'Counselling is not just providing information': perceptions of caregivers and stakeholders on the design of nutrition and health counselling interventions for families with young children in rural Kenya.

BACKGROUND: Globally, a fifth of the children continue to face chronic undernutrition with a majority of them situated in the Low- and Middle-Income Countries (LMIC). The rising numbers are attributed to aggravating factors like limited nutrition knowledge, poor feeding practices, seasonal food insecurity, and diseases. Interventions targeting behaviour change may reduce the devastating nutrition situation of children in the LMICs. OBJECTIVE: For the co-design of a Behaviour Change Communication (BCC) intervention for young children in rural Kenya, we aimed to identify the experiences, barriers, facilitators, and preferences of caregivers and stakeholders regarding nutrition and health counselling. DESIGN: We employed a qualitative study design and used a semi-structured interview guide. The in-depth interviews were recorded, transcribed, and analysed using content analysis, facilitated by the software NVivo. SETTING: Health and Demographic Surveillance System (HDSS) area in Siaya County, rural Kenya. PARTICIPANTS: We interviewed 30 caregivers of children between 6 and 23 months of age and 29 local stakeholders with experience in implementing nutrition projects in Kenya. RESULTS: Nutrition and health counselling (NHC) was usually conducted in hospital settings with groups of mothers. Barriers to counselling were long queues and delays, long distances and high travel costs, the inapplicability of the counselling content, lack of spousal support, and a high domestic workload. Facilitators included the trust of caregivers in Community Health Volunteers (CHVs) and counselling services offered free of charge. Preferences comprised (1) delivering of counselling by CHVs, (2) offering individual and group counselling, (3) targeting male and female caregivers. CONCLUSION: There is a disconnect between the caregivers' preferences and the services currently offered. Among these families, a successful BCC strategy that employs nutrition and health counselling should apply a community-based communication channel through trusted CHVs, addressing male and female caregivers, and comprising group and individual sessions.

Yeast Platforms for Production and Screening of Bioactive Derivatives of Rauwolscine.

Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.

teemi: An open-source literate programming approach for iterative design-build-test-learn cycles in bioengineering.

Synthetic biology dictates the data-driven engineering of biocatalysis, cellular functions, and organism behavior. Integral to synthetic biology is the aspiration to efficiently find, access, interoperate, and reuse high-quality data on genotype-phenotype relationships of native and engineered biosystems under FAIR principles, and from this facilitate forward-engineering strategies. However, biology is complex at the regulatory level, and noisy at the operational level, thus necessitating systematic and diligent data handling at all levels of the design, build, and test phases in order to maximize learning in the iterative design-build-test-learn engineering cycle. To enable user-friendly simulation, organization, and guidance for the engineering of biosystems, we have developed an open-source python-based computer-aided design and analysis platform operating under a literate programming user-interface hosted on Github. The platform is called teemi and is fully compliant with FAIR principles. In this study we apply teemi for i) designing and simulating bioengineering, ii) integrating and analyzing multivariate datasets, and iii) machine-learning for predictive engineering of metabolic pathway designs for production of a key precursor to medicinal alkaloids in yeast. The teemi platform is publicly available at PyPi and GitHub.

Bacillus subtilis-597 induces changes in lung pathology and inflammation during influenza A virus infection in pigs.

In recent years, it has become apparent that imbalances in the gastrointestinal system can impact organs beyond the intestine such as the lungs. Given the established ability of probiotics to modulate the immune system by interacting with gastrointestinal cells, our research aimed to investigate whether administering the probiotic strain Bacillus subtilis-597 could mitigate the outcome of influenza virus infection in pigs. Pigs were fed a diet either with or without the probiotic strain B. subtilis-597 for 14 days before being intranasally inoculated with a swine influenza A H1N2 strain (1 C.2 lineage). Throughout the study, we collected fecal samples, blood samples, and nasal swabs to examine viral shedding and immune gene expression. After seven days of infection, the pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination. Our findings indicate that the administration of B. subtilis-597 exhibit potential in reducing lung lesions, possibly attributable to a general suppression of the immune system as indicated by reduced C-reactive protein (CRP) levels in serum, decreased expression of interferon-stimulated genes (ISGs), and localized reduction of the inflammatory marker serum amyloid A (SAA) in ileum tissue. Notably, the immune-modulatory effects of B. subtilis-597 appeared to be unrelated to the gastrointestinal microbiota, as the composition remained unaltered by both the influenza infection and the administration of B. subtilis-597.

Conference Report: The FutuRE oF MinimalLy InvasivE GI and Capsule DiagnosTics (REFLECT) Nyborg, Denmark, October 2023.

The gastrointestinal (GI) tract, particularly the small bowel (SB), can be challenging for novel investigation tools [...].

Factors associated with negative colonoscopy in participants with a positive faecal immunochemical test from the Danish Colorectal Cancer Screening Program - a population-based study.

AIM: In the Danish Colorectal Cancer Screening Program (DCCSP), 37% of participants undergoing colonoscopy have a negative result with no obvious findings that can be attributed to a positive faecal immunochemical test (FIT). The aim of this work was to identify predictors for a negative colonoscopy in DCCSP participants with a positive FIT. METHOD: We included 73 655 FIT-positive DCCSP participants using the Danish Colorectal Cancer Screening Database and linked their screening results with data from several other national health registers. We stratified participants by all predictors, and compared them using multivariate logistic regression analysis. Results are reported as odds ratios (ORs). RESULTS: We found that having a condition linked to gastrointestinal bleeding, for example fissures, haemorrhoids and inflammatory bowel disease, was strongly associated with the probability of having a negative colonoscopy [OR 2.77 (95% CI 2.59, 2.96)]. FIT concentration was inversely related to the probability of a negative colonoscopy, the OR decreased steadily from 0.79 (95% CI 0.75, 0.83) in the 40-59 µg/g group, to 0.44 (95% CI 0.42, 0.46) in the ≥200 µg/g group. Women had a 1.64 (95% CI 1.59, 1.70) times higher probability of a negative colonoscopy than men. CONCLUSION: Our findings indicate that baseline conditions linked to gastrointestinal bleeding are an associating factor with having a negative colonoscopy. The same is true for low FIT concentration and female sex. Further studies with similar findings could suggest that an incorporation of these factors into a personalized screening approach by differentiating between diagnostic modalities could improve the process for the participant while alleviating the health care system.

The Effect of Enzymatic Treatment with Mutanase, Beta-Glucanase, and DNase on a Saliva-Derived Biofilm Model.

INTRODUCTION: The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase, and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model. METHODS: Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides. RESULTS: The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0% ± 1.3% SD vs. 36.9% ± 21.9% SD and 48.2% ± 14.9% SD). For biofilm removal, significant reductions of up to 73.2% ± 5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes. CONCLUSION: Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.

Effect of Supplementing a Bacillus Multi-Strain Probiotic to a Post-Weaning Diet on Nutrient Utilisation and Nitrogen Retention of Piglets.

Probiotics are suggested to improve pig health, nutrient utilisation, performance, and they may reduce nitrogen (N) pollution. However, the effectivity of a single strain might be different from that of a multi-strain. The study was conducted to investigate the effect of a novel Bacillus multi-strain on nutrient digestibility, energy utilisation, and N retention in weaned piglets using an European diet. The experiment consisted of a control diet (CD) and a supplemented diet (SD). The probiotic used for SD consisted of B. amyloliquefaciens-516 and B. subtilis-541. A total of eight boars/treatment were weaned (day 0; 8.5 kg body weight). Only boars were used to ease the collection of urine. Until day 10, piglets were fed ad libitum and were housed in pairs; from day 11, piglets were fed semi ad libitum (feeding level 3.2× metabolic body weight) and were housed individually. From day 14, faecal and urine were collected twice daily. Piglets were humanely euthanised at day 19 (15.0 kg bodyweight) after which the jejunum, ileum, and colon content were collected. In faeces, the apparent total tract digestibility (ATTD) of, amongst others, DM, organic matter (OM), crude protein (CP), non-starch polysaccharides (NSP), and subsequently net energy (NE) were calculated using titanium dioxide as an indigestible marker. In the jejunum and ileum, the apparent digestibility of CP was estimated, and in the ileum, the apparent AA digestibility was measured. In urine, the N content was measured to determine N retention. The volatile fatty acid (VFA), branched-chain fatty acid (BCFA), and lactic acid content were analysed in the colon and faeces. The apparent CP digestibility in the jejunum and ileum was not affected by treatment (p > 0.05), and no effect was observed on the apparent ileal digestibility of AA (p > 0.05). Supplementation with the multi-strain probiotic improved the ATTD of DM (p = 0.01; +1.3%) and OM (p = 0.02; +1.2%) and tended to improve the ATTD of CP (p = 0.10; +2.2%) and NSP (p = 0.07; +1.9%). The multi-strain probiotic also improved the NE value (p = 0.02; +0.2 MJ/kg DM) and improved N retention (p = 0.05; +1.6%). Supplementation did not influence the VFA, BCFA, and lactic acid content in the faeces (p > 0.05). However, in the colon, supplementation did influence the lactic acid content (lower; p = 0.01) and tended to influence the valeric acid content (higher; p = 0.09). In conclusion, results from the current study suggest that the multi-strain probiotic has the potential to contribute to improve nutrient efficiency in weaned piglets. More research needs to be conducted to identify the impact of the improved nutrient utilisation on gut health in post-weaned pigs as well as environmental pollution.

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

The Effect of a Bacillus-Based Probiotic on Sow and Piglet Performance in Two Production Cycles.

The aim of this study was to assess the impact of Bacillus-based probiotic diets on reproduction performance, fecal scores, microflora, and economic factors in lactating sows and suckling piglets across two productive cycles. A total of 96 sows, reared in a continuous farrowing system for two full cycles, were divided into two groups: a control group and an experimental group. Sows were fed a basal diet without the probiotic or a diet supplemented with viable bacterial spores. At seven days of age, control group piglets were offered standard creep feed, whereas piglets in the experimental (probiotic) group received a diet containing the probiotic fed to their dams. Sows receiving probiotic-supplemented diets were characterized by significantly higher (p ≤ 0.05) average daily feed intake in lactation, lower (p ≤ 0.01) body weight (BW) loss during lactation, and reduced loss of backfat thickness as well as higher body condition score after lactation. Dietary probiotic supplementation increased (p ≤ 0.01) birth weight, total creep feed consumption, litter weight gain, and piglet weaning weight. The probiotic also improved (p ≤ 0.01) overall fecal scores, decreased total E. coli count on day seven and Clostridium perfringens count (trend) in sucking piglets. The total feed cost per weaned piglet was lower in the experimental (probiotic) group. Supplementing the diet with a probiotic containing Bacillus strains improved the reproductive performance of sows and the performance and health of piglets.

A New Device for In Situ Dental Biofilm Collection Additively Manufactured by Direct Metal Laser Sintering and Vat Photopolymerization.

Dental biofilms are complex medical biofilms that cause caries, the most prevalent disease of humankind. They are typically collected using handcrafted intraoral devices with mounted carriers for biofilm growth. As the geometry of handcrafted devices is not standardized, the shear forces acting on the biofilms and the access to salivary nutrients differ between carriers. The resulting variability in biofilm growth renders the comparison of different treatment modalities difficult. The aim of the present work was to design and validate an additively manufactured intraoral device with a dental bar produced by direct metal laser sintering and vat photopolymerized inserts with standardized geometry for the mounting of biofilm carriers. Additive manufacturing reduced the production time and cost, guaranteed an accurate fit of the devices and facilitated the handling of carriers without disturbing the biofilm. Biofilm growth was robust, with increasing thickness over time and moderate inter- and intraindividual variation (coefficients of variance 0.48-0.87). The biofilms showed the typical architecture and composition of dental biofilms, as evidenced by confocal microscopy and 16S rRNA gene sequencing. Deeper inserts offering increased protection from shear tended to increase the biofilm thickness, whereas prolonged exposure to sucrose during growth increased the biofilm volume but not the thickness. Ratiometric pH imaging revealed considerable pH variation between participants and also inside single biofilms. Intraoral devices for biofilm collection constitute a new application for medical additive manufacturing and offer the best possible basis for studying the influence of different treatment modalities on biofilm growth, composition, and virulence. The Clinical Trial Registration number is: 1-10-72-193-20.

Effect of a Bacillus-Based Probiotic on Performance and Nutrient Digestibility When Substituting Soybean Meal with Rapeseed Meal in Grower-Finisher Diets.

The objective of the present study was to test the hypothesis of B. subtilis and B. licheniformis supplementation to a negative control diet in comparison to a standard control diet, had the potential to improve the performance and nutrient digestibility of growing-finishing pigs. For this purpose, 384 fattening pigs of 85 d of age were allotted to three treatments: a standard diet, a negative control (NC) diet (5% soybean meal replaced by 5% rapeseed meal), or a NC diet + probiotic. After reaching a body weight of approximately 110 kg, all animals going to the slaughterhouse (87% of total pigs) were selected to measure carcass quality. Moreover, the apparent total tract digestibility of protein was evaluated at the end of the grower period. The results of this study indicate that supplementation of the tested Bacillus-based probiotic significantly improved average daily gain (ADG, +14.6%) and Feed:gain ratio (F:G, -9.9%) during the grower phase compared to the NC diet. The improvement observed during the grower phase was maintained for the whole fattening period (ADG, +3.9%). Probiotic supplementation significantly improved the total apparent faecal digestibility of dry matter and crude protein in pigs at the end of the grower period. The improvements observed with the additive tested could indicate that supplementation of the Bacillus-based probiotic was able to counteract the lower level of crude protein and standardised ileal digestible amino acids in the NC diet by means of improved protein digestibility.

Bacillus velezensis DSM 33864 reduces Clostridioides difficile colonization without disturbing commensal gut microbiota composition.

Up to 25% of the US population harbor Clostridioides difficile in the gut. Following antibiotic disruption of the gut microbiota, C. difficile can act as an opportunistic pathogen and induce potentially lethal infections. Consequently, reducing the colonization of C. difficile in at-risk populations is warranted, prompting us to identify and characterize a probiotic candidate specifically targeting C. difficile colonization. We identified Bacillus velezensis DSM 33864 as a promising strain to reduce C. difficile levels in vitro. We further investigated the effects of B. velezensis DSM 33864 in an assay including human fecal medium and in healthy or clindamycin-treated mouse models of C. difficile colonization. The addition of B. velezensis DSM 33864 to human fecal samples was shown to reduce the colonization of C. difficile in vitro. This was supported in vivo where orally administered B. velezensis DSM 33864 spores reduced C. difficile levels in clindamycin-treated mice. The commensal microbiota composition or post-antibiotic reconstitution was not impacted by B. velezensis DSM 33864 in human fecal samples, short-, or long-term administration in mice. In conclusion, oral administration of B. velezensis DSM 33864 specifically reduced C. difficile colonization in vitro and in vivo without adversely impacting the commensal gut microbiota composition.

Feeding Bacillus-based probiotics to gestating and lactating sows is an efficient method for improving immunity, gut functional status and biofilm formation by probiotic bacteria in piglets at weaning.

The effects of dietary probiotic supplementation with viable Bacillus subtilis and Bacillus amyloliquefaciens spores on sow performance, immunity, gut functional status and biofilm formation by probiotic bacteria in piglets at weaning were investigated. Ninety-six sows reared in a continuous farrowing system for one full cycle were fed gestation diets during the first 90 d of pregnancy and lactation diets until the end of lactation. The sows were fed a basal diet without probiotics (control; n = 48) or a diet supplemented with viable spores (1.1 × 109 CFU/kg of feed) (probiotic; n = 48). At 7 d of age, sucking piglets (n = 12/group) were provided prestarter creep feed until weaning at 28 d of age. The piglets in the probiotic group were supplemented with the same probiotic and dosage as their dams. Blood and colostrum collected from sows and ileal tissues collected from piglets on the day of weaning were used for analyses. Probiotics increased the weight of piglets (P = 0.077), improved the weaning weight (P = 0.039) and increased both the total creep feed consumption (P = 0.027) and litter gain (P = 0.011). Probiotics also improved the faecal score in the second (P = 0.013) week of life. The immunoglobulin G (IgG) concentrations in sow blood at farrowing and the IgM concentrations in piglet blood at weaning were higher in the probiotic group than in the control group (P = 0.046). The piglets from the probiotic-treated sows showed a higher IgM concentration in the ileal mucosa (P = 0.050) and a lower IgG concentration in the ileal mucosa (P = 0.021) compared with the piglets from control sows. The probiotic-treated piglets had a thicker ileal mucosa (P = 0.012) due to the presence of longer villi and larger Peyer's patches (P < 0.001). B. subtilis and B. amyloliquefaciens were detected in the probiotic-treated piglets but not the control piglets; these bacteria were present in the digesta and villus structures and formed structures resembling biofilms. Overall, Bacillus-based probiotic supplementation improves the health indices of sows and their piglets.

Risk factors for and pregnancy outcomes after SARS-CoV-2 in pregnancy according to disease severity: A nationwide cohort study with validation of the SARS-CoV-2 diagnosis.

INTRODUCTION: We identified risk factors and outcomes associated with SARS-CoV-2 infection in pregnancy in a universally tested population according to disease severity and validated information on SARS-CoV-2 during pregnancy in national health registers in Denmark. MATERIAL AND METHODS: Cohort study using data from national registers and medical records including all pregnancies between March 1, 2020 and February 28, 2021. We compared women with a validated positive SARS-CoV-2 test during pregnancy with non-infected pregnant women. Risk factors and pregnancy outcomes were assessed by Poisson and Cox regression models and stratified according to disease severity defined by hospital admission status and admission reason (COVID-19 symptoms or other). Using medical record data on actual period of pregnancy, we calculated predictive values of the SARS-CoV-2 diagnosis in pregnancy in the registers. RESULTS: SARS-CoV-2 infection was detected in 1819 (1.6%) of 111 185 pregnancies. Asthma was associated with infection (relative risk [RR] 1.63, 95% confidence interval [CI] 1.28-2.07). Risk factors for severe COVID-19 disease requiring hospital admission were high body mass index (median ratio 1.06, 95% CI 1.04-1.09), asthma (RR 7.47, 95% CI 3.51-15.90) and gestational age at the time of infection (gestational age 28-36 vs < 22: RR 3.53, 95% CI 1.75-7.10). SARS-CoV-2-infected women more frequently had hypertensive disorders in pregnancy (adjusted hazard ratio [aHR] 1.31, 95% CI 1.04-1.64), early pregnancy loss (aHR 1.37, 95% CI 1.00-1.88), preterm delivery before gestational age 28 (aHR 2.31, 95% CI 1.01-5.26), iatrogenically preterm delivery before gestational age 37 (aHR 1.49, 95% CI 1.01-2.19) and small-for-gestational age children (aHR 1.28, 95% CI 1.05-1.54). The associations were stronger among women admitted to hospital for any reason. The validity of the SARS-CoV-2 diagnosis in relation to pregnancy in the registers compared with medical records showed a negative predictive value of 99.9 (95% CI 99.9-100.0) and a positive predictive value of 82.1 (95% CI 80.4-83.7). CONCLUSIONS: Women infected with SARS-CoV-2 during pregnancy were at increased risk of hypertensive disorders in pregnancy, early pregnancy loss, preterm delivery and having children small for gestational age. The validity of Danish national registers was acceptable for identification of SARS-CoV-2 infection during pregnancy.

Effect of an enzyme-containing mouthwash on the dental biofilm and salivary microbiome in patients with fixed orthodontic appliances: a randomized placebo-controlled pilot trial.

BACKGROUND: Mouthwashes containing oral antiseptics or enzymes are suggested suitable for controlling biofilm accumulation in patients with fixed appliances and thereby limiting unwanted side effects during the orthodontic treatment. OBJECTIVES: To evaluate the effect of an enzyme-based mouthwash on the amount of dental biofilm and the composition of the salivary microbiome in patients undergoing treatment with fixed orthodontic appliances. TRIAL DESIGN: Randomized double-blind placebo-controlled trial. MATERIAL AND METHODS: In total, 35 young adolescents (14-18 years) under treatment with fixed appliances were consecutively enrolled and randomly allocated to an experimental or a placebo group by opening a computer-generated numbered envelope. The subjects were instructed to rinse twice daily during an intervention period of 8 days with experimental mouthwash or placebo without active enzymes. Unstimulated whole saliva samples were collected at baseline and after 8 days. The participants and examiner were blinded for the allocation. The primary outcome was the Orthodontic Plaque Index (OPI) and the secondary was the composition of the salivary microbiome. RESULTS: In total, 28 adolescents (21 females and 7 males) completed the trial and there were no differences in age, clinical, or microbial findings between the test (n = 14) and the placebo group (n = 14) at baseline. We found a decreased OPI in the test group after 8 days and the difference was statistically significant compared with the placebo group (P < 0.05). There were no significant treatment effects on the richness and global composition of the salivary microbiome. HARMS: In total, one participant in the test group claimed nausea and abandoned the project. In total, two participants did not like the taste of the mouthwash but used it as instructed. No other adverse events or side effects were reported. LIMITATIONS: Short-term pilot trials may by nature be sensitive for selection and performance biases and are not designed to unveil persisting effects. CONCLUSION: Daily use of enzyme-containing mouthwash reduced the amount of dental biofilm in adolescents under treatment with the fixed orthodontic appliances, without affecting the composition of the salivary microbiota. ETHICAL APPROVAL: Approved by the Regional Ethical Board, Lund, Sweden (Dnr 2020-05221). CLINICAL TRIAL REGISTRATION: NCT05033015.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Fungal lysozyme leverages the gut microbiota to curb DSS-induced colitis.

Colitis is characterized by colonic inflammation and impaired gut health. Both features aggravate obesity and insulin resistance. Host defense peptides (HDPs) are key regulators of gut homeostasis and generally malfunctioning in above-mentioned conditions. We aimed here to improve bowel function in diet-induced obesity and chemically induced colitis through daily oral administration of lysozyme, a well-characterized HDP, derived from Acremonium alcalophilum.C57BL6/J mice were fed either low-fat reference diet or HFD ± daily gavage of lysozyme for 12 weeks, followed by metabolic assessment and evaluation of colonic microbiota encroachment. To further evaluate the efficacy of intestinal inflammation, we next supplemented chow-fed BALB/c mice with lysozyme during Dextran Sulfate Sodium (DSS)-induced colitis in either conventional or microbiota-depleted mice. We assessed longitudinal microbiome alterations by 16S amplicon sequencing in both models.Lysozyme dose-dependently alleviated intestinal inflammation in DSS-challenged mice and further protected against HFD-induced microbiota encroachment and fasting hyperinsulinemia. Observed improvements of intestinal health relied on a complex gut flora, with the observation that microbiota depletion abrogated lysozyme's capacity to mitigate DSS-induced colitis.Akkermansia muciniphila associated with impaired gut health in both models, a trajectory that was mitigated by lysozyme administration. In agreement with this notion, PICRUSt2 analysis revealed specific pathways consistently affected by lysozyme administration, independent of vivarium, disease model and mouse strain.Taking together, lysozyme leveraged the gut microbiota to curb DSS-induced inflammation, alleviated HFD-induced gastrointestinal disturbances and lowered fasting insulin levels in obese mice. Collectively, these data present A. alcalophilum-derived lysozyme as a promising candidate to enhance gut health.

SARS-CoV-2 infection in pregnancy in Denmark-characteristics and outcomes after confirmed infection in pregnancy: A nationwide, prospective, population-based cohort study.

INTRODUCTION: Assessing the risk factors for and consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy is essential to guide clinical care. Previous studies on SARS-CoV-2 infection in pregnancy have been among hospitalized patients, which may have exaggerated risk estimates of severe outcomes because all cases of SARS-CoV-2 infection in the pregnant population were not included. The objectives of this study were to identify risk factors for and outcomes after SARS-CoV-2 infection in pregnancy independent of severity of infection in a universally tested population, and to identify risk factors for and outcomes after severe infection requiring hospital admission. MATERIAL AND METHODS: This was a prospective population-based cohort study in Denmark using data from the Danish National Patient Register and Danish Microbiology Database and prospectively registered data from medical records. We included all pregnancies between March 1 and October 31, 2020 and compared women with a positive SARS-CoV-2 test during pregnancy to non-infected pregnant women. Cases of SARS-CoV-2 infection in pregnancy were both identified prospectively and through register linkage to ensure that all cases were identified and that cases were pregnant during infection. Main outcome measures were pregnancy, delivery, maternal, and neonatal outcomes. Severe infection was defined as hospital admission due to coronavirus disease 2019 (COVID-19) symptoms. RESULTS: Among 82 682 pregnancies, 418 women had SARS-CoV-2 infection during pregnancy, corresponding to an incidence of 5.1 per 1000 pregnancies, 23 (5.5%) of which required hospital admission due to COVID-19. Risk factors for infection were asthma (odds ratio [OR] 2.19, 95% CI 1.41-3.41) and being foreign born (OR 2.12, 95% CI 1.70-2.64). Risk factors for hospital admission due to COVID-19 included obesity (OR 2.74, 95% CI 1.00-7.51), smoking (OR 4.69, 95% CI 1.58-13.90), infection after gestational age (GA) 22 weeks (GA 22-27 weeks: OR 3.77, 95% CI 1.16-12.29; GA 28-36 weeks: OR 4.76, 95% CI 1.60-14.12), and having asthma (OR 4.53, 95% CI 1.39-14.79). We found no difference in any obstetrical or neonatal outcomes. CONCLUSIONS: Only 1 in 20 women with SARS-CoV-2 infection during pregnancy required admission to hospital due to COVID-19. Risk factors for admission comprised obesity, smoking, asthma, and infection after GA 22 weeks. Severe adverse outcomes of SARS-CoV-2 infection in pregnancy were rare.