one of regeneration leading to the reconstruction of the epidermal barrier. The regulatory mechanism of gene expression underpinning this key switch during human skin wound healing is enigmatic. Long noncoding RNAs (lncRNAs) constitute a new horizon in the understanding of the regulatory programs encoded in the mammalian genome. By comparing the transcriptome of an acute human wound and skin from the same donor as well as keratinocytes isolated from these paired tissue samples, we generated a list of lncRNAs showing changed expression in keratinocytes during wound repair. Our study focused on HOXC13-AS, a recently evolved human lncRNA specifically expressed in epidermal keratinocytes, and we found that its expression was temporally downregulated during wound healing. In line with its enrichment in suprabasal keratinocytes, HOXC13-AS was found to be increasingly expressed during keratinocyte differentiation, but its expression was reduced by EGFR signaling. After HOXC13-AS knockdown or overexpression in human primary keratinocytes undergoing differentiation induced by cell suspension or calcium treatment and in organotypic epidermis, we found that HOXC13-AS promoted keratinocyte differentiation. Moreover, RNA pull-down assays followed by mass spectrometry and RNA immunoprecipitation analysis revealed that mechanistically HOXC13-AS sequestered the coat complex subunit alpha (COPA) protein and interfered with Golgi-to-endoplasmic reticulum (ER) molecular transport, resulting in ER stress and enhanced keratinocyte differentiation. In summary, we identified HOXC13-AS as a crucial regulator of human epidermal differentiation.

Circular RNA Signatures of Human Healing and Nonhealing Wounds.

Venous ulcers (VUs) have complex and obscure pathogenicity, and effective VU therapies are still lacking. Circular RNAs (circRNAs) have emerged as powerful gene regulators with important roles in health and disease. In this study, we used paired total RNA and small RNA sequencing to profile circRNAs, protein-coding mRNAs, and microRNAs expression in a unique collection of clinical samples: healthy skin and acute wounds at inflammatory and proliferative phases and wound-edge VU biopsies. We unravel a dynamically changed expression pattern of circRNAs during human skin repair and their abnormal expression signature in VU, which are presented as a searchable web resource (www.xulandenlab.com/humanwounds-circrna). We analyzed the coexpression relationship between the circRNAs and mRNAs with weighted correlation network analysis and constructed circRNA‒mRNA‒microRNA networks. This allowed us to expose the regulatory networks specific to the inflammatory and proliferative phases of wound repair and VU, the biological processes the circRNAs may regulate, and the circRNAs that could sponge microRNAs in human wounds. Importantly, we found that hsa-CHST15_0003 and hsa-TNFRSF21_0001, two circRNAs upregulated in VU, hampered epidermal keratinocyte migration while promoting proliferation by modulating gene networks underpinning these cellular processes. This study paves the way to decipher the functional significance of circRNAs in tissue repair.

Integrative small and long RNA omics analysis of human healing and nonhealing wounds discovers cooperating microRNAs as therapeutic targets.

MicroRNAs (miR), as important epigenetic control factors, reportedly regulate wound repair. However, our insufficient knowledge of clinically relevant miRs hinders their potential therapeutic use. For this, we performed paired small and long RNA-sequencing and integrative omics analysis in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic nonhealing venous ulcers (VUs). On the basis of the findings, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte migration and growth while promoting inflammatory response. By combining miR expression patterns with their specific target gene expression context, we identified miRs highly relevant to VU pathology. Our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.

Single-Cell Analysis Reveals Major Histocompatibility Complex II‒Expressing Keratinocytes in Pressure Ulcers with Worse Healing Outcomes.

Pressure ulcer (PU) is a chronic wound often seen in patients with spinal cord injury and other bed-bound individuals, particularly in the elderly population. Despite its association with high mortality, the pathophysiology of PU remains poorly understood. In this study, we compared single-cell transcriptomic profiles of human epidermal cells from PU wound edges with those from uninjured skin and acute wounds in healthy donors. We identified significant shifts in the cell composition and gene expression patterns in PU. In particular, we found that major histocompatibility complex class II‒expressing keratinocytes were enriched in patients with worse healing outcomes. Furthermore, we showed that the IFN-γ in PU-derived wound fluid could induce major histocompatibility complex II expression in keratinocytes and that these wound fluid‒treated keratinocytes inhibited autologous T-cell activation. In line with this observation, we found that T cells from PUs enriched with major histocompatibility complex II+ keratinocytes produced fewer inflammatory cytokines. Overall, our study provides a high-resolution molecular map of human PU compared with that of acute wounds and intact skin, providing insights into PU pathology and the future development of tailored wound therapy.

Isolation and Identification of Natural Colorant Producing Soil-Borne Aspergillus niger from Bangladesh and Extraction of the Pigment.

Natural colorants have been used in several ways throughout human history, such as in food, dyes, pharmaceuticals, cosmetics, and many other products. The study aimed to isolate the natural colorant-producing filamentous fungi Aspergillus niger from soil and extract pigments for its potential use specially for food production. Fourteen soil samples were collected from Madhupur National Park at Madhupur Upazila in the Mymensingh district, Bangladesh. The Aspergillus niger was isolated and identified from the soil samples by following conventional mycological methods (cultural and morphological characteristics), followed by confirmatory identification by a polymerase chain reaction (PCR) of conserved sequences of ITS1 ribosomal DNA using specific oligonucleotide primers. This was followed by genus- and species-specific primers targeting Aspergillus niger with an amplicon size of 521 and 310 bp, respectively. For pigment production, a mass culture of Aspergillus niger was conducted in Sabouraud dextrose broth in shaking conditions for seven days. The biomass was subjected to extraction of the pigments following an ethanol-based extraction method and concentrated using a rotary evaporator. Aspergillus niger could be isolated from three samples. The yield of extracted brown pigment from Aspergillus niger was 0.75% (w/v). Spectroscopic analysis of the pigments was carried out using a UV-VIS spectrophotometer. An in vivo experiment was conducted with mice to assess the toxicity of the pigments. From the colorimetric and sensory evaluations, pigment-supplemented products (cookies and lemon juice) were found to be more acceptable than the control products. This could be the first attempt to use Aspergillus niger extracted pigment from soil samples in food products in Bangladesh, but for successful food production, the food colorants must be approved by a responsible authority, e.g., the FDA or the BSTI. Moreover, fungal pigments could be used in the emerging fields of the food and textile industries in Bangladesh.

Circular RNA hsa_circ_0084443 Is Upregulated in Diabetic Foot Ulcer and Modulates Keratinocyte Migration and Proliferation.

Objective: Insufficient knowledge about the molecular pathology of diabetic foot ulcer (DFU) impedes the development of effective wound treatment. Circular RNAs (circRNAs) are a novel class of RNA recently discovered to be widely expressed and have important biological functions; however, their role in skin wound healing remains largely unexplored. In this study, we investigated the role of circRNAs in DFU. Approach: CircRNA expression was profiled in normal wounds (NWs) and DFUs by microarray analysis, and hsa_circ_0084443 was identified as differentially expressed. The circularity and subcellular localization of hsa_circ_0084443 were characterized by northern blotting, real-time PCR, and fluorescence in situ hybridization. Cell migration, cell growth, and the transcriptome of human primary keratinocytes were analyzed after overexpression or RNA interference of hsa_circ_0084443. Results: hsa_circ_0084443 is downregulated in NWs compared with intact skin, and its level is higher in DFUs than NWs. We confirmed its circularity and presence in the cytoplasm of human epidermal keratinocytes. We showed that hsa_circ_0084443 reduced motility while enhancing the growth of keratinocytes. Furthermore, we identified a gene network with the potential to mediate the biological effect of hsa_circ_0084443. Innovation: CircRNAs have a functional role and a potential clinical significance in skin wound healing. Conclusions: We identified hsa_circ_0084443, a circRNA downregulated during NW healing, as a negative regulator of keratinocyte migration. Higher levels of hsa_circ_0084443 were detected in DFU samples, suggesting that it plays a role in pathology. These findings pave the way to understanding the functional role of circRNAs in human skin wound healing.

