Except for Robust Outliers, Rapamycin Increases Lesion Burden in a Murine Model of Cerebral Cavernous Malformations.

Cerebral cavernous malformation (CCM) is a hemorrhagic cerebrovascular disease where lesions develop in the setting of endothelial mutations of CCM genes, with many cases also harboring somatic PIK3CA gain of function (GOF) mutations. Rapamycin, an mTORC1 inhibitor, inhibited progression of murine CCM lesions driven by Ccm gene loss and Pik3ca GOF, but it remains unknown if rapamycin is beneficial in the absence of induction of Pik3ca GOF. We investigated the effect of rapamycin at three clinically relevant doses on lesion development in the Ccm3-/-PDGFb-icreERPositive murine model of familial CCM disease, without induction of Pik3ca GOF. Lesion burden, attrition, and acute and chronic hemorrhaging were compared between placebo and rapamycin-treated mice. Plasma miRNome was compared to identify potential biomarkers of rapamycin response. Outlier, exceptionally large CCM lesions (> 2 SD above the mean lesion burden) were exclusively observed in the placebo group. Rapamycin, across all dosages, may have prevented the emergence of large outlier lesions. Yet rapamycin also appeared to exacerbate mean lesion burden of surviving mice when outliers were excluded, increased attrition, and did not alter hemorrhage. miR-30c-2-3p, decreased in rapamycin-treated mouse plasma, has gene targets in PI3K/AKT and mTOR signaling. Progression of outlier lesions in a familial CCM model may have been halted by rapamycin treatment, at the potential expense of increased mean lesion burden and increased attrition. If confirmed, this can have implications for potential rapamycin treatment of familial CCM disease, where lesion development may not be driven by PIK3CA GOF. Further studies are necessary to determine specific pathways that mediate potential beneficial and detrimental effects of rapamycin treatment, and whether somatic PIK3CA mutations drive particularly aggressive lesions.

Lactobacillus johnsonii N6.2 and Blueberry Phytophenols Affect Lipidome and Gut Microbiota Composition of Rats Under High-Fat Diet.

Obesity is considered a primary contributing factor in the development of many diseases, including cancer, diabetes, and cardiovascular illnesses. Phytochemical-rich foods, associated to healthy gastrointestinal microbiota, have been shown to reduce obesity and associated comorbidities. In the present article, we describe the effects of the probiotic Lactobacillus johnsonii N6.2 and blueberry extracts (BB) on the gut microbiota and lipid profile of rats under a high-fat (HF) or low-calorie (LC) diet. L. johnsonii was found to increase the levels of long chain fatty acids (LCFA) in the serum of all animals under HF diet, while reduced LCFA concentrations were observed in the adipose tissue of animals under HF diet supplemented with BB extracts. All animals under HF diet also showed lower protein levels of SREBP1 and SCAP when treated with L. johnsonii. The gut microbiota diversity, ß-diversity was significantly changed by L. johnsonii in the presence of BB. A significant reduction in α-diversity was observed in the ileum of animals under HF diet supplemented with L. johnsonii and BB, while increased α-diversity was observed in the ilium of animals under LC diet supplemented with L. johnsonii or BB. In summary, L. johnsonii and BB supplementation induced significant changes in gut microbiota diversity and lipid metabolism. The phospholipids pool was the lipidome component directly affected by the interventions. The ileum and colon microbiota showed clear differences depending on the diet and the treatments examined.

Genomic and Epigenetic Foundations of Neocentromere Formation.

Centromeres are essential to genome inheritance, serving as the site of kinetochore assembly and coordinating chromosome segregation during cell division. Abnormal centromere function is associated with birth defects, infertility, and cancer. Normally, centromeres are assembled and maintained at the same chromosomal location. However, ectopic centromeres form spontaneously at new genomic locations and contribute to genome instability and developmental defects as well as to acquired and congenital human disease. Studies in model organisms have suggested that certain regions of the genome, including pericentromeres, heterochromatin, and regions of open chromatin or active transcription, support neocentromere activation. However, there is no universal mechanism that explains neocentromere formation. This review focuses on recent technological and intellectual advances in neocentromere research and proposes future areas of study. Understanding neocentromere biology will provide a better perspective on chromosome and genome organization and functional context for information generated from the Human Genome Project, ENCODE, and other large genomics consortia.

Human milk microbes: Strategies to improve delivery to the infant.

Mother's own milk provides personalized nutrition and immune protection to the developing infant. The presence of healthy microbes plays an important role in the infant's gut by programming the microbiota and excluding potential pathogens. This review describes the important components in mother's own milk that contribute to its superiority for infant nutrition and suggest potential strategies to replicate these factors in alternative feedings when sufficient milk is unavailable. Current strategies to supplement, substitute and replicate mother's own milk including microbial restoration, use of unpasteurized donor human milk, probiotics and fortification are discussed. Critical work remains to be done in understanding the human milk microbiome and metabolome and in improving lactation support for mothers of preterm infants. Increasing delivery of mother's own milk and milk components to infants would likely positively impact infant mortality and health worldwide.

Frozen Mother's Own Milk Can Be Used Effectively to Personalize Donor Human Milk.

Feeding preterm infants mother's own milk (MOM) lowers rates of sepsis, decreases necrotizing enterocolitis, and shortens hospital stay. In the absence of freshly expressed MOM, frozen MOM (FMOM) is provided. When MOM is unavailable, preterm infants are often fed pasteurized donor human milk (DHM), rendering it devoid of beneficial bacteria. We have previously reported that when MOM is inoculated into DHM to restore the live microbiota [restored milk (RM)], a similar microbial diversity to MOM can be achieved. Yet, it is unknown if a similar diversity to MOM can be obtained when FMOM is inoculated into DHM. The goal of this study was to determine whether a similar microbial composition to MOM could be obtained when FMOM is used to personalize DHM. To this end, a fresh sample of MOM was obtained and divided into fresh and frozen fractions. MOM and FMOM were inoculated into DHM at different dilutions: MOM/FMOM 10% (RM/FRM10) and MOM/FMOM 30% (RM/FRM30) and incubated at 37°C. At different timepoints, culture-dependent and culture-independent techniques were performed. Similar microbiota expansion and alpha diversity were observed in MOM, RM10, and RM30 whether fresh or frozen milk was used as the inoculum. To evaluate if microbial expansion would result in an abnormal activation on the innate immune system, Caco-2 epithelial cells were exposed to RM/FRM to compare interleukin 8 levels with Caco-2 cells exposed to MOM or DHM. It was found that RM samples did not elicit a significant increase in IL-8 levels when compared to MOM or FMOM. These results suggest that FMOM can be used to inoculate DHM if fresh MOM is unavailable or limited in supply, allowing both fresh MOM and FMOM to be viable options in a microbial restoration strategy.

Metabolomic Profile of Personalized Donor Human Milk.

Human milk could be considered an active and complex mixture of beneficial bacteria and bioactive compounds. Since pasteurization drastically reduces the microbial content, we recently demonstrated that pasteurized donor human milk (DHM) could be inoculated with different percentages (10% and 30%) of mother's own milk (MOM) to restore the unique live microbiota, resulting in personalized milk (RM10 and RM30, respectively). Pasteurization affects not only the survival of the microbiota but also the concentration of proteins and metabolites, in this study, we performed a comparative metabolomic analysis of the RM10, RM30, MOM and DHM samples to evaluate the impact of microbial restoration on metabolite profiles, where metabolite profiles clustered into four well-defined groups. Comparative analyses of DHM and MOM metabolomes determined that over one thousand features were significantly different. In addition, significant changes in the metabolite concentrations were observed in MOM and RM30 samples after four hours of incubation, while the concentration of metabolites in DHM remained constant, indicating that these changes are related to the microbial expansion. In summary, our analyses indicate that the metabolite profiles of DHM are significantly different from that of MOM, and the profile of MOM may be partially restored in DHM through microbial expansion.

Sex Modulates Lactobacillus johnsonii N6.2 and Phytophenol Effectiveness in Reducing High Fat Diet Induced mTOR Activation in Sprague-Dawley Rats.

Metabolic syndrome (MetS) is the underlying cause of some devastating diseases, including type 2 diabetes and cardiovascular disease. These diseases have been associated with over-activation of the mechanistic Target of Rapamycin (mTOR) pathway. This study utilizes a high fat diet (HFD) to induce MetS and to dissect the effects of a beneficial bacterium, L. johnsonii N6.2, and natural phenolics on mTOR complex 1 (mTORC1) expression compared to a reduced energy density diet (REDD). HFD significantly elevated MetS markers in males, as noted through an increase in weight, glucose levels, and triglyceride levels. Treatments were effective in reducing mTORC1-activating phosphorylation of pAKT-T308 and pAKT-S473 (p = 0.0012 and 0.0049, respectively) in HFD-fed females, with the combined treatments of L. johnsonii and phytophenols reducing phosphorylation below REDD-fed control levels, and significantly below HFD-fed control levels. Meanwhile, diet was the significant factor influencing male mTORC1-activating phosphorylation (p < 0.0001), as treatments were only effective in reducing phosphorylation in REDD-fed animals. Downstream analysis of mTORC1 activated genes phosphogluconate dehydrogenase (pgd) and phosphofructose kinase (pfk) followed this similar trend, enforcing the significant effect sex has on a treatments' ability to modulate diet induced abnormalities. Analyzing mTORC1 stimulators such as insulin, inflammatory cytokines, and tryptophan, revealed no significant differences among groups. These results indicate that the effects observed on mTORC1 are a direct consequence of the treatments, and not exerted indirectly via the modulation of stimuli. This study highlights the potential use of commensal microorganisms and natural compounds in reducing the onset of metabolic diseases through mTORC1.