Nematode mitochondrial metagenomics: A new tool for biodiversity analysis.

DNA barcoding approaches have greatly increased our understanding of biodiversity on the planet, and metabarcoding is widely used for classifying members of the phylum Nematoda. However, loci typically utilized in metabarcoding studies are often unable to resolve closely related species or are unable to recover all taxa present in a sample due to inadequate PCR primer binding. Mitochondrial metagenomics (mtMG) is an alternative approach utilizing shotgun sequencing of total DNA to recover the mitochondrial genomes of all species present in samples. However, this approach requires a comprehensive reference database for identification and currently available mitochondrial sequences for nematodes are highly dominated by sequences from the order Rhabditida, and excludes many clades entirely. Here, we analysed the efficacy of mtMG for the recovery of nematode taxa and the generation of mitochondrial genomes. We first developed a curated reference database of nematode mitochondrial sequences and expanded it with 40 newly sequenced taxa. We then tested the mito-metagenomics approach using a series of nematode mock communities consisting of morphologically identified nematode species representing various feeding traits, life stages, and phylogenetic relationships. We were able to identify all but two species through the de novo assembly of COX1 genes. We were also able to recover additional mitochondrial protein coding genes (PCGs) for 23 of the 24 detected species including a full array of 12 PCGs from five of the species. We conclude that mtMG offers a potential for the effective recovery of nematode biodiversity but remains limited by the breadth of the reference database.

Long-term survival outcomes associated with robotic-assisted minimally invasive esophagectomy (RAMIE) for esophageal cancer.

BACKGROUND: Minimally Invasive esophagectomy for esophageal cancer is associated with less morbidity compared to open approach. Whether robotic-assisted minimally invasive esophagectomy (RAMIE) results in better long-term survival compared with open esophagectomy (OE) and minimally invasive esophagectomy (MIE) is unclear. METHODS: We analyzed data from the National Cancer Database (NCDB) for patients with primary esophageal cancers who underwent esophagectomy in 2010-2017. Those with unknown staging, distant metastasis, or diagnosed with another cancer were excluded. Patients were stratified by RAMIE, MIE, and OE operative techniques. The Kaplan-Meier method and associated log-rank test were employed to compare unadjusted survival outcomes by surgical technique, our primary outcome. Multivariable Cox proportional hazards regression model was employed to discern factors independently contributing to survival. RESULTS: A total of 5170 patients who underwent esophagectomy were included in the analysis; 428 underwent RAMIE, 1417 underwent MIE, and 3325 underwent OE. Overall median survival was 42 months. In comparison to RAMIE, there was an increased risk of death for those that underwent either MIE [Hazard Ratio (HR) = 1.19; 95% Confidence Interval (CI): > 1.00 to 1.41; P < 0.047)] or OE (HR = 1.22; 95% CI: 1.04 to 1.43; P < 0.017). Academic vs community program facility type was associated with decreased risk of death (HR = 0.84; 95% CI: 0.76 to 0.93; P < 0.001). In general, males from areas of lower income with advanced stages of cancer who received neoadjuvant chemotherapy or radiation were at increased risk of death. Factors that were not associated with survival included race and ethnicity, Charlson-Devo Score, type of health insurance, zipcode level education, and population density. CONCLUSIONS: Overall survival was significantly longer in patients with esophageal cancers that underwent RAMIE in comparison to either MIE or OE in a 7-year NCDB cohort study.

Reversal of loss of bone mass in old mice treated with mefloquine.

Aging is accompanied by imbalanced bone remodeling, elevated osteocyte apoptosis, and decreased bone mass and mechanical properties; and improved pharmacologic approaches to counteract bone deterioration with aging are needed. We examined herein the effect of mefloquine, a drug used to treat malaria and systemic lupus erythematosus and shown to ameliorate bone loss in glucocorticoid-treated patients, on bone mass and mechanical properties in young and old mice. Young 3.5-month-old and old 21-month-old female C57BL/6 mice received daily injections of 5 mg/kg/day mefloquine for 14 days. Aging resulted in the expected changes in bone volume and mechanical properties. In old mice mefloquine administration reversed the lower vertebral cancellous bone volume and bone formation; and had modest effects on cortical bone volume, thickness, and moment of inertia. Mefloquine administration did not change the levels of the circulating bone formation markers P1NP or alkaline phosphatase, whereas levels of the resorption marker CTX showed trends towards increase with mefloquine treatment. In addition, and as expected, aging bones exhibited an accumulation of active caspase3-expressing osteocytes and higher expression of apoptosis-related genes compared to young mice, which were not altered by mefloquine administration at either age. In young animals, mefloquine induced higher periosteal bone formation, but lower endocortical bone formation. Further, osteoclast numbers were higher on the endocortical bone surface and circulating CTX levels were increased, in mefloquine- compared to vehicle-treated young mice. Consistent with this, addition of mefloquine to bone marrow cells isolated from young mice led to increased osteoclastic gene expression and a tendency towards increased osteoclast numbers in vitro. Taken together our findings identify the age and bone-site specific skeletal effects of mefloquine. Further, our results highlight a beneficial effect of mefloquine administration on vertebral cancellous bone mass in old animals, raising the possibility of using this pharmacologic inhibitor to preserve skeletal health with aging.

Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice.

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca2+ channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad-/- calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity.