Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

Background: The upper gastrointestinal tract plays a prominent role in human physiology as the primary site for enzymatic digestion and nutrient absorption, immune sampling, and drug uptake. Alterations to the small intestine microbiome have been implicated in various human diseases, such as non-alcoholic steatohepatitis and inflammatory bowel conditions. Yet, the physiological and functional roles of the small intestine microbiota in humans remain poorly characterized because of the complexities associated with its sampling. Rodent models are used extensively in microbiome research and enable the spatial, temporal, compositional, and functional interrogation of the gastrointestinal microbiota and its effects on the host physiology and disease phenotype. Classical, culture-based studies have documented that fecal microbial self-reinoculation (via coprophagy) affects the composition and abundance of microbes in the murine proximal gastrointestinal tract. This pervasive self-reinoculation behavior could be a particularly relevant study factor when investigating small intestine microbiota. Modern microbiome studies either do not take self-reinoculation into account, or assume that approaches such as single housing mice or housing on wire mesh floors eliminate it. These assumptions have not been rigorously tested with modern tools. Here, we used quantitative 16S rRNA gene amplicon sequencing, quantitative microbial functional gene content inference, and metabolomic analyses of bile acids to evaluate the effects of self-reinoculation on microbial loads, composition, and function in the murine upper gastrointestinal tract. Results: In coprophagic mice, continuous self-exposure to the fecal flora had substantial quantitative and qualitative effects on the upper gastrointestinal microbiome. These differences in microbial abundance and community composition were associated with an altered profile of the small intestine bile acid pool, and, importantly, could not be inferred from analyzing large intestine or stool samples. Overall, the patterns observed in the small intestine of non-coprophagic mice (reduced total microbial load, low abundance of anaerobic microbiota, and bile acids predominantly in the conjugated form) resemble those typically seen in the human small intestine. Conclusions: Future studies need to take self-reinoculation into account when using mouse models to evaluate gastrointestinal microbial colonization and function in relation to xenobiotic transformation and pharmacokinetics or in the context of physiological states and diseases linked to small intestine microbiome and to small intestine dysbiosis

A Quantitative Sequencing Framework for Absolute Abundance Measurements of Mucosal and Lumenal Microbial Communities

A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies

Control of Initiation, Rate, and Routing of Spontaneous Capillary-Driven Flow of Liquid Droplets through Microfluidic Channels on SlipChip

This Article describes the use of capillary pressure to initiate and control the rate of spontaneous liquid–liquid flow through microfluidic channels. In contrast to flow driven by external pressure, flow driven by capillary pressure is dominated by interfacial phenomena and is exquisitely sensitive to the chemical composition and geometry of the fluids and channels. A stepwise change in capillary force was initiated on a hydrophobic SlipChip by slipping a shallow channel containing an aqueous droplet into contact with a slightly deeper channel filled with immiscible oil. This action induced spontaneous flow of the droplet into the deeper channel. A model predicting the rate of spontaneous flow was developed on the basis of the balance of net capillary force with viscous flow resistance, using as inputs the liquid–liquid surface tension, the advancing and receding contact angles at the three-phase aqueous–oil–surface contact line, and the geometry of the devices. The impact of contact angle hysteresis, the presence or absence of a lubricating oil layer, and adsorption of surface-active compounds at liquid–liquid or liquid–solid interfaces were quantified. Two regimes of flow spanning a 104-fold range of flow rates were obtained and modeled quantitatively, with faster (mm/s) flow obtained when oil could escape through connected channels as it was displaced by flowing aqueous solution, and slower (micrometer/s) flow obtained when oil escape was mostly restricted to a micrometer-scale gap between the plates of the SlipChip (“dead-end flow”). Rupture of the lubricating oil layer (reminiscent of a Cassie–Wenzel transition) was proposed as a cause of discrepancy between the model and the experiment. Both dilute salt solutions and complex biological solutions such as human blood plasma could be flowed using this approach. We anticipate that flow driven by capillary pressure will be useful for the design and operation of flow in microfluidic applications that do not require external power, valves, or pumps, including on SlipChip and other droplet- or plug-based microfluidic devices. In addition, this approach may be used as a sensitive method of evaluating interfacial tension, contact angles, and wetting phenomena on chip

Quantitative microbiome profiling in lumenal and tissue samples with broad coverage and dynamic range via a single-step 16S rRNA gene DNA copy quantification and amplicon barcoding

Current methods for detecting, accurately quantifying, and profiling complex microbial communities based on the microbial 16S rRNA marker genes are limited by a number of factors, including inconsistent extraction of microbial nucleic acids, amplification interference from contaminants and host DNA, different coverage of PCR primers utilized for quantification and sequencing, and potentially biases in PCR amplification rates among microbial taxa during amplicon barcoding. Here, we describe a single-step method that enables the quantification of microbial 16S rRNA gene DNA copies with wide dynamic range and broad microbial diversity, and simultaneous amplicon barcoding for quantitative 16S rRNA gene amplicon profiling of microbiota. The method is suitable for a variety of sample types and is robust in samples with low microbial abundance, including samples containing high levels of host mammalian DNA, as is common in human clinical samples. We demonstrate that our modification to the Earth Microbiome Project (EMP) V4 16S rRNA gene primers expands their microbial coverage while dramatically reducing non-specific mammalian mitochondrial DNA amplification, thus achieving wide dynamic range in microbial quantification and broad coverage for capturing high microbial diversity in samples with or without high host DNA background. The approach relies only on broadly available hardware (real-time PCR instruments) and standard reagents utilized for conventional 16S rRNA gene amplicon library preparation both of which make it amenable for immediate and widespread adoption. Simultaneous 16S rRNA gene DNA copy quantification and amplicon barcoding for multiplexed next-generation sequencing from the same analyzed sample, performed in a combined workflow, reduces the amount of sample needed and reduces time and reagent costs. Additionally, we demonstrate that using our modified 16S rRNA gene primers in a digital PCR (dPCR) format enables precise and exact microbial quantification in samples with very high host DNA background levels without the need for quantification standards. Potential future applications of this approach include: (1) quantitative microbiome profiling in human and animal microbiome research; (2) detection of monoinfections and profiling of polymicrobial infections in tissues, stool, and bodily fluids in human and veterinary medicine; (3) environmental sample analyses (e.g., soil and water); and (4) broad-coverage detection of microbial food contamination in products high in mammalian DNA, such as meat products. We predict that utilization of this approach primarily for quantitative microbiome profiling will be invaluable to microbiome studies, which have historically been limited to analysis of relative abundances of microbes

Charge-localized p-phenylenedihydrazine radical cations: ESR and optical studies of intramolecular electron transfer rates

1,4-Bis(2-tert-butyl-2,3-diazabicyclo[2.2.2]oct-3-yl)benzene-1,4-diyl (2) its 2,5-dimethyl and 2,3,5,6-tetramethyl derivatives (3 and 4), their radical cations, and bis-radical dications are studied. Crystal structures including those of 2^+BPh_4^-, 3^(2+)(BPh_4^-)_2, 4^+BPh_4^-, and 4^(2+)(BPh_4^-)_2 establish that ring methylation causes more N-lone pair, aryl π twist without changing the NAr,NAr distance significantly and that both 2^+ and 4^+ have the charge localized in one hydrazine unit. NMR measurements show that 3^+ has about 6% of its spin at the four aryