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Introduction: The gut-brain axis The gut-brain axis is a two-way communication network between the digestive system and the brain, involving hormones, metabolism, the immune system, and other pathways. Key components include the autonomic nervous system, the hypothalamic-pituitary-adrenal (HPA) axis, and gut nerves that link the brain to digestion and immune responses. The gut can also influence mood, cognition, and mental health (Appleton, 2018). Gut bacteria play a crucial role in this relationship, affecting mental health, emotional regulation, and the HPA axis. Changes in the gut microbiome have been linked to mood disorders like anxiety and depression, as well as gastrointestinal (GI) diseases like irritable bowel syndrome, which often coincide with psychological conditions. Gut microbiota also impact brain development in fetuses and newborns, and diet influences how the gut microbiome affects cognitive functions (Appleton, 2018). Depression Depression is a mood disorder characterized by feelings of sadness, emptiness, or irritability, leading to a loss of interest in daily activities. It can cause both mental and physical changes that interfere with everyday life. In the U.S., depression contributes to nearly 40,000 suicides each year, with older men being at the highest risk (Chand & Arif, 2023). Depression can affect anyone, regardless of age or social background, impacting both women and men. If symptoms persist for more than two weeks, it may indicate depression ( InformedHealth.org , 2020). Major Depressive Disorder (MDD) has multiple causes, including biological, genetic, environmental, and psychological factors. While it was initially thought to be due to imbalances in neurotransmitters like serotonin, norepinephrine, and dopamine, newer research suggests disruptions in complex neural circuits. Other neurotransmitters, such as GABA and glutamate, as well as hormonal imbalances, also play a role. Early life stress and trauma can result in lasting brain changes that contribute to depression. Genetics have a strong influence, especially in twins, and life events, personality traits, and cognitive distortions further increase the risk (Chand & Arif, 2023). Diagnosis of major depression is primarily made through a clinical evaluation, including a detailed interview and mental status examination, and is found to be as reliable as many medical tests (Goldman et al., 1999). The DSM-5 provides specific criteria for diagnosing depression (Regier et al., 2013). Treatment typically involves medication and brief psychotherapy, such as cognitive-behavioral therapy (CBT) or interpersonal therapy. Combining these treatments improves symptom relief and quality of life. CBT is particularly effective in preventing relapse. Despite effective treatments, about 50% of people may not initially respond, and full recovery is uncommon. However, around 40% of patients experience partial improvement within a year (Chand & Arif, 2023). Selective Serotonin Reuptake Inhibitors (SSRIs) Selective Serotonin Reuptake Inhibitors (SSRIs) are the most commonly prescribed medications for treating depression. They are usually the first choice for pharmacotherapy because of their safety, effectiveness, and good tolerance in both adults and children (Chu & Wadhwa, 2023).  SSRIs work by increasing the levels of serotonin, also known as 5-hydroxytryptamine (5-HT), in the brain, which is often low in individuals with depression. They do this by blocking the serotonin transporter (SERT) at nerve endings, which prevents the reabsorption (reuptake) of serotonin. This action keeps more serotonin in the brain's synapses, allowing it to have a more prolonged effect on mood (Figure 1). Unlike other antidepressants, SSRIs primarily target serotonin and have minimal impact on other neurotransmitters like dopamine or norepinephrine (Chu & Wadhwa, 2023). Figure 1: A schematic diagram illustrating the mechanism of SSRIs: These medications block the reuptake of serotonin at the presynaptic membrane, leading to increased serotonin levels at the postsynaptic nerve terminal membrane. Image taken from (Lattimore et al., 2005) . Serotonin is also present in the gut mainly at the enterochromaffin (EC) cells of the mucosa. It interacts with various receptors in the gut, the 5-HT3 and 5-HT4 receptors have been most extensively studied for their role in gut motility. It has also been seen that, 5-HT released from EC cells is capable of inducing the mucosal peristaltic reflex and hence propulsive peristalsis (Kendig & Grider, 2015). 5-HT synthesis and gut-brain interaction As mentioned above, serotonin (5-HT) in the gut is produced from tryptophan in enterochromaffin (EC) cells and serotonergic neurons through the actions of tryptophan hydroxylase 1 and 2 (TPH1 and TPH2), converting tryptophan into 5-hydroxytryptophan (5-HTP), which is then turned into serotonin by L-amino acid decarboxylase (L-AADC). This serotonin, along with chromogranin A (CGA), is stored in vesicles via vesicular monoamine transporter 1 (VMAT1). EC cells release serotonin into the extracellular space in response to various stimuli, including chemical and mechanical changes. Most serotonin is released into the extracellular space, with an amount going into the gut lumen. Serotonin is taken up by surrounding enterocytes via the serotonin reuptake transporter (SERT) and then metabolized into 5-hydroxyindoleacetic acid (5-HIAA) by monoamine oxidase (MAO). In serotonergic neurons, serotonin is released into the synaptic cleft, where it acts on postsynaptic receptors and is reabsorbed by SERT. Serotonin is also taken up by endothelial cells and platelets, where it is either converted into 5-HIAA or transported to other tissues (Figure 2) (Liu et al ., 2021). Figure 2: Schematic representation of 5-HT biosynthesis and metabolism. Image taken from (Liu et al., 2021) . The brain-gut hypothesis suggests that serotonin (5-HT) plays a key role in the communication between the gut and the brain. While most serotonin is found in the gut, it also influences brain functions like mood, sleep, and appetite. Imbalances in serotonin levels or receptor activity can result in gastrointestinal issues, such as irritable bowel syndrome (IBS), as well as symptoms in other parts of the body. Research indicates that therapies targeting serotonin can help manage IBS symptoms and related central nervous system dysfunctions (Crowell & Wessinger, 2007). A study investigating the effects of selective serotonin reuptake inhibitors (SSRIs) and the probiotic Lactobacillus rhamnosus  JB-1 on gut-brain signaling through the vagus nerve found some interesting results. Using mouse models, researchers observed that oral SSRI treatment activated vagal pathways, which were crucial for reducing anxiety and depression-like behaviors. Similarly, Lactobacillus rhamnosus  JB-1 alleviated these behaviors, but its effectiveness was reduced in mice with a severed vagus nerve. This highlights the importance of vagal signaling in gut-brain communication. Both SSRIs and the probiotic also affected the gut microbiota and serotonin levels, suggesting the potential of targeting the gut-brain axis in treating mood disorders like anxiety and depression (McVey et al ., 2019). Comprehensive review: Depressive gut microbiota Depressive Gut Microbiota refers to an imbalanced composition and reduced diversity of microorganisms in the gastrointestinal tract, which has been connected to the development and progression of depression.  Studies have shown that individuals with depression exhibit differences in the microbiota community across various taxonomic levels when compared to those without depression, including variations in both α-diversity and β-diversity. At the phylum level, there were inconsistencies in the abundance of Firmicutes , Bacteroidetes , and Proteobacteria , but higher levels of Actinobacteria  and Fusobacteria  were consistently observed in depressed individuals. On the family level, people with depression had increased levels of families like Actinomycineae , Bifidobacteriaceae , Clostridiaceae , and Streptococcaceae , while families such as Veillonellaceae  and Prevotellaceae  were less abundant. At the genus level, those with depression showed higher levels of genera like Oscillibacter , Blautia , Streptococcus , and Klebsiella , with lower levels of beneficial bacteria like Coprococcus , Lactobacillus , and Escherichia/Shigella . These changes in gut microbiota composition are evident in those living with depression, and they are associated with inflammation, gut barrier dysfunction, and disruption of gut-brain communication, contributing to depressive symptoms (Barandouzi et al ., 2020). Study 1: Gut Microbiome Patterns Associated With Treatment Response in Patients With Major Depressive Disorder (Bharwani et al ., 2020) The first long-term study to explore a potential link between major depressive disorder (MDD) and the gut microbiome by analyzing microbial patterns before starting antidepressant treatment and during 6 months of therapy. No other research has looked at the microbiota in individuals who have not yet begun antidepressant use (Bharwani et al ., 2020). Results: Microbial differences Fifteen participants (average age 36.9, SD = 12.9; 12 women) were studied. At the start, their average Montgomery–Åsberg Depression Rating Scale (MADRS) score was 22.53 (SD = 6.63). After 6 months, 11 participants were classified as "remitters" (MADRS < 12) and 4 as "nonremitters" (MADRS > 13). Most participants took Escitalopram, while a few took Citalopram (Bharwani et al ., 2020). Baseline diversity of gut microbiota was higher in remitters than in nonremitters (Figure 3). However, there were no differences in variability or community clustering based on treatment response. At baseline, 22 gut microbiota types of operational taxonomic units (OTUs) were different between the two groups. No significant changes in diversity were observed at 3 months, but differences reappeared at 6 months. Within-subject diversity and community composition showed no significant changes over time for either group (Bharwani et al ., 2020). One OTU, a Clostridiales, increased in remitters at 6 months, while no OTUs changed in nonremitters. At 3 months, 35 OTUs were different between the groups, and by 6 months, 42 OTUs showed differences, with some also differing at 3 months. Diet and antidepressant type had no impact on these results (Bharwani et al ., 2020). Figure 3: Baseline differences between eventual responders and nonresponders in alpha-diversity metrics. Image taken from (Bharwani et al., 2020) . Conclusion The study shows that antidepressant treatment impacts the microbiota at the OTU level, based on how well patients respond to treatment. Although overall diversity and microbiota profiles did not change significantly in either group, remitters continued to have higher diversity compared to nonremitters after 6 months. This indicates a potential microbial community signature that differentiates between treatment responders and nonresponders (Bharwani et al ., 2020). Study 2: Gut Microbial Signatures Can Discriminate Unipolar from Bipolar Depression (Zheng et al ., 2020) Gut microbiome changes are linked to major depressive disorder (MDD) and bipolar disorder (BD), but their specific