Comparative study of droplet-digital PCR and absolute Q digital PCR for ctDNA detection in early-stage breast cancer patients.

BACKGROUND: Analysis of circulating tumor DNA (ctDNA) is increasingly used for clinical decision-making in oncology. However, ctDNA could represent ≤ 0.1 % of cell-free DNA in early-stage tumors and its detection requires high-sensitive techniques such as digital PCR (dPCR). METHODS: In 46 samples from patients with early-stage breast cancer, we compared two leading dPCR assays for ctDNA analysis: QX200 droplet digital PCR (ddPCR) system from Bio-Rad which is the gold-standard in the field, and Absolute Q plate-based digital PCR (pdPCR) system from Thermo Fisher Scientific which has not been reported before. We analyzed 5 mL of baseline plasma samples prior to any treatment. RESULTS: Both systems displayed a comparable sensitivity with no significant differences observed in mutant allele frequency. In fact, ddPCR and pdPCR possessed a concordance > 90 % in ctDNA positivity. Nevertheless, ddPCR exhibited higher variability and a longer workflow. Finally, we explored the association between ctDNA levels and clinicopathological features. Significantly higher ctDNA levels were present in patients with a Ki67 score > 20 % or with estrogen receptor-negative or triple-negative breast cancer subtypes. CONCLUSION: Both ddPCR and pdPCR may constitute sensitive and reliable tools for ctDNA analysis with an adequate agreement in early-stage breast cancer patients.

Fermented Goat's Milk Contributes to the Recovery of Iron Deficiency Anemia via Modulation of the Gut Microbiome.

Iron deficiency anemia (IDA) is a global public health concern affecting 1.6 billion people worldwide. The administration of iron supplements during the treatment of IDA adversely affects the intestinal barrier function and the composition and functionality of the intestinal microbiome, both of which are already altered during IDA. For this reason, it is of great interest to develop nutritional strategies aimed at alleviating these gut alterations associated with IDA and its treatment. In this sense, fermented goat's milk (FGM) was studied due to its nutritional quality. Our findings showed that in anemic animals the consumption of a FGM-based diet, compared to a standard diet, had positive modulatory effects on the intestinal microbiome. FGM-based diet restored intestinal dysbiosis, the intestinal barrier functionality, and bacterial translocation, contributing to a more efficient recovery of IDA. Therefore, FGM is a useful nutritional tool to ease intestinal alterations occurring during IDA and during its treatment.

Burden in caregivers of primary care patients with dementia: influence of neuropsychiatric symptoms according to disease stage (NeDEM project).

BACKGROUND: Caregiver burden is related to personal factors and patient characteristics and is greater when neuropsychiatric symptoms (NPSs) are present. OBJECTIVE: Estimate the prevalence of burden among caregivers of dementia patients and its association with NPSs and identify NPSs causing greater caregiver distress according to dementia stage. METHODS: A cross-sectional observational study in caregivers of noninstitutionalized dementia patients was conducted. Caregiver variables were sociodemographic, time of care, NPS-associated distress based on the Neuropsychiatric Inventory Caregiver Distress Scale (NPI-D) and burden based on the Zarit Burden Interview (ZBI). Patient variables were time since disease onset, Global Deterioration Scale (GDS) disease stage, functional assessment and NPS presence and intensity according to the Neuropsychiatric Inventory (NPI). The mean ZBI score, prevalence of burden and NPI-D score with 95% CIs at each dementia stage were estimated. Factors associated with burden were identified by multivariate analysis. RESULTS: Of the 125 caregivers included, 77.6% were women, with a mean age of 60.7 (± 14.3) years; 78.4% (95%CI: 71.0; 86.0) experienced burden. The mean ZBI score was 12.3 (95%CI: 11.6; 12.9) and increased according to NPS number (p = 0.042). The NPSs causing the most burden were disinhibition (93.5%), irritability (87.3%) and agitation (86.1%). Agitation, apathy, and sleep disorders were the NPSs generating the greatest overall caregiver distress; depression (max NPI-D 1.9), hyperactivity (max NPI-D 2.1), and psychosis symptoms (max NPI-D 1.6) generated the greatest distress at stage GDS 3, stages GDS 4-5, and stages GDS 6-7, respectively. The NPI score (OR = 1.0, 95%CI 1.0; 1.1), intensity of irritability (OR = 1.2, 95%CI 1.0; 1.6), disinhibition (OR = 2.6, 95%CI 1.1; 5.8) and hyperactivity subsyndrome (OR = 1.1, 95%CI 1.0; 1.2) were associated with caregiver burden. Other associated factors were female gender (OR = 6.0, 95%CI 1.6; 22.8), ≥ 8 h daily care (OR = 5.6, 95%CI 1.4; 22.8), working outside the home (OR = 7.6, 95%CI 1.8; 31.8), living with the patient (OR = 4.5, 95%CI 1.1; 19.6), kinship (OR = 5.4, 95%CI 1.0; 28.2) and lower patient education (OR = 8.3, 95%CI 2.3; 30.3). CONCLUSIONS: The burden on caregivers of dementia patients is high and associated with NPS presence and intensity. Disinhibition and irritability caused the highest burden. Depression, hyperactivity and psychosis produce more distress in mild, mild-moderate and severe dementia, respectively.

Benefits for cardiovascular system, bone density, and quality of life of a long-term hormone therapy in hysterectomized women: a 20-year follow-up study.

OBJECTIVE: The safety, consequences, and dosage of long-term hormone therapy (HT) for postmenopausal women remain unclear. Our aim was to analyze the effects of HT after 20 years of therapy in women after hysterectomy, focusing on the symptoms of menopause, blood pressure, lipid profiles, and bone density. METHODS: A prospective observational longitudinal study was designed. The initial transdermal estradiol dose was reduced in half (0.025 mg/d) at 60 years of age. Different parameters including demographic, cardiovascular, bone density, and metabolic variables, as well as quality of life characteristics, were analyzed using bivariate analyses. Multivariate generalized estimating equations for longitudinal data were fitted for differences over time and between doses (<60 vs ≥60 y) using the R package geepack. RESULTS: After 20 years of HT, the mean age of 56 studied hysterectomized women was 67.1 years. The mean Kupperman index score decreased from 26.7 to 12.0 ( P < 0.001). A trend with total and low-density lipoprotein cholesterol reduction and high-density lipoprotein cholesterol increase was observed over time. A decrease in very-low-density lipoprotein cholesterol ( P = 0.05) and an increase in T score vertebral densitometry ( P = 0.014) were detected after HT. No changes in health outcome were detected in women older than 60 years with the reduced dose of HT. Breast cancer was the reason for dropouts in 0.02% women. CONCLUSIONS: HT for up to 20 years after hysterectomy may be beneficial for bone and cardiovascular health and for the overall quality of life. Our data suggest the importance of evaluating the dose and the timing of HT.

Sulforaphane Reduces the Chronic Inflammatory Immune Response of Human Dendritic Cells.

BACKGROUND: Sulforaphane (SFN) is an isothiocyanate of vegetable origin with potent antioxidant and immunomodulatory properties. The characterization of its pleiotropic activity in human dendritic cells (DCs) is poorly summarized. The aim of this work was to study the immunomodulatory power of SFN in response to an inflammatory microenvironment on human monocyte-derived DCs (moDCs). METHODS: We studied the immunological response induced by SFN. Apoptosis and autophagy assays were performed using flow cytometry on moDCs and a cancer cell line (THP-1). These included moDC maturation, lymphocyte proliferation and cytokine production under different experimental conditions. We investigated whether these results were associated with an inflammatory microenvironment induced by lipopolysaccharides (LPSs). RESULTS: Our results demonstrated that SFN could interact with moDCs, significantly reducing the autophagy process and enhancing apoptosis similarly to cancer cell line THP-1 cells in a chronic inflammatory microenvironment. Under chronic inflammation, SFN modulated the phenotypical characteristics of moDCs, reducing the expression of all markers (CD80, CD83, CD86, HLA-DR and PD-L1). SFN significantly reduced the Th2 proliferative response, with a decrease in the IL-9 and IL-13 levels. Although we did not observe any changes in the regulatory proliferative response, we noted an increase in the IL-10 levels. CONCLUSIONS: These findings demonstrate that SFN exerts protective effects against LPS-induced inflammation via the modulation of moDCs/T cells towards a regulatory profile. SFN may be a potential candidate for the treatment of pathologies with an inflammatory profile.

A novel drug specific mRNA biomarker predictor for selection of patients responding to dovitinib treatment of advanced renal cell carcinoma and other solid tumors.

PURPOSE: Dovitinib is a receptor tyrosine kinase inhibitor of VEGFR1-3, PDGFR, FGFR1/3, c-KIT, FLT3 and topoisomerase 1 and 2. The drug response predictor (DRP) biomarker algorithm or DRP-Dovitinib is being developed as a companion diagnostic to dovitinib and was applied retrospectively. PATIENTS AND METHODS: Archival tumor samples were obtained from consenting patients in a phase 3 trial comparing dovitinib to sorafenib in renal cell carcinoma patients and the DRP-Dovitinib was applied. The biomarker algorithm combines the expression of 58 messenger RNAs relevant to the in vitro sensitivity or resistance to dovitinib, including genes associated with FGFR, PDGF, VEGF, PI3K/Akt/mTOR and topoisomerase pathways as well as ABC drug transport, and provides a likelihood score between 0-100%. RESULTS: The DRP-Dovitinib divided the dovitinib treated RCC patients into two groups, sensitive (n = 49, DRP score >50%) or resistant (n = 86, DRP score ≤ 50%) to dovitinib. The DRP sensitive population was compared to the unselected sorafenib arm (n = 286). Median progression-free survival (PFS) was 3.8 months in the DRP sensitive dovitinib arm and 3.6 months in the sorafenib arm (hazard ratio 0.71, 95% CI 0.51-1.01). Median overall survival (OS) was 15.0 months in the DRP sensitive dovitinib arm and 11.2 months in the sorafenib arm (hazard ratio 0.69, 95% CI 0.48-0.99). The observed clinical benefit increased with increasing DRP score. At a cutoff of 67% the median OS was 20.6 months and the median PFS was 5.7 months in the dovitinib arm. The results were confirmed in five smaller phase II trials of dovitinib which showed a similar trend. CONCLUSION: The DRP-Dovitinib shows promise as a potential biomarker for identifying advanced RCC patients most likely to experience clinical benefit from dovitinib treatment, subject to confirmation in an independent prospective trial of dovitinib in RCC patients.

Factors influencing age of common allergen introduction in early childhood.

Objectives: We evaluated factors influencing the timing of allergen introduction in the U.S., including updated peanut introduction guidelines. Study design: The Gastrointestinal Microbiome and Allergic Proctocolitis (GMAP) study is a prospective observational cohort in suburban Massachusetts. Infants' caregivers enrolled between 2014 and 2017, and they reported when they introduced common allergens to their child. Multivariable linear and survival regression analyses were used to examine factors influencing time of introduction of allergens. Results: By 9 months, children old enough to be potentially affected by NIAID's 2017 peanut introduction guidelines were more often introduced to peanut than children enrolled well before guidelines publication [54% vs. 42%, OR: 1.63, CI: (1.03, 2.57), P = 0.03]. At any given time, Black children were 73% [HR: 0.27, CI: (0.11, 0.69), P = 0.006] less likely to be introduced to peanut as early as White children. Asian children were, respectively, 36% [HR: 0.64, CI: (0.47, 0.86), P = 0.003] and 26% [HR: 0.74, CI: (0.55, 0.97), P = 0.03] less likely to be introduced to peanut and egg as early as White children. A first child was 27% [HR: 1.27, CI: (1.04, 1.56), P = 0.02] more likely to have been introduced to peanut earlier than a non-first child. There was no association between age of introduction and sex, gestational age, family history of food allergy, or other allergic comorbidities. Conclusion: Updated introduction guidelines, race, and birth order all influenced earlier introduction of peanut. Further studies to evaluate current practices for allergen introduction with a focus on potential disparities are needed.

