Exclusive breastfeeding (EBF) plays a crucial role in infant gut microbiome assembly and development. However, few studies have investigated the effects of EBF in restoring a perturbed microbiome. In this study, we applied whole metagenomic sequencing to assess the gut microbiome assembly in 525 Brazilian infants from 3 to 9 months of age of the Germina Cohort, demonstrating the early determinants of microbial taxonomy and function modulation. Our analysis shows that EBF alters the relative abundance of genes related to the microbiome taxonomy and function, with effects varying by delivery mode. EBF alters the pattern of carbohydrates, lipid metabolism, and cell structure pathways depending on the delivery mode. The microbiome age is closer to chronological infant age in EBF than in non-EBF infants, meaning a lower microbiome maturation index (MMI). Using a complementary machine learning approach, we show that Escherichia coli, Ruminococcus gnavus, and Clostridium neonatale, as well as vitamin K and o-antigen pathways contribute strongly to EBF prediction. Moreover, EBF influences the microbiome maturation in early life, toward a microbiome age more similar to the chronological infant's age.

Breast milk, rich in human milk oligosaccharides (HMOs), supports the early-life colonization of beneficial bacteria such as bifidobacteria and lactobacilli, potentially reducing early-life antibiotic resistance. However, antibiotic treatment may interfere with the beneficial functions of HMO-degrading bacteria. This study investigated the metabolism of HMOs by bifidobacteria and lactobacilli isolated from human milk and mother-infant paired fecal samples, along with their antibiotic resistance profiles. Understanding these species- and sample-type-specific interactions will provide valuable insights into how bioactive components in human milk may shape the infant resistome during early life. A total of 39 Bifidobacterium and 14 Lactobacillaceae strains were isolated from paired mother-infant fecal and breast milk samples. Whole genome sequencing (WGS) allowed functional predictions on the HMO metabolism abilities and the resistance genotype of each strain. In vitro HMO utilization was assessed using growth kinetics assays combined with HMO glycoprofiling in culture supernatant. The minimum inhibitory concentration (MIC) was also determined for each strain. HMO metabolism by the bifidobacteria was species-specific. Bifidobacterium bifidum (B. bifidum) and Bifidobacterium longum subsp. infantis (B. infantis) exhibited the highest capacity for HMO degradation, consistent with genomic predictions. In contrast, lactobacilli were unable to degrade HMOs in vitro but were predicted to metabolize the by-products of HMO degradation. Phenotypic analysis revealed that B. bifidum strains had the lowest levels of antibiotic resistance, while Bifidobacterium animalis subsp. lactis (B. lactis) strains were resistant to most tested antibiotics. Overall, B. bifidum demonstrated the strongest HMO-degrading ability while remaining the most antibiotic-susceptible species. Early-life colonizing bifidobacterial species possess the essential machinery required to degrade HMOs and are highly susceptible to antibiotics. A better understanding of these dynamics could inform clinical strategies to protect and restore the infant gut microbiome, particularly in neonates exposed to antibiotics.

Human milk oligosaccharides (HMOs) are a major component of human milk and colostrum, yet they are non-digestible and thus not utilized directly by the infant. Nevertheless, they are important for infant health and development and have been implicated in immune development, pathogen deflection, cognitive development and the development of healthy microbiome. To understand how HMOs may be utilized it is important to be able to measure them both in milk and faeces. Many methods for the determination of HMOs in milk have been published. However, there are fewer reports of methods describing the quantitative determination of oligosaccharides in faeces. Here we report a validated method for the determination of 30 oligosaccharides in faeces. Oligosaccharides are labelled with 2-aminobenzamide and determined by liquid chromatography with fluorescence detection. The method precision determined as relative standard deviation under intermediate reproducibility conditions is below 12 % for all of the oligosaccharides. Recoveries were in the range 86.6-115 % for the 8 oligosaccharides for which quantitative standards were available, and are estimated to be in the range 81-117 % when using 2'-fucosyllactose as a universal calibrant assuming equimolar response factors of the 2-aminobenzamide labelled oligosaccharides.

Sulfated polysaccharides (SPS) derived from seaweeds are precious bioactive compounds of diverse biological activities. Fucoidan is a complex SPS composed of L-fucose and sulfate groups, can be extracted from brown seaweeds, as well as microbial, insect, plant glycans, and marine invertebrates. It has gained considerable attention due to its anti-inflammatory, anticancer, antiviral, antithrombotic, hypolipidemic, and immune-modulatory properties. Recent research has focused on the extraction and extensive characterization of fucoidan. Its structural complexity, influenced by species, sources, and harvesting conditions, directly influences its bioactivity, with higher sulfation and lower molecular weight enhancing its activity. Interestingly, fucoidan oligosaccharides (FOs) play a critical role in various metabolic processes and hold significant potential in disease diagnostics. This comprehensive review explores the current status of fucoidan research, covering its sources, extraction and purification techniques, structural variations and biological activities. Additionally, we highlight its potential health benefits, providing insights for researchers interested in sulfated polysaccharides.

α-L-Fucosidases are attractive biocatalysts for the production of bioactive fucosylated oligosaccharides, however, poor regioselectivity and activity for transglycosylation have significantly limited their applications. We have recently derived an α-L-Fucosidase, BF3242, from Bacteroides fragilis NCTC9343, which could efficiently synthesize a mixture of Fuc-α-1,3/1,6-GlcNAc, but its 1,3/1,6-regioselectivity was observably affected by reaction temperature. Here, we integrated loop-targeted random mutagenesis and site-directed mutagenesis to engineer the regioselectivity and transglycosylation activity of BF3242. Loop-targeted random mutagenesis revealed that L266 in the loop-4 (H242-S267) within the model of BF3242 was a key residue for the regioselectivity for transglycosylation, and the saturation mutagenesis at residue L266 uncovered a mutant L266H with a significantly increased 1,3-regioselectivity of 97 % from 69 % of WT BF3242. Subsequently, five designed single-site mutations at the putative aglycone subsites were performed, resulting in a double-site mutant L266H/M285C that increased the overall yield of Fuc-α-1,3/1,6-GlcNAc to 76 % from 68 % of WT BF3242. The saturation mutagenesis at residue M285 finally generated a double-site mutant L266H/M285T with the maximal overall yield of Fuc-α-1,3/1,6-GlcNAc of 85 % and 1,3-regioselectivity of 98 %. The R T/H of L266H/M285T was approximately 2.7-fold higher than that of the WT BF3242. Molecular dynamics simulations revealed that the structural flexibility of the loop-4 was substantially reduced in mutant L266H, and the hydrogen bond formation and binding affinity between mutant L266H/M285T and Fuc-α-1,3-GlcNAc was significantly enhanced. The semi-rationally engineered enzyme L266H/M285T would be a promising biocatalyst for highly 1,3-regioselective synthesis of fucosyl-N-acetylglucosamine disaccharide.