differences are unclear. This study identifies unique microbial patterns for MDD and BD and offers markers to differentiate between the two based on gut microbiome signatures (Zheng et al ., 2020). Results: Distinct gut microbiome signature in MDD and BD The study compared gut microbiomes across individuals with bipolar disorder (BD), major depressive disorder (MDD), and healthy controls (HC). At the phylum level (Figure 4a), BD had lower Bacteroidetes  and higher Proteobacteria  compared to MDD and HC. At the family level (Figure 4b), BD showed elevated Pseudomonadaceae , while MDD had higher Bacteroidaceae  and Bifidobacteriaceae  but lower Enterobacteriaceae  than HC. Comparing BD and MDD, BD had more Enterobacteriaceae  and Pseudomonadaceae , while MDD showed higher Bacteroidaceae  and Veillonellaceae  (Zheng et al ., 2020). Figure 4: (a) At the phylum level. (b) At the family level. Image taken from (Zheng et al., 2020) . Results: Gut Microbial Biomarkers for Discriminating MDD, BD, and HC  They identified four microbial OTUs, mostly from the  Lachnospiraceae  family, that were strongly linked to Hamilton Depression Rating Scale (HAMD) scores in MDD and BD patients (Figure 5). These microbial markers helped distinguish between MDD, BD, and healthy controls and also indicated the severity of MDD or BD in patients (Zheng et al ., 2020). Figure 5: Four microbial OTUs, primarily from the Lachnospiraceae  family, showed significant links to HAMD scores in MDD and BD patients. Red lines represent positive correlations, while blue lines represent negative correlations, with thicker lines indicating stronger statistical significance (p < 0.05). Image taken from (Zheng et al., 2020) . Conclusion In summary, they identified distinct gut microbiota differences between MDD, BD, and healthy controls. The researchers also found markers that effectively distinguishes MDD from BD and HC.  Looking at all these studies, they confirm that there is a connection between the gut and the brain. Role of the Vagus Nerve The vagus nerve is a key part of the parasympathetic nervous system, responsible for regulating essential functions like mood, immune response, digestion, and heart rate. It serves as a communication link between the brain and the gastrointestinal tract, transmitting information about the condition of internal organs to the brain through afferent fibers (Breit et al ., 2018). Issues with the vagus nerve or changes in its function have been linked to a range of gastrointestinal and psychiatric disorders (Breit et al ., 2018). Importance of Vagus Nerve A study demonstrated that the vagus nerve impacts the anxiety-reducing effects of Lactobacillus rhamnosus  by acting as a conduit for communication between the gut microbiota and the brain. The study shows that when the vagus nerve is severed, the beneficial effects of L. rhamnosus  on anxiety and behavior are lost, indicating that the vagus nerve is essential for transmitting signals from the gut microbiota to the brain. This underscores the nerve’s crucial role in modulating the gut-brain axis, affecting how gut microbiota influence mental health and stress responses (Bravo et al ., 2011). BDNF (Brain-derived neurotrophic factor) BDNF is a protein essential for the growth, maintenance, and survival of neurons in the brain, especially in regions such as the hippocampus, which are critical for memory, learning, and mood regulation. Changes in gut microbiota composition may affect BDNF levels, thereby influencing cognitive functions and neuroplasticity. This underscores the bidirectional relationship between the gut and brain (Agnihotri & Mohajeri, 2022). Lactobacillus in digestive system (Capuco et al., 2020) A study found that patients with major depressive disorder (MDD) who took SSRIs along with the probiotic Lactobacillus plantarum  299v had significantly lower levels of kynurenine, a neurotoxic compound linked to cognitive decline and mood disorders. This group also showed improved cognitive functions, including better attention, perception, and verbal learning, compared to a placebo group. Kynurenine, which can harm the central nervous system, is produced during inflammation triggered by the immune system. The probiotic may have reduced intestinal inflammation, leading to lower kynurenine production and contributing to improved cognition and mood (Capuco et al ., 2020). (Han & Kim, 2019) This study found that isolated Lactobacillus mucosae  NK41 and Bifidobacterium longum  NK46, derived from human feces, increased levels of brain-derived neurotrophic factor (BDNF) in cells stressed with corticosterone. In mice, both probiotics, alone and in combination, reduced anxiety and depression related to stress, lowered inflammation and stress markers in the brain and blood, and improved gut health. They also decreased populations of Proteobacteria  and Bacteroidetes  in the gut, as well as bacterial lipopolysaccharide production. These findings suggest that these probiotics may alleviate anxiety, depression, and colitis by mitigating gut dysbiosis (Han & Kim, 2019). (Yong et al., 2020) Lactobacillus rhamnosus , has shown antidepressant effects in both healthy and stressed mice. Studies in postpartum women and obese individuals also reported reduced depressive thoughts with its use. Its positive effects are linked to signaling through the vagus nerve, influencing brain activity and the stress-regulating hypothalamic-pituitary-adrenal (HPA) axis. L. rhamnosus  produces Gamma-aminobutyric acid (GABA), a neurotransmitter that helps regulate mood, and can cross the gut barrier to interact with neurons. This probiotic also lowers stress hormones and may help prevent