Longitudinal assessment of early growth in children with IgE- and non-IgE-mediated food allergy in a healthy infant cohort.

BACKGROUND: There are conflicting associations reported between food allergies (FAs) and poor growth, with some indication that children with multiple FAs are at highest risk. OBJECTIVE: We analyzed longitudinal weight-for-length (WFL) trajectories from our healthy cohort to evaluate growth in children with IgE-mediated FAs and food protein-induced allergic proctocolitis (FPIAP), a non-IgE-mediated FA. METHODS: Our observational cohort of 903 healthy newborn infants was prospectively enrolled to evaluate the development of FAs. Longitudinal mixed effects modeling was used to compare differences in WFL among children with IgE-FA and FPIAP, compared with unaffected children, through age 2. RESULTS: Among the 804 participants who met inclusion criteria, FPIAP cases had significantly lower WFL than unaffected controls during active disease, which resolved by 1 year of age. In contrast, children with IgE-FA had significantly lower WFL than unaffected controls after 1 year. We also found that children with IgE-FA to cow's milk had significantly lower WFL over the first 2 years of age. Children with multiple IgE-FAs had markedly lower WFL over the first 2 years of age. CONCLUSION: Children with FPIAP have impaired growth during active disease in the first year of age which resolves, whereas children with IgE-FA, particularly those with multiple IgE-FA, have impaired growth more prominently after the first year of age. It may be appropriate to focus nutritional assessment and interventions accordingly during these higher risk periods in these patient populations.

Early infancy dysbiosis in food protein-induced enterocolitis syndrome: A prospective cohort study.

BACKGROUND: The microbiome associations of food protein-induced enterocolitis syndrome (FPIES) are understudied. We sought to prospectively define the clinical features of FPIES in a birth cohort, and investigate for the evidence of gut dysbiosis. METHODS: We identified children diagnosed with FPIES in the Gastrointestinal Microbiome and Allergic Proctocolitis Study, a healthy infant cohort. Children were assessed and stools were collected at each well child visit. The clinical features of the children with FPIES were summarized. Stool microbiome was analyzed using 16S rRNA sequencing comparing children with and without FPIES. RESULTS: Of the 874 children followed up for 3 years, 8 FPIES cases (4 male) were identified, yielding a cumulative incidence of 0.92%. The most common triggers were oat and rice (n = 3, each) followed by milk (n = 2). The children with FPIES were more likely to have family history of food allergy (50% vs. 15.9% among unaffected, p = .03). The average age of disease presentation was 6 months old. During the first 6 months of life, stool from children with FPIES contained significantly less Bifidobacterium adolescentis, but more pathobionts, including Bacteroides spp. (especially Bacteroides fragilis), Holdemania spp., Lachnobacterium spp., and Acinetobacter lwoffii. The short-chain fatty acid (SCFA)-producing Bifidobacterium shunt was expressed significantly less in the stool from FPIES children. CONCLUSIONS: In this cohort, the cumulative incidence over the 3-year study period was 0.92%. During the first 6 months of life, children with FPIES had evidence of dysbiosis and SCFA production pathway was expressed less in their stool, which may play an important role in the pathogenesis of FPIES.

Both abundant and rare fungi colonizing Fagus sylvatica ectomycorrhizal root-tips shape associated bacterial communities.

Ectomycorrhizal fungi live in close association with their host plants and form complex interactions with bacterial/archaeal communities in soil. We investigated whether abundant or rare ectomycorrhizal fungi on root-tips of young beech trees (Fagus sylvatica) shape bacterial/archaeal communities. We sequenced 16S rRNA genes and fungal internal transcribed spacer regions of individual root-tips and used ecological networks to detect the tendency of certain assemblies of fungal and bacterial/archaeal taxa to inhabit the same root-tip (i.e. modularity). Individual ectomycorrhizal root-tips hosted distinct fungal communities associated with unique bacterial/archaeal communities. The structure of the fungal-bacterial/archaeal association was determined by both, dominant and rare fungi. Integrating our data in a conceptual framework suggests that the effect of rare fungi on the bacterial/archaeal communities of ectomycorrhizal root-tips contributes to assemblages of bacteria/archaea on root-tips. This highlights the potential impact of complex fine-scale interactions between root-tip associated fungi and other soil microorganisms for the ectomycorrhizal symbiosis.