The AlfB α-L-fucosidase from Lacticaseibacillus paracasei exhibits high specificity on fucosyl-α1,3-N-acetylglucosamine, achieving yields of 30 % in transfucosylation reactions for its synthesis. By random mutagenesis we selected AlfB variants with enhanced transfucosylation activity. Expression of a collection of alfB mutants in E. coli resulted in the isolation of eighteen clones with reduced activity on p-nitrophenyl-α-L-fucopyranoside. The AlfB variants carried diverse amino substitutions, leading to modifications in their enzymatic parameters. In some cases, these changes increased transfucosylation yields, although no direct correlation was observed between kcat or Km values and the yields. One particular AlfB mutant (M58) achieved 100 % yield in the synthesis of fucosyl-α1,3-N-acetylglucosamine. This enzyme contained three amino acid substitutions (N196S, V261M and N346K); however, further analysis confirmed that the N346K mutation was sufficient to generate the maximum yield. Elucidation of the tridimensional structure of AlfB and AlfBM58 through X-ray crystallography allowed us to propose a mechanism by which the mutation at position 346, located in a loop close to the active site of an adjacent monomer in the protein tetramer, enhanced transfucosylation over hydrolysis of fucosyl-α1,3-N-acetylglucosamine. This study paves the way for designing novel AlfB variants as tools for the efficient enzymatic synthesis of regio-specific fucosyl-oligosaccharides of biotechnological interest.

A selective enzymatic acetylation method for the protection of the second and the sixth positions of thio-d-galactopyranoside was found using immobilized Candida antarctica lipase-B (CAL-B). Unfortunately, it was determined that the immobilized enzyme cannot be recycled effectively. The optimized acetylation method was screened with different thioglycosides and with fully unprotected saccharides. New methods for several new partially protected saccharides were found, while the synthesis of some known saccharides, e.g., the third and the sixth position-protected d-glucose or the fourth position-protected l-rhamnose, was improved. Furthermore, an enzymatic acetal formation between the fourth and the sixth positions was discovered. The main limitation for acetylation reactions with CAL-B has been determined to be the substrate solubility.

Human milk (HM) is regarded as the gold standard for infant nutrition. The metabolite profiles of mature human milk (MHM) have been reported to be affected by maternal physiological conditions, lactation stage, and maternal diets. We collected MHM (3-6 months postpartum) from 32 healthy mothers with different parities and delivery modes. Then, GC-MS and LC-MS were used to perform an untargeted metabolomics study. A clear distinction can be observed in terms of MHM metabolites of mothers with different delivery modes and parities with a 95% confidence interval. A total of 119 differentially expressed metabolites (DEMs) were identified in MHM of women with different delivery modes. Metabolic pathway analyses indicated that these DEMs are mainly associated with fatty acid biosynthesis. The higher abundances of these DEMs in MHM of cesarean women may be due to the differing levels of cortisol and oxytocin between mothers with different delivery modes. Meanwhile, 284 DEMs were identified in MHM of women with different parities. These DEMs are primarily related to ABC transporters, center carbon metabolism in cancer, and D-amino acid metabolism. These findings highlighted the impact of delivery modes and parity on HM metabolite composition. Further research is needed to explore the long-term health implications of these metabolic differences and optimize infant nutrition strategies.

Food allergies, like cow's milk allergy, significantly impact children, with sensitization often beginning during the first year of life. Human milk oligosaccharides (HMOs) may influence this process, as specific HMOs differentially affect mucosal immune responses in vitro. Given the distinct HMO profiles of secretor (Se+) and non-secretor (Se-) milk, we investigate how the full HMO profiles from Se+ and Se- milk affect immune responses in the absence or presence of a cow's milk allergen.

Monocyte-derived dendritic cells (moDCs) were exposed to isolated Se+ and Se- pooled HMOs (pHMOs), and subsequently co-cultured with naïve T cells to confirm immune modulation. We compared the type 2-activation capability of several cow's milk proteins via direct exposure to moDCs or via intestinal epithelial cells (IECs) co-cultured with moDCs. Finally, we studied the effect of pHMOs in the presence of cow's milk allergen β-lactoglobulin (BLG) (via (IECs)) on moDCs and subsequent T cell response.

Both Se+ and Se- pHMOs dose-dependently activated moDCs, indicated by increased IL8 release and %CD80+ moDCs. Se+ pHMOs tended to increase type 2-associated markers, while also increasing regulatory IL10 release. Se+ pHMOs-pre-exposed moDCs instructed T cells to produce type 2 cytokines like IL13. Se- pHMOs reduced the %CD86+ moDCs but did not drive a type 2 signature in T cells. In the presence of BLG, Se+ pHMOs-pre-exposed moDCs also instructed IL13 release by T cells, while increasing the percentage regulatory T cells. In contrast, co-exposure of BLG with Se- pHMOs only slightly affected moDC phenotype, and these moDCs did not modify T cell phenotypes.

Se+ and Se- pHMOs with BLG differentially affected moDC activation. Se+ pHMO-pre-exposed moDCs induced a type 2- and regulatory-associated T cell phenotype. These data suggest that depending on the secretor status, HMOs differentially modulate immune responsiveness in vitro.

Mother's own milk is the preferred source of nutrition for preterm infants due to its beneficial compounds, including human milk oligosaccharides (HMOs). HMOs support microbiota and immune development, but their effect on the preterm brain remains unstudied. Here, we examined the therapeutic potential of HMOs and short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS) in a preclinical model for encephalopathy of prematurity. Pregnant Wistar rats were injected with 10 μg/kg lipopolysaccharides at embryonic day 20, and pups were exposed to hypoxia (8% O2, 140 min) at postnatal day (P)4 (fetal inflammation and postnatal hypoxia; FIPH). From P1, FIPH-pups of both sexes were treated intragastrically with HMOs, scGOS/lcFOS (9:1), or water. Transcriptomic analysis of CD11b/c + microglia was performed at P6, while immunohistochemical, microbial and short-chain fatty acid (SCFA) analyses were performed at P20. Decreased cortical myelin in FIPH animals was rescued exclusively by HMOs. Furthermore, both HMOs and scGOS/lcFOS treatments normalized reduced parvalbumin+ interneuron numbers in the hippocampus, potentially through promoting beneficial bacteria, including Lactobacillus and Bifidobacterium, and cecal acetic acid content. Interestingly, treatment with HMOs more effectively restored FIPH-induced upregulation of microglial genes associated with immune activation and normalized persistent activated microglial morphology in FIPH-males. HMOs supplementation holds promise to improve neurodevelopmental outcomes following preterm birth.

Human milk oligosaccharides (HMOs) may shape intestinal homeostasis, although the optimal form of HMOs to restore the gut microbiota in antibiotic-induced dysbiosis remains unclear. Here, we found that HMOs with various structures modulate microbial communities differently after antibiotic exposure. Lacto-N-neotetraose (LNnT) better promotes the recovery of intestinal microbiota (chiefly Lactobacillus) and increases the level of Bifidobacterium compared to 3'-sialyllactose, 2'-fucosyllactose, and the mixture. Additionally, LNnT decreases the potential pathogenic bacteria Klebsiella level and the microbial dysbiosis index. Although supplementation with LNnT does not decrease the Clostridioides difficile burden or alleviate the decline in the fecal numbers of Lactobacillus and Bifidobacterium after C. difficile infection (CDI), LNnT attenuates intestinal epithelial damage, decreases inflammatory status, and alters metabolome profiles after CDI. Collectively, LNnT may function as a promising prebiotic to promote gut microbiota recovery in the context of antibiotic-induced dysbiosis.

Background: Human milk oligosaccharides (HMOs) serve as critical bioactive components supporting infant growth and development. However, the influence of maternal metabolic factors during lactation on HMOs remains to be fully elucidated. This study aimed to investigate the association between maternal metabolic factors and HMOs, as well as the potential mediating effects of these factors. Methods: An observational cross-sectional study was conducted in Central South China, enrolling 196 lactating mothers. HMOs were quantified using liquid chromatography-tandem mass spectrometry. Maternal metabolic factors were assessed through physical examinations. Associations between metabolic factors and HMOs were analyzed using linear regression, and mediation effects were evaluated. Results: HMOs from Central South China were predominantly composed of neutral fucosylated HMOs. Significant differences were observed in the levels of several HMOs across maternal age groups and lactation periods. The concentration of 3'-sialyllactose (3'-SL) exhibited a negative association with the pre-pregnancy body mass index (BMI) (β = -0.16, 95% CI: -0.29, -0.03; p = 0.02), while a positive association was found with maternal heart rate (β = 0.14, 95% CI: 0.01, 0.27; p = 0.04). However, these associations were different between secretor and non-secretor mothers. Associations of 3'-SL with pre-pregnancy BMI and maternal HR were only found in the secretor mothers. Triglycerides and low-density lipoprotein cholesterol mediated the associations between maternal pre-pregnancy BMI and 3'-sialyllactose (3'-SL). Conclusions: The variations of several HMOs among mothers from Central South China were associated with maternal age and lactation period. The concentration of 3'-SL was negatively correlated with maternal pre-pregnancy BMI. The potential mechanism underlying the influence of maternal BMI on 3'-SL levels may involve maternal lipid metabolism and genetic factors.

The function of the immune system is not only dependent on factors like genetics, age, the environment, and exposure to infectious agents and allergens but also on our microbiota and our diet. It has been known for centuries that food can influence health and vulnerability to infection. This is especially true for infants, young children, and the elderly. This review focuses on how nutrition can support immune function from gestation to school-aged children. Immune support begins during pregnancy by the mother's diet and transfer of nutritional components as well as antibodies to her fetus. After birth, breastfeeding is of crucial importance for immune development as well as for the development of the intestinal microbiota of an infant. If breastfeeding is not possible, early-life formulas are an alternative. These can provide several of the functionalities of breastmilk, as well as the key nutrients a child needs. New foods are introduced during and after weaning, and after this period, children switch to consuming a normal diet. However, due to circumstances, children can be malnourished. This can range from severe protein/energy malnutrition to micronutrient deficiencies and obesity, all of which can affect the function of the immune system. This narrative review describes the immune challenges in early life, explores breastfeeding and early life nutrition, and provides mechanistic insight into the relative contribution of macronutrients, micronutrients and other immunomodulatory food components that can support immune function in early life.

Glycans present in human milk have been the focus of many studies due to challenges associated with their synthesis. The development of new methods for obtaining individual glycans found in human milk has been a vibrant area of research in glycosciences. This study reports the synthesis of two fucosylated glycans found in human milk, 3-fucosyllactose, which was previously synthesized by both chemical and enzymatic methods, and the first chemical synthesis of lacto-N-fucopentaose V. Screening different protecting and leaving groups, as well as optimizing the glycosylation reaction conditions helped us to achieve efficient assembly and deprotection of these two fucosylated human milk oligosaccharides in good yields.

Sialyllactose (SL), a bioactive trisaccharide abundant in porcine colostrum, demonstrates multifunctional properties including antimicrobial activity, immune regulation, and apoptosis inhibition. This research uncovers the mechanisms by which SL mitigates enterotoxigenic Escherichia coli (ETEC)-mediated damage to intestinal barrier integrity, employing IPEC-J2 porcine epithelial models. SL pre-treatment effectively blocked pathogen adhesion by competitively binding to cellular receptors, concurrently mitigating inflammation through significant suppression of TNF-α, IL-1β, and IL-6 expression (p < 0.05). Notably, SL exhibited functional parallels to the NF-κB inhibitor BAY11-7082, jointly enhancing tight junction integrity via ZO-1 protein stabilization and inhibiting pro-inflammatory signaling through coordinated suppression of IκB-α/NF-κB phosphorylation cascades. The dual-action mechanism combines molecular interception of microbial attachment with intracellular modulation of the TLR4/MyD88/NF-κB pathway, effectively resolving both pathogenic colonization and inflammatory amplification. These findings position SL as a potential therapeutic application nutraceutical for livestock, with the capacity to address post-weaning porcine enteritis through functional feed formulations that synergistically enhance intestinal barrier resilience while curbing ETEC-mediated inflammatory pathogenesis.

Lacto-N-triose II (LNT II) represents a significant neutral human milk oligosaccharide (HMO) that functions as a fundamental structural framework for the synthesis of more complex HMOs. In this study, we utilize GlcNAc and lactose as substrates, employing a whole-cell catalytic approach for the synthesis of LNT II. First, we have identified and successfully expressed three genes associated with the production of LNT II: Nahk, EcGlmU, and LgtA, synthesized LNT II through a whole-cell catalytic system utilizing multistrain coupling. Then, to further enhance the yield, we incorporated yeast cells for energy regeneration, employed coexpression strategies to minimize cell density, following a series of systematic optimizations resulting in a 7-fold increase in LNT II production. Finally, a two-step catalytic process was conducted in a 5L bioreactor, and the maximum LNT II production reached 52.34 g/L with a conversion rate of lactose 95.95%.

Goat lactoferrin is an N-glycoprotein, and its glycan moieties are essential for its biological activity. However, the structures of these glycans, particularly the sialylated isomers with α-2,3- or α-2,6-linkages, remain poorly characterized. Utilizing online hydrophilic liquid chromatography-tandem mass spectrometry, our study characterized N-glycans in goat lactoferrin across different lactation stages, and the putative structures of the 86 N-glycans of goat lactoferrin were presented, including 53 previously undetected ones. The content and variety of N-glycans decreased from colostrum to mature milk, with transitional milk exhibiting the highest levels of neutral and high-mannose N-glycans. Colostrum was particularly rich in fucose- and sialylated N-glycans, especially those with α-2,6-linkages. Notably, the α-2,6-linked sialylated N-glycan H5N4F1A1-1 was 7.09-fold and 12.85-fold more abundant in colostrum compared to transitional and mature milk, respectively. These findings provide valuable insights into the structure of lactoferrin and could facilitate the development of functional goat milk products.

Campylobacter jejuni is a common foodborne pathogen worldwide that is associated with high rates of morbidity and mortality among infants in low- to middle-income countries (LMICs). Human milk provides infants with an important source of nutrients and contains antimicrobial components for protection against infection. However, recent studies, including our own, have found significantly higher levels of Campylobacter in diarrheal stool samples collected from breastfed infants compared to non-breastfed infants in LMICs. We hypothesized that C. jejuni has unique strategies to resist the antimicrobial properties of human milk. Transcriptional profiling found human milk exposure induces genes associated with ribosomal function, iron acquisition, and amino acid utilization in C. jejuni strains 81-176 and 11168. However, unidentified proteinaceous components of human milk prevent bacterial growth. Evolving both C. jejuni isolates to survive in human milk resulted in mutations in genes encoding the acyl carrier protein (AcpP) and the major outer membrane porin (PorA). Introduction of the PorA/AcpP amino acid changes into the parental backgrounds followed by electron microscopy showed distinct membrane architectures, and the AcpP changes not only significantly improved growth in human milk, but also yielded cells surrounded with outer membrane vesicles. Analyses of the phospholipid and lipooligosaccharide (LOS) compositions suggest an imbalance in acyl chain distributions. For strain 11168, these changes protect both evolved and 11168∆acpPG33R strains from bacteriophage infection and polymyxin killing. Taken together, this study provides insights into how C. jejuni may evolve to resist the bactericidal activity of human milk and flourish in the hostile environment of the gastrointestinal tract.

In this study, we evolved C. jejuni strains which can grow in the presence of human milk and found that cell membrane alterations may be involved in resistance to the antimicrobial properties of human milk. These bacterial membrane changes are predominantly linked to amino acid substitutions within the acyl carrier protein, AcpP, although other bacterial components, including PorA, are likely involved. This study provides some insights into possible strategies for C. jejuni survival and propagation in the gastrointestinal tract of breastfed infants.

Neurodegenerative diseases, including Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), and amyotrophic lateral sclerosis (ALS), are characterized by progressive neuronal loss and pose significant global health challenges. Glycosphingolipids (GSLs), critical components of neuronal membranes, regulate signal transduction, membrane organization, neuroinflammation, and lipid raft functionality. This review explores GSL roles in neural development, differentiation, and neurogenesis, along with their dysregulation in neurodegenerative diseases. Aberrations in GSL metabolism drive key pathological features such as protein aggregation, neuroinflammation, and impaired signaling. Specific GSLs, such as GM1, GD3, and GM3, influence amyloid-beta aggregation in AD, α-synuclein stability in PD, and mutant huntingtin toxicity in HD. Therapeutic strategies targeting GSL metabolism, such as GM1 supplementation and enzyme modulation, have demonstrated potential to mitigate disease progression. Further studies using advanced lipidomics and glycomics may support biomarker identification and therapeutic advancements. This work aims to highlight the translational potential of GSL research for diagnosing and managing devastating neurodegenerative conditions.

Prophylactic iron fortification in infant formula effectively prevents iron deficiency anemia. However, the low absorption rate results in excess unabsorbed iron accumulates in colon, where it has been linked to harmful microbiota changes and increased diarrheal incidence. Prebiotic oligosaccharides have shown promise in mitigating these adverse effects, but the role of inulin or synbiotic supplementation with inulin-fermenting lactic acid bacteria in modulating early gut microbiome under iron fortification remains understudied.

This study used a neonatal pig model to investigate the effects of iron fortification and inulin supplementation, with or without Ligilactobacillus agilis YZ050 (L. agilis), on gut microbiome.

Twenty-four piglets were stratified and randomly assigned into 1 of the 4 dietary treatments from postnatal day (PD) 2: iron-adequate milk (AI), high-iron milk (HI), high-iron milk with 5% inulin (HIP), or HIP milk with oral gavage of L . agilis every third day (HIS). Piglets were individually housed and fed milk in proportion to body weight in 14 meals daily, simulating formula feeding in infants. Fecal and colonic microbiome were analyzed via 16S rRNA sequencing, with microbial diversity and relative abundance analyzed using QIIME2 and R.

Iron fortification, regardless of inulin supplementation, decreased α-diversity compared with AI. β-Diversity showed clustering of HIP and HIS samples, which were distinct from AI and HI. Although iron fortification had minor impact on microbial composition, inulin supplementation significantly modified microbiome diversity, increasing Prevotella, Megasphaera, and Lachnospiraceae_NK3A20_group species, while reducing Bacteroides and Ruminococcus. Colonic microbiome shifted from Bacteroides-dominant enterotype in AI and HI groups to Prevotella-dominant enterotype in HIP and HIS groups, indicating enhanced fiber degrading capacity. Despite its inulin-fermenting property, L . agilis showed limited colonization and minimal microbiome impact.

Inulin supplementation significantly influenced gut microbiome, shifting enterotype from Bacteroides to Prevotella. dominance and overriding the effect of high-iron fortification in a milk-fed piglet model.

Infants should be provided with effective feeding skills with evidence-based care practices to ensure nutritional tolerance and maximize the growth and development in preterm infants.

This study aimed to examine the effects of "oral administration of breast milk droplets" and "palatal stimulation with a finger" methods on feed tolerance in preterm newborns fed via an orogastric tube.

This randomized controlled trial was conducted in the neonatal intensive care unit of a private hospital. The study included 90 premature newborns born at the 28th-36th gestational weeks and admitted to intensive care. We applied breast milk droplets inside the oral cavity of newborns (30) in one of the intervention groups and stimulated the palate of newborns (30) by using a finger in the other. We performed these interventions every 3 hours for 5 minutes at the feeding times of the newborns for 7 days. Babies in the control group were not applied any intervention. We used SPSS (Statistical Package for Social Sciences) 22.0 for Windows software for statistical analyses.

The number of defecations, frequencies of residuals, body weight, and abdominal circumference were significantly different between the groups (P < 0.05). The increments in body weight and reductions in abdominal circumference were significantly different between the groups in the study (P < 0.05).

Palatal stimulation with a finger acted on feed tolerance more favorably than the oral administration of breast milk droplets or no intervention in preterm infants fed via OGT. We suggest primarily that palatal stimulation with a finger and secondarily the oral administration of breast milk droplets as the two methods to be employed to overcome feeding intolerance, which is a significant problem in premature infants.

Infant formula with human milk oligosaccharides (HMOs) and increased β-palmitate mimics breast milk nutritional composition and clinical benefits. We aimed to assess formula-fed infant growth, gastrointestinal tolerance, infections, and parental satisfaction with a partly fermented infant formula with an improved lipid profile (enriched with β-palmitate and docosahexaenoic/arachidonic acid) and short and long-chain oligosaccharides (scGOS/lcFOS [9:1]) and HMOs.

A prospective descriptive observational study in healthy infants with formula feeding or breastfeeding (reference population) was conducted in six Spanish primary care centres following routine clinical practice. In the first, second and fourth month of life visits sociodemographic, clinical, and anthropometric variables (weight, length, head circumference), stool consistency (Brussels Infant and Toddler Stool Scale [BITSS]), gastrointestinal symptoms, infections incidence and associated healthcare resource utilisation, and caregivers' satisfaction with formula were collected. A descriptive statistical analysis was performed (STATA-v.14). Growth was estimated as the mean (standard deviation) increase in the anthropometric variables and z-scores.

A total of 61 formula-fed and 65 breastfed infants were included in the study (50.8% male). The average increase in weight, length and head circumference in the formula feeding and in the breastfeeding groups from the first to the fourth month of life was 2,566 (496) g, 9.7 (1.7) cm and 4.4 (1.0) cm, and 2,571 (702) g, 9.8 (1.8) cm and 4.4 (1.1) cm, respectively. The weight z-score was -0.1 (0.7) for formula-fed and 0.1 (1.1) for breastfed infants. In all visits, more than 88% of infants had loose/watery stools and most infants suffered gastrointestinal symptoms with low/medium frequency. In the fourth month of life visit, 16 (26.2%) formula-fed and 16 (24.6%) breastfed infants had infections, mainly respiratory, with 16% of formula-fed and 12% of breastfed infants requiring treatment. Most formula-feeding caregivers had a good/very good opinion of formula (85.2%). 75.4% infants drank the whole feeding bottle.

The growth, gastrointestinal tolerance, and incidence of infections of healthy formula-fed infants during the first four months of life were appropriate and in line with WHO standards. Formula feeding caregivers were satisfied with this partly fermented infant formula with an improved lipid profile and oligosaccharides.

Glycans regulate a vast spectrum of disease-related processes, yet effectively leveraging these important mediators in a therapeutic context remains a frontier in contemporary medicine. Unlike many other classes of clinically important biopolymers, carbohydrates derive from discrete biosynthetic pathways and are not produced directly from genes. The conspicuous absence of a biological blueprint to achieve amplification creates a persistent challenge in obtaining well-defined glycostructures for therapeutic translation. Isolating purified sugars from biological sources is not without challenge, rendering synthetic organic chemistry the nexus of this advancing field. Chemical synthesis has proven to be an unfaltering pillar in the production of complex glycans, but laborious syntheses coupled with purification challenges frequently introduce reproducibility issues. In an effort to reconcile these preparative challenges with the societal importance of glycans, automated glycan synthesis was conceptualised at the start of the 21st century. This rapidly expanding, multifaceted field of scientific endeavor has effectively merged synthetic chemistry with technology and engineering to expedite the precision synthesis of target glycans. This minireview describes the structural diversity and function of glycans generated by automated glycan synthesis platforms over the last five years. The translational impact of these advances is discussed together with current limitations and future directions.

Lacto-N-neotetraose (LNnT) is a prevalent neutral core human milk oligosaccharides (HMOs) recognized for its numerous benefits to infant health. In infant formula, galactooligosaccharide (GOS) are frequently used as substitutes for HMOs. However, the regulatory roles of LNnT and GOS in early intestinal development are not yet fully understood. This study aims to elucidate the effects of LNnT and GOS on intestinal development during early life. Our findings show that administering LNnT or GOS significantly increased the spleen and liver indices of infant mice at postnatal day 21. Immunofluorescence and qPCR analysis showed that feeding LNnT significantly promoted the proliferation and differentiation of intestinal stem cells (ISCs) in the colon of infant mice at postnatal day 21, and increased the expression of differentiation markers of goblet cells, intestinal epithelial cells, Paneth cells, and intestinal endocrine cells. Conversely, feeding GOS had no significant effect on the proliferation and differentiation of ISCs. Furthermore, intestinal microbiota analysis showed that LNnT increased the microbiota associated with intestinal regeneration and ISCs proliferation and differentiation in infant mice at postnatal day 21. In conclusion, LNnT promoted ISCs proliferation and differentiation in the colon and alters the composition and function of the intestinal microbiota to support intestinal development in infant mice.

Human milk oligosaccharides (HMOs) are integral to infant health. Yet, their complex biosynthesis pathways in the mammary gland during lactation remain under characterized. To address this knowledge gap, we performed integrated analyses of single-cell RNA-sequencing (scRNA-seq) datasets combined with select HMO concentration measures. We identify differential expression patterns of known HMO synthesis genes in epithelial subsets and nominate several candidate genes that vary with HMO concentration. Additionally, we identify novel gene patterns and transcription factors that may regulate the expression of HMO biosynthesis genes and the cellular pathways supporting HMO production. Finally, we demonstrate that co-expression of HMO synthesis genes and milk fat synthesis genes is limited, suggesting distinct epithelial cell subtypes may be responsible for the production of different milk components. Our study suggests that HMO synthesis may be achieved through cell type specialization within the lactocyte compartment.

The ability to identify isomers of human milk oligosaccharides (HMOs) remains a challenge. Typically, HMOs are analyzed using a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). Yet, separation methods have been unable to resolve several HMO isomers, necessitating the need for fragmentation methods that can differentiate these structures within mixtures. Common fragmentation methods, such as collision-induced dissociation (CID), often fail to form fragments that can differentiate the linkage and branching isomers. Previously, we determined that electron transfer dissociation (ETD) yields distinct fragmentation products when using certain metal-charge carriers, such as Co2+. Here, we compare fragmentation methods, including CID, higher-energy collisional dissociation (HCD), ETD, and electron transfer, higher-energy collisional dissociation (EThcD) for Co2+- and Na+-adducted HMO isomers. The formation of several fragments using only ETD and EThcD, but not CID and HCD, indicates that certain fragmentation products only form from electron dissociation. EThcD enabled differentiation of Co2+-adducted tri-, tetra-, and pentasaccharide isomers, while CID, HCD, and ETD were incapable of differentiating all the examined HMO isomers as Co2+, Na+, and 2Na2+ adducts. We also show that Co2+ adduction combined with EThcD generates fragments that are related to the core structure and fucose branching location, allowing for further validation of the HMO structures. This work establishes a general method using fragmentation that can be used to differentiate HMOs, which should enable improved identification and structural validation of various carbohydrate linkage and branching isomers.

Human milk oligosaccharides (HMOs) represent the third most abundant fraction of biomolecules in human milk (HM) and play a crucial role in infant health and development. The unique contributions of HMOs to healthy development of breast-fed infants are assumed to rely on the extraordinary complexity and diversity of HMO isomeric structures, which in turn still cause a huge analytical challenge. Many contemporary analytical methods aiming for more detailed HMO characterization combine ion mobility (IM) with LC-MS for enhanced structural resolution but are typically lacking the robustness necessary for application to HM cohorts with hundreds of samples. To overcome these challenges, we introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MS method integrating four analytical dimensions: high-resolution LC separation, IM drift time, accurate mass precursor, and fragment ion measurements. This four-dimensional (4D) analytical characterization is sufficient for resolving various HMO structural isomers in an efficient way. Thereby, up to 200 HMO compounds with a maximum degree of polymerization of 13 could be simultaneously identified and relatively quantified. We devised two methods using this 4D analytical approach. One intended for in-depth characterization of multiple known but also novel HMO structures and the second is designed for robust, increased-throughput analyses. With the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected before in HM, as well as additional difucosyl-lacto-N-heaose isomers (DF-LNHs), were revealed and structures fully elucidated by AIF and IM. This exemplifies the potential of our method for in-depth characterization of novel complex HMO structures. Furthermore, the increased-throughput method featuring a shorter LC gradient was applied to real-world HM samples. Here, we could differentiate the HM types I-IV based on a broader range of partly new marker HMOs. We could also derive valuable new insights into variations of multiple and rare HMOs up to DP 11 across lactational stages. Overall, our AIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confident identification of a broad array of distinct and simple to very complex HMOs. We envision this robust AIF LC-ESI-IM-MS approach to advance HMO research by facilitating the characterization of a broad range of HMOs in high numbers of HM samples. This may help to further extend our understanding about HMOs structure-function relationships relevant for infants' healthy development.

The health status of the growing infant is closely related to the development of the gut microbiota during infancy, which is also a major stimulator of the immune system. Goat milk oligosaccharides (gMOs) are a class of bioactive compounds in goat milk, which have attracted extensive research interest in recent years. Recent studies have highlighted that gMOs as prebiotics can regulate the gut microbiota, exhibit multiple health effects, and act as immunomodulators. This article outlines the structure, classification, and functions of gMOs. In addition, we also deeply explored the mechanism of gMO interaction with infant gut microbiota and regulation of infant immunity. Finally, the possibility of gMOs as an effective substitute for natural prebiotics in breast milk is revisited. We concluded that gMOs improve infant immune function by regulating intestinal beneficial bacteria (Bifidobacteria, Lactobacilli, etc.) and their metabolism. Therefore, gMOs are significant to infant immune health and are expected to become a substitute for human milk oligosaccharides (HMOs).

Human milk oligosaccharides (HMOs) are crucial for promoting neonatal health, with sialylated oligosaccharides, a significant subclass, offering a variety of health benefits such as prebiotic effects, anti-inflammatory and antimicrobial properties, antiviral defense, and cognitive development support. Among these, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) have received "GRAS" status from the U.S. Food and Drug Administration and approval from the European Food Safety Authority for use as novel food additives in infant formula and supplements. This review focuses on the synthesis methods of sialylated human milk oligosaccharides (SHMOs), their functional properties, downstreaming developments and application technologies. Given the challenges associated with achieving sufficient availability for food and medical applications, the review emphasizes the viability and efficiency of various production strategies. The review also highlights recent research advancements and offers insights for optimizing large-scale production to support future applications in the food and pharmaceutical industries.

The emergence and global spread of carbapenem-resistant Enterobacter cloacae complex species present a pressing public health challenge. Carbapenem-resistant Enterobacter spp. cause a wide variety of infections, including septic shock fatalities in newborns and immunocompromised adults. The intestine may be a major reservoir for these resistant strains, either by facilitating contamination of fomites and transfer to susceptible individuals, or through translocation from the gut to the bloodstream. For this reason, we sought to establish a neonatal mouse model to investigate the mechanisms underpinning gut colonization by carbapenem-resistant Enterobacter hormaechei. We describe a new mouse model to study gut colonization by Enterobacter spp., leading to vital insights into the adaptation of carbapenem-resistant E. hormaechei to the gut environment during the early stages of intestinal colonization. We observed successful colonization and proliferation of E. hormaechei in the 5-day-old infant mouse gut, with primary localization to the colon following oral inoculation. We also uncovered evidence that E. hormaechei uses mucus as a carbon source during colonization of the colon. Our findings underscore the importance of oxygen-dependent metabolic pathways, including the pyruvate dehydrogenase complex and N-acetyl-D-glucosamine metabolism, in gut colonization and proliferation, which aligns with previous human studies. These insights are essential for developing novel therapeutic strategies that can serve as decolonization therapies in at-risk populations.IMPORTANCEBloodstream infections caused by Enterobacter spp. pose a significant clinical threat. The intestine acts as the primary site for colonization and serves as a reservoir for infection. To combat this pathogen, it is crucial to understand how carbapenem-resistant Enterobacter spp. colonize the gut, as such knowledge can pave the way for alternative therapeutic targets. In this study, we developed a novel neonatal mouse model for gastrointestinal colonization by Enterobacter spp. and discovered that mucus plays a key role as a carbon source during colonization. Additionally, we identified two mucus catabolism pathways that contribute to intestinal colonization by carbapenem-resistant E. hormaechei. This new mouse model offers valuable insights into host-pathogen interactions and helps identify critical gastrointestinal fitness factors of Enterobacter, potentially guiding the development of vaccines and alternative therapeutic strategies to minimize intestinal carriage in patient populations at risk of infection with Enterobacter spp.

This study presents an engineered strain of Escherichia coli specifically designed to enhance the production of l-fucose while minimizing residues of 2'-fucosyllactose. The optimization strategies employed include the selection of key enzymes, optimization of gene copy numbers, and fermentation using mixed carbon sources. The metabolic flux was directed toward l-fucose synthesis by integrating preferred 1,2-fucosyltransferase and α-l-fucosidase into the genome. Furthermore, the gene copy numbers were optimized to enhance enzyme expression, thereby increasing l-fucose production. Additionally, the supply of guanosine 5'-triphosphate was improved, and cofactors were regenerated to better regulate metabolism. Modifications to transporter proteins effectively reduced the accumulation of 2'-fucosyllactose. The implementation of a glucose/glycerol co-fermentation strategy enhanced carbon flux distribution and strain efficiency. The optimized strain achieved a yield of 91.90 g/L of l-fucose in a 5 L bioreactor, representing an 80.01% increase over previous yields, with a productivity of 1.18 g L-1 h-1. This yield is the highest reported for l-fucose, demonstrating its potential for industrial production.

Breast milk is rich in bioactive components, especially human milk oligosaccharides (HMOs), which are crucial for establishing gut microbiota. The 2'-FL (2-Fucosyllactose), one of the most abundant oligosaccharides in breast milk, functions as a selective prebiotic. Objective: To examine the effect of adding 2'-FL (2-Fucosyllactose) to an infant formula containing prebiotic galacto-oligosaccharides (GOSs) and fructo-oligosaccharides (FOSs) on the gut microbiome of healthy formula-fed infants. Methods: This study enrolled infants from three groups: an HMO experimental group (n = 29), a GOS/FOS control group (n = 30), and an exclusively breastfed (breast milk [BM]) reference group (n = 28). Fecal samples from the three groups in the first and fourth months of life were analyzed. The V3 and V4 regions of the 16S rRNA gene were amplified and sequenced on the Illumina MiSeq. ANOVA, Kruskal-Wallis, richness indices (Chao1, Shannon), UniFrac distances, and the Adonis tests were used to perform statistical analyses on the relative abundance of phyla and genera, as well as the alpha and beta-diversity of the gut microbiota. Results: After intervention, Actinobacteriota emerged as the predominant phylum in both the HMO (60.4%) and BM (46.6%) groups. Bifidobacterium and Escherichia-Shigella were identified as the two most abundant bacterial genera in both groups. Nevertheless, the statistical analysis showed that the relative abundance of Bifidobacterium in the HMO formula-fed group after intervention was similar to that in the BM group (p > 0.05). Infants in the HMO and GOS/FOS groups showed higher relative abundance of [Ruminococcus]_gnavus_group bacteria compared to those in the BM group. Groups fed with infant formula demonstrated higher alpha-diversity of gut microbiota compared to breastfed infants (p < 0.05), at the time of admission as well as after the intervention. Beta-diversity was significantly different among the three groups, according to type of feeding. Infants fed a 2'-FL-supplemented infant formula exhibited growth comparable to that of breastfed infants throughout the intervention period, demonstrating that the formula was both safe and well tolerated. Conclusions: Adding 2'-FL to an infant formula containing 4 g/L of GOS + FOS resulted in a stronger bifidogenic effect compared to the formula without 2'-FL.

Background/Objectives: Individuals with HIV on combined antiretroviral therapy (ART) with virologic suppression exhibit chronic immune activation and immune dysfunction. Numerous studies have shown that human milk oligosaccharide (HMO) controls the postnatal transmission of HIV-1, but its effect on adult HIV-1 infection is not known. The purpose of this study was to investigate the anti-HIV activity of Lacto-N-fucopentaose III (LNFPIII) in adult blood-borne macrophages. Methods: Primary human monocyte-derived macrophages from the blood of HIV-seronegative individuals were infected with HIV and treated with or without dextran-conjugated LNFPIII (P3DEX). HIV replication was measured by quantifying the accumulation of HIV Gag p24 in the culture supernatants by ELISA. The quantities of chemokines MIP-1α, MIP-1β, and CCL5 in the culture supernatant were also measured by ELISA. The expression of IL-1β, IL-18, TNFα, IL-10, BECN1, and housekeeping gene HuPO in the macrophages was determined by qRT PCR. The expression of NF-kB, LC3, p62, and β-actin was measured by immunoblotting. Results: We found that P3DEX controls HIV replication without affecting HIV binding and/or internalization by human macrophages. The treatment of HIV-infected macrophages with P3DEX increased the quantity of beta (β)-chemokines MIP-1α, CCL5, and MIP-1β, which are known to have anti-HIV activity. Furthermore, the treatment of HIV-infected macrophages with P3DEX increased autophagic flux in a TLR8-dependent manner and ameliorated the expression of proinflammatory cytokines. These results suggest that P3DEX is a prominent milk-derived sugar that simultaneously augments anti-viral mechanisms and controls immune activation. These findings prudently justify the use and clinical development of P3DEX as a host-directed therapeutic option for people living with HIV.

Recently, the non-intestinal functions of human milk oligosaccharides (HMOs) have been widely documented, including their roles in promoting brain development and growth, as well as ameliorating anxiety, allergies, and obesity. Understanding their mechanisms of action is becoming increasingly critical. Furthermore, these effects are frequently associated with the type and structure of HMOs. As an innovative technology, "plant factory" is expected to complement traditional synthesis technology. This study reviews the novel "plant factory" synthesis techniques. Particular emphasis is placed on the processes, advantages, and limitations of "plant factory" synthesis of HMOs. This technology can express genes related to HMO synthesis instantaneously in plant leaves, thereby enabling the rapid and cost-effective generation of HMOs. However, "plant factory" technology remains underdeveloped, and challenges related to low yield and unsustainable production must be addressed. Furthermore, we present an overview of the most recent clinical and preclinical studies on the non-intestinal functions of HMOs. This review emphasizes the mechanisms of action underlying the non-intestinal functions of HMOs. HMOs primarily exert non-intestinal functions through the cleavage of beneficial monomer components, metabolism to produce advantageous metabolites, and regulation of immune responses.

This study aims to investigate the volatile and α-dicarbonyl compounds (α-DCs) formed in Maillard reactions between 2'-fucosyllactose (2'-FL) and amino acids, with the goal of exploring their potential as flavoring agents and enhancing food quality and safety. The effects of pH, temperature, reaction time, and amino acid concentration on α-DC production were evaluated. Fucose generated the most α-DCs, whereas 2'-FL produced the least. α-DC formation increased with increasing pH, reaction time, temperature, and amino acid concentration. Among the amino acids evaluated, threonine elicited the highest α-DC production. In total, 50 volatile compounds were identified, with 2'-FL and lactose primarily forming furan and furan derivatives. In particular, 2'-FL yielded greater amounts of 2-furfural, 2-acetylfuran, 5-methylfurfural, furfuryl alcohol, and 2-furanmethanol than other monosaccharides. These findings highlight the potential of 2'-FL as a flavouring agent and enhance our understanding of α-DC formation during food processing and storage.

2'-Fucosyllactose (2'-FL), a predominant human milk oligosaccharide, is widely employed as a commercial infant prebiotic. Nevertheless, the benefits of 2'-FL on complex gut microbiota need further exploration. The modulation of 2'-FL on adult gut microbiota composition and metabolome, particularly short-chain fatty acids (SCFAs) and tryptophan metabolism, was investigated via colonic fermentation, single bacterial strains cultivation, and prebiotic activity scoring evaluation. The 2'-FL fermentation in a complex microbial community indicated promising effects, including an increase in Bifidobacterium levels and changes in metabolic levels of tryptophan, SCFAs, and vitamins. Correlation analysis of microbiota and metabolome highlighted a positive correlation between Bifidobacterium and metabolites derived from SCFAs and indole. Scoring formulas for bacterial growth and substrate utilization were introduced to compare the overall response of 15 bacterial strains from key genera that were identified by 16S rRNA profiling to several prebiotics. In the cultivation of single bacterial strains, 2'-FL was selectively used by Bifidobacteria infantis and Enterococcus faecalis and was not degraded by potential pathogenic strains. This selective promotion of probiotics by 2'-FL was observed according to the scoring results, as 2'-FL showed higher scores for bacterial growth and substrate utilization when compared to galacto-oligosaccharides and fucose. This study demonstrated beneficial bifidogenic effect of 2'-FL in an adult complex microbial community and the potential of scoring evaluation as a quantitative tool for measuring the prebiotic effects of different oligosaccharides.

Probiotic use has increased in preterm infants and may reduce the risk of necrotizing enterocolitis. Probiotic-associated infection is a concern for infants receiving probiotic supplementation in the neonatal intensive care unit, as highlighted by a recent case and subsequent action by the United States Food and Drug Administration. Based on reports to date, invasive infection is an infrequent but known risk of probiotic supplementation. In this article, we discuss the epidemiology and pathophysiology of invasive infection in preterm infants, review the benefits and risks of probiotic as regulations and available products continue to evolve.

Background: Preventing neurodevelopmental impairment after extremely preterm birth remains challenging. While breast milk feeding is linked to better neurodevelopment, the underlying mechanisms are unclear. This study explored the association between individual human milk oligosaccharides (HMO) and neurodevelopment at two years of age in extremely preterm children. Methods: Milk samples from mothers of 76 extremely preterm infants collected at two weeks after birth were analyzed for 15 dominant HMOs. Register data from examination and Bayley-III neurodevelopmental assessment at two years' corrected age was retrieved and categorized into levels of impairment. An exploratory analysis examined associations between the HMO composition and neurodevelopment. Results: Bioinformatic volcano plots revealed associations between specific HMOs and outcomes: 3FL with less neurodevelopmental impairment, LSTb with higher Bayley-III cognitive scores, and LSTa with worse neurodevelopmental impairment outcomes. Spearman correlations indicated LSTa was linked to more neurodevelopmental impairment (p = 0.018), lower language (p = 0.009), and motor (p = 0.02) scores, whereas 3FL correlated with less neurodevelopmental impairment (p = 0.02). Dichotomized analysis showed LSTa was associated with more neurodevelopmental impairment and lower language scores (p < 0.05), 3FL with milder neurodevelopmental impairment (p < 0.05), and LSTb with better cognitive (p < 0.01) and language (p < 0.05) scores. No significant associations were found for HMO diversity, total sialic acid content, or secretor/Lewis patterns. Conclusions: In this explorative hypothesis-generating study, certain HMOs appeared to be associated with both potentially beneficial and adverse neurodevelopmental outcomes in extremely preterm infants. However, these findings should be interpreted with caution, as they do not constitute evidence but rather serve as a preliminary foundation for future hypothesis-driven research.