Overview on biotics development.

Although probiotics have been used in food products and supplements for decades, there has been a considerable increase in their use more recently. Recent technological advances have thus led to major advances in knowledge of the gut microbiota, enabling a significant development of biotics. In this review, we discuss the uses of traditional probiotics but also the discovery of next-generation probiotics that could be used as live biotherapeutics. These novel preventive and therapeutic strategies hold promise for the treatment of numerous diseases such as inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Probiotic bacteria can be consumed alone, or in combination with prebiotics as synbiotics, or mixed with other probiotic strains to form a consortium for enhanced effects. We also discuss the benefits of using postbiotics.

Microbiota analysis for risk assessment of xenobiotic exposure and the impact on dysbiosis: identifying potential next-generation probiotics.

On-going projects of the team are currently dealing with microbiota, xenobiotics, endocrine-disrupting chemicals (EDCs), obesity, inflammation and probiotics. The combination of diet, lifestyle and the exposure to dietary xenobiotics categorised into microbiota-disrupting chemicals (MDCs) could determine obesogenic-related dysbiosis. This modification of the microbiota diversity impacts on individual health-disease balance, inducing altered phenotypes. Specific, complementary, and combined prevention and treatments are needed to face these altered microbial patterns and the specific misbalances triggered. In this sense, searching for next-generation probiotics (NGP) by microbiota culturing, and focusing on their demonstrated, extensive scope and well-defined functions could contribute to counteracting and repairing the effects of obesogens. Therefore, EU-FORA project contributes to present a perspective through compiling information and key strategies for directed taxa searching and culturing of NGP that could be administered for preventing obesity and endocrine-related dysbiosis by (i) observing the differential abundance of specific microbiota taxa in obesity-related patients and analysing their functional roles, (ii) developing microbiota-directed strategies for culturing these taxa groups, and (iii) design and applying the successful compiled criteria from recent NGP clinical studies. New isolated or cultivable microorganisms from healthy gut microbiota specifically related to xenobiotic obesogens' neutralisation effects might be used as an NGP single strain or in consortia, both presenting functions and the ability to palliate metabolic-related disorders. Identification of holistic approaches for searching and using potential NGP, key aspects, the bias, gaps and proposals of solutions were also considered in this workplan.

Ligilactobacillus salivarius CNCM I-4866, a potential probiotic candidate, shows anti-inflammatory properties in vitro and in vivo.

Introduction: The aim of this work was to characterize a new strain of Ligilactobacillus salivarius (CNCM I-4866) (CNCM I-4866) to address its potential as probiotic with a special focus on intestinal inflammation. Potential anti-inflammatory abilities of this strain were evaluated through in vivo and in vitro experiments. Methods: Firstly, the strain was tested in a murine acute inflammation colitis model induced by DNBS. In vitro characterization was then performed with diverse tests: modulation capability of intestinal permeability; study of the impact on immunity profile through cytokines dosage; capacity to inhibit pathogens and adhere to intestinal cells lines. Production of metabolites, antibiotic resistance and survival to gastro-intestinal tract conditions were also tested. Results: In vitro assay has shown a reduction of colonic damage and markers of inflammation after treatment with CNCM I-4866. Transcriptomic analysis performed on colons showed the capacity of the strain to down-regulate pro-inflammatory cytokines. L. salivarius CNCM I-4866 exerted anti-inflammatory profile by reducing IL-8 production by TNF-α stimulated cell and modulated cytokines profile on peripheral blood mononuclear cells (PBMC). It protected intestinal integrity by increasing trans-epithelial electrical resistance (TEER) on Caco-2 TNF-α inflamed cells. Additionally, L. salivarius CNCM I-4866 displayed inhibition capacity on several intestinal pathogens and adhered to eukaryotic cells. Regarding safety and technical concerns, CNCM I-4866 was highly resistant to 0.3% of bile salts and produced mainly L-lactate. Finally, strain genomic characterization allowed us to confirm safety aspect of our strain, with no antibiotic gene resistance found. Discussion: Taken together, these results indicate that L. salivarius CNCM I-4866 could be a good probiotic candidate for intestinal inflammation, especially with its steady anti-inflammatory profile.

A randomized, double-blind study on the efficacy of oral domperidone versus placebo for reducing SARS-CoV-2 viral load in mild-to-moderate COVID-19 patients in primary health care.

INTRODUCTION: The clinical effect of domperidone against COVID-19 has been investigated in a double-blind phase III clinical trial (EudraCT number 2021-001228-17). Domperidone has shown in vitro antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential immudolatory properties through the stimulation of prolactin secretion. PATIENTS AND METHODS: The efficacy of oral domperidone plus standard of care (SOC; n = 87) versus placebo plus SOC (n = 86) was evaluated in a 28-day randomized double-blind multicentre study in primary health care centres. A total of 173 outpatients with mild-to-moderate COVID-19 were included. Three daily doses of 10 mg (30 mg/day) of domperidone or placebo were administered for 7 days. Reduction of viral load on day 4 was the primary efficay endpoint. It was estimated in saliva samples by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), as the cycle thresholds detected ORF1ab, N Protein and S Protein genes. RESULTS: A significant reduction in the viral load was observed (p < 0.001) from baseline to days 4, 7 and 14 of the three genes studied with non-significant differences between domperidone and placebo groups. Twenty-three patients (13.3%) experienced adverse events, 14 patients in the domperidone group (16.1%) and 9 patients in the placebo group (10.5%). No patients needed to be hospitalized. CONCLUSION: Results do not prove the use of domperidone as antiviral in patients with COVID-19.

A 28-day double-blind clinical trial was performed to investigate the antiviral effect of domperidone, 30 mg/day for 7 days (n = 87) versus placebo (n = 86) in outpatients with mild-to-moderate COVID-19.The primary efficacy endpoint was the reduction of viral load on day 4 as compared with baseline, estimated as the cycle thresholds to detect ORF1ab, N Protein and S Protein genes by RT-qPCR in saliva samples.The study findings do not prove the use of domperidone as antiviral in patients with COVID-19.

Faecalibacterium: a bacterial genus with promising human health applications.

In humans, many diseases are associated with alterations in gut microbiota, namely increases or decreases in the abundance of specific bacterial groups. One example is the genus Faecalibacterium. Numerous studies have underscored that low levels of Faecalibacterium are correlated with inflammatory conditions, with inflammatory bowel disease (IBD) in the forefront. Its representation is also diminished in the case of several diseases, including colorectal cancer (CRC), dermatitis, and depression. Additionally, the relative presence of this genus is considered to reflect, at least in part, intestinal health status because Faecalibacterium is frequently present at reduced levels in individuals with gastrointestinal diseases or disorders. In this review, we first thoroughly describe updates to the taxonomy of Faecalibacterium, which has transformed a single-species taxon to a multispecies taxon over the last decade. We then explore the links discovered between Faecalibacterium abundance and various diseases since the first IBD-focused studies were published. Next, we examine current available strategies for modulating Faecalibacterium levels in the gut. Finally, we summarize the mechanisms underlying the beneficial effects that have been attributed to this genus. Together, epidemiological and experimental data strongly support the use of Faecalibacterium as a next-generation probiotic (NGP) or live biotherapeutic product (LBP).

Differential contributions of nuclear lamina association and genome compartmentalization to gene regulation.

Chromatin regions that interact with the nuclear lamina are often heterochromatic, repressed in gene expression, and in the spatial B compartment. However, exceptions to this trend allow us to examine the relative impact of lamin association and spatial compartment on gene regulation. Here, we compared lamin association, gene expression, Hi-C, and histone mark datasets from cell lines representing different states of differentiation across different cell-type lineages. With these data, we compare, for example, gene expression differences when a B compartment region is associated with the nuclear lamina in one cell type but not in another. In general, we observed an additive rather than redundant effect of lamin association and compartment status. But, whether compartment status or lamin association had a dominant influence on gene expression varied by cell type. Finally, we identified how compartment and lamin association influence the likelihood of gene induction or repression in response to physicochemical treatment.

From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders.

Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

New gene markers for classification and quantification of Faecalibacterium spp. in the human gut.

Faecalibacterium prausnitzii is a promising biomarker of a healthy human microbiota. However, previous studies reported the heterogeneity of this species and found the presence of several distinct groups at the species level among F. prausnitzii strains. Our recent study revealed that methods previously developed for quantification of F. prausnitzii were not specific to the species level because of the heterogeneity within the F. prausnitzii species and the application of 16S rRNA gene, which is an invalid genetic marker for the species. Therefore, previously available data failed to provide information on different groups, which limits our understanding of the importance of this organism for host health. Here, we propose an alternative gene marker for quantification of F. prausnitzii-related taxa. A total of nine group-specific primer pairs were designed by targeting rpoA gene sequences. The newly developed rpoA-based qPCR successfully quantified targeted groups. Application of the developed qPCR assay in six healthy adults revealed marked differences in abundance and prevalence among the different targeted groups in stool samples. The developed assay will facilitate detailed understanding of the impact of Faecalibacterium populations at the group level on human health and to understand the links between depletion of specific groups in Faecalibacterium and different human disorders.

Amorphous calcium phosphate-coated surfaces as a model for bone microenvironment in prostate cancer.

Bone metastasis remains one of the biggest challenges in the treatment of prostate cancer, and other solid tumors such as breast, lung, and colon. Modeling a complex microenvironment in-vitro, such as the bone niche, requires interrogation of cell-cell interactions, specific extracellular matrix proteins and a high calcium environment. Here, we present a fast and cost-effective system in which commercially available, non-adhesive, cell culture vessels are coated with amorphous calcium phosphate (ACP) as a surrogate for bone matrix. We further present modified protocols for subculturing cells, as well as nucleic acid and protein collection in high calcium samples. We find that prostate epithelial cell lines show increased adhesion and proliferation when cultured in these surfaces, as well as independence from androgen starvation. We observe gene expression changes on ACP surfaces in early adenocarcinoma cell lines which may reflect alterations relevant to prostate cancer progression. Summary statement: To model the role of calcium in the microenvironment of the metastatic bone niche, we developed a cost-effective way to coat cell culture vessels in bioavailable calcium, and show that it has an effect on prostate cancer cell survival.

Recruiting women faculty through inclusive, family-friendly practices.

Recruitment of STEM faculty is biased against parents and caregivers. Specifically, women experience discrimination associated with childrearing and marriage. Underestimating the value of these candidates leads to a tremendous loss of talent. Here, we present a toolkit to facilitate the recruitment of talented women caregivers by providing guidelines for hiring.

Comparing technology and regulatory landscape of probiotics as food, dietary supplements and live biotherapeutics.

Application of beneficial microorganisms as probiotics targets a broad range of intended uses, from maintaining health and supporting normal bodily functions to curing and preventing diseases. Currently, three main regulatory fields of probiotic products can be defined depending on their intended use: the more similar probiotic foods and probiotic dietary supplements, and live biotherapeutic products. However, it is not always straightforward to classify a probiotic product into one of these categories. The regulatory nuances of developing, manufacturing, investigating and applying each category of probiotic products are not universal, and not always apparent to those unfamiliar with the various global probiotic regulatory guidelines. Various global markets can be significantly different regarding legislation, possible claims, market value and quality requirements for the development and commercialization of probiotic products. Furthermore, different probiotic product categories are also linked with variable costs at different stages of product development. This review outlines the current landscape comparing probiotic foods, probiotic dietary supplements, and live biotherapeutics as probiotic products from a regulatory lens, focusing on product development, manufacturing and production, and clinical research agenda. The aim is to inform and promote a better understanding among stakeholders by outlining the expectations and performance for each probiotic product category, depending on their intended use and targeted geographical region.

Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification.

The expression of a mutant Lamin A, progerin, in Hutchinson-Gilford Progeria Syndrome leads to alterations in genome architecture, nuclear morphology, epigenetic states, and altered phenotypes in all cells of the mesenchymal lineage. Here, we report a comprehensive analysis of the transcriptional status of patient derived HGPS fibroblasts, including nine cell lines not previously reported, in comparison with age-matched controls, adults, and old adults. We find that Progeria fibroblasts carry abnormal transcriptional signatures, centering around several functional hubs: DNA maintenance and epigenetics, bone development and homeostasis, blood vessel maturation and development, fat deposition and lipid management, and processes related to muscle growth. Stratification of patients by age revealed misregulated expression of genes related to endochondral ossification and chondrogenic commitment in children aged 4-7 years old, where this differentiation program starts in earnest. Hi-C measurements on patient fibroblasts show weakening of genome compartmentalization strength but increases in TAD strength. While the majority of gene misregulation occurs in regions which do not change spatial chromosome organization, some expression changes in key mesenchymal lineage genes coincide with lamin associated domain misregulation and shifts in genome compartmentalization.

SnapShot: Chromosome organization.

Chromosomes in higher eukaryotes are folded at different length scales into loop extrusion domains, spatial compartments, and chromosome territories and exhibit interactions with nuclear structures such as the lamina. Microscopic methods can probe this structure by measuring positions of chromosomes in the nuclear space in individual cells, while sequencing-based contact capture approaches can report the frequency of contacts of different regions within these structural layers. To view this SnapShot, open or download the PDF.

Chromosome compartmentalization alterations in prostate cancer cell lines model disease progression.

Prostate cancer aggressiveness and metastatic potential are influenced by gene expression and genomic aberrations, features that can be influenced by the 3D structure of chromosomes inside the nucleus. Using chromosome conformation capture (Hi-C), we conducted a systematic genome architecture comparison on a cohort of cell lines that model prostate cancer progression, from normal epithelium to bone metastasis. We describe spatial compartment identity (A-open versus B-closed) changes with progression in these cell lines and their relation to gene expression changes in both cell lines and patient samples. In particular, 48 gene clusters switch from the B to the A compartment, including androgen receptor, WNT5A, and CDK14. These switches are accompanied by changes in the structure, size, and boundaries of topologically associating domains (TADs). Further, compartment changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation. These results suggest that discrete 3D genome structure changes play a deleterious role in prostate cancer progression. .

From Short- to Long-Term Effects of C-Section Delivery on Microbiome Establishment and Host Health.

The establishment of gut microbiota has been proven to be impacted by several factors during pregnancy, delivery, and neonate periods. The body of evidence describing C-section delivery (CSD) as one of the most disruptive events during early life has expanded in recent years, concluding that CSD results in a drastic change in microbiota establishment patterns. When comparing the gut microbiota composition of CSD babies with vaginally delivered (VD) babies, the former show a microbiome that closely resembles that found in the environment and the mother's skin, while VD babies show a microbiome more similar to the vaginal microbiome. Although these alterations of normal gut microbiota establishment tend to disappear during the first months of life, they still affect host health in the mid-long term since CSD has been correlated with a higher risk of early life infections and non-transmissible diseases, such as inflammatory diseases, allergies, and metabolic diseases. In recent years, this phenomenon has also been studied in other mammals, shedding light on the mechanisms involved in the effects of a CSD on host health. In addition, strategies to revert the disruptions in gut microbiomes caused by a CSD are currently in the process of development and evaluation. In this review, we discuss the recent advances in CSD research, from the alteration of gut microbiota establishment to the possible effects on host health during early life and development.

Overstretched and overlooked: solving challenges faced by early-career investigators after the pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has had a detrimental effect on research. However, little has been done to identify and solve the unique challenges faced by early career investigators (ECIs). As a group of American Cancer Society-funded ECIs, we provide recommendations for solving these challenges in the aftermath of the pandemic.

From Nanoparticle Heteroclusters to Filament Networks by Self-Assembly at the Water-Oil Interface of Reverse Microemulsions.