MicroRNA-34 Family Enhances Wound Inflammation by Targeting LGR4.

Venous ulcers are the most common type of human chronic nonhealing wounds and are stalled in a constant and excessive inflammatory state. The molecular mechanisms underlying the chronic wound inflammation remain elusive. Moreover, little is known about the role of regulatory RNAs, such as microRNAs, in the pathogenesis of venous ulcers. We found that both microRNA (miR)-34a and miR-34c were upregulated in the wound-edge epidermal keratinocytes of venous ulcers compared with normal wounds or the skin. In keratinocytes, miR-34a and miR-34c promoted inflammatory chemokine and cytokine production. In wounds of wild-type mice, miR-34a-mimic treatment enhanced inflammation and delayed healing. To further explore how miR-34 functions, LGR4 was identified as a direct target mediating the proinflammatory function of miR-34a and miR-34c. Interestingly, impaired wound closure with enhanced inflammation was also observed in Lgr4 knockout mice. Mechanistically, the miR-34-LGR4 axis regulated GSK-3ß-induced p65 serine 468 phosphorylation, changing the activity of the NF-κB signaling pathway. Collectively, the miR-34-LGR4 axis was shown to regulate keratinocyte inflammatory response, the deregulation of which may play a pathological role in venous ulcers.

Clinical Trials with Herbal Products for the Prevention of Dental Caries and Their Quality: A Scoping Study.

It is currently recognized that an injudicious strategy about caries in the last decades has been not only focusing of research mostly in children, but also the narrow focusing on fluoride, because despite sufficient availability of fluoride in water and oral healthcare products, caries levels escalate steadily as people get older and caries remain a main public health issue to be settled. In the last two decades the scientific community intensified efforts of exploring other products for caries prevention, herbal products being one of these approaches. Preliminary evidence indicated that clinical trials for caries prevention with herbal products are heterogeneous in design, quality and products evaluated, we therefore performed a scoping review intended to explore the main characteristics of such clinical trials. From an initial collection of 1986 unique papers from different literature databases, 56 articles satisfied the inclusion and exclusion criteria. The species investigated, dosage forms, study designs, duration of intervention, controls, endpoints, quality of reporting, and risk of bias are discussed. Of the trials reviewed here, 85.71% reported positive results but given the methodological flaws and biases affecting them, it is difficult to conclude on the efficacy of those products based on the studies published thus far.

WAKMAR2, a Long Noncoding RNA Downregulated in Human Chronic Wounds, Modulates Keratinocyte Motility and Production of Inflammatory Chemokines.

Chronic wounds represent a major and growing health and economic burden worldwide. A better understanding of molecular mechanisms of normal as well as impaired wound healing is needed to develop effective treatment. Herein we studied the potential role of long noncoding RNA LOC100130476 in skin wound repair. LOC100130476 is an RNA polymerase II-encoded polyadenylated transcript present in both cytoplasm and nucleus. We found that its expression was lower in wound-edge keratinocytes of human chronic wounds compared to normal wounds of healthy donors and intact skin. In cultured keratinocytes, LOC100130476 expression was induced by TGF-ß signaling. By reducing LOC100130476 expression with antisense oligos or activating its transcription with CRISPR/Cas9 Synergistic Activation Mediator system, we showed that LOC100130476 restricted the production of inflammatory chemokines by keratinocytes, while enhancing cell migration. In line with this, knockdown of LOC100130476 impaired re-epithelization of human ex vivo wounds. Based on these results, we named LOC100130476 wound and keratinocyte migration-associated long noncoding RNA 2 (WAKMAR2). Moreover, we identified a molecular network that may mediate the biological function of WAKMAR2 in keratinocytes using microarray. In summary, our data suggest that WAKMAR2 is an important regulator of skin wound healing and its deficiency may contribute to the pathogenesis of chronic wounds.