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Acne is a prevalent and multifactorial skin disease affecting teenagers and adults, generally stemming from an imbalance in the skin microbiome. Studies have shown that various essential oils (EOs) possess antimicrobial properties that can inhibit the growth of acne-causing bacteria strains like Cutibacterium acnes and Staphylococcus epidermidis. What We Know: Current acne treatments often involve topical or oral antibiotics, raising concerns about antibiotic resistance in C. acnes strains (Sardana et al., 2015). EOs are natural, concentrated liquids derived from plants, known for their aromatic properties and diverse biological characteristics, including bactericidal, virucidal and fungicidal properties (Cossetin et al., 2021). These include oregano, lavender, lemon grass, myrtle, lemon, thyme, eucalyptus, rosemary and tea tree EOs, which have all demonstrated anti-inflammatory, antioxidant and antimicrobial properties (Bungau et al., 2023). EOs are able to permeate the skin and facilitate the enhanced penetration of active compounds into deeper skin layers (Bungau et al., 2023). Their antimicrobial property occurs as their bioactive components target multiple cellular sites and interact with cell membranes, disrupting microbial integrity and ultimately causing cell death (Bungau et al., 2023). Industry Impact & Potential: Incorporating English lavender and peppermint EOs into gelatine nanofibers created effective topical patches for localised acne treatment by diminishing C. acnes and S. epidermidis (Uhlířová et al., 2023). Therefore, English lavender and peppermint EOs may be beneficial in the gentle and focused treatment of acne, as well as potentially other microbial-related skin conditions (Uhlířová et al., 2023). However, a challenge with EO use is their standardisation, which arises due to different cultivation conditions that produce EOs of varied quality, quantity and composition (Uhlířová et al., 2023). Methods for assessing antioxidant potential, such as determining total phenolic content, evaluating reducing power, measuring ferrous ion chelating activity, among other methods, are vital for gauging the efficacy and safety of essential oil use in acne treatment (Bungau et al., 2023). Future investigations could focus on developing innovative acne treatments incorporating essential oils and evaluating their effectiveness through large-scale clinical trials. Moreover, further research is warranted to elucidate the mechanisms of action of essential oils and identify their optimal doses and safety profiles for effective management of acne vulgaris (Bungau et al., 2023). Our Solution: At Sequential, we offer a comprehensive Microbiome Product Testing Solution, separately or in conjunction with guided product development and formulation services. This holistic approach enables your business to explore innovative methods for addressing skin conditions, like acne, using essential oils treatment strategies. Reference List: Bungau, A. F., Radu, A. F., Bungau, S. G., Vesa, C. M., Tit, D. M., Purza, A. L., & Endres, L. M. (2023). Emerging Insights into the Applicability of Essential Oils in the Management of Acne Vulgaris. Molecules (Basel, Switzerland) , 28(17), 6395. https://doi.org/10.3390/molecules28176395 Castellanos Lorduy, H. J., Pérez Cely, H. C., Casadiego Rincón, E. J., Henao Riveros, S. C., & Colorado, C. L. (2021). Cutibacterium acnes tetracycline resistance profile in patients with acne vulgaris, in a colombian dermatologic center. Actas Dermo-Sifiliográficas (English Edition), 112(10), 873-880. doi:10.1016/j.adengl.2021.09.003 Cossetin, L. F., Garlet, Q. I., Velho, M. C., Gündel, S., Ourique, A. F., Heinzmann, B. M., & Monteiro, S. G. (2021). Development of nanoemulsions containing lavandula dentata or myristica fragrans essential oils: Influence of temperature and storage period on physical-chemical properties and chemical stability. Industrial Crops and Products, 160, 113115. doi:10.1016/j.indcrop.2020.113115 Sardana, K., Gupta, T., Garg, V.,K., & Ghunawat, S. (2015). Antibiotic resistance to propionobacterium acnes: Worldwide scenario, diagnosis and management. Expert Review of Anti-Infective Therapy, 13(7), 883-896. doi:10.1586/14787210.2015.1040765 Uhlířová, R., Langová, D., Bendová, A., Gross, M., Skoumalová, P., & Márová, I. (2023). Antimicrobial Activity of Gelatin Nanofibers Enriched by Essential Oils against Cutibacterium acnes and Staphylococcus epidermidis. Nanomaterials (Basel, Switzerland), 13(5), 844. https://doi.org/10.3390/nano13050844

Elucidating the relationship between ceramides and the skin microbiome could revolutionise the treatment of skin disease. Ceramides, the lipid molecules abundant in the stratum corneum, play a crucial role in skin barrier function and alterations to their levels or profiles are associated with impaired barrier function and, in some cases, skin disease. What We Know: Ceramides are responsible for preventing water loss, maintaining skin moisture levels and ultimately maintaining skin barrier function (Baker et al., 2023). The breakdown of the skin barrier is a common feature of numerous inflammatory skin conditions and is well-understood in diseases like atopic dermatitis (AD) and psoriasis (Rajkumara et al., 2023). In the case of AD, patients generally have reduced ceramide levels (Elias et al., 2019). Regarding psoriasis, there is a reduction in the enzymes involved in ceramide biosynthesis and metabolism, causing a decrease in overall ceramides levels (Choi & Maibach, 2005). Staphylococcus epidermidis plays a role in skin barrier maintenance by secreting sphingomyelinase, an enzyme that facilitates the conversion of host cell sphingomyelin into ceramides (Zheng et al., 2022). Ceramides also play a role in maintaining the acidic mantle: a protective film that covers that surface of the skin with a pH 4.5-5.5, which is crucial for the proliferation of beneficial microorganisms like S. epidermidis (Baker et al., 2023). Industry Impact & Potential: Probiotic-type topical treatments, containing microbiota like S. epidermidis, show potential in treating skin diseases by leveraging their role in maintaining skin barrier integrity (Zheng et al., 2022). Researchers propose a targeted approach of screening the existing skin microbiome composition for deficiencies in S. epidermidis enzymes and subsequently treating the skin with strains that produce the lacking enzyme(s) in combination with direct bacterial interference mechanisms to eliminate harmful pathogens (Zheng et al., 2022). pH-optimised moisturisers enhance the chemical skin barrier by promoting optimal enzymatic activity that boosts ceramide synthesis, as well as creating favourable environments for beneficial microorganisms (Rajkumara et al., 2023). Mice models have shown that through the bacterial phenomenon of quorum sensing, commensal microbiota can inhibit Staphylococcus aureus toxin production (which is highly associated with AD symptoms), thus preventing skin barrier damage and inflammation (Williams et al., 2019). Our Solution: Sequential offers an end-to-end Microbiome Product Testing Solution, alongside guided product development and formulation services. Leveraging our expertise, we assist businesses in devising novel approaches to topical ceramide and/or probiotic-based treatments for various skin diseases and inflammatory conditions. Reference List: Baker, P., Huang, C., Radi, R., Moll, S. B., Jules, E., & Arbiser, J. L. (2023). Skin Barrier Function: The Interplay of Physical, Chemical, and Immunologic Properties. Cells, 12(23), 2745. https://doi.org/10.3390/cells12232745 Bouwstra, J. A., Helder, R. W. J., & El Ghalbzouri, A. (2021). Human skin equivalents: Impaired barrier function in relation to the lipid and protein properties of the stratum corneum. Advanced drug delivery reviews, 175, 113802. https://doi.org/10.1016/j.addr.2021.05.012 Choi, M. J., & Maibach, H. I. (2005). Role of ceramides in barrier function of healthy and diseased skin. American journal of clinical dermatology, 6(4), 215–223. https://doi.org/10.2165/00128071-200506040-00002 Elias, P. M., Wakefield, J. S., & Man, M. Q. (2019). Moisturizers versus Current and Next-Generation Barrier Repair Therapy for the Management of Atopic Dermatitis. Skin pharmacology and physiology, 32(1), 1–7. https://doi.org/10.1159/000493641 Rajkumar, J., Chandan, N., Lio, P., & Shi, V. (2023). The Skin Barrier and Moisturization: Function, Disruption, and Mechanisms of Repair. Skin pharmacology and physiology, 36(4), 174–185. https://doi.org/10.1159/000534136 Williams, M. R., Costa, S. K., Zaramela, L. S., Khalil, S., Todd, D. A., Winter, H. L., . . . Gallo, R. L. (2019). Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis. Science Translational Medicine, 11(490), eaat8329. doi:10.1126/scitranslmed.aat8329 Zheng, Y., Hunt, R. L., Villaruz, A. E., Fisher, E. L., Liu, R., Liu, Q., Cheung, G. Y. C., Li, M., & Otto, M. (2022). Commensal Staphylococcus epidermidis contributes to skin barrier homeostasis by generating protective ceramides. Cell host & microbe, 30(3), 301–313.e9. https://doi.org/10.1016/j.chom.2022.01.004

Alopecia (hair loss) can be caused by factors such as reduced physiological function, scalp tension-induced blood flow disorders, genetic predisposition and poor scalp nutrition. While microbiome-focused research into alopecia is limited, biomolecules of fermented fruits and fermenting microbes show promise as a potential solution. What We Know: Studies investigating the scalp microbiome determined that Cutibacterium spp. and Staphylococcus spp. make up approximately 90% of a healthy scalp microbiome, with the remaining 10% consisting of Corynebacterium spp., Streptococcus spp., Acinetobacter spp. and Prevotella spp (Jo et al., 2022). The scalp microbiome may impact scalp health and alopecia. Research indicates no variance in species diversity or abundance between alopecia-affected and healthy scalps. However, a study found a >10% difference in Proteobacteria and Actinobacteria distributions. Healthy scalps showed higher Proteobacteria abundance, while alopecia-affected scalps had higher Actinobacteria levels (Jo et al., 2022). Fermented aloe vera and kimchi were successfully used to treat burn wounds and improve the skin microbiota pattern, maintaining probiotic (saprophytic) bacteria, including Lactobacillus, and reducing pathogenic bacteria (such as Prevotella and Cutibacterium acnes) (Yoon et al., 2022; Park et al., 2020). Industry Impact and Potential: Hair care products containing fermented papaya and mangosteen improved hair shaft conditions and reduced hair loss compared to control groups, showing potential for alopecia (Mayer et al., 2023). These experimental products improved the scalp microbiota, correcting the alopecia-associated altered microbiota pattern by decreasing pathogen content whilst maintaining probiotic (saprophyte) levels (Mayer et al., 2023). Fermented papaya's probiotic activity may involve selectively inhibiting catalase in microbial pathogens, which they rely on for enzymatic antioxidant defence against the host's immune response (Mayer et al., 2023). Further studies are needed to explore the preclinical and clinical effects of using food-grade fermented products in cosmetics, aiming to better understand their mechanisms and potential benefits for hair health. Integrating phytochemical and biological experiments could help identify specific plant- and microbe-derived substances with hair loss prevention and hair quality enhancement properties (Mayer et al., 2023). Our Solution: With a vast database of over 20,000 microbiome samples and 4,000 ingredients, coupled with a global network of over 10,000 testing participants, Sequential offers comprehensive services to evaluate product impacts and formulations. Our customisable microbiome studies provide real-life context testing, while formulation support ensures products maintain biome integrity. Therefore, we are your ideal candidate to utilise our solutions for your product development and efficacy. References: Jo, H., Kim, S.Y., Kang, B.H., Baek, C., Kwon, J.E., Jeang, J.W., Heo, Y.M., Kim, H.-B., Heo, C.Y., Kang, S.M., Shin, B.H., Nam, D.Y., Lee, Y.-G., Kang, S.C. & Lee, D.-G. (2022) Staphylococcus epidermidis Cicaria, a Novel Strain Derived from the Human Microbiome, and Its Efficacy as a Treatment for Hair Loss. Molecules. 27 (16). doi:10.3390/molecules27165136. Mayer, W., Weibel, M., De Luca, C., Ibragimova, G., Trakhtman, I., Kharaeva, Z., Chandler, D.L. & Korkina, L. (2023) Biomolecules of Fermented Tropical Fruits and Fermenting Microbes as Regulators of Human Hair Loss, Hair Quality, and Scalp Microbiota. Biomolecules. 13 (4), 699. doi:10.3390/biom13040699. Park, D.-W., Lee, H.S., Shim, M.-S., Yum, K.J. & Seo, J.T. (2020) Do Kimchi and Cheonggukjang Probiotics as a Functional Food Improve Androgenetic Alopecia? A Clinical Pilot Study. The World Journal of Men’s Health. 38 (1), 95–102. doi:10.5534/wjmh.180119. Yoon, Y.C., Ahn, B.H., Min, J.W., Lee, K.R., Park, S.H. & Kang, H.C. (2022) Stimulatory Effects of Extracellular Vesicles Derived from Leuconostoc holzapfelii That Exists in Human Scalp on Hair Growth in Human Follicle Dermal Papilla Cells. Current Issues in Molecular Biology. 44 (2), 845–866. doi:10.3390/cimb44020058.

At Sequential, we have developed a complete end-to-end solution to support microbiome R&D, formulation support and claims support. In 2019, we launched the world’s first at-home skin microbiome test kit (here) through Sequential Skin, and since then we have developed our B2B offering under Sequential (here). Sequential is formed by an award-winning team of scientists that is leading the way in microbiome analysis and detection: we are the first skin microbiome company to carry out in vivo testing (testing on the human skin) via an end-to-end service – from consultation, testing kits, to analysis and results, post-study consultation including claims substantiation and “Maintains the Microbiome in vivo” certification, as well as formulation optimisation. To date, we have analysed over 12,000 skin microbiome samples, deriving an AI-driven platform to improve formulation design, through working with 30 personal care, and skincare companies – such as Johnson & Johnson, Arcaea, Walgreens Boots Alliance, Clinique La Prairie, Vavin, and the Wella Company. Our microbiome technology is backed by some of the world's most prominent investors such as Metaplanet, Scrum Ventures, SOSV, Genedant, Ben Holmes (ex. Index Ventures), Innovate UK, and we are a resident company of Johnson & Johnson Innovation – JLABS. Sequential officially launched in March 2022 (here) and since then we have developed scalp, oral, and vulva microbiome testing kits as new products. We are headquartered in London UK, with testing labs in NYC and Singapore. Why is Sequential innovative? Sequential is developing first-in-class innovative microbiome solutions for the cosmetic and skincare industry. Since conception in 2017, we’ve launched first-of-its-kind products to help drive forward and advance R&D on the human microbiome. The human skin microbiome is incredibly complex and relies on a systems biology approach to understand the presence, function, and relationships between microbes on the skin. In medical research, in vitro experiments have been used for the past century in understanding the basic function of the cell, however, they often fail in translating what's really happening in – or on – the human body. In the context of the skin microbiome, this is because the microbe behaves very differently when they’re taken out of their natural environment – and network with other microbes and biofilm. The only way to test a specific species or strain of microbe is to test it within its ‘real-world’ context – or in vivo. This is where the innovative solutions of Sequential come in. We have developed rigorous experimental designs for longitudinal studies that allow us to measure the specific amount of micro-organisms using qPCR, giving us quantitative information on species and strain level of over 20+ microbes found on the skin in vivo. By testing at several time points over 2-4 weeks of product usage, we can have confidence in how the skin microbiome is truly being affected. Our database on the skin microbiome and products are extensive, which allows us to have a clear understanding of how products and ingredients are having an effect on the microbiome. Our Sequential Patch is a skin microbiome testing solution that can be employed to ensure more consistent collection and we have shown more reliable results than using a swab for collection in longitudinal studies. We’ve also shown higher DNA and RNA amounts, which improves the quality of data we can present to our clients, and to their customers. Industry Challenges & Our Solutions 16S/ITS Sequencing on the microbiome doesn’t resolve species or strain level information Sequential uses shotgun metagenomic sequencing in our R&D, to understand critical species and strains that are influenced by cosmetic products and influence skin health. We have developed highly specific probes to measure the absolute quantity of these species or strains, down to the copy number. This gives us much more confidence that the microbiome is changing, not just at the community diversity level but at the individual species and strain level. A challenge has also been to have a sufficient database to know which strains of micro-organisms are critical for skin health Sequential has leveraged early datasets from our key advisors, Dr Paola de Sessions, Dr Tom Dawson and Dr Niranjan Nagarajan, to start with a strong foundation of a skin microbiome database. Since then, we have worked with 30+ clients to collect over 12,000 skin microbiome samples which give us a solid understanding of skin microbiome and how products and ingredients affect it. Human biological data is highly sensitive and individuals are concerned about data privacy Sequential uses highly secure encrypted AWS servers to store our microbiome data, in order to perform analysis and results. Only two people in Sequential Bio have direct access to this database and it is securely protected from anyone else having access. Finally, the personal details (name, address, contact details) of the individual samples are de-identified so there’s no way of being able to trace who each microbiome sample comes from. Some companies outsource microbiome testing to third parties A challenge here for the end client is that the price is higher, the quality isn’t always ensured, and the data is shared with another group. Sequential develops all our microbiome probes to test species and strains of micro-organisms in-house and therefore owns the entire process. This ensures higher quality service and a lower end cost to the client we work with. If you are interested in carrying out any research with us and testing products, you can reach us at team@sequential.bio. Our Press Releases https://www.personalcareinsights.com/news/microbiome-start-up-sequential-bio-debuts-first-in-vivo-testing-for-personal-care-industry.html https://www.cosmeticsandtoiletries.com/testing/microbiology/news/22105697/sequential-skin-debuts-sequential-bio-testing-solution