Food-specific immunoglobulin A does not correlate with natural tolerance to peanut or egg allergens.

ImmunoglobulinA (IgA) is the predominant antibody isotype in the gut, where it regulates commensal flora and neutralizes toxins and pathogens. The function of food-specific IgA in the gut is unknown but is presumed to protect from food allergy. Specifically, it has been hypothesized that food-specific IgA binds ingested allergens and promotes tolerance by immune exclusion; however, the evidence to support this hypothesis is indirect and mixed. Although it is known that healthy adults have peanut-specific IgA in the gut, it is unclear whether children also have gut peanut-specific IgA. We found in a cohort of non-food-allergic infants (n = 112) that there is detectable stool peanut-specific IgA that is similar to adult quantities of gut peanut-specific IgA. To investigate whether this peanut-specific IgA is associated with peanut tolerance, we examined a separate cohort of atopic children (n = 441) and found that gut peanut-specific IgA does not predict protection from development of future peanut allergy in infants nor does it correlate with concurrent oral tolerance of peanut in older children. We observed higher plasma peanut-specific IgA in those with peanut allergy. Similarly, egg white-specific IgA was detectable in infant stools and did not predict egg tolerance or outgrowth of egg allergy. Bead-based epitope assay analysis of gut peanut-specific IgA revealed similar epitope specificity between children with peanut allergy and those without; however, gut peanut-specific IgA and plasma peanut-specific IgE had different epitope specificities. These findings call into question the presumed protective role of food-specific IgA in food allergy.

Longitudinal disease-associated gut microbiome differences in infants with food protein-induced allergic proctocolitis.

BACKGROUND: Complex interactions between the gut microbiome and immune cells in infancy are thought to be part of the pathogenesis for the marked rise in pediatric allergic diseases, particularly food allergies. Food protein-induced allergic proctocolitis (FPIAP) is commonly the earliest recognized non-immunoglobulin E (IgE)-mediated food allergy in infancy and is associated with atopic dermatitis and subsequent IgE-mediated food allergy later in childhood. Yet, a large prospective longitudinal study of the microbiome of infants with FPIAP, including samples prior to symptom onset, has not been done. RESULTS: Here, we analyzed 954 longitudinal samples from 160 infants in a nested case-control study (81 who developed FPIAP and 79 matched controls) from 1 week to 1 year of age by 16S rRNA ribosomal gene sequencing as part of the Gastrointestinal Microbiome and Allergic Proctocolitis (GMAP) study. We found key differences in the microbiome of infants with FPIAP, most strongly a higher abundance of a genus of Enterobacteriaceae and a lower abundance of a family of Clostridiales during the symptomatic period. We saw some of these significant taxonomic differences even prior to symptom onset. There were no consistent longitudinal differences in richness or stability diversity metrics between infants with FPIAP and healthy controls. CONCLUSIONS: This study is the first to identify differences in the infant gut microbiome in children who develop FPIAP, some even before they develop symptoms, and provides a foundation for more mechanistic investigation into the pathogenesis of FPIAP and subsequent food allergic diseases in childhood. Video abstract.

Curvicollide D Isolated from the Fungus Amesia sp. Kills African Trypanosomes by Inhibiting Transcription.

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on chemotherapy. Although the current drugs are effective, most of them are far from the modern concept of a drug in terms of toxicity, specificity and therapeutic regime. In a search for new molecules with trypanocidal activity, a high throughput screening of 2000 microbial extracts was performed. Fractionation of one of these extracts, belonging to a culture of the fungus Amesia sp., yielded a new member of the curvicollide family that has been designated as curvicollide D. The new compound showed an inhibitory concentration 50 (IC50) 16-fold lower in Trypanosoma brucei than in human cells. Moreover, it induced cell cycle arrest and disruption of the nucleolar structure. Finally, we showed that curvicollide D binds to DNA and inhibits transcription in African trypanosomes, resulting in cell death. These results constitute the first report on the activity and mode of action of a member of the curvicollide family in T. brucei.

Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

EasyMap - An Interactive Web Tool for Evaluating and Comparing Associations of Clinical Variables and Microbiome Composition.

One of the most common tasks in microbiome studies is comparing microbial profiles across various groups of people (e.g., sick vs. healthy). Routinely, researchers use multivariate linear regression models to address these challenges, such as linear regression packages, MaAsLin2, LEfSe, etc. In many cases, it is unclear which metadata variables should be included in the linear model, as many human-associated variables are correlated with one another. Thus, multiple models are often tested, each including a different set of variables, however the challenge of selecting the metadata variables in the final model remains. Here, we present EasyMap, an interactive online tool allowing for (1) running multiple multivariate linear regression models, on the same features and metadata; (2) visualizing the associations between microbial features and clinical metadata found in each model; and (3) comparing across the various models to identify the critical metadata variables and select the optimal model. EasyMap provides a side-by-side visualization of association results across the various models, each with additional metadata variables, enabling us to evaluate the impact of each metadata variable on the associated feature. EasyMap's interface enables filtering associations by significance, focusing on specific microbes and finding the robust associations that are found across multiple models. While EasyMap was designed to analyze microbiome data, it can handle any other tabular data with numeric features and metadata variables. EasyMap takes the common task of multivariate linear regression to the next level, with an intuitive and simple user interface, allowing for wide comparisons of multiple models to identify the robust microbial feature associations. EasyMap is available at http://yassour.rcs.huji.ac.il/easymap.

Glycine Receptor Subtypes and Their Roles in Nociception and Chronic Pain.

Disruption of the inhibitory control provided by the glycinergic system is one of the major mechanisms underlying chronic pain. In line with this concept, recent studies have provided robust proof that pharmacological intervention of glycine receptors (GlyRs) restores the inhibitory function and exerts anti-nociceptive effects on preclinical models of chronic pain. A targeted regulation of the glycinergic system requires the identification of the GlyR subtypes involved in chronic pain states. Nevertheless, the roles of individual GlyR subunits in nociception and in chronic pain are yet not well defined. This review aims to provide a systematic outline on the contribution of GlyR subtypes in chronic pain mechanisms, with a particular focus on molecular pathways of spinal glycinergic dis-inhibition mediated by post-translational modifications at the receptor level. The current experimental evidence has shown that phosphorylation of synaptic α1ß and α3ß GlyRs are involved in processes of spinal glycinergic dis-inhibition triggered by chronic inflammatory pain. On the other hand, the participation of α2-containing GlyRs and of ß subunits in pain signaling have been less studied and remain undefined. Although many questions in the field are still unresolved, future progress in GlyR research may soon open new exciting avenues into understanding and controlling chronic pain.

Negative schizotypy attenuates the effect of momentary stress on social dysfunction related to COVID-19 social distancing.

Schizotypy is associated with a broad range of motivational and social dysfunctions. However, there is reason to suspect that negative schizotypy may limit social dysfunction in specific contexts that typically increase isolation and loneliness. We analyzed whether positive, negative and disorganized facets of schizotypy would mitigate detrimental reactions to stress in the context of a global stressor (the COVID-19 pandemic) that has widely necessitated social and physical isolation. Responses to two measures of schizotypy were compared to ecological momentary assessments of social dysfunction for 85 undergraduates using multilevel modelling techniques. Negative schizotypy moderated the relation between momentary stress and loss of social support, such that negative schizotypy limited the magnitude of social dysfunction stemming from increases in momentary stress. This pattern was not seen for other facets of schizotypy. Implications for the treatment and measurement of schizotypy symptoms are discussed.