depression by boosting GABA levels and protecting against HPA axis overactivity (Yong et al ., 2020). These studies demonstrate that a specific Lactobacillus strain effectively treats major depressive disorder (MDD) by lowering inflammation and increasing serotonin production. Strength and limitations Strengths Clinical Relevance : Understanding the gut-brain axis has key clinical implications for treating various conditions, including mental health disorders like depression, anxiety, and irritable bowel syndrome (IBS), as well as neurological diseases like Parkinson’s and Alzheimer’s. Therapeutic Potential : Research on the gut-brain axis has led to innovative treatments targeting gut microbiota, including probiotics, prebiotics, and fecal microbiota transplantation (FMT), offering potential for managing gastrointestinal and psychiatric disorders. Advanced Technologies : Cutting-edge tools such as metagenomics, metabolomics, and neuroimaging have enhanced the study of the gut-brain axis, helping researchers uncover the mechanisms of gut-brain communication with greater precision. Limitations Correlational Nature : Much of the research is based on associations between gut microbiota and brain function or behavior, making it difficult to establish causality and deeper understanding. Animal Models : Many gut-brain axis studies use animal models, which may not fully reflect human physiology and behavior. Translating these findings to humans requires careful attention to species differences and limitations. Long-term Effects and Safety : The long-term safety and effects of gut microbiota-targeted interventions for mental health are still unclear. More research is needed to evaluate potential risks and benefits over extended periods. Implications and Applications Probiotic Supplements : Recommending probiotics with beneficial bacteria to help balance gut microbiota. Psychotherapy : Using therapies like cognitive-behavioral therapy (CBT) that address both mental health and gut-brain axis interactions. Medication : Considering medications that influence the gut-brain axis, including specific antidepressants or treatments targeting gut microbial imbalances. Education and Awareness : Informing patients about the link between gut health and mental well-being to help them make better lifestyle choices. Related Research and Future Directions Role in Neurological and Psychiatric Disorders : Exploring the influence of the gut-brain axis on a variety of neurological and psychiatric conditions beyond depression and anxiety, such as Alzheimer's, Parkinson's, autism spectrum disorders, schizophrenia, and bipolar disorder. This research could reveal common underlying mechanisms and highlight new therapeutic options. Gut Microbiota and Brain Development : Examining how gut microbiota impacts brain development and function during key stages like infancy, childhood, and adolescence. Insights into early microbial exposures could guide strategies for enhancing neurodevelopment and brain health. Technological Advances : Utilizing cutting-edge tools like microbiome sequencing, multi-omics profiling, neuroimaging, and computational modeling to delve deeper into gut-brain interactions. Combining diverse data sources will allow for more detailed analyses of complex biological systems. Conclusion With the growing pressures of modern life, depression has become increasingly prevalent, yet a complete cure remains unknown. Research on the gut-brain axis suggests a strong connection between depression and changes in the gut microbiota. Depression can disrupt the gut's microbial balance and potentially trigger complications like IBS. IBS, linked to gut microbiota imbalance, also increases the risk of depression. Adjusting and restoring the gut microbiota composition may help alleviate depressive symptoms. This highlights the importance of investigating microbiota-based treatments for depression (Zhu et al ., 2022). References Agnihotri N, Mohajeri MH. Involvement of Intestinal Microbiota in Adult Neurogenesis and  the Expression of Brain-Derived Neurotrophic Factor. Int J Mol Sci. 2022 Dec 14;23(24):15934. doi: 10.3390/ijms232415934. PMID: 36555576; PMCID: PMC9783874. Appleton J. The Gut-Brain Axis: Influence of Microbiota on Mood and Mental Health. 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In the era of elaborate skincare routines, consumer trends often outpace evidence-based science. Research on popular high frequency (HF) devices, such as the wands commonly used for acne treatment, may offer valuable insights into their true effects on the skin microbiome. What We Know: Developed in the 19th century, HF therapy gained attention for its potential benefits in supporting lymphatic drainage, preventing hair loss and reducing wrinkles. By the early 20th century, it was recognised as a versatile treatment for various conditions, including skin infections, eczema and wounds, as well as ailments like migraines, neuralgia and even tuberculosis (Napp et al., 2015) . HF devices, also known as Violet Wands, function by delivering Cold Atmospheric Pressure Plasma (CAPP) to the site of application, such as the face. This process releases bioactive components, including charged particles and reactive species like ozone and nitrogen oxides (Frommherz et al., 2022). HF therapy ultimately lost popularity in the mid-20th century due to the rise of antibiotics and limited efficacy data. However, with increasing antibiotic resistance, plasma medicine (or CAPP) is gaining renewed interest. Recent studies suggest that HF devices may outperform traditional antiseptics in targeting wound pathogens, raising questions about their potential benefits for