Genes journal focusing on our current understanding of RNA splicing alterations associated with tumor initiation, progression, current and emerging technologies targeting RNA splicing for cancer therapy, critical roadblocks and technical problems, and future challenges in the field.Since the discovery of RNA splicing as a fundamental step to remove introns from pre-mRNA to produce mature mRNAs, substantial research in the past decades has highlighted RNA splicing as a critical mediator of gene expression and proteome diversity, also being important in many developmental and biological processes. In addition, critical insights have transpired regarding the interplay between splicing and other steps in mRNA metabolism, including transcription and the various relevant features of chromatin biology, processing at the 5′ and 3′ ends, RNA editing or modifications, mRNA export and localization, and mRNA turnover or decay. Genetic and cellular alterations impairing the correct complements of RNA and RNA-binding proteins compromise the fidelity of splicing and splicing-associated processes and often cause pathological consequences, including genetic defects, neurological disorders, and cancer ,2. AdvanWan et al., in their review , highligRNA editing is an important type of post-transcriptional processing, which changes the primary RNA sequence through the insertion/deletion or modification of specific nucleotides. Piazzi et al., in their review , highligNMD is another post-transcriptional RNA processing mechanism. It was initially identified as a surveillance mechanism to safeguard the fidelity of mRNA transcripts. NMD ensures the selective recognition and rapid degradation of erroneous transcripts containing a premature translation-termination codon (PTC). One third of mutated and disease-causing mRNAs were reported to be recognized and degraded by NMD, highlighting the importance of this intricate mechanism in maintaining cellular integrity. In addition to surveillance, NMD also elicits the decay of many endogenous mRNAs (~10% of the human transcriptome) without mutations, therefore also fine-tuning transcript abundance. It is interesting to note that about one third of all alternative splicing (AS) events result in PTC-containing transcripts and are subsequently degraded by NMD. Substantial evidence has disclosed a coordinated regulatory link between AS and NMD to regulate gene expression. This coordinated action is commonly termed as AS coupled to NMD (AS–NMD), which controls the ratio of productive to unproductive transcript isoforms. By modulating gene expression, NMD regulates diverse biological functions during development and differentiation and expedites cellular responses to adaptation, physiological changes, stresses, environmental insults, etc. NMD is a complex pathway regulated by the dynamic interactions between mRNA and a series of NMD-associated proteins and upon a complex choreography of events between mRNA-bound proteins. AS–NMD is further regulated by the splicing pathway. Mutations or alterations  that impair the function or expression of proteins associated with the NMD (or AS–NMD) pathway can be detrimental to cells and may cause pathological consequences. Nagar and Islam et al., in their review , extensiOne critical aspect of targeting RNA splicing for cancer therapy is to identify potential target gene(s) associated with tumorigenesis. Unlike monogenic genetic defects, many genes are simultaneously dysregulated at the splicing level in cancer due to heterogeneous and complex genetic backgrounds. Among these dysregulated splicing events, only a few are directly associated with tumorigenesis (cancer driver events), and the remaining have no relevance to cancer (passenger events). Advancements in high-throughput sequencing, including whole genome sequencing, whole exome sequencing, whole transcriptome sequencing, and non-coding RNA sequencing, and the development of these techniques to identify modified nucleic acids have helped us to identify many cancer-driver splicing events. In addition, they have permitted a global analysis of gene expression, identified novel pathway interactions, and provided critical insights assessing both disease progression and therapeutic response. However, there are significant caveats to these analyses, which are crucial roadblocks against our progress in obtaining the complete picture. Piazzi et al., in their review , discussConsidering the importance of RNA splicing dysregulation in cancer initiation and progression, there is immense interest in targeting RNA splicing for cancer therapy. Bonner et al., in their review , extensiTo conclude, research on RNA splicing should be continued with creativity, novel discoveries, and the courage to challenge existing roadblocks or limitations, which will likely advance the journey of RNA therapeutics in cancer, revolutionizing novel tools for medicine and science.


RNA-based therapies are currently used for immunisation against infections and to treat metabolic diseases. They can modulate gene expression in immune cells and hepatocytes, but their use in other cell types has been limited by an inability to selectively target specific tissues. Potential solutions to this targeting problem involve packaging therapeutic RNA molecules into delivery vehicles that are preferentially delivered to cells of interest. In this review, we consider why the kidney is a desirable target for RNA-based therapies in cardiovascular disease and discuss how such therapy could be delivered. Because the kidney plays a central role in maintaining cardiovascular homeostasis, many extant drugs used for preventing cardiovascular disease act predominantly on renal tubular cells. Moreover, kidney disease is a major independent risk factor for cardiovascular disease and a global health problem. Chronic kidney disease is projected to become the fifth leading cause of death by 2040, with around half of affected individuals dying from cardiovascular disease. The most promising strategies for delivering therapeutic RNA selectively to kidney cells make use of synthetic polymers and engineered extracellular vesicles to deliver an RNA cargo. Future research should focus on establishing the safety of these novel delivery platforms in humans, on developing palatable routes of administration and on prioritising the gene targets that are likely to have the biggest impact in cardiovascular disease. RNA-based therapies are emerging as an effective and versatile class of medications in a range of diseases. So far, they have been largely limited to small interfering RNAs (siRNAs) that mediate gene silencing in hepatocytes and mRNA vaccines. Their full potential will be unlocked when we learn how to target them effectively to other cell types. In this review, we consider recent progress towards developing RNA-based therapies that can selectively target kidney cells and in particular renal tubular epithelial cells (rTECs). Given the central role of the renal tubules in maintaining cardiovascular homeostasis, such novel therapies could have the greatest impact in treating cardiovascular disease (CVD).We begin by briefly introducing the concepts of RNA-based therapy and reviewing the causative relationship between kidney disease and CVD. We then discuss attempts to deliver RNA-based therapies selectively to rTECs in animal models and humans. We conclude by positing the potential clinical impact that these novel therapeutics could have in cardiovascular disease.There are several different classes of RNA therapeutic, including mRNAs, siRNAs, RNA aptamers and CRISPR guide RNAs . In prinC elegans  or anti-sense oligonucleotides (ASOs) silence the expression of a pathogenic mRNA through RNA interference (RNAi). These molecules directly bind their cognate mRNA and inhibit protein synthesis. This process is the mammalian correlate of a phenomenon that was first characterised in plants and  elegans . In the  elegans . This ca elegans . It coulOur current approach to treating kidney and cardiovascular disease relies almost entirely on conventional small molecule drugs. These drugs have been developed over decades, often more by accident than design. They tend to have rapid metabolism—and so require daily or twice-daily dosing—and to target receptors that have been chosen predominantly because they are easily “druggable”, rather than because they are necessarily the most important points in a disease pathway. Drug development is slow, in part because of the relatively unpredictable safety profile of small molecules.RNA-based therapies could potentially solve all of these problems , 8. RNA-However, for the successful clinical application of RNA-based therapies in kidney disease, we must first learn how to direct these drugs selectively to kidney cells. After intravenous injection, naked RNA is widely distributed amongst different body tissues . There aThe kidneys maintain cardiovascular homeostasis by regulating the circulating volume and blood pressure . Therefoper se will reduce cardiovascular disease.The other rationale for targeting the kidney to treat cardiovascular disease, is that chronic kidney disease (CKD) is a leading risk factor for cardiovascular disease. Therefore, treating kidney disease CKD is the eighth leading cause of death worldwide , with juRNA-based therapies are already being used in the clinic to treat kidney disease, but not by directly targeting kidney cells. For example, Lumasiran is an siRNA that targets glycolate oxidase in hepatocytes that is effective at treating primary hyperoxaluria . SimilarThere are some other metabolic diseases in which targeting gene expression in the liver will improve kidney outcomes, but for the vast majority of kidney diseases, it will be necessary to develop therapies that can directly regulate gene expression in kidney cells. The capacity for treating cardiovascular disease will be hugely augmented if we can develop drugs that target the kidneys, given their central role in cardiovascular homeostasis. Therefore, potential strategies for kidney-specific RNA delivery are being tested extensively in pre-clinical models. These are summarised in Naked RNA is a negatively-charged macromolecule that does not easily cross the hydrophobic, negatively-charged cell membrane. In the absence of modifications, naked RNA is subject to nuclease degradation and clearance by the kidneys and reticuloendothelial system , 46. TheBroadly, RNA can be delivered as “naked” nucleic acid or in delivery systems that are classified as viral or non-viral .Naked nucleic acids, injected systemically, are excreted in the urine and accumulate within the kidney, particularly in renal tubular cells –51. ThisThe stability and biodistribution of siRNA can be altered by chemical modification of the RNA backbone. Phosphorothioate siRNA accumulates preferentially in the kidneys, relative to other organs . PhosphoIn contexts where rapid clearance and off-target RNA delivery is not problematic, naked RNA could be used to therapeutic effect. Naked siRNA targeting the tumour suppressor gene p53 has been tested in pre-clinical and clinical trials of acute kidney injury. Intravenous administration gave effective delivery to rTECs and prevented kidney injury in rat models of ischemia-reperfusion injury and cisplatin toxicity . This siSome oligonucleotides appear to be selectively delivered to kidney cells when delivered in their naked form. For example, an ASO directed against miRNA-17 performed well in pre-clinical trials of polycystic kidney disease (ADPKD) . IntriguViral vectors are highly effective at delivering genetic material to target cells . Viral vWhilst the risks of cellular toxicity and immune reactions are minimised with third generation adenoviral vectors , there iLipid-based nanoparticles (LNPs) are the most established non-viral RNA delivery system –63. Thesminimise renal clearance  that they ought to cross the glomerular filtration barrier. In mice, a PEG-PLL carrier significantly enhanced delivery of fluorescently-tagged siRNA to kidneys in mice, compared to a naked siRNA control . A MAPK-Chitosan particles showed promise as a kidney-targeting strategy in an early murine biodistribution study, with siRNAs accumulating in the kidney, 24 h after intravenous administration . The likvβ3 integrin) respectively in murine kidney disease models  and carry with them a subset of cellular macromolecules, including RNA , 70, 71.Furthermore, EVs can be readily engineered to express surface receptors to enhance delivery to specific target cells. For example, EVs engineered to express the rabies virus glycoprotein (RGV) peptide gave enhanced delivery of an siRNA cargo to kidney cells , 74, becdiseased rTECs. When erythrocyte-derived vesicles were engineered to express a KIM-1 ligand, they delivered cargo siRNA preferentially to injured rTECs in murine ischaemia-reperfusion and ureteral obstruction models ? A variety of ligand classes have been tested: peptides, antibodies, aptamers, carbohydrates, and small molecules . LigandsNanoparticles delivered into the systemic circulation face significant hurdles to reaching the kidney. They may be degraded by the reticuloendothelial system or—depending on their chemical composition—be preferentially delivered to the liver. Furthermore, there is uncertainty as to the extent to which nanoparticles can cross an intact glomerular filtration barrier. Some provocative studies suggest that fluorescently-labelled extracellular vesicles can traverse this barrier in healthy mice and humans, even though their size and charge profile ought to be prohibitive , 87.In pre-clinical models, direct administration into the renal artery, renal vein, ureter or kidney parenchyma have been used to deliver RNA to the kidney, but the translational potential of these routes is obviously limited , 60. OneRNA-based therapies offer a powerful opportunity to target new pathways in cardiovascular disease. Existing RNA-based therapies largely target gene expression in hepatocytes and are therefore limited in their ability to treat cardiovascular disease. The kidney is an attractive target in cardiovascular disease because the kidney plays a central role in maintaining cardiovascular homeostasis and because CKD is a leading risk factor for cardiovascular disease.Various approaches to delivering RNA selectively to kidney cells are being tested in pre-clinical models. Polymer and vesicle-based delivery systems show promise, particularly in their ability to deliver siRNA selectively to rTECs, without inciting toxicity or immune activation. Extracellular vesicles can be engineered to express surface ligands that interact with receptors on specific kidney cells, permitting highly selective delivery of RNA to target cells. Several lines of evidence suggest that megalin  mediates uptake of exogenous RNA from the tubular space, whether the RNA is delivered as naked nucleic acid or complexed to polymeric delivery vehicles. Delineating the precise molecular details of this pathway and its relative contribution to total kidney RNA uptake will be important areas of study.Future research should also focus on establishing the safety of these novel delivery platforms in humans and on developing palatable routes of administration. In particular, strategies that induce long-lasting gene silencing could offer advances in drug adherence, which would help to reduce the prevalence of cardiovascular disease. Therefore, the potential targets of novel RNA-based therapies include established targets of conventional small-molecule drugs  as well as a theoretically unlimited array of novel targets.


The rise in non-communicable diseases over the last two decades highlights the environment as a major risk factor. Changes in dietary habits in western countries have significantly reshaped the composition of the gut microbiota. This has resulted in the loss of gut bacterial species that have co-evolved with humans for millennia and changing the balance of microbial-derived metabolites crucial for maintaining the symbiotic relationship between gut bacteria and their host.via fecal microbiota transplantation, by reshaping the gut microbiota through dietary intervention (prebiotics), by introducing beneficial bacteria (probiotics) or by administering by-products released by healthy gut microbiota (postbiotics).Non-communicable diseases are commonly associated with a detrimental shift in the gut microbiota composition known as dysbiosis. Dysbiosis contributes to increased gut permeability and overexposure of the host to microbial products, which triggers inflammation. Targeting the gut microbiota to restore a healthy gut environment and to control the immune response is thus an attractive strategy to treat inflammatory diseases. There are different strategies to target the gut microbiota, either by introducing a healthy gut microbiota Corrêa et al. highlighted that the beneficial reshaping of the gut microbiota by dietary fiber reduced the development of pulmonary diseases, including COPD, pulmonary fibrosis, allergic asthma, lung cancer, and acute respiratory distress syndrome in mice, confirming that targeting the gut microbiota could improve host's health.Dietary fiber, a prebiotic, is a complex carbohydrate indigestible to the host but represents a major source of energy for gut bacteria. Zeng et al. presented the effects of probiotics in six different types of arthritis through a meta-analysis of 34 randomized clinical trials. Inflammatory arthritis is characterized by chronic joint inflammation as well as dysbiosis and increased gut permeability. A wide range of probiotics was tested mostly from the Lactobacillus and Bifidobacterium genera. The main conclusion of this analysis is that probiotics are generally safe for use in humans but the lack of reproducibility and the variability between trials make it inconclusive whether probiotics are effective for arthritis. Some potential benefits of probiotics were reported in gout, osteoporosis and osteoarthritis, while no benefits were observed in spondyloarthritis and juvenile idiopathic arthritis. The inconsistency of response included either decreased inflammatory markers (CRP), decreased swollen joints or no differences with mild adverse events reported by a few patients. This variability may be explained by the intrinsic differences in the gut microbiota of the patients. A study has shown that people could be subclassified as responders or non-responders depending on the engraftment ability of the probiotics , particularly deoxycholic acid and lithocholic acid and derivatives in suppressing IL-6 production by macrophages, which is elevated in RA.Postbiotic intervention is another strategy to improve the host's health, acting independently of the gut microbiota and reducing interindividual response variability. While postbiotics show promise in preclinical studies, their efficacy in the clinic is poorly known. Preclinical studies show the benefit of short-chain fatty acids, by-products of dietary fiber fermentation, in pulmonary diseases like COPD, pulmonary fibrosis, allergic asthma, lung cancer, and acute respiratory distress syndrome in mice. SCFA also promote anti-inflammatory regulatory T cell generation, gut homeostasis and increase bone density. Rheumatoid arthritis (RA) patients had lower or unchanged levels of SCFA and preclinical studies have shown potential benefits of dietary fiber and SCFA in RA. in vitro, their role in RA patients may be limited as a study has shown that TGR5, a bile acid receptor, is downregulated in PBMC of patients with RA  and TNF. They also mention a lack of association between protein intake and microbial L-tryptophan-derived metabolites that could be due to host metabolism, sex or gut microbiota composition.Extensive work in preclinical models showed that conjugated BA promoted anti-inflammatory regulatory T cells while inhibiting Th17 cells. Of note, not all conjugated BA have anti-inflammatory effects, with some having pro-Th17 effects discussed elsewhere . While s with RA . L-tryptFernandes Rodrigues et al. showed that A. municiphila-derived proteins were more beneficial than the live bacteria, opening safer and more controlled therapeutic opportunities.Other than metabolites, proteins, outer-membrane vesicles or even pasteurized organisms may be more beneficial than live probiotics. Altogether, these articles highlight the gut microbiota as a promising therapeutic target for numerous diseases. Their efficacy in humans and the parameters that may influence their efficacy, including microbiota composition, genetics, gender and type of disease, are largely undefined. A holistic understanding is needed to optimize their therapeutic potential.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Amyloidosis is a protein deposition disorder in which insoluble fibril structures accumulate in the bodily tissues damaging the organ function. Cardiac amyloidosis is a severe but under-reported medical condition characterized by the accumulation of amyloid in the extracellular area of the myocardium, which results in thickening and stiffening of ventricular walls. Cardiac amyloidosis has recently gained much attention with its slowly surging incidence. With this study, we seek to comprehensively compile the pathophysiology and clinical picture of cardiac amyloidosis subtypes, extending a clinically oriented, up-to-date clinical approach to diagnosis and therapy. Cardiac amyloidosis can be caused by rare genetic mutations which may be inherited or acquired. The growing incidence can be attributed to advancements in imaging methods and other diagnostic modalities. Most occurrences of cardiac amyloidosis result from two forms of precursor protein: transthyretin [TTR] amyloid and immunoglobulin-derived light-chain amyloid. Prompt identification of cardiac amyloidosis can facilitate the implementation of evolving therapeutic interventions to enhance the outcomes. The modalities for the management of CA have evolved significantly in the last ten years. Apart from therapies for modifying disease and heart failure, a myriad of novel therapeutic approaches that target specific aspects of the disease, including gene therapies, are being researched. These aim at impeding its progression and improving clinical outcomes.See also Figure 1 The abnormal accumulation of insoluble polymeric fibrillar proteins outside the cells  is the hallmark of amyloidosis . Amyloi. . Transtal., 1983; Tanskanal., 1983). Free ral., 1983; Wieczoral., 1983)ATTR-CM typically has an insidious onset with mild symptoms, but unchecked progression can have a poor prognosis . At theSeveral new therapies are being developed at the moment, including patisiran, a gene-silencing RNA therapy, and Inotersenan antisense oligonucleotide, which are inhibitors of TTR protein production, and tafamidis, a drug that helps preserve the soluble non-amyloid conformation of the TTR protein.  amyloidosis, ATTRwt (previously known as "senile cardiac amyloidosis"), and ATTRM/ mutated ATTR that can result in hereditary cardiac amyloidosis.The frequency of cardiac manifestation is different in different variants of amyloidosis. The AL variant, also called primary amyloidosis, is the most frequent among these, in which approximately 90 % of the patients show cardiac presentation. The rarest to show cardiac presentations is the SA variant or secondary amyloidosis. In ATTRwt, the heart typically presents as the only organ demonstrating clinical manifestations.In hereditary ATTR, both cardiac and neurological presentations dominate . IndiviIn most patients, cardiac manifestations occur late and are preceded by extracardiac manifestations. The extracardiac manifestations can aid in the early diagnosis of this vastly underdiagnosed condition. In AL amyloidosis most common findings are macroglossia  , periorAmyloid formation occurs when a protein loses its normal functional fold or fails to adopt it correctly. Various factors contribute to protein misfolding and aggregation, including abnormal proteolysis, point mutations, and post-translational modifications such as phosphorylation, oxidation, and glycation. The misfolded protein or peptide subsequently interacts with other misfolded proteins or peptides, leading to the formation of oligomers. These oligomers circulate in the bloodstream and eventually deposit as well-structured fibrils in the interstitial spaces of specific target organs . Systemic amyloidosis is categorized into three main types: (a) Primary systemic amyloidosis, also known as plasma-cell dyscrasia, (b) Amyloidosis associated with multiple myeloma, and (c) Secondary systemic amyloidosis resulting from chronic inflammatory or infectious conditions. In primary systemic amyloidosis, insoluble deposits of monoclonal immunoglobulin (Ig) light (L) chains or L-chain segments accumulate in various tissues and structures, including smooth and striated muscles, connective tissues, blood vessel walls, and peripheral nerves .Cardiac amyloidosis is a condition characterized by the deposition of amyloid in the parenchymal tissue of the heart, leading to a restrictive pathology and resulting in diastolic dysfunction. This extracellular amyloid deposition disrupts the tissue structure mechanically and induces proteotoxicity, inflammation, generation of reactive oxygen species, apoptosis, and autophagy . The inThe liver produces a plasma transfer protein called transthyretin . It funATTRv amyloidosis presents with various clinical phenotypes, including primary polyneuropathy associated with the V30M mutation, cardiomyopathy linked to the V20I, V122I, L111M, and I68L mutations, and a mixed phenotype observed in cases of the E89Q and T60A mutations . Among AA amyloidosis, dialysis-associated amyloidosis (the precursor protein is beta 2 microglobulin), and isolated atrial amyloidosis  are other less prevalent forms of CA. Along with inflammatory bowel disease, rheumatoid arthritis, and hidradenitis suppurativa, AA amyloidosis is linked to other auto-inflammatory diseases .Protein Unfolding and Misfolding: Proteins have a perfectly folded, three-dimensional conformation that keeps their quantity and function intact. Protein folding happens spontaneously in accordance with the amino acid sequence following the endoplasmic reticulum's synthesis of the protein polypeptide chain . ChaperTypically, misfolded proteins undergo degradation and elimination by the proteasome . HoweveThe process of amyloid fibril formation can be divided into two stages: nucleation and subsequent elongation.Nucleation phase: During the nucleation phase, the initial nucleus of amyloid fibrils is formed as misfolded protein monomers assemble into a soluble oligomer. This process of nucleation is not spontaneous but rather requires specific conditions. The nucleation phase plays a crucial role in regulating the overall rate of fibril formation. Elongation phase: Once nuclei have formed, the subsequent elongation phase occurs, characterized by the sequential binding and growth of monomers, also known as "nucleation-dependent polymerization." This allows amyloid fibrils to propagate and extend their structure along the fiber axis . Commonjyo, 1999).Minor variations in the amino acid sequences of precursor proteins can significantly impact the organs targeted by amyloid accumulation. In the case of transthyretin (TTR) mutations in ATTRv, the specific location of the mutation influences the clinical characteristics and timing of amyloid deposition. Furthermore, the properties of target organs can change due to the fragmentation of amyloid transthyretin (ATTR) caused by proteolysis . The coIn recent times, diagnoses of cardiac amyloidosis variants-ATTR amyloidosis and systemic monoclonal immunoglobulin light chain amyloidosis (AL) amyloidosis, the most frequently diagnosed form of cardiac amyloidosis-have risen. The latter is caused by the accumulation of either variant or wild-type TTR amyloid in the heart's interstitium.AL amyloidosis: Accumulation of light immunoglobulin chains due to a plasma-cell dyscrasia causes AL amyloidosis. With about 2,500 to 5,000 new instances reported in the USA each year on average, it is a rare disorder. According to a recent study, the prevalence and incidence of AL amyloidosis among people aged 18 to 64 years is increasing significantly .ATTR amyloidosis: A mutated gene that makes transthyretin's tetramer more prone to instability is the underlying cause of hereditary ATTR amyloidosis. A liver-produced protein called transthyretin transfers retinol and thyroid hormone. More than 120 transthyretin mutations related to amyloidosis have been found documented, with some genetic variations confined to geographical areas or ethnic groupings. Diverse transthyretin mutations have varying onset ages and cardiomyopathy risk. The Val30Met variant is the transthyretin mutation that has been described the most frequently, but the Leu111Met and Ile68Leu variants are primarily associated with a cardiac phenotype . The VaATTR amyloidosis mainly affects older males with a cardiac predominance phenotype . VirtuaCardiac amyloidosis is a difficult-to-diagnose disorder with a dismal outcome, but new treatments and the promise for newer drug targets are improving its prognosis. Echocardiography is generally the preferred imaging technique. If any abnormalities are detected, non-invasive techniques like cardiac magnetic resonance imaging [CMR] and nuclear imaging [NI] can be used to diagnose and evaluate it. For AL-type cardiac amyloidosis, it may be possible to diagnose the condition without resorting to regular cardiac imaging techniques by noting levels of NT-pro BNP or troponins which are abnormally high, provided that other potential causes of abnormally high NT-pro BNP or troponins have been eliminated. Interestingly, if the serologic markers for AL are negative and the results of the nuclear imaging tests meet the criteria, then a formal medical diagnosis of ATTR CA can be established .ECG: QRS complexes of low voltage-5 millimeters in the limb leads or 10 millimeters in the precordial leads-are usually seen as an electrocardiographic indication of CA . This ial., 2019; Mussineal., 2019). The thal., 2019). Aroundal., 2019; Ruberg al., 2019). Atrialal., 2019).In AL amyloidosis, ECG can reveal left ventricular hypertrophy in about 10-15 % of patients, but this is probably caused by pre-existing hypertension in conjunction with amyloidosis. Conduction system diseases and Bradyarrhythmia is common, likely caused by progressive infiltration of amyloids and autonomic system dysfunction, particularly in AL and ATTRv-cardiac amyloidosis  can offer identification of tissue properties in CA. Consequently, these multiple imaging methods should be used together to maximize the diagnosis of CA .Echocardiography: Echocardiography is still the leading imaging tool due to its accessibility, safety, affordability, portability, and extensive history of usage when assessing diastolic performance and cardiac deformation, though it often necessitates verification by additional tests . Imaginal., 2009). Echocaal., 2009). The deal., 2009; Yamamotal., 2009). When the left ventricle of the heart has thickened but the heart remains of normal size, in absence of other apparent causes like hypertension or hypertrophic cardiomyopathy, then cardiac amyloidosis must be considered . In thiThere is usually a spectrum of impairment in diastolic function, varying from slight to severe, with signs of augmented left ventricular filling pressures . It is MRI: Magnetic Resonance Imaging has a major advantage over other forms of imaging since it can provide a detailed analysis of tissue and accurately measure the degree of infiltration. However, the need for access to the equipment, expense, fear of enclosed spaces, use of contrast dyes, and presence of metal objects may prevent its use . Patienal., 2020).An investigation of 36 people with heart failure (HF) and either a myocardial biopsy or autopsy showing cardiac amyloidosis (CA) (n = 11) or additional non-cardiac biopsy along with left ventricular hypertrophy (LVH) on echocardiogram revealed that close to a third of the patients had normal left ventricular mass index (LVMI) based on cardiac magnetic resonance (CMR) . This iCMR: Cardiac Magnetic Resonance imaging is an effective tool for distinguishing CA from other ailments causing Left Ventricular Hypertrophy (LVH). In comparison with healthy individuals with Hypertensive Heart Disease (HHD), those with CA have been found to have a reduced Left Ventricular Ejection Fraction (LVEF) and Right Ventricular Ejection Fraction (RVEF), increased Left Ventricular Mass Index (LVMI), heightened Right Ventricular Mass (RVM), and a higher magnitude of Right Ventricular Hypertrophy (RVH) on CMR scanning . Nuclear Imaging: Currently, nuclear imaging is the sole non-invasive method capable of distinguishing between ATTR and AL cardiac amyloidosis . Nucleaal., 2023). The pral., 2023).Cardiac scintigraphy: Examining pictures, both flat and SPECT is the initial step in understanding each study. If there is wide uptake of the tracer in the heart muscle, this is an indication of ATTR cardiac amyloidosis . In cural., 1998). The usal., 1998).Genotyping and immunohistochemistry were used as the reference standard, and the positivity of the scan was evaluated by analysing the uptake using a rating system still utilized in medical practice. This rating system consists of four grades: grade 0 represents no uptake in the heart and normal uptake in the bones, grade 1 indicates lower uptake in the heart compared to the bones, grade 2 signifies similar uptake in the heart and bones, and grade 3 indicates higher uptake in the heart compared to the bones, or even the absence of bone uptake .PET: A comprehensive research study with 1217 participants believed to have cardiac amyloidosis showed that using phosphate-based tracers through cardiac scintigraphy had positive predictive value and 100 % specificity for ATTR cardiac amyloidosis. A systematic review validated that the scintigraphy accuracy in identifying ATTR CA was over 90 % in both sensitivity and specificity . 18F-flal., 2018). A piloal., 2018; Jung etal., 2018). Qualital., 2018). Nucleaal., 2018). The Real., 2018; Cuddy eal., 2018; Kero etal., 2018). This tal., 2018; Manwanial., 2018) Biomarkers: Diagnosis of CA poses a significant challenge, as the clinical presentation is often non-specific, and with late onset of symptoms in the disease course. Imaging techniques have proven essential in diagnosing CA, but given the disease's progressive nature, early detection is crucial to optimizing outcomes. Biomarkers have emerged as a promising tool to aid in the prognosis and diagnosis of CA, with several studies shedding light on their role in the disease.Biomarkers such as B-type natriuretic peptide (BNP), N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP), and high sensitivity cardiac troponin have demonstrated utility in detecting cardiac involvement, predicting prognosis, and evaluating treatment response in patients with systemic immunoglobulin light chain amyloidosis (AL)  is significantly elevated in patients with amyloid light-chain (AL) cardiac amyloidosis compared to other groups, including those with non-cardiac systemic amyloidosis , and haBiomarkers have a prominent role in predicting prognosis in (ATTR-CA) patients. Two biomarker-based staging systems are available, one including estimated glomerular filtration rate (eGFR) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), and the other including troponin I (TnI) and NT-proBNP. A comparison of the two systems found that the NT-proBNP and eGFR systems had better prognostic accuracy and resulted in effective survival stratification, while the combination of TnI and NT-proBNP was not effective in successfully differentiating survival . HoweveA systematic review also highlighted the role biomarkers play in diagnosing and staging CA, with investigators focusing on quantification and standardization of amyloid burden. The review points to the potential of combining imaging techniques and biomarkers for the early detection and monitoring of CA . FurtheIn summary, biomarkers such as hs-cTn and NT-proBNP have proven useful in diagnosing and prognosticating cardiac involvement in systemic light chain amyloidosis. Further, biomarkers and imaging techniques play a complementary role in diagnosing and staging CA, with investigators focusing on standardization and quantification of amyloid burden. Monitoring biomarker levels in CA patients can also aid in measuring treatment efficacy and overall disease status. The use of biomarkers and imaging techniques in CA diagnosis is an active area of research, with much promise for early detection and improved outcomes. A summary of these biomarkers is described in Table 1It is challenging to detect rare cases of cardiac amyloidosis (CA) as the symptoms and signs are often similar to those of more common ailments, which results in the delayed application of appropriate treatments. In such situations, Artificial Intelligence (AI) has the potential to serve as a valuable tool for early diagnosis .ML [machine learning] models are capable of sourcing the appropriate data from the Electronic Health Record (EHR) systems and can skilfully examine the intricate interactions among numerous input factors , in an entirely automated way. This process would be less time-consuming than traditional statistical strategies and can also furnish clinical decision aid for physicians or be employed as web-based risk estimators .In a research study, the discovery of CA was done with the help of Artificial Intelligence models and ECG or echocardiogram as inputs. The ECG model had a good precision rate based on the C-statistics from the ECG-test set from different medical facilities. The examination of cardiac amyloidosis types indicated a better performance of the model on ATTR amyloid in contrast to AL amyloidosis .Examining the global longitudinal strain (GLS) and left ventricular ejection fraction (LVEF) is a critical part of diagnosing cardiac amyloidosis (CA). The research was conducted to analyze if an automated artificial intelligence (AI) technique of gauging global longitudinal strain (GLS) and left ventricular ejection fraction (LVEF) could match estimations and detect anomalies in agreement with standard manual procedures in patients with pre-clinical and clinical cardiac amyloidosis (CA). The study's findings indicated that there were no significant distinctions between manually measured and automatically calculated values of left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) during both pre-CA and CA stages at the time of diagnosis. The application of automated LVEF and GLS could allow for a quicker evaluation in diverse medical conditions with similar exactness and repeatability to manual techniques .Combining AI with sophisticated cardiovascular imaging provides more reliable outcomes without the utilization of contrast agents. Zhang et al. created a Cardiac Magnetic Resonance (CMR) Virtual Native Enhancement (VNE) imaging system powered by artificial intelligence to detect fibrosis in myocardium without using any contrast agents. This new approach was tested on 1348 people with hypertrophic cardiomyopathy, and they had both late gadolinium enhancement (LGE) scans with contrast and VNE scans. The two methods were found to be in good agreement, and the VNE images were of higher quality than the LGE. Furthermore, VNE images can be created in less than one second, reducing the routine CMR protocol for cine and tissue characterization to fifteen minutes .A normal echocardiogram consists of approximately 70 videos taken from various perspectives, and the viewpoints are not marked in every examination. This means that physicians have to go through all the important features in a lot of videos in every study, and this task can be made simpler with the help of artificial intelligence models. Additionally, measurements can fluctuate from one video to another due to the intrinsic variability in cardiac performance and the issue of representing a 3D object with 2D cross-sectional images. Considering the extent of this variability and the vast amount of information present in each study, which often goes unnoticed, it can be said that echocardiography can benefit from automated learning to facilitate the interpretation .Scientists have shown that incorporating machine learning (ML) algorithms into a cardiovascular risk assessment tool, which combines standardized peri-coronary fat attenuation index (FAI) mapping with clinical risk factors and plaque metrics, is a highly accurate method for predicting the prognosis of cardiovascular risk. A diagnosis of all subtypes of cardiac amyloidosis can be made through observation of apple-green birefringence of endomyocardial biopsy sample under polarized light with Congo red staining, mass spectrometry typing or immunohistochemistry. While a positive extracardiac biopsy sample has to be supported by the typical features in cardiac imaging for a diagnosis to be reached .Over the last ten years, significant progress has been made in the diagnosis and treatment of various types of amyloidosis. Notably, the introduction of a gene-editing technique utilizing the CRISPR-Cas9 nuclease has been a ground-breaking development in this field . A compThe mainstay therapy of supportive care is aimed at symptomatic relief. This includes therapies for congestive heart failure symptoms, arrhythmias, and conduction disorders.Heart failure: Initially, it is recommended to implement general measures to manage symptoms of heart failure (HF). These measures include providing diet counselling, restricting sodium  intake, and managing weight. The mainstay treatment for CA patients with heart failure in CA is loop diuretics  along with aldosterone antagonists (Eplerenoneor spironolactone). Conventional treatment of HF is not used because of the following reasons -Angiotensin-converting enzyme inhibitors (ACE inhibitors) and angiotensin receptor blockers are associated with a higher risk of severe hypotension, particularly in AL amyloidosis patients. This is because the impaired sympathetic nervous system function in these individuals leads to a greater reliance on angiotensin receptors for vascular tone regulation .Digoxin is avoided as it has a high affinity for the amyloid fibrils and can lead to arrhythmias .CCBs are also not used because of their negative inotropic effect and affinity for fibrils  the oveal. (2019) it was Cardiac amyloid patients often have poor tolerance to most rate control agents such as beta-blockers, calcium channel blockers, and digoxin. However, if rate control is attempted, experts advise starting with low doses and closely monitoring hemodynamics. In the case of digoxin, regular monitoring of drug levels, electrolytes, and renal function is recommended . If atrConduction disorders: Conduction disorders in cardiac amyloidosis mostly involve the His-Purkinje system . The moIn a study conducted by Donnellan et al. it was observed that the implementation of cardiac resynchronization therapy led to enhancements in several aspects, including the severity of mitral regurgitation, NYHA functional class, and left ventricular ejection fraction. .Chemotherapy followed by stem cell transplantation is the preferred treatment option for individuals diagnosed with AL amyloidosis, and in some cases, proteasome inhibitors are used. The current standard of therapy is based on antimyeloma therapies and focuses on achieving a significant hematological response. The effectiveness of a treatment is evaluated based on the hematologic and organ response.The criteria for this response is described in Table 2SCT (stem cell transplantation) has shown promising results in terms of hematological and cardiac response rates, with rates of 66 % and 41 % respectively. The most frequently employed treatment approach is administering a high dose of melphalan, followed by autologous stem cell transplantation (HDM/ SCT). Furthermore, there has been a decrease in mortality associated with the transplantation procedure. Alkylating agents: Alkylating agents used are melphalan, cyclophosphamide, and bendamustine. The most commonly used is melphalan. A high dose of melphalan (100 to 200 mg/m2 of intravenous dose divided over 2 days) is given prior to SCT .In cases where SCT is not viable, oral melphalan can be combined with dexamethasone for treatment. Cyclophosphamide, however, tends to be administered alongside proteasome inhibitors and CD38 monoclonal antibodies despite potential adverse effects such as hemorrhagic cystitis and hair loss. Bendamustine serves mainly as a tertiary line of rescue therapy coupled with dexamethasone .Immunomodulators: Immunomodulatory medications such as thalidomide, lenalidomide, and pomaidomide are currently utilized to treat AL amyloidosis. These drugs work by inhibiting the activity of interleukin-6, which is a growth factor that promotes the proliferation of myeloma cells (Anderson 2005[rson 2005). LenaliProteosome inhibitors: Cell cycle arrest and cellular apoptosis can be induced by inhibiting proteasome activity, making proliferating malignant cells more vulnerable compared to normal cells  Bortezoal., 2005).Monoclonal antibodies: CD38 and SLAMF7 are cell surface antigens being evaluated as targets for AL amyloidosis as they are specific to MM cells. Daratumumab is an anti-CD38 monoclonal antibody used in the Dara-CyBorD regimen. It has now become the standard method of care for individuals who are newly diagnosed with AL amyloidosis . Anotheal., 2021).Doxycycline: Doxycycline is a type of tetracycline antibiotic that inhibits matrix metalloproteinases activity. Doxycycline is categorized as a tetracycline antibody, which hinders the action of Matrix metalloproteinases. Evidence suggests that increased serum levels and tissue expression of MMP and tissue inhibitor of matrix metalloproteinase are linked to renal and cardiac impairment in AL. It suppresses the production of amyloid fibrils formed due to light chains. The effectiveness varies depending on the dosage administered ex vivo or in vivo. Research studies are currently underway to evaluate its efficacy when used concurrently with SCT.Other new modalities that are coming up are the Small molecule inhibitors - venetoclax. They lead to the modulation of intrinsic apoptotic pathways.Clearance of amyloid deposits- 11-1F4 and NEOD001 are being tested on mouse models. These are exogenous antibodies that are targeted to kappa and light chain extracts.Different type of therapies using different mechanisms of action are present for ATTR type of amyloidosis.Stabilizers: They stabilize the TTR tetramer. These include Tafamidis, Diflunisal, and AG-10. Tafamidis and AG10 are selective stabilizers whereas diflunisal is a non-selective agent. Tafamidis is an FDA-approved drug for ATTR cardiomyopathy. Tafamidis is selective for thyroxine-binding sites of the TTR protein. The Transthyretin Amyloidosis Cardiomyopathy Clinical Trail (ATTR-ACT) evaluated the efficacy and safety in hereditary and wild-type cases. The rate of cardiovascular-related hospitalizations was lower in the treatment, with 32 % less risk when compared to the placebo  . Diflual., 2018) . In a al., 2018). AG10 ial., 2018).Silencers: These drugs affect the production of TTR by interrupting the stages of protein synthesis on a molecular level. In these, the first-generation drugs are - Inotersen and Patisiran. Patisiran is a small intervering RNA blocking the expression of both variant and wt-TTR. It targets the 3′ untranslated region of the TTR mRNA. In phase 3 trials, patisiran significantly improved neuropathy, leading to a statistically significant reduction in NT-proBNP left ventricular wall thickness and reduced worsening in GLS (suggestive of cardiac benefit) . Inoteral., 2018). The seDegraders: These drugs destabilize and disaggregate the amyloid fibers. The combination of doxycycline and tauroursodeozycholic acid (TUDCA) is the most effective regime. Trials have shown that TUDCA combined with doxycycline has shown some ability to decrease left ventricular strain after a year but does not entirely halt the disease .Liver and cardiac transplantation: The best treatment for ATTR type of amyloidosis is combined heart and liver transplantation. Fibrils are formed in the liver therefore transplanting the liver would remove the source of mutated TTR molecules and prolongs survival. But this type of transplantation is most feasible in younger patients who got the diagnosis earlier in the disease course.The latest modalities targeted by monoclonal antibodies are plasma cells. A human monoclonal antibody called daratumumab is directed against CD38, prominently expressed in abnormal plasma cells. Daratumumab demonstrated promising outcomes in early trials in patients with AL amyloidosis. The hematological response was obtained in 76 % of cases (36 % with complete remission) in a retrospectively conducted trial involving 25 patients who had extensive pretreatment with the dosing such as once every week for two months, administering 16 mg/kg weight in 1 l for the initial dose and 500 mL for subsequent doses . AmyloiFor ATTR amyloidosis, Tafamidis is a tiny medication that binds to the transthyretin's thyroxin binding site, stabilizing TTR tetramers in both wild-type and genetically modified TTR , taken 80 mg (four 20-mg tafamidis meglumine capsules) orally once daily. Tafamidis primarily underwent evaluation in polyneuropathy trials, with encouraging outcomes regarding the degradation of nerve function .Among the therapeutic agents that target transthyretin mRNA, Patisiran (administered intravenously at a dose of 0.3 mg/kg every three weeks) and Inotersen (subcutaneously administered once a week at a dose of 284 mg) are two oligonucleotide-based drugs currently available. By targeting the 3' untranslated region of transthyretin mRNA, Patisiran acts as double-stranded RNA in circulation but eventually dissociates into single-stranded molecules within cells. The sense strand subsequently binds to TTR mRNA forming an RNA-induced silencing complex resulting in hindering protein synthesis through cleavage inhibition, thereby controlling both normal and mutant TTR protein levels. Similarly, Inotersen is an antisense oligonucleotide that primarily works by binding with its target or complementary sequence on mRNA inside the nucleus followed by degradation facilitated by ribonuclease H enzyme action after translation regulation has been affected effectively ensuring efficient reduction on overall protein production from either normal or pathological variants .Tafamidis functions as a particular TTR stabilizer by occupying one of the thyroxine-binding sites present in the TTR tetramer, thereby preventing the initial stage of amyloidogenic cascade dissociation. It has been approved for individuals with polyneuropathy at an oral dosage quantity of 20 mg per day. For those having cardiomyopathy, they are advised to consume a daily dose quantity ranging from 80 mg/61 mg once .Diflunisal is a type of analgesic medication derived from salicylic acid and is typically taken at 250 mg twice daily. Its mechanism of action involves interfering with the binding site located at the dimer-dimer interface of TTR tetramers, ultimately leading to TTR stabilization. This stabilizing effect induces complete kinetic stabilization in wild-type and variant TTR forms, thus effectively inhibiting fibril formation . Vutrisiran, also known as ALN-TTRsc02, is a newer type of RNAi-based medicine that aims to block the production of transthyretin protein by targeting a specific segment in TTR messenger RNA. This approach works by impeding the formation of TTR protein altogether instead of reducing its levels like many first-generation therapie .Streptococcus pyogenes Cas9 protein technology .A clinical experiment on mice yielded a knockdown rate of more than 97 % of the mouse TTR. LNP-INT01 was given to mice in doses 0.3-3 mg/kg. TTR levels were shown to be consistently lower after a year of follow-up. By day 28, the TTR level decreased by 52 % in the group administered with a dosage of 0.1 mg/kg, while experiencing an 87 % decrease in TTR levels was observed in the group receiving the minimum dose .Acoramidis, formerly AG10, is a TTR stabilizer with high selectivity that emulates the configuration of Thr119Met mutation's protective qualities. Its mechanism inhibits tetramer dissociation by encouraging hydrogen bonds to form between serine residues . This iTolcapone can stabilize TTR tetramers by engaging with both binding sites for thyroxine within the TTR tetramer .Selective targeting of dissociative monomers, oligomers or TTR aggregates by monoclonal antibodies can effectively hinder fibril formation and further aggregation. Additionally, these antibodies can target amyloid deposits which initiate phagocytic mechanisms that spare normal tetrameric TTR from removal .Dezamizumab, an IgG1 antibody, effectively eliminates amyloid deposits containing SAP by triggering complement activation and macrophage-mediated phagocytosis .It has been observed that they can interfere with TTR fibrils in amyloid deposits present in ATTRv and -wt patients. Although doxycycline, a derivative of tetracycline, is demonstrated to be the most effective method for disrupting pre-existent TTR fibrils, it was not completely successful with non/pre-fibrillar TTR deposits. Strong synergistic effects were noted when combining both doxycycline and tauroursodeoxycholic acid, which facilitated destruction and absorption of the deposited TTR molecules . A phasDue to the diverse pathogenesis of CA, it often presents with various clinical features, making it hard to diagnose. This is further complicated by its similarity to more common disorders. However, owing to the advances in diagnostic techniques, drug therapies, and AI, it is now possible to diagnose early and begin the treatment immediately. In light of the current awareness of the significance of ATTR-CM, medical professionals should be on the lookout for cardiac amyloidosis when patients show symptoms of CA with adequate clinical suspicion. Additionally, serologic markers testing, nuclear imaging, and tissue biopsy should be employed promptly to confirm the diagnosis and identify the type of CA. Endomyocardial biopsy is particularly important when the subtype of CA is unclear or suspicion persists despite the results of serologic and imaging tests. All diagnostic methods should be used together to ensure the most accurate diagnosis. Risk assessment for unfavorable outcomes can be achieved by utilizing various imaging modalities. Furthermore, certain CA biomarkers, such as troponin and NT-proBNP, also help in risk stratification and monitoring the effectiveness of treatment outcomes in CA patients. Diagnosing CA and differentiating its subtype is necessary to begin a suitable therapeutic regimen with disease-modifying agents.Several problems regarding the clinical approach to CA-including uncertainties in screening, progression evaluation, AT-TRv asymptomatic carriers management, and the changes that should be done with treatment strategies for non-responders-have yet to be resolved. Additionally, the most effective treatment, single therapy or a combination of gene silencer and stabilizer is currently purely based on the clinical judgement of the physician. More research is required to identify the optimal medical treatment for ATTR-CM ensuring affordability and accessibility.Mohammad Amjad Kamal and Nirja Kaka  contributed equally as corresponding author.The study was conducted under the supervision of PearResearch (pearresearch.com). The authors declare no conflict of interest. There are no relevant financial or non-financial competing interests to report. Not applicable.The study did not receive any funding from any authority or organization.Not applicable.NK, NP, YS: Conceptualization, methodology, project administration, resourses, and supervision. All authors: Visualization, roles/writing; MAK, NK, NP, YS: Writing - review & editing.


The development of infant gut microbiome is a pivotal process affecting the ecology and function of the microbiome, as well as host health. While the establishment of the infant microbiome has been of interest for decades, the focus on gut microbial metabolism and the resulting small molecules (metabolites) has been rather limited. However, technological and computational advances are now enabling researchers to profile the plethora of metabolites in the infant gut, allowing for improved understanding of how gut microbial-derived metabolites drive microbiome community structuring and host-microbial interactions. Here, we review the current knowledge on development of the infant gut microbiota and metabolism within the first year of life, and discuss how these microbial metabolites are key for enhancing our basic understanding of interactions during the early life developmental window. The gut microbiome thus has a profound influence on both early and long-term health and development5. In addition to influencing development and maturation of the host immune system, the gut microbiota has a number of important functions in the host, including breakdown of ingested complex carbohydrates, protection against pathogenic bacteria and metabolic functions including production of vitamins, short chain fatty acids (SCFAs) and bile acid biotransformation6. The importance of neonatal bacterial colonization for the growth of a healthy individual is clear and exhibits the power that the microbiome has on its host from an early stage in development.A relatively simple microbial community at birth develops into a diverse and complex microbial ecosystem across the early life window8. In this early phase, the gut microbiota is more vulnerable to modulation than in the adult state9, suggesting that the first 1000 days of life is a critical window of opportunity for optimal bacterial colonization. Disruption to microbial development during this period is believed to have long-lasting consequences, impacting on normal immune and neurocognitive development and has been associated with increased risk of developing diseases such as asthma and metabolic syndrome later in life11.The gut microbiota composition is initially known to be in a state of flux and consequently stabilizes, so that by age 2.5–4 years, it begins to resemble the composition and characteristics of an adult gut12 or postbiotics13. The most promising results for probiotics have been seen in the prevention or treatment of diarrhea in children and in necrotizing enterocolitis in preterm infants15. Pharmabiotics including microbially produced bacteriocins, bacteriophages and SCFAs have gained interest for potential application for microbiota modulation16–19. Furthermore, there has been a shift in recent years to expand microbiome research beyond profiling taxonomic composition using 16S rRNA gene sequencing and to focus on gut microbial functionality and activity using whole metagenome sequencing (WMS) and metabolomics. WMS while more costly than 16S rRNA sequencing provides insight on the functions encoded by the genes present in the microbiome. WMS also facilitates community structure to be examined at a far greater and reliable taxonomic resolution beyond genus level to examine species and subspecies20. In addition, metabolomics is a useful complementary approach to measure metabolites in the gut derived from the diet, host or the microbiota (collectively denoted the gut metabolome)21. Advances in informatics such as machine learning have also provided superior insight and facilitated meaningful interpretations of functional microbiome research23. This review briefly discusses the factors influencing the gut microbiota in early life, summarizes current literature available on the diet-dependent development of the infant gut metabolome, and discusses how microbiota-derived metabolites may be key for early life microbiome and host development.The gut microbiota may also be modulated at this critical phase in a beneficial manner. Strategies to optimize early microbial acquisition during this key developmental window of opportunity may help to prevent and treat disease. Interventions to modulate the early microbiome include the use of probiotic and prebiotic interventions or combinations of both, as either synbiotics24. Passage of the infant through the birth canal exposes it to the mother’s vaginal and fecal bacteria thereby contributing to microbial and metabolic diversity in the infant gut25. Cesarean section impairs the transfer of certain maternal strains resulting in an infant gut microbiota with increased abundance of potentially pathogenic microbes, and a lack of beneficial taxa such as Bifidobacterium26. Other factors reported to influence the development of the infant gut microbiome include gestational age, host genetics, feeding regime, geographical location and perinatal antibiotic usage28. Gestational age is an important factor in gut microbiota development with preterm birth associated with reduced diversity and delayed colonization with Bifidobacterium compared with full-term infants29. Exposure to antibiotics in early life has been reported to induce an increased abundance of members of the Enterobacteriaceae family and decrease in abundance of Bifidobacterium31. Antibiotic exposure has also been linked with the development of metabolic and inflammatory diseases later in life32. In recent years, evidence also supports the existence of a breast milk microbiome, which is a source of beneficial microbes to the infant34. Infants also acquire microbes that colonize their gut from the environment including siblings, pets and the hospital35.Delivery mode exerts a significant influence of gut microbiota composition, exposing the infant to various microbes associated with the maternal gut and vaginal tract36. Breast milk contains essential nutrients such as caseins, whey, fatty acids, and lactose as well as micronutrients including Immunoglobulin A, Immunoglobulin G, Immunoglobulin M, calcium and vitamin A37. Furthermore, breast milk is a source of an array of bioactive components such as human milk oligosaccharides (HMOs). HMOs consist of > 150 known complex glycans with the most common being 2′-fucosyllactose (2′FL), accounting for 20–30% of all HMOs37–39. HMOs are not digested in the upper gastrointestinal (GI) tract and are degraded in the colon by specific HMO-degrading bacteria, mostly Bifidobacterium, into metabolites such as acetate and lactate with potential beneficial host health effects38. The ability of some species of Bifidobacterium to efficiently degrade HMOs makes them dominant members of the microbiota of breastfed infants. Host secretor status, based on active or inactive copy of the fucosyltransferase 2 (FUT2) gene, determines a mother’s ability to produce HMOs40–43. Non-secretors have been associated with reduced Bifidobacterium and Bacteroides abundances and lower microbial diversity in their infant’s gut microbiota44–47. The absence/decrease of 2′FL in non-secretor women has potential implications for infant short and long term health and development48–50.Human breast milk can meet all nutritional requirements essential for infant growth, health and development52. Higher abundance of potentially pathogenic bacteria such as Enterobacteriaceae and Clostridium, as well as antimicrobial resistance genes, is observed in formula-fed infants compared to exclusively breastfed infants54. Conversely, breastmilk feeding is associated with low microbial diversity, dominated by HMO-utilizing Bifidobacterium species and saccharolytic fermentation compared to formula-feeding55. Due to formula milk’s higher protein content, formula fed infants may also exhibit a higher degree of proteolytic metabolism in the gut57. Proteolytic microbiota-derived metabolites have been linked to detrimental cardiovascular and metabolic health effects in adults58–61, as discussed in greater details below. Yet, whether changes in colonic fermentation observed in formula fed infants influence the risk of developing diseases later remains unknown. Nonetheless, many infant formulas have in recent years been supplemented with prebiotics in an attempt to mimic health benefits of HMOs in breast milk. Prebiotic supplementation has predominantly consisted of short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS), and HMOs such as 2-fucosyllactose and Lacto-N-neotetraose62–64.Compositional analysis of the infant gut microbiota has shown altered community structures in formula-fed infants compared to breastfed infantsB. longum subsp. infantis typically decrease in this period65. Due to the increased consumption of protein and dietary fibers during this period, microbes capable of digesting more complex nutrients increase in abundance such as Lachnospiraceae, Bacteroidaceae and Ruminococcaceae66–68. Microbiota maturation occurs continuously even beyond five years of age where the children have acquired a diverse, stable and adult-like configuration, but with a lower community richness compared to the adult microbiota69.During the transition period to solid foods, typically between 4–6 months of age, changes appear in the richness and diversity of the infant gut microbiota. Milk adapted bacterial species such as 70, which may contribute to impairment of child growth72. The disturbance in microbial succession in malnourished children could contribute to lifelong deficits in growth and development as reviewed elsewhere73. Intriguingly, a recent 3-month-long intervention study showed that a microbiota-directed complementary food prototype in comparison with an existing ready-to-use supplementary food promoted weight gain in children with moderate acute malnutrition between the ages of 12 months and 18 months of age74. The study suggested that the improved weight gain could be mediated by a more complete repair of the gut microbiota74. Supporting this hypothesis, a placebo-controlled trial in 2- to 6-month-old undernourished Bangladeshi infants with severe acute malnutrition found that supplementation with a Bifidobacterium longum subspecies infantis strain promoted weight gain that was associated with reduced levels of intestinal inflammation markers75. Globally in 2020, 149 million children under the age of five years were estimated by be stunted76, emphasizing the need for also understanding the role of the immature gut microbiome in malnourished children.Food is a major factor shaping the gut microbiota in early life. This is also evident in children who do not receive adequate nutrition during infancy and early childhood. Malnourished infants and children develop an immature gut microbiome77 not only shapes gut microbiome composition78, but also shapes gut microbial metabolism in early life resulting in the generation of a plethora of small molecules (metabolites) (The transitioning from a sole source of milk to a diverse range of foodsbolites) .Figure 79. SCFAs also serve as energy substrates both for the resident gut microbes (as discussed further below), as well as human cells with butyrate being a primary energy substrate for colonocytes80, and propionate being a substrate for gluconeogenesis in the intestine81 and liver83. In adults, the major SCFAs acetate, propionate and butyrate are typically found in a 3:1:1 ratio, whereas succinate and lactate appear as intermediates and therefore do not appear in consistent levels. However, in early life, SCFA profiles change concordant with the child’s progression in dietary and microbiome diversity85. During breastfeeding (early phase), the SCFA profile is characterized by low acetate and high succinate, during complementary feeding (middle phase) by high lactate, pyruvate and formate, and after cessation of breastfeeding (late phase) by high propionate and butyrate84. The high abundances of lactate, pyruvate and formate measured in the infant gut during the early phase reflect a less-developed microbiome since these metabolites, in the context of a developed gut microbiome, typically would have been converted into other metabolites86. Compared to non-breast infants, exclusive breastfed children have been reported to have lower absolute concentrations of total SCFAs including acetate, butyrate, propionate, valerate, isobutyrate, and isovalerate, yet higher concentrations of lactate at four months of age87. Bifidobacterium are highly abundant in breastfed infants88 due to their ability to metabolize HMOs89, which results in the production of acetate and lactate90. Yet, with the cessation of breastfeeding and development of the gut microbiome1, butyrate production increases in the infant gut84. Despite the large dynamics in SCFAs observed in early life and their potential importance as signaling molecules, the relationships between SCFAs, the gut microbiome and given carbohydrate substrates are rather limited. Furthermore, with over 200 HMO varieties identified in human milk and with large variations in HMO profiles between mothers and throughout lactation92, it also remains to be elucidated how differences in breastmilk HMO profiles affect the infant’s gut microbial metabolism.The most studied microbiota-derived metabolites are the SCFAs. SCFAs are products of colonic fermentation of HMOs, dietary fiber and proteins, and known to affect human metabolism in numerous ways via activation of several host receptors in different organs93–95. However, given that intake of dietary fiber and proteins increases with the complementary diet96, which is associated with increased gut microbial diversity68 and capacity to transport amino acids during infancy78, a multitude of microbial-derived metabolites of dietary fiber and proteins97–104 are likely to change with this dietary transition. For example, it was recently reported that HMO-degrading Bifidobacterium species convert aromatic amino acids  into aromatic lactic acids  in the infant gut88. In agreement with breastfed infants being dominated by Bifidobacterium68, aromatic lactic acids have been found in higher levels in feces of breastfed infants compared to formula-fed infants105 and weaned infants88, emphasizing the strong interplay between early life nutrition and specific gut microbes affecting the levels of microbial aromatic amino acid catabolites in early life.Studies on gut microbial metabolites in the context of early life have mainly been limited to the study of SCFAs21, which are implicated in insulin resistance106. The levels of proteins and amino acids could likely play a role in dictating the levels of BCFA, since a study found that breastfed infants had significantly lower levels of fecal BCFAs compared to infants fed two infant formulas with extensively hydrolyzed proteins and free amino acids, respectively, at one and two months of age107. Infant formula typically contains more protein and lower free amino acids than breast milk109, suggesting that more proteins are being fermented in the colon of formula-fed infants. In line herewith, a study in newborn rhesus monkeys found that by reducing the protein content in formula, urinary levels of microbial-derived proteolytic compounds  could be reduced110. Fecal BCFA levels have also been associated with variations in breastmilk lipid and HMO compositions111. Furthermore, BCFA have been reported to increase in the infant gut over time independent of early feeding (breastfeeding versus formula feeding)93, suggesting that the lack of HMO following a progression toward solid food promotes a shift toward amino acid fermentation in the infant gut. Also biogenic amines (i.e. cadaverine and tyramine) have been reported in higher levels in feces of formula-fed infants compared to breastfed infants113. These differences could be due to differences in the gut microbiome composition, as infant formula-associated genera include Streptococcus and Enterococcus114, which are producers of putrescine, tyramine and cadaverine116. Another prevalent commensal in early life as children progress toward a solid diet is Ruminococcus gnavus117, which is also able to produce tryptamine in vitro118. However, it remains to be investigated whether R. gnavus indeed produces tryptamine in the infant gut. In fact, many microbial proteolytic-derived metabolites associated with detrimental effects on cardiovascular and metabolic health in adults, such as trimethylamine-N-oxide 58, imidazole propionate (derivative of histidine)59, phenylacetylglutamine 60, p-cresol sulfate and indoxyl sulfate 61, remain understudied in the context of early life. These metabolites would be expected to arise when children transition toward adult-like solid foods. Yet, very few studies have looked into the impact of the complementary diet on the infant gut microbiome68.Other examples of microbiota-derived metabolites changing in early life are the branched-chain fatty acids . The BCFA reflect increased proteolytic fermentation as they originate from conversion of the branched-chain amino acids valine, leucine and isoleucine77. Besides having direct effects on the gut microbiome composition and activity119, the polyunsaturated fatty acids can be metabolized by colonic microbes into hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates120–122. Interestingly, specific Bifidobacterium strains have been found to be able to convert LA to conjugated linoleic acid (CLA)123, which has been associated with a variety of systemic health promoting effects124. Although the production and importance of CLA in the infant gut are currently unknown, another LA-derivate measured in feces, 12,13-dihydroxy-9Z-octadecenoic acid  has been suggested to impede immune tolerance in early life126. This metabolite was recently reported to be produced by common early-life gut microbiome members, namely B. bifidum and E. faecalis strains125. Furthermore, a recent randomized, controlled, clinical trial reported that infants receiving formula milk containing HMOs (i.e. 2-fucosyllactose and LNnT) had reduced fecal levels of two dihydroxy fatty acids ) compared to infants receiving control formula milk without HMOs, suggesting that HMO-induced alterations in the microbiome may affect microbial conversion of fatty acids127. Therefore, more studies are warranted to investigate intestinal fatty acid metabolism in the infant gut, which is likely to depend on the amount of fatty acids ingested, the efficacy of absorption in the small intestine, as well as the individual gut microbiome composition.Dietary fat is also metabolized by microbes in the gastrointestinal tract. Infants are, through breast milk and to some extent formula milk, provided with the essential fatty acids linoleic acid (LA) and alpha-linoleic acid (ALA), as well as the long chain polyunsaturated fatty acids (LC-PUFA): eicosapentaenoic acid (EPA), arachidonic acid (AA) and docosahexaenoic acid 128. While approximately 95% of the bile acids are reabsorbed in the ileum and returned to the liver via the portal vein, unabsorbed bile acids end up in the colon where they undergo chemical transformations by the microbiota resulting in secondary bile acids129. Microbial deconjugation of bile acids reduces their reuptake in the small intestine, which affects the pool of bile acids entering the colon129. Furthermore, microbial modification of bile acids affects their chemical structure leading to altered signaling via host receptors including the nuclear farnesoid X receptor (FXR) and G protein coupled bile acid receptor 1 (TGR5)128, which may have major impact on host metabolism as discussed elsewhere131. Concordant with the succession of the gut microbial community in early life, the intestinal bile acid profiles develop according to both the microbiota-stage and nutritional stage. A longitudinal study with 10 healthy Japanese subjects found that during the first six months of life, infants dominated by Bifidobacterium species had high fecal levels of unconjugated primary bile acids whereas infants dominated by Enterobacteriaceae had high fecal levels of conjugated primary bile acids132. Indeed, bile salt hydrolase activity, catalyzing deconjugation of conjugated bile acids, is common in Bifidobacterium133, suggesting that Bifidobacterium is a key player deconjugating bile acids during breastfeeding. Yet, as infants progress toward a solid diet, dietary energy intake goes up77, dietary complexity and gut microbial diversity increase68. Consequently, both the total excretion of bile acids in the feces as well as the fecal levels of secondary bile acids increase132. Furthermore, decreasing level of primary bile acids and an increase of secondary bile acids in feces have consistently been observed after weaning135. Differences in fecal bile acid profiles have also been observed when comparing breastfed and formula-fed infants with breastfed infants showing higher levels of sulfated bile acids and formula fed infants showing higher levels of secondary bile acids85. Finally, a study in mice has shown that oral administration of bile acids to newborn mice accelerate postnatal microbiome maturation136, suggesting that the progression in intestinal bile acid profiles during infancy also drives maturation of the postnatal gut microbiome.Upon intake of fat, bile is released into the small intestine to aid digestion of lipids. Bile acids constitute the primary component of bile and is conjugated by glycine (dominant in humans) and taurine, respectively138. HMOs are poorly digested by the infant but are favored growth substrates for certain Bifidobacterium species and strains that can enzymatically degrade these complex dietary components that include LN(n)T, fucosylated and sialylated structures139. Along with Bifidobacterium, Bacteroides, Ruminococcus and more recently Akkermansia have also been shown to degrade these complex carbohydrates, although in most cases this relates to enzymatic cleavage of sugar side chains and correlates with associated mucin degradation pathways140–142. Certain Bifidobacterium species seem to have limited impact on wider ecosystem structuring due to their ‘selfish’ digestion of HMOs. This is particularly true for B. longum subsp. infantis strains where they actively transport (via ABC transporters) and internalize whole (intact) HMOs to perform intracellular breakdown, utilizing the monosaccharide metabolites for their own cellular processes, rather than sharing among other early life microbes143. Other species such as B. bifidum, Bifidobacterium pseudocatenulatum, B. longum subsp. longum and B. breve also carry out extracellular hydrolysis of complex HMOs, and then import and metabolize the resulting mono- and di-saccharides. These strains therefore provide an important keystone function during early life by providing simpler metabolites for cross-feeding145. Metabolites that can be used by other secondary degraders include the SCFAs acetate and lactate, formate, succinate, pyruvate, fucose, and 1,2-propanediol 146. Previous work has indicated that within ‘mini’ Bifidobacterium ecosystems , there is often a keystone member that actively breakdown different HMOs, which provides key metabolites such as fucose, galactose, acetate and N-acetylglucosamine then utilized by other non-HMO degrading strains138. Other work supports this concept, showing that during growth on HMOs, cross-feeding interactions between multiple bifidobacterial strains increase overall cell numbers, which was linked to production of formate, acetate, 1,2-propanediol, and lactate during co-cultivation147. Most recently, a study has indicated how ‘timing’ of sugars in the infant gut shape the wider Bifidobacterium ecosystem, with B. bifidum and B. longum subsp. infantis dominating through inhibitory priority effects based on their ability to digest HMOs, whilst B. breve benefits from facilitative priority effects and dominates by utilizing fucose 148. These studies highlight the importance of cross-feeding term ‘syntrophy’ for Bifidobacterium, which has been defined as ‘obligately mutualistic metabolism’ whereby without a complete mini ecosystem all or combinations of strains cannot survive without the other. This collective approach to HMO utilization may indicate additional processes by which multiple Bifidobacterium species and strains dominate the breastfed infant gut, and successfully outcompete other bacterial taxa. Indeed, acetate and lactate production by Bifidobacterium (in response to HMO-metabolism) is associated with lowering intestinal pH, which also correlates with changes in microbial composition, such as reducing levels of potential pathogens like Enterobacteriaceae, thus enhancing colonization resistance in the early life gut150. In agreement, in vitro experiments have found that SCFAs directly inhibit growth of enteric pathogens belonging to the Enterobacteriaceae family including Escherichia coli, Klebsiella and Salmonella in a pH-dependent manner151–153.Gut microbial conversion of dietary substrates into metabolites represents a potential regulatory mechanism by which specific gut microbes impact the wider gut ecosystem . In partBifidobacterium, HMO-derived metabolites also cross-feed other early life taxa. In vitro studies have shown that growth of Limosilactobacillus reuteri is enhanced through 1,2-propanediol produced by B. breve UCC2003146. There has also been a number of studies exploring the cross-feeding impacts on a range of butyrate-producing bacteria, given their key health-promoting role. Specifically, butyrate-producing bacteria can utilize acetate and lactate, and HMO-derived monosaccharides as carbon sources, which serves to increase their levels in the infant gut. For example, Anaerostipes caccae has previously shown to only grow in co-culture with B. longum subsp. infantis, leading butyrate production154. Additional SCFAs production by Eubacterium hallii in response to (fucosylated) HMO breakdown by B. breve and/or B. longum subsp. infantis enables utilization of L-fucose (for production of butyrate), and 1,2-PD uptake for propionate production137, although the latter has yet to be demonstrated in the infant gut. The availability of L-fucose also supports growth of other microbes encoding Fuc catabolic pathways such as B. thetaiotaomicron and A. muciniphila, but these microbes can also cleave fucose from fucosylated HMOs, according to their fucosidase activity155. Notably, some pathogenic bacteria have evolved strategies to use fucose to their advantage, including Salmonella and C. jejuni, which is the latter case appears to be enriched in breastfed infants when compared to those on formula156. Thus, the availability of different metabolites in the gut may both have beneficial and detrimental consequences depending on the ecological circumstances.Looking beyond 2S), or SCFAs (lowering the pH), or via production of metabolites with direct antimicrobial activity such as bacteriocins. Bacteriocins are ribosomally-synthesized antimicrobial peptides, which are active against other bacteria, and can be either narrow spectrum (i.e. targeting similar species) or broad spectrum (i.e. across diverse genera), with the host bacterium being resistant157. A number of early life microbiota members, including Bifidobacterium, are known to produce these antimicrobials which may be linked to their ability to modulate the wider microbiota, and also inhibit overgrowth of potentially pathogenic bacteria that would otherwise be harmful to the infant158. The gene clusters required for biosynthesis appear to be very strain specific; previous work has indicated that the bacteriocin bifidin (produced by Bifidobacterium bifidum NCDC 1452) has activity against a range of Gram positive and Gram negative’s including Escherichia coli and Staphylococcus aureus158, which are common members of the early life gut microbiota. There are other Bifidobacterium-associated bacteriocins, however these have shown activity against specific food-borne pathogens including Salmonella spp. and Bacillus cereus, thus may not be so relevant in the context of the first 12 months of life. More recently, Bifidobacterium longum subsp. infantis LH_664 has been shown to encode and produce a narrow spectrum bacteriocin (putatively named Bifidococcin_664) that whilst not impacting growth of Lactobacillus and Enterococcus strains, does have discrete activity against a potentially important pathobiont in the (preterm) gut, Clostridium perfringens159. The ability of these active peptides to target specific bacteria, may be particularly important in the context of early life perturbations , when levels of pathobionts may be higher given the absence of these key metabolite/peptide modulating factors that would otherwise be produced by Bifidobacterium which may be depleted in these contexts. However at this stage it is unclear if the dietary (e.g. HMO) milieu in the gut plays a role in shaping the production of these peptides, and further work is required to disentangle these relationships.In some cases there may be competitive interactions via production of metabolites know to inhibit metabolic reactions and growth of other microbiota members e.g. hydrogen sulfide  that enables the diverse array of bonds between monosaccharide units to be ‘broken’, thus allowing these simple sugars to enter into different metabolism pathways to provide energy (ATP) for cellular responses and growth160. Fermentation end products are further metabolized by cross-feeders which often results in the production of SCFAs, with increasing production of butyrate and propionate, in comparison to the acetate dominant SCFA profile observed during exclusive milk-based feeding161. Additional key components that can be produced as a result of protein fermentation are amino acids, which play a central metabolism (enzyme co-factors) role for many bacteria, driving growth and changes in motility, biofilm formation and virulence factor expression162. Furthermore, there are strain level differences with respect to B vitamins biosynthesis, which is required by the host, and by other strains unable to de novo synthesize these key metabolites163. For example, several early life taxa are predicted, and have been experimentally confirmed, to produce vitamin B1 including some Bacteroides spp., Clostridium spp., Lactobacillus spp., and Bifidobacterium spp.164. These strains are expected to play a key role for other species and strains that lack a vitamin B1 synthesis , highlighting the cross-feeding and also competition for this essential metabolite in the infant gut165. It is clear that this time-period represents a highly dynamic period of ecosystem development, with burgeoning cross-feeding networks being established, and like other underdeveloped ecosystems, may be particularly sensitive to diet-derived perturbations166. However, clear evidence on the longer-term effects during this time period are required via both longitudinal cohorts and experimental studies. For a comprehensive review of this with a more complex (adult-like) ecosystem see a review by Oliphant and Allen-Verco161.With the introduction of additional complex dietary components (particularly plant-derived) during weaning there is a significant expansion in cross-feeding networks due to the resultant complex milieu of metabolites produced by primary degradersFinally, the spatial context of these potential metabolite interactions should be considered. Clearly the levels and diffusability of key metabolites would be expected to have the most pronounced impact on ecosystem structure on those microbes in close proximity to each other. However, given the complex ‘geography’ of the gut, including the overall length, mucus layers, and micro-structure e.g crypts, it is likely that the studies undertaken so far may not completely replicate these spatial considerations. Therefore, additional model systems e.g. organoids and mice, may be required to pinpoint relative contributions of microbial metabolites as modulators of the infant gut microbiome.167, these microbiota-derived molecules not only shape the gut microbiome ecosystem, but also likely play a central role in mediating host-microbial cross-talk in early life as they may communicate with host cells via host receptors in the intestine and distant organs  can be consumed by IECs, however this is a less efficient process than for butyrate. Nevertheless, acetate is also associated with inducing additional anti-inflammatory responses in the gut epithelium such as enhancing tight junction protein expression and anti-inflammatory cytokine production171. This appears to be mediated through Toll-like receptors (TLRs) and G protein-coupled receptors (GCPRs), including FFA2 pathway stimulation, as defined using KO mouse models. This acetate-ligand interaction can lead to NLRP3 inflammasome activation172, which also links to a more efficient clearance of enteric pathogens from the gut173, and enhanced production of sIgA (via Dendritic Cell-B cell activation). Of the other metabolites discussed above, such as B vitamins, it is appreciated these also directly modulate host immune responses, including the epithelial barrier  and hydroxycarboxylic acid receptor 3 (HCA3)176. During the last decade, tryptophan catabolites have emerged as potential key AhR-ligands playing a role in regulating intestinal barrier and immune function98. It was recently shown that fecal levels of indolelactic acid were positively correlated with the fecal samples’ ability to activate AhR in an AhR-reporter cell line88, suggesting that indolelactic acid is a key AhR-ligand in early life. However, it remains to be determined whether indolelactic acid and/or other microbiota-derived AhR ligands indeed play a role for immune barrier development in the context of early life. For all of these studies, it is important to note that most work in this area has concentrated on adult models. Previous work has suggested that microbes (including Bifidobacterium) drive distinct responses in the neonate vs. later life time points177, thus there is a pressing need to determine how microbial metabolites such as aromatic lactic acids and SCFAs modulate host intestinal responses during the early life developmental window.Although a ‘strong’ barrier is considered essential for host health, during very early life a balance is required; allowing key microbial metabolites to cross and initiate immune programming in the underlying mucosal and at more distal and systemic sites. Butyrate is the main ‘fuel’ used by intestinal epithelial cells (IECs) for growth178. Despite this, very little is known about the interplay between the diet, the developing gut microbiota, and the ever-changing bowel habits in early life. Several microbial metabolites, which have been measured in the infant gut, could potentially play a role in affecting gut motility in early life, although this has not yet been investigated. For example, tryptamine has been found to accelerate transit time in vivo through the 5-HT4 receptor (5-HT4R), a receptor uniquely expressed in the colonic epithelium179. Other tryptophan-derived catabolites may also affect intestinal motility via AhR signaling in enteric neurons in the distal gastrointestinal tract180. Furthermore, SCFAs can stimulate the release of serotonin (5-HT) in enterochromaffin cells by activating GPCRs such as FFA3 (GPR41), FFA2 (GPR43), and HCA2 (GPR109A). Serotonin has multiple paracrine and endocrine roles including promotion of peristalsis via the enteric nervous system181. Secondary bile acids and SCFAs may also through TGR5 and FFA2/FFA3, respectively, stimulate release of GLP-1 and PYY in enteroendocrine cells and thereby affect colonic motility183. Variations in intestinal transit time have been associated with the gut microbiome composition and activity in adults184. Furthermore, given the large fluctuations and changes in SCFAs, secondary bile acids and proteolytic metabolites during the first year of life, it is possible that the gut microbiome contributes to inter- and intra-individual variations in bowel habits during infancy. Such variations could potentially be linked to common functional gastrointestinal disorders in infants such as infantile colic and functional constipation185.Stool frequency and consistency vary significantly according to early life nutrition between and within infants during the first year of life169. Furthermore, SCFAs can indirectly affect whole body metabolism by stimulating secretion of gut hormones (e.g. GLP-1 and PYY) in enteroendocrine cells affecting appetite and through stimulation of immune cells that regulate whole body metabolism79. The high gut luminal levels of SCFAs, lactate, and succinate measured during infancy could possibly be important for recruitment of macrophages and neutrophils acting through FFA2, HCA1, and GPR91, respectively168. However, in the context of early life, little is known about the role of SCFAs regulating whole body metabolism and inflammation. High fecal levels of formate during early infancy (3–4 months of age) have been associated with lower BMI z-score, whereas high fecal levels of butyrate have been associated with a higher BMI z-score at 3 years of age in Canadian infants186. Based on these observations one could speculate that the development of the infant gut metabolome from high formate (reflecting a low-diversity microbiome) to high butyrate (reflecting a more diverse microbiome) could play a role in growth trajectories186. Yet, the timing of the gut metabolome development may be important, since another study found that Danish infants with normal weight gain at nine months of age had significantly higher fecal concentrations of butyrate compared to infants with high weight gain111. In line with this, both exclusive formula feeding as well as early introduction to solid foods (vs later) in infancy has been associated with altered gut microbiota and increased risk of being overweight in early childhood187. Prospective studies focusing on the infant gut metabolome development are warranted to understand whether the gut microbiota via production of metabolites is indeed casually involved in the programming of host metabolism, which may trigger long-lasting physiological effects that promote or prevent diseases.SCFAs are proposed to affect whole body metabolism and inflammation upon absorption and circulation in the blood stream to distant organs through interactions with host receptorsBifidobacterium-metabolism of HMOs and the generation of SCFAs have been studied. Therefore, prospective infant cohort studies following infants’ progression in diet, concurrent with their progression in gut microbiome composition and activity, are needed to further advance the field. By collecting and analyzing longitudinal fecal samples by untargeted metabolomics, unpreceded insights into known and novel microbial-derived metabolites, as well as their dynamics across and within infants during early life, can be provided88. Combined with high-resolution microbiome profiling, enabling strain level classification20, as well as immune cell profiling176, this may take the field to a whole new level. Furthermore, there is a need for relevant animal models and human organoids mimicking the context of early life to uncover the functional and mechanistic impact of metabolites on host development and specific pathways. Such insights are needed to develop/establish targeted strategies that contribute to healthy growth and development in infants, which may include manipulation of the infant gut microbiota using prebiotics, probiotics or postbiotics. However, before we can tackle these more translational aspects, we need to gain deeper insights into the range and mode-of-action of key microbial metabolites in the infant gut.Together, there is a clear need to uncover and explore microbial metabolites and their interactions with both gut microbes and host cells within early life. These microbial interactions may be pivotal for establishment of the gut microbiome and appropriate cross-talk between gut commensals and host cells with both short- and long-term physiological consequences. Currently, much of the work with regards to microbial metabolites and ecosystem structuring have been done in the context of the adult gut. Furthermore, only few aspects of cross-feeding in the infant gut such as