Deep Illumina-Based Shotgun Sequencing Reveals Dietary Effects on the Structure and Function of the Fecal Microbiome of Growing Kittens

Background

Previously, we demonstrated that dietary protein:carbohydrate ratio dramatically affects the fecal microbial taxonomic structure of kittens using targeted 16S gene sequencing. The present study, using the same fecal samples, applied deep Illumina shotgun sequencing to identify the diet-associated functional potential and analyze taxonomic changes of the feline fecal microbiome.

Methodology & Principal Findings

Fecal samples from kittens fed one of two diets differing in protein and carbohydrate content (high–protein, low–carbohydrate, HPLC; and moderate-protein, moderate-carbohydrate, MPMC) were collected at 8, 12 and 16 weeks of age (n = 6 per group). A total of 345.3 gigabases of sequence were generated from 36 samples, with 99.75% of annotated sequences identified as bacterial. At the genus level, 26% and 39% of reads were annotated for HPLC- and MPMC-fed kittens, with HPLC-fed cats showing greater species richness and microbial diversity. Two phyla, ten families and fifteen genera were responsible for more than 80% of the sequences at each taxonomic level for both diet groups, consistent with the previous taxonomic study. Significantly different abundances between diet groups were observed for 324 genera (56% of all genera identified) demonstrating widespread diet-induced changes in microbial taxonomic structure. Diversity was not affected over time. Functional analysis identified 2,013 putative enzyme function groups were different (p<0.000007) between the two dietary groups and were associated to 194 pathways, which formed five discrete clusters based on average relative abundance. Of those, ten contained more (p<0.022) enzyme functions with significant diet effects than expected by chance. Six pathways were related to amino acid biosynthesis and metabolism linking changes in dietary protein with functional differences of the gut microbiome.

Conclusions

These data indicate that feline feces-derived microbiomes have large structural and functional differences relating to the dietary protein:carbohydrate ratio and highlight the impact of diet early in life.     

Young age as a prognostic factor in cervical cancer: analysis of population based data from 10 022 cases

The effect of young age on survival in cervical cancer is not fully known, although evidence has suggested that it is a poor prognostic factor and that young patients should therefore be treated differently from older patients. All 10 022 cases of invasive cervical cancer in the west Midlands during 1957-81, which comprised 10% of the cases in England and Wales, were analysed to determine the prognostic effect of age. Univariate analysis showed a median survival time of 54 months for all cases, with survival rates at five years of 69% for patients aged under 40 and 45% for those aged 40 or older (χ₁² (log rank)=331·4; p<0·0001). This difference remained significant after stratification for stage (χ₁² (log rank)=7·1; p=0·008). Cox regression analysis with nine covariables, including age and year of registration, reaffirmed the importance of conventional prognostic factors such as stage of disease, size of tumour, state of lymph nodes, and differentiation of the tumour. After allowance was made for the effects of other prognostic factors young age was found to be a small but significant favourable factor that did not change during the period of the study. Estimated survival distributions obtained from the Cox model showed that for women presenting with the common characteristics associated with stage Ib disease who were treated with radical radiotherapy the survival rate at five years fell non-linearly from 71% in the group aged 25-29 to 65% in the group aged 65-69.Young age alone is not a reason to alter existing policies for treatment for patients with invasive cervical cancer.     

Population structure in a saproxylic funnelweb spider (Hexathelidae: Hadronyche) along a forested rainfall gradient

We present research undertaken at Tallaganda State Forest, Australia, describing how current climatic conditions impact upon the saproxylic (decaying wood) habitat and the relationship between this habitat and the demography and morphology of a saproxylic funnelweb spider ( Hadronyche sp.). Climatic data support a north–south habitat cline and, to a lesser extent, a short-range, aspect-driven habitat cline. Rainfall and log moisture content increased with latitude through the forest, and aspect affected the amount of solar radiation penetrating to ground level. The distribution and abundance of Hadronyche varied among sites along both clines in response to several variables. The decay state of logs was highly influential, with wet, highly decayed logs favoured over dry, hard ones. Population density was highest in wetter, southern sites where these logs were abundant, but rare, suitably decomposed logs in dry, northern sites still typically hosted a comparable number of individuals. Morphological measurements showed some phenotypic variation along the north–south habitat gradient, but not over the short-range, aspect-driven gradients. Hadronyche would not be expected to show similarly strong patterns of molecular variation as seen in saproxylic Collembola and Onychophora within Tallaganda as it appears to be more vagile.     

Aging induces muscle-specific impairment of endothelium-dependent dilation in skeletal muscle feed arteries.

We tested the hypothesis that aging decreases endothelium-dependent vasodilation in feed arteries perfusing rat skeletal muscle. In addition, we tested the hypothesis that attenuated vasodilator responses are associated with decreased endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) expression. Soleus feed arteries (SFA) and gastrocnemius feed arteries (GFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. Feed arteries from the right hindlimb were cannulated with two glass micropipettes for examination of endothelium-dependent [acetylcholine (ACh)] and endothelium-independent [adenosine (Ado) or sodium nitroprusside (SNP)] vasodilator function. Feed arteries from the left hindlimb were frozen and used to assess eNOS and SOD-1 protein and mRNA expression. In SFA, endothelium-dependent dilation to ACh was reduced in old rats (0.9 +/- 0.04 vs. 0.8 +/- 0.03), whereas dilator responses to Ado and SNP were similar in SFA of young and old rats. In GFA, vasodilator responses to ACh, Ado, and SNP were not altered by age. eNOS and SOD-1 protein expression declined with age in SFA (-71 and -54%, respectively) but not in GFA. eNOS and SOD-1 mRNA expression were not altered by age in SFA or GFA. Collectively, these data indicate aging induces muscle-specific impairment of endothelium-dependent vascular function in SFA.     

Covalent attachment of ferrocene to soybean peroxidase glycans: electron transfer mediation to redox enzymes

The electrochemical properties of native soybean peroxidase (SBP) and of SBP modified with covalently attached ferrocene electron-transfer mediators within microcavities etched at the tip of 25 mum radius platinum microelectrodes are reported. The microcavities incorporate approximately 50 fmol of SBP. In the microcavity, native SBP undergoes a relatively fast reduction but a very slow oxidation. Ferrocene mediators have been attached to the SBP glycans (Fc-SBP) (approximately 1.5 ferrocene mediators per SBP molecule). Cyclic voltammetry reveals that these centers are capable of mediating the reduction of oxidized SBP and increase the rate of heterogeneous electron transfer between the enzyme and the electrode by >10-fold. Micromolar concentrations of H2O2 chemically oxidize the SBP and Fc-SBP systems leading to an electrocatalytic reduction at approximately -0.1 V. Successive additions of 2.5 micromol of H2O2 at a constant applied potential of -0.1 V gave a steady-state constant current of approximately 60 nA within 20 s. The steady-state current increased linearly with peroxide concentration for 2.5 相似文献   

The Dipeptidyl Peptidase-4 Inhibitor Linagliptin Preserves Endothelial Function in Mesenteric Arteries from Type 1 Diabetic Rats without Decreasing Plasma Glucose

The aim of the study was to investigate the effect of the DPP-4 inhibitor linagliptin on the mechanism(s) of endothelium-dependent relaxation in mesenteric arteries from STZ-induced diabetic rats. Both normal and diabetic animals received linagliptin (2 mg/kg) daily by oral gavage for a period of 4 weeks. To measure superoxide generation in mesenteric arteries, lucigenin-enhanced chemiluminescence was used. ACh-induced relaxation of mesenteric arteries was assessed using organ bath techniques and Western blotting was used to investigate protein expression. Pharmacological tools (1μM TRAM-34, 1μM apamin, 100 nM Ibtx, 100 μM L-NNA, 10 μM ODQ) were used to distinguish between NO and EDH-mediated relaxation. Linagliptin did not affect plasma glucose, but did decrease vascular superoxide levels. Diabetes reduced responses to ACh but did not affect endothelium-independent responses to SNP. Linagliptin improved endothelial function indicated by a significant increase in responses to ACh. Diabetes impaired the contribution of both nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation and linagliptin treatment significantly enhanced the contribution of both relaxing factors. Western blotting demonstrated that diabetes also increased expression of Nox2 and decreased expression and dimerization of endothelial NO synthase, effects that were reversed by linagliptin. These findings demonstrate treatment of type 1 diabetic rats with linagliptin significantly reduced vascular superoxide levels and preserved both NO and EDH-mediated relaxation indicating that linagliptin can improve endothelial function in diabetes independently of any glucose lowering activity.     

Nek5 promotes centrosome integrity in interphase and loss of centrosome cohesion in mitosis

Nek5 is a poorly characterized member of the NIMA-related kinase family, other members of which play roles in cell cycle progression and primary cilia function. Here, we show that Nek5, similar to Nek2, localizes to the proximal ends of centrioles. Depletion of Nek5 or overexpression of kinase-inactive Nek5 caused unscheduled separation of centrosomes in interphase, a phenotype also observed upon overexpression of active Nek2. However, separated centrosomes that resulted from Nek5 depletion remained relatively close together, exhibited excess recruitment of the centrosome linker protein rootletin, and had reduced levels of Nek2. In addition, Nek5 depletion led to loss of PCM components, including γ-tubulin, pericentrin, and Cdk5Rap2, with centrosomes exhibiting reduced microtubule nucleation. Upon mitotic entry, Nek5-depleted cells inappropriately retained centrosome linker components and exhibited delayed centrosome separation and defective chromosome segregation. Hence, Nek5 is required for the loss of centrosome linker proteins and enhanced microtubule nucleation that lead to timely centrosome separation and bipolar spindle formation in mitosis.