acne-prone skin and their impact on the skin microbiome (Daeschlein et al., 2015) . Industry Impact and Potential: Recent research has demonstrated that HF therapy possesses a microbicidal effect on skin microbiota and pathogens in vitro , significantly reducing bacterial and fungal counts after just a brief treatment. Notably, one minute of HF application led to a significant reduction in C. acnes levels (Frommherz et al., 2022) . It is hypothesised that HF therapy increases ozone formation during application, suggesting that its primary antimicrobial effects stem from ozone and the oxidative stress it induces in microbes. Ozone is also recognised for its anti-inflammatory properties, further enhancing its efficacy in treating skin conditions (Frommherz et al., 2022) . Ultimately, the antiseptic properties of HF therapy present a promising alternative to antibiotics for managing conditions like acne ( Frommherz et al., 2022) . However, it is important to remember that not all HF devices available, especially cheaper options, are created equal; variations in voltage, frequency and design can affect their efficacy and safety.  Our Solution: With a database of 25,000 microbiome samples and 4,000 ingredients, plus a global network of testing participants, Sequential provides customised solutions for microbiome studies and product formulation. Our commitment to developing microbiome-safe products ensures the preservation of biome integrity, making us an ideal partner investigating the skin, scalp, oral and vaginal microbiome. References: Daeschlein, G., Napp, M., von Podewils, S., Scholz, S., Arnold, A., Emmert, S., Haase, H., Napp, J., Spitzmueller, R., Gümbel, D. & Jünger, M. (2015) Antimicrobial Efficacy of a Historical High-Frequency Plasma Apparatus in Comparison With 2 Modern, Cold Atmospheric Pressure Plasma Devices. Surgical Innovation . 22 (4), 394–400. doi:10.1177/1553350615573584. Frommherz, L., Reinholz, M., Gürtler, A., Stadler, P.-C., Kaemmerer, T., French, L. & Clanner-Engelshofen, B.M. (2022) High-frequency devices effect in vitro: promissing approach in the treatment of acne vulgaris? Anais Brasileiros de Dermatologia . 97, 729–734. doi:10.1016/j.abd.2021.09.015. Napp, J., Daeschlein, G., Napp, M., von Podewils, S., Gümbel, D., Spitzmueller, R., Fornaciari, P., Hinz, P. & Jünger, M. (2015) On the history of plasma treatment and comparison of microbiostatic efficacy of a historical high-frequency plasma device with two modern devices. GMS hygiene and infection control . 10. doi:10.3205/dgkh000251.

Recent research into aroma perception - the process by which the brain detects scent molecules from food - has primarily focused on the physical and chemical properties of these substances and their release during chewing. However, the role of the oral microbiome in this process is still underexplored, offering new insights into how microorganisms shape the flavours we experience. What We Know: While taste is detected by the tongue’s taste buds, aroma involves volatile compounds released from food that are detected by olfactory receptors in the nose. Aroma significantly enhances flavour, allowing us to distinguish foods with similar tastes. During chewing, aroma compounds travel from the mouth to the nasal cavity (retronasal olfaction), blending with taste to create a complete sensory experience (Xi et al., 2024) . The oral microbiome plays a crucial role in aroma perception. These microorganisms interact with food compounds in various ways, such as breaking down odourless molecules and transforming them into volatile compounds that we perceive as aroma. Moreover, the oral microbiota influences how taste and smell signals are processed by the brain, further affecting overall flavour perception (Xi et al., 2024) . Emerging research indicates that oral microbiota help metabolise complex food precursors, such as glycosides, into flavour-active molecules during chewing. A balanced oral microbiome enhances flavour perception, while an imbalance may dull or alter this sensory experience (Xi et al., 2024) . Industry Impact and Potential: Research is still in infancy regarding the role of the oral microbiome in aroma perception. Therefore, harnessing the power of oral bacteria to enhance flavours or create novel sensory experiences is a promising avenue of exploration. Furthermore, understanding how the oral microbiome contributes to the perception of scent and taste could also lead to the development of targeted oral care products that maintain or improve flavour perception by supporting a healthy microbiome. A company that harnesses the intricate relationship between taste, scent, and retronasal olfaction is @air up®, which has developed innovative water bottles featuring built-in scent-releasing pods. This design allows users to enjoy unflavoured water while experiencing specific flavours, as the released scents interact with the olfactory system to create a taste sensation in the mouth. Our Solution: Sequential is a leading authority in microbiome product testing and formulation, offering customisable solutions that empower businesses to innovate oral hygiene products while preserving microbiome integrity. We ensure the efficacy and compatibility of products for a healthier oral microbiome, making us the ideal partner to help your company explore the potential of oral, as well as skin, scalp and vaginal, microbiome studies and product development.  References: Xi, Y., Yu, M., Li, X., Zeng, X. & Li, J. (2024) The coming future: The role of the oral–microbiota–brain axis in aroma release and perception. Comprehensive Reviews in Food Science and Food Safety . 23 (2), e13303. doi:10.1111/1541-4337.13303.

What is Sequential's testing platform? Sequential has developed the gold standard test for microbiome-friendly products, in vivo (in, or on, humans). Finally, we can give some certainty about if a product is truly affecting the microbiome. We offer a complete end-to-end solution to support microbiome-friendly claims. From consultancy and study design to our proprietary microbiome testing kits. We analyse, interpret and report our findings to meet your needs. Why is it necessary to test the microbiome in vivo? At present, there are no regulations for microbiome-related formulas that brands and formulators can follow, however, it has been universally acknowledged that the in vivo method of conducting clinical studies is becoming critical and paramount to getting marketing claims through. When regulations are introduced, which may be imminent, the in vitro system will find itself lacking, resulting in limited claims and certifications that do not hold their value. This is why, we at Sequential strive to offer an in vivo approach, knowing full well that we want our client's claims to be significantly backed by scientific and quantifiable data. What type of sequencing technology does Sequential use for analysis? We offer four types of sequencing techniques including qPCR with our Smart Probes™, 16S, ITS and Shotgun Metagenomics. Using next-generation sequencing of the collection of microorganisms found on the body, during product usage, Sequential investigates the microbial diversity, and particular microorganisms we know are important and play a role in a healthy microbiome. Does Sequential offer claims certification for tested products? We provide our clients with a certification to claim “Maintains the Microbiome” subject to in vivo testing results which can be used in communication efforts. Once your product is tested with our qPCR Smart Probes™ and has shown favourable results in supporting the microbiome, we can certify your product with our Maintains the Microbiome certification seal. We have ensured that our seal and certification are backed by quantifiable data and scientifically significant markers. The aim is to ensure our clients feel confident in making their claims and can communicate the true benefit of their microbiome formulations.

We are a Team of Award-Winning Scientists Creating a World with Healthier Microbiomes Our platform is the result of our team’s combined expertise in genetics, epigenetics, and microbiome research. We utilise deep molecular analysis and next-generation sequencing (NGS) technology to understand the impact of product usage on an individual’s microbiome. Through our efforts, we hope to revolutionise the way in which the industry develops and tests its products to deliver optimal results to those utilising them. Our Mission Sequential is the industry leader in clinical microbiome research and testing offering a comprehensive end-to-end platform designed to bring science-backed solutions to the personal care and pharmaceutical industry. Our mission is to understand the impact of the microbiome on the host (humans) and how the host impacts the microbiome in order to characterise human health fully. We offer an extensive platform to conduct research on personal care products through microbiome testing, and biophysical assessments, and offer full recruitment services for studies. We are keen to publish our findings with our partners to increase the literature within this space. At present our database of over 20,000 human microbiome samples is one of the most sophisticated within the industry and is growing rapidly. Innovation Pioneering the forefront of biological science, we consistently introduce groundbreaking advancements to redefine industry standards. Transparency Our commitment to openness ensures a clear understanding of our human microbiome testing processes and analysis. Reliability We guarantee dependable results, fostering trust in the accuracy of our analyses. Scientific Board of Advisors Our advisors are world leaders in the skin microbiome and have extensive experience in bringing forward solutions for skin concerns Prof. Tom Dawson Senior Principal Investigator at Skin Research Institute of Singapore. Over 30 years experience in biotechnology innovations, and expert in the skin and hair microbiome. Doctor of Philosophy (PhD), Pharmacology at the Univer sity of North Carolina. Dr Kimberly Capone Dr Kimberly Capone is a pioneer and established expert in microbiology and the human microbiome field where she created new business opportunities across multiple brands over 13 years at Johnson & Johnson Consumer, Inc. Areas of concentration included infant and adult skin, vaginal, gut, and oral health. Prof. Phillip Bennett Phillip Bennett is Professor of Obstetrics and Gynaecology and Director of the Institute of Reproductive and Developmental Biology. Professor Bennett has been one of the key pioneers in researching the vaginal microbiome. In particular, to understand and characterise the impact of the vaginal microbiome on preterm labour. Bennett has published over 400 peer-reviewed research articles over his career. Dr Natalya Fox Dr Natalya Fox is a Dermatologist at the NHS - St George's Hospital, London. Previously, Fox did her MBChB at the University of Edinburgh 201 4 and has her Full MRCP UK in Dermatology. Fox is passionate about the skin microbiome and its place in dermatology. Prof. Elena Lurie-Luke A senior R&D, Innovation and Entrepreneurship Executive with extensive technical, strategic business development. Proven leadership experience in both global FMCG and public health sector environments. Prof. Niranjan Nagarajan Associate Director & Senior Group Leader at Genome Institute of Singapore. Expert in computation biology, in particular the study of microbial communities resident on the human skin. Doctor of Philosophy (PhD), Computer Science at Cornell University. Dr Alexander Lezhava Senior Group Leader & Associate Director at Genome Institute of Singapore. Expert in the commercial development of medical diagnostics and clinical-grade molecular assays. Doctor of Philosophy (PhD), Microbiology at Hiroshima University. Please find listed a selection of relevant peer-reviewed publications from our advisors. Wu G, TL Dawson, et al. (2015) Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin. PLOS Genetics 11(11): e1005614. Chng, K., Nagarajan, N., et al. (2016) Whole metagenome profiling reveals skin microbiome-dependent susceptibility to atopic dermatitis flare. Nat Microbiol 1, 16106. Tay, A.S., Nagarajan, N., et al (2018). 1039 Skin microbiome profiles of atopic dermatitis patients segregate into two community composition types that are stable before and after therapy. Journal of Investigative Dermatology. 138. S176. 10.1016/j.jid.2018.03.1051. Ramasamy S., Barnard, E., Dawson, TL, and Huiying Li. (2019). Role of the skin microbiota in acne pathophysiology. British Journal of Dermatology, https://doi.org/10.1111/bjd.18230. Dawson, TL. (2019) Malassezia: The Forbidden Kingdom Opens. Cell Host Microbe https://doi.org/10.1016/j.chom.2019.02.010 Tay, A.S., Nagarajan, N., et al (2020). Atopic dermatitis microbiomes stratify into ecologic dermotypes enabling microbial virulence and disease severity. The Journal of allergy and clinical immunology. 10.1016/j.jaci.2020.09.031. Dawson, TL. (2021) Malassezia: A Skin Commensal Yeast Impacting Both Health and Disease. Front. Cell. Infect. Microbiol., doi.org/10.3389/fcimb.2021.659219 Bissonnette, Robert & FAAD, & Palijan, Ana & Salem, Youssef & Maari, Catherine & Proulx, Etienne & Edjekouane, Lydia & Joly-Chevrier, Maxine & Devis, Andrew & Dashi, Albert & Worsley, Oliver. (2024). 50694 Gut microbiome differences between patients with moderate to severe Chronic Hand Eczema and healthy subjects. Journal of the American Academy of Dermatology. 91. AB224. 10.1016/j.jaad.2024.07.889. Supported By Our Team at Sequential Dr. Oliver Worsley CEO & CO-FOUNDER Oliver is the co-founder and CEO of Sequential. He completed his PhD in molecular genetics as a scholar at the Genome Institute of Singapore from 2014-2018, and has won multiple awards including the P&G Young Entrepreneurship Scheme, presented at the Royal Society in London in 2017; and the top prize at the L’Oréal Innovation Runway 2018. Oliver has previously founded Anya Consulting, a healthcare communications company that has published >150 articles and has produced several technical whitepapers for clients like Fierce Health. Prior, Oliver completed his BSc at Edinburgh University, including six months at Leiden University Medical Centre through the Erasmus Programme. Sibora Peca CLINICAL OPERATIONS LEAD Grace Robinson ASSOCIATE SCIENTIST Shalindri Jayawardene RESEARCH ASSOCIATE Kajal Patel RESEARCH ASSOCIATE Dr. Albert Dashi CHIEF SCIENCE OFFICER & CO-FOUNDER Albert is the co-founder and CSO of Sequential. He completed his PhD in molecular genetics, epigenetics and stem cell research at the National University of Singapore (NUS) and the Genome Institute of Singapore in 2019. In 2014, he received the Singapore International Graduate Award from A*STAR for his PhD research and was also awarded the “Young Investigator” award. He also won the “Young Entrepreneur Scheme” award by P&G for his innovative and business driven ideas. Prior moving to Singapore for his doctor program, Albert obtained his Masters in Biomedical Sciences at University of Bern, Switzerland. Andrew Davis SENIOR BIOINFORMATICIAN Forest Wong SENIOR LAB TECHNICIAN Ashley LaSalle JUNIOR LAB TECHNICIAN Petronille Houdart, PharmD SKINCARE DIRECTOR Petronille is Sequential's lead skincare director, focused on translating the latest in skin science to personalised skincare recommendations. She has over a decade in the industry, working with international brands to lead and consult on R&D, creative projects and brand innovation. Petronille also led her own award-winning dermocosmetic brand, Petronille Dermo Cosmetic, that produced customisable products for men and women. Petronille holds an MSc in Cosmetology Sciences and a professional doctorate in pharmacy (specialising in dermo-pharmacy) from Paris Descartes University. Marya Sheikh-Ahmed SENIOR MARKETING ASSOCIATE Dr. Sija Sajibu RESEARCH ASSOCIATE Melissa Ople ADMINISTRATIVE ASSISTANT Annabelle Schaefer RESEARCH ASSOCIATE Our Labs Sequential has microbiome testing labs in New York City, London and Singapore. Being close to our customers has allowed us to reduce turnaround time, whilst retaining the intellectual property in-house. Proud to Have Worked With

Your Clinical Microbiome Testing Partner A trusted leader in microbiome testing and research for the personal care industry, Sequential provides cutting-edge solutions to elevate product development. Our extensive microbiome research delivers effective and actionable insights, empowering brands to create innovative, science-backed personal care products that meet consumer demands and set new standards in the market. View Services Brochure 20,000+ Microbiome Sample Database 4,000+ Ingredient Database 10,000+ Testing Participants Globally 60+ Industry Partners Who We Are Sequential is a leading provider of clinical microbiome analysis and microbiome testing services, offering a comprehensive end-to-end platform with science-backed solutions tailored for the personal care and pharmaceutical industries. Our team of award-winning scientists is dedicated to understanding the intricate relationship between the microbiome and its human host, exploring how the microbiome impacts human health and how humans, in turn, influence their microbiome. This holistic approach enables us to fully characterise human health and provide actionable insights for product innovation and efficacy. Learn More What We Offer Microbiome Testing A fully customizable microbiome study to test your product's impact in a real-life context. Formulation Support Allow our formulation experts to guide you through the process of creating a product that maintains the biome. Claims & Certification Test your formulation to understand what marketing claims you can attribute to your product. Strategic Partnerships Join us in full end-to-end partnership (testing to collaborating on white papers). Clinical Assessments Understand your product's impact on transepidermal water loss, pH, and elasticity. Study Recruitment Let us recruit candidates and carry out in-lab testing for your study to ensure controlled collection. Our Testing Capabilities Differentiate your brand by leveraging the power of skin microbiome science to deliver innovative, research-backed personal care products. Stand out from competitors with solutions supported by robust clinical research and gold-standard microbiome certification, appealing to customers seeking credible and cutting-edge personal care innovations. Harness the science of the microbiome to build trust, drive customer loyalty, and position your brand as a leader in the personal care industry. Skin Scalp Vaginal Oral What Our Clients Say “Sequential is one of the world’s most innovative Microbiome companies. The resolution at subspecies level, and to perform quantification of key vaginal microbes, in vivo , was exactly what we wanted at Curive Healthcare to know intricately how our product is working to improve women’s health.” - Matthew Line, Chief Marketing Officer at Curive Healthcare Supporting World-Class Clients & Partners Join Our Partners! Microbiome's Impact on Human Health Aromatic Answers: Can the Oral Microbiome Alter How We Perceive Scent and Taste? That’s Fishy: The Powerful Role of Marine Collagen in Wound Healing Unveiling the Microbial Truth: The Unpleasant Reality of Scalp Malodour Read More Articles FAQ What is Sequential's testing platform? Sequential has developed the gold standard test for microbiome-friendly products, in vivo (in, or on, humans). Finally, we can give some certainty about if a product is truly affecting the microbiome. We offer a complete end-to-end solution to support microbiome-friendly claims. From consultancy and study design to our proprietary microbiome testing kits. We analyse, interpret and report our findings to meet your needs. Why is it necessary to test the microbiome in vivo? At present, there are no regulations for microbiome-related formulas that brands and formulators can follow, however, it has been universally acknowledged that the in vivo method of conducting clinical studies is becoming critical and paramount to getting marketing claims through. When regulations are introduced, which may be imminent, the in vitro system will find itself lacking, resulting in limited claims and certifications that do not hold their value. This is why, we at Sequential strive to offer an in vivo approach, knowing full well that we want our client's claims to be significantly backed by scientific and quantifiable data. What type of sequencing technology does Sequential use for analysis? We offer four types of sequencing techniques including qPCR with our Smart Probes™, 16S, ITS and Shotgun Metagenomics. Using next-generation sequencing of the collection of microorganisms found on the body, during product usage, Sequential investigates the microbial diversity, and particular microorganisms we know are important and play a role in a healthy microbiome. Does Sequential offer claims certification for tested products? We provide our clients with a certification to claim “Maintains the Microbiome” subject to in vivo testing results which can be used in communication efforts. Once your product is tested with our qPCR Smart Probes™ and has shown favourable results in supporting the microbiome, we can certify your product with our Maintains the Microbiome certification seal. We have ensured that our seal and certification are backed by quantifiable data and scientifically significant markers. The aim is to ensure our clients feel confident in making their claims and can communicate the true benefit of their microbiome formulations.