Proprioception, Emotion and Social Responsiveness in Children with Developmental Disorders: An Exploratory Study in Autism Spectrum Disorder, Cerebral Palsy and Different Neurodevelopmental Situations.

Proprioception has long been linked with emotional dysregulation in neurotypical adults. Neuropediatric disorders such as autism spectrum disorder (ASD) and cerebral palsy (CP) are distinct entities and yet both present with deficits and challenges in sensory processing and the regulation of emotions. This study aimed to explore the relationship between proprioception and emotional-social performance in children and to compare proprioception and emotional-social performance in different underlying neurodevelopmental conditions. For this purpose, this cross-sectional study included 42 children with ASD, 34 children with CP and 50 typically developing peers. Proprioceptive acuity, proprioceptive reactive behavior as well as emotion regulation and social responsiveness were assessed. The results show a significant correlation between proprioceptive deficits and emotional difficulties in this pediatric sample, with distinct proprioceptive impairment patterns according to the underlying neurological disorder. Children with CP showed significant emotional knowledge deficits, while children with ASD predominantly showed challenges in social responsiveness. These data thus suggest a differentiated impact of proprioception on emotional-social performance in neurodevelopmental disorders and highlight proprioception as a potential therapeutic target for balancing emotion regulation in children with neurodevelopmental conditions.

Rumen microbial degradation of bromoform from red seaweed (Asparagopsis taxiformis) and the impact on rumen fermentation and methanogenic archaea.

BACKGROUND: The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane (CH4) analogues, primarily bromoform (CHBr3). This study aimed to investigate the degradation process of CHBr3 from A. taxiformis in the rumen and whether this process is diet-dependent. An in vitro batch culture system was used according to a 2 × 2 factorial design, assessing two A. taxiformis inclusion rates [0 (CTL) and 2% DM diet (AT)] and two diets [high-concentrate (HC) and high-forage diet (HF)]. Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0, 0.5, 1, 3, 6, 8, 12, 16, 24, 48 and 72 h to assess the pattern of degradation of CHBr3 into dibromomethane (CH2Br2) and fermentation parameters. Additionally, an in vitro experiment with pure cultures of seven methanogens strains (Methanobrevibacter smithii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanosarcina barkeri, Methanobrevibacter millerae, Methanothermobacter wolfei and Methanobacterium mobile) was conducted to test the effects of increasing concentrations of CHBr3 (0.4, 2, 10 and 50 µmol/L). RESULTS: The addition of AT significantly decreased CH4 production (P = 0.002) and the acetate:propionate ratio (P = 0.003) during a 72-h incubation. The concentrations of CHBr3 showed a rapid decrease with nearly 90% degraded within the first 3 h of incubation. On the contrary, CH2Br2 concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation. Neither CHBr3 degradation nor CH2Br2 synthesis were affected by the type of diet used as substrate, suggesting that the fermentation rate is not a driving factor involved in CHBr3 degradation. The in vitro culture of methanogens showed a dose-response effect of CHBr3 by inhibiting the growth of M. smithii, M. ruminantium, M. stadtmanae, M. barkeri, M. millerae, M. wolfei, and M. mobile. CONCLUSIONS: The present work demonstrated that CHBr3 from A. taxiformis is quickly degraded to CH2Br2 in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr3 degradation.

Multi-omics in vitro study of the salivary modulation of the goat rumen microbiome.

Ruminants are able to produce large quantities of saliva which enter into the rumen and salivary components exert different physiological functions. Although previous research has indicated that salivary immunoglobulins can partially modulate the rumen microbial activity, the role of the salivary components other than ions on the rumen microbial ecosystem has not been thoroughly investigated in ruminants. To investigate this modulatory activity, a total of 16 semi-continuous in vitro cultures with oats hay and concentrate were used to incubate rumen fluid from four donor goats with autoclaved saliva (AUT) as negative control, saliva from the same rumen fluid donor (OWN) as positive control, and either goat (GOAT) or sheep (SHEEP) saliva as experimental interventions. Fermentation was monitored throughout 7 days of incubation and the microbiome and metabolome were analysed at the end of this incubation by Next-Generation sequencing and liquid chromatography coupled with mass spectrometry, respectively. Characterisation of the proteome and metabolome of the different salivas used for the incubation showed a high inter-animal variability in terms of metabolites and proteins, including immunoglobulins. Incubation with AUT saliva promoted lower fermentative activity in terms of gas production (-9.4%) and highly divergent prokaryotic community in comparison with other treatments (OWN, GOAT and SHEEP) suggesting a modulatory effect derived from the presence of bioactive salivary components. Microbial alpha-diversity at amplicon sequence variant (ASV) level was unaffected by treatment. However, some differences were found in the microbial communities across treatments, which were mostly caused by a greater abundance of Proteobacteria and Rikenellacea in the AUT treatment and lower of Prevotellaceae. These bacteria, which are key in the rumen metabolism, had greater abundances in GOAT and SHEEP treatments. Incubation with GOAT saliva led to a lower protozoal concentration and propionate molar proportion indicating a capacity to modulate the rumen microbial ecosystem. The metabolomics analysis showed that the AUT samples were clustered apart from the rest indicating different metabolic pathways were promoted in this treatment. These results suggest that specific salivary components contribute to host-associated role in selecting the rumen commensal microbiota and its activity. These findings could open the possibility of developing new strategies to modulate the saliva composition as a way to manipulate the rumen function and activity.

Enhancing rumen microbial diversity and its impact on energy and protein metabolism in forage-fed goats.

Introduction: This study explores if promoting a complex rumen microbiota represents an advantage or a handicap in the current dairy production systems in which ruminants are artificially reared in absence of contact with adult animals and fed preserved monophyte forage. Methods: In order to promote a different rumen microbial diversity, a total of 36 newborn goat kids were artificially reared, divided in 4 groups and daily inoculated during 10 weeks with autoclaved rumen fluid (AUT), fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate (RFC) diets, or absence of inoculation (CTL). At 6 months of age all animals were shifted to an oats hay diet to determine their ability to digest a low quality forage. Results and discussion: Early life inoculation with fresh rumen fluid promoted an increase in the rumen overall microbial diversity which was detected later in life. As a result, at 6 months of age RFF and RFC animals had higher bacterial (+50 OTUs) and methanogens diversity (+4 OTUs) and the presence of a complex rumen protozoal community (+32 OTUs), whereas CTL animals remained protozoa-free. This superior rumen diversity and presence of rumen protozoa had beneficial effects on the energy metabolism allowing a faster adaptation to the forage diet, a higher forage digestion (+21% NDF digestibility) and an energetically favourable shift of the rumen fermentation pattern from acetate to butyrate (+92%) and propionate (+19%) production. These effects were associated with the presence of certain rumen bacterial taxa and a diverse protozoal community. On the contrary, the presence of rumen protozoa (mostly Entodinium) had a negative impact on the N metabolism leading to a higher bacterial protein breakdown in the rumen and lower microbial protein flow to the host based on purine derivatives urinary excretion (-17% to -54%). The inoculation with autoclaved rumen fluid, as source of fermentation products but not viable microbes, had smaller effects than using fresh inoculum. These findings suggest that enhancing rumen microbial diversity represents a desirable attribute when ruminants are fed forages in which the N supply does not represent a limiting factor for the rumen microbiota.

Presence of Adult Companion Goats Favors the Rumen Microbial and Functional Development in Artificially Reared Kids.

Newborn dairy ruminants are usually separated from their dams after birth and fed on milk replacer. This lack of contact with adult animals may hinder the rumen microbiological and physiological development. This study evaluates the effects of rearing newborn goat kids in contact with adult companions on the rumen development. Thirty-two newborn goat kids were randomly allocated to two experimental groups which were reared either in the absence (CTL) or in the presence of non-lactating adult goats (CMP) and weaned at 7 weeks of age. Blood and rumen samples were taken at 5, 7, and 9 weeks of age to evaluate blood metabolites and rumen microbial fermentation. Next-generation sequencing was carried out on rumen samples collected at 7 weeks of age. Results showed that CTL kids lacked rumen protozoa, whereas CMP kids had an abundant and complex protozoal community as well as higher methanogen abundance which positively correlated with the body weight and blood ß-hydroxybutyrate as indicators of the physiological development. CMP kids also had a more diverse bacterial community (+132 ASVs) and a different structure of the bacterial and methanogen communities than CTL kids. The core rumen bacterial community in CMP animals had 53 more ASVs than that of CTL animals. Furthermore, the number of ASVs shared with the adult companions was over 4-fold higher in CMP kids than in CTL kids. Greater levels of early rumen colonizers Proteobacteria and Spirochaetes were found in CTL kids, while CMP kids had higher levels of Bacteroidetes and other less abundant taxa (Veillonellaceae, Cyanobacteria, and Selenomonas). These findings suggest that the presence of adult companions facilitated the rumen microbial development prior to weaning. This accelerated microbial development had no effect on the animal growth, but CMP animals presented higher rumen pH and butyrate (+45%) and ammonia concentrations than CTL kids, suggesting higher fibrolytic and proteolytic activities. CMP kids also had higher blood ß-hydroxybutyrate (+79%) and lower blood glucose concentrations (-23%) at weaning, indicating an earlier metabolic development which could favor the transition from pre-ruminant to ruminant after the weaning process. Further research is needed to determine the effects of this intervention in more challenging farm conditions.

Peripheral blood mononuclear cells (PBMC) microbiome is not affected by colon microbiota in healthy goats.

BACKGROUND: The knowledge about blood circulating microbiome and its functional relevance in healthy individuals remains limited. An assessment of changes in the circulating microbiome was performed by sequencing peripheral blood mononuclear cells (PBMC) bacterial DNA from goats supplemented or not in early life with rumen liquid transplantation. RESULTS: Most of the bacterial DNA associated to PBMC was identified predominantly as Proteobacteria (55%) followed by Firmicutes (24%), Bacteroidetes (11%) and Actinobacteria (8%). The predominant genera found in PBMC samples were Pseudomonas, Prevotella, Sphingomonas, Acinetobacter, Corynebacterium and Ruminococcus. Other genera such as Butyrivibrivio, Bifidobacterium, Dorea and Coprococcus were also present in lower proportions. Several species known as blood pathogens or others involved in gut homeostasis such as Faecalibacterium prausnitzii were also identified. However, the PBMC microbiome phylum composition differed from that in the colon of goats (P ≤ 0.001), where Firmicutes was the predominant phylum (83%). Although, rumen liquid administration in early-life altered bacterial community structure and increased Tlr5 expression (P = 0.020) in colon pointing to higher bacterial translocation, less than 8% of OTUs in colon were also observed in PBMCs. CONCLUSIONS: Data suggest that in physiological conditions, PBMC microbiome differs from and is not affected by colon gut microbiota in small ruminants. Although, further studies with larger number of animals and covering other animal tissues are required, results point to a common circulating bacterial profile on mammals being phylum Proteobacteria, and genera Pseudomonas and Prevotella the most abundants. All suggest that PBMC microbiome in healthy ruminants could be implicated in homeostatic condition. This study expands our knowledge about PBMC microbiome contribution to health in farm animals.

A novel ammoniation treatment of barley as a strategy to optimize rumen pH, feed degradability and microbial protein synthesis in sheep.

BACKGROUND: Meeting the energy and nitrogen (N) requirements of high-performing ruminants at the same time as avoiding digestive disturbances (i.e. rumen acidosis) is a key priority in ruminant nutrition. The present study evaluated the effect of a cereal ammoniation treatment, in which barley grains are combined with urea and enzymes that catalyze the conversion of urea to ammonia to optimize rumen function. Twelve rumen cannulated sheep were randomly divided into two groups and fed a diet containing 60% of ammoniated barley (AMM) or untreated barley supplemented with urea (CTL) to investigate the impact on rumen fermentation and feed utilization. RESULTS: AMM had higher total N content and effective rumen degradable N than untreated barely. AMM sheep had a consistently higher rumen pH throughout the day (6.31 versus 6.03) and tended to have a lower post-prandial ammonia peak and higher acetate molar proportion (+5.1%) than CTL sheep. The rumen environment in AMM sheep favored the colonization and utilization of agro-industrial by-products (i.e. orange pulp) by the rumen microbes leading to a higher feed degradability. AMM sheep also had higher total tract apparent N digestibility (+21.7%) and urinary excretion of purine derivatives (+34%), suggesting a higher N uptake and microbial protein synthesis than CTL sheep. CONCLUSION: The inclusion of AMM in the diet of ruminants represents a valid strategy for maintaining rumen pH within a physiological range and improving N utilization by the rumen microbes, which could have positive effects on the health and productivity of animals in intensive production systems. These findings warrant further studies under conventional farm conditions. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Inoculation with rumen fluid in early life accelerates the rumen microbial development and favours the weaning process in goats.

BACKGROUND: Newborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function. RESULTS: Repetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+ 234 OTUs), methanogens (+ 6 OTUs) and protozoal communities (+ 25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favoring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favored a more rapid microbial development during the post-weaning than observed for CTL kids. CONCLUSIONS: This study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.

In vitro assessment of the factors that determine the activity of the rumen microbiota for further applications as inoculum.

BACKGROUND: The rumen microbiota has been used as inoculum for in vitro studies and as a probiotic to improve productivity in young animals. However, great variability across studies has been noted depending on the inoculum considered. The present study aims to assess the relevance of different factors (microbial fraction, collection time, donor animal diet, fermentation substrate and inoculum preservation method) to maximize the rumen inoculum activity and set the standards for further in vitro and in vivo applications. RESULTS: Rumen inoculum sampled at 3 h after feeding led to greater microbial growth and activity [+12% volatile fatty acid (VFA), +17% ammonia] compared to before feeding. Similar results were noted when rumen liquid or rumen content were used as inocula. Rumen inoculum adapted to concentrate diets increased microbial activity (+19% VFA) independently of the substrate used in vitro. Freezing-thawing the inoculum, in comparison to fresh inoculum, decreased microbial activity (-14% VFA, -96% ammonia), anaerobic fungi and protozoa, with holotrichs protozoa being particularly vulnerable. Inoculum lyophilization had a stronger negative effect on microbial activity (-51% VFA) and delayed re-activation of the microbes, leading to lower levels of methanogens and anaerobic fungi, as well as almost complete wipe out of rumen protozoa. CONCLUSIONS: Fresh rumen fluid sampled at 3 h after feeding from donor animals that were fed concentrate diets should be chosen when the aim is to provide the most diverse and active rumen microbial inoculum. © 2018 Society of Chemical Industry.

Targeting EGFR in Lung Cancer: Current Standards and Developments.

Lung cancer is the second most common malignant tumor and the leading cause of cancer death. Epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) is a distinct subtype of lung cancer comprising approximately 15-40% of non-squamous tumors. The development of first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) has been a significant step forward in the treatment of patients with EGFR-mutant tumors, and over the last few years has been the therapy of choice in the initial management of patients with activating mutations in EGFR, with some differences in efficacy and toxicity profile. Up to 50% of patients treated with first- and second-generation TKIs develop an EGFR exon 20 T790M mutation at the time of progression. In this context, osimertinib has shown a great benefit in terms of progression-free survival (PFS) in the second-line setting, including central nervous system metastasis control. The FLAURA trial, which compared osimertinib to first-generation inhibitors as first-line therapy, showed a clear PFS advantage for osimertinib and a trend towards an increased overall survival (OS) assessed by investigator review. Although T790M mutation is the most common mechanism of resistance to first- and second-generation EGFR TKIs, other EGFR-dependent and -independent mechanisms have been described, such as HER2 and MET amplifications or BRAF and MEK mutations. Some mechanisms of resistance to osimertinib and other third-generation TKIs have also been described. Several fourth-generation TKIs, targeted drug combinations and immunotherapy strategies are under investigation to overcome resistance to EGFR TKIs in order to improve EGFR-mutant NSCLC patient outcomes.

Prospective Clinical Integration of an Amplicon-Based Next-Generation Sequencing Method to Select Advanced Non-Small-Cell Lung Cancer Patients for Genotype-Tailored Treatments.

INTRODUCTION: A substantial fraction of non-small-cell lung cancers (NSCLCs) harbor targetable genetic alterations. In this study, we analyzed the feasibility and clinical utility of integrating a next-generation sequencing (NGS) panel into our routine lung cancer molecular subtyping algorithm. PATIENTS AND METHODS: After routine pathologic and molecular subtyping, we implemented an amplicon-based gene panel for DNA analysis covering mutational hot spots in 22 cancer genes in consecutive advanced-stage NSCLCs. RESULTS: We analyzed 109 tumors using NGS between December 2014 and January 2016. Fifty-six patients (51%) were treatment-naive and 82 (75%) had lung adenocarcinomas. In 89 cases (82%), we used samples derived from lung cancer diagnostic procedures. We obtained successful sequencing results in 95 cases (87%). As part of our routine lung cancer molecular subtyping protocol, single-gene testing for EGFR, ALK, and ROS1 was attempted in nonsquamous and 3 squamous-cell cancers (n = 92). Sixty-nine of 92 samples (75%) had sufficient tissue to complete ALK and ROS1 immunohistochemistry (IHC) and NGS. With the integration of the gene panel, 40 NSCLCs (37%) in the entire cohort and 30 NSCLCs (40%) fully tested for ALK and ROS1 IHC and NGS had actionable mutations. KRAS (24%) and EGFR (10%) were the most frequently mutated actionable genes. Ten patients (9%) received matched targeted therapies, 6 (5%) in clinical trials. CONCLUSION: The combination of IHC tests for ALK and ROS1 and amplicon-based NGS is applicable in routine clinical practice, enabling patient selection for genotype-tailored treatments.

Natural and artificial feeding management before weaning promote different rumen microbial colonization but not differences in gene expression levels at the rumen epithelium of newborn goats.

The aim of this work was to evaluate the effect of feeding management during the first month of life (natural with the mother, NAT, or artificial with milk replacer, ART) on the rumen microbial colonization and the host innate immune response. Thirty pregnant goats carrying two fetuses were used. At birth one kid was taken immediately away from the doe and fed milk replacer (ART) while the other remained with the mother (NAT). Kids from groups received colostrum during first 2 days of life. Groups of four kids (from ART and NAT experimental groups) were slaughtered at 1, 3, 7, 14, 21 and 28 days of life. On the sampling day, after slaughtering, the rumen content was sampled and epithelial rumen tissue was collected. Pyrosequencing analyses of the bacterial community structure on samples collected at 3, 7, 14 and 28 days showed that both systems promoted significantly different colonization patterns (P = 0.001). Diversity indices increased with age and were higher in NAT feeding system. Lower mRNA abundance was detected in TLR2, TLR8 and TLR10 in days 3 and 5 compared to the other days (7, 14, 21 and 28). Only TLR5 showed a significantly different level of expression according to the feeding system, presenting higher mRNA abundances in ART kids. PGLYRP1 showed significantly higher abundance levels in days 3, 5 and 7, and then experienced a decline independently of the feeding system. These observations confirmed a highly diverse microbial colonisation from the first day of life in the undeveloped rumen, and show that the colonization pattern substantially differs between pre-ruminants reared under natural or artificial milk feeding systems. However, the rumen epithelial immune development does not differentially respond to distinct microbial colonization patterns.

Second-line Treatment of Non-Small Cell Lung Cancer: Focus on the Clinical Development of Dacomitinib.

Dacomitinib is a second-generation, irreversible, covalent pan-HER tyrosine-kinase inhibitor (TKI). It showed potent EGFR signaling inhibition in experimental models, including first-generation TKI-resistant non-small cell lung cancer (NSCLC) cell lines. This preclinical efficacy did not translate into clinically meaningful treatment benefits for advanced, pretreated, molecularly unselected NSCLC patients enrolled in two parallel phase III trials. Dacomitinib and erlotinib showed overlapping efficacy data in chemotherapy-pretreated EGFR wild-type (WT) patients in the ARCHER 1009 trial. Similarly, it failed to demonstrate any survival benefits as compared to placebo in EGFR WT subsets progressing on chemotherapy and at least one previous first-generation TKI (erlotinib or gefitinib) in the BR.26 trial. In the case of EGFR-mutant NSCLCs, a pooled analysis of the ARCHER 1009 and ARCHER 1028 trials comparing the efficacy of dacomitinib vs. erlotinib in chemotherapy-pretreated, EGFR TKI-naïve patients showed a trend to a longer progression-free survival (PFS) and overall survival in favor of dacomitinib that did not reach statistical significance, with a higher rate of treatment related adverse events (mainly skin rash, paronychia, and gastrointestinal toxicities). On the other hand, the clinical activity in patients with EGFR-mutant NSCLCs with acquired TKI resistance that were included in phase II/III trials was equally poor (response rate <10%; PFS 3-4 months). Therefore, with the results of the ARCHER 1050 trial (NCT01774721) still pending, the current clinical development of dacomitinib is largely focused on EGFR-mutant, TKI-naïve patients. Here, we review the most relevant clinical data of dacomitinib in advanced NSCLC. We discuss the potential role of dacomitinib in pretreated EGFR WT and EGFR-mutant (TKI-naïve and TKI-resistant) patients. Finally, we briefly comment the available clinical data of dacomitinib in HER2-mutant NSCLC patients.

The anti-proliferative effect of TI1B, a major Bowman-Birk isoinhibitor from pea (Pisum sativum L.), on HT29 colon cancer cells is mediated through protease inhibition.

Bowman-Birk inhibitors (BBI) from legumes, such as soyabean, pea, lentil and chickpea, are naturally occurring plant protease inhibitors which have potential health-promoting properties within the mammalian gastrointestinal tract. BBI can survive both acidic conditions and the action of proteolytic enzymes within the stomach and small intestine, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. In a previous study, we reported the ability of a recombinant form of TI1B (rTI1B), representing a major BBI isoinhibitor from pea, to influence negatively the growth of human colorectal adenocarcinoma HT29 cells in vitro. In the present study, we investigate if this effect is related directly to the intrinsic ability of BBI to inhibit serine proteases. rTI1B and a novel engineered mutant, having amino acid substitutions at the P1 positions in the two inhibitory domains, were expressed in the yeast Pichia pastoris. The rTI1B proved to be active against trypsin and chymotrypsin, showing K i values at nanomolar concentrations, whereas the related mutant protein was inactive against both serine proteases. The proliferation of HT29 colon cancer cells was significantly affected by rTI1B in a dose-dependent manner (IC50 = 31 (sd 7) µm), whereas the inactive mutant did not show any significant effect on colon cancer cell growth. In addition, neither recombinant protein affected the growth of non-malignant colonic fibroblast CCD-18Co cells. These findings suggest that serine proteases should be considered as important targets in investigating the potential chemopreventive role of BBI during the early stages of colorectal carcinogenesis.

The cytotoxic effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases.

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.

Anti-carcinogenic soyabean Bowman-Birk inhibitors survive faecal fermentation in their active form and do not affect the microbiota composition in vitro.

Bowman-Birk inhibitor (BBI) from soyabeans is a naturally occurring protease inhibitor with potential anti-inflammatory and chemopreventive properties within the gastrointestinal tract (GIT). In a previous paper, we reported that significant amounts of BBI-related proteins reach the terminal ileum functionally and biologically active. We have now investigated: (a) if soyabean BBI is biotransformed by faecal microbiota which would reduce its potential colorectal chemopreventive properties and (b) the potential influence of this protease inhibitor on the modulation of faecal microbiota. In vitro incubation studies of native soyabean BBI at a physiological level (93 microM) with mixed faecal samples of pigs for 24 h at 37 degrees C demonstrated that BBI remains active and its intrinsic trypsin and chymotrypsin inhibitory activities were not significantly influenced by the enzymic or metabolic activity of faecal microbiota. Soyabean BBI did not affect the growth of the different bacterial groups studied (lactobacilli, bifidobacteria, bacteroides, coliforms, enterobacteria, clostridia and total anaerobes). It was concluded that protease inhibitory activities, intrinsically linked to the chemopreventive properties of soyabean BBI, were largely unaffected by faecal microbiota in vitro. BBI retains significance, therefore, as a bioactive compound in the human GIT.