Surface self-assembly of spherical nanoparticles of sizes below 10 nm into hierarchical heterostructures is under arising development despite the inherent difficulties of obtaining complex ordering patterns on a larger scale. Due to template-mediated interactions between oil-dispersible superparamagnetic nanoparticles (MNPs) and polyethylenimine-stabilized gold nanoparticles (Au(PEI)NPs) at the water-oil interface of microemulsions, complex nanostructured films can be formed. Characterization of the reverse microemulsion phase by UV-vis absorption revealed the formation of heteroclusters from Winsor type II phases (WPII) using Aerosol-OT (AOT) as the surfactant. SAXS measurements verify the mechanism of initial nanoparticle clustering in defined dimensions. XPS suggested an influence of AOT at the MNP surface. Further, cryo-SEM and TEM visualization demonstrated the elongation of the reverse microemulsions into cylindrical, wormlike structures, which subsequently build up larger nanoparticle superstructure arrangements. Such WPII phases are thus proven to be a new form of soft template, mediating the self-assembly of different nanoparticles in hierarchical network-like filaments over a substrate during solvent evaporation.

The Keystone commensal bacterium Christensenella minuta DSM 22607 displays anti-inflammatory properties both in vitro and in vivo.

Christensenellaceae is a family of subdominant commensal bacteria found in humans. It is thought to play an important role in gut health by maintaining microbial symbiosis. Indeed, these bacteria occur at significantly lower levels or are absent in individuals suffering from inflammatory bowel diseases (IBDs). Here, we explored if type species Christensenella minuta (strain: DSM 22607) could have the potential to help treat IBDs. We assessed key properties displayed by the bacterium using a combination of in vitro and in vivo assays. We found that while C. minuta is a strict anaerobe, it is also oxygen tolerant. Additionally, we observed that the species produces high levels of acetate and moderate levels of butyrate. We performed deep phenotyping using Biolog microarrays. Using human intestinal cell lines, we discovered that C. minuta demonstrated strong anti-inflammatory activity, resulting in reduced levels of proinflammatory IL-8 cytokines via the inhibition of the NF-κB signaling pathway. Furthermore, C. minuta protected intestinal epithelial integrity in vitro. Finally, in two distinct animal models of acute colitis, C. minuta prevented intestinal damage, reduced colonic inflammation, and promoted mucosal healing. Together, these results indicate that C. minuta has potent immunomodulatory properties, underscoring its potential use in innovative microbiome-based IBD biotherapies.

Antioxidant and Anti-Inflammatory Properties of Probiotic Candidate Strains Isolated during Fermentation of Agave (Agave angustifolia Haw).

Agave species are a source of diverse products for human use, such as food, fiber, and beverages, which include mezcal, a distilled beverage produced by spontaneous fermentation. Agave is an excellent source of high amounts of sugars, minerals, and phenolic compounds, which favor the growth of lactic acid bacteria (LAB) and yeast communities. In this work, 20 promising LAB strains with probiotic characteristics were isolated from the agave fermentation stage in mezcal production. The strains belonged to Lactobacillus plantarum (15), Lactobacillus rhamnosus (2), Enterococcus faecium (2), and Lactococcus lactis (1). These isolates were characterized for their resistance under gastrointestinal conditions, such as lysozyme, acid pH, and bile salts. In addition, the adherence of these LABs to human intestinal epithelial cells (Caco-2 and HT-29 cells) was tested in vitro and their antioxidant and immunomodulatory profile was determined using cellular models. Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 and LM19 strains were selected for their antioxidant properties, and their capacities in an oxidative stress model in intestinal epithelial cells IECs (Caco-2 and HT-29 cells) in the presence of hydrogen peroxide were evaluated. Interestingly, Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 and LM19 strains showed anti-inflammatory properties in TNF-α-stimulated HT-29 cells. Subsequently, bacterial strains exhibiting antioxidant and anti-inflammatory properties were tested in vivo in a mouse model with dinitrobenzene sulfonic acid (DNBS)-induced chronic colitis. Weight loss, intestinal permeability, and cytokine profiles were measured in mice as indicators of inflammation. One of the selected strains, Lactobacillus plantarum LM17, improved the health of the mice, as observed by reduced weight loss, and significantly decreased intestinal permeability. Altogether, our results demonstrate the potential of LAB (and lactobacilli in particular) isolated from the agave fermentation stage in mezcal production. Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 strains represent potential candidates for developing new probiotic supplements to treat inflammatory bowel disease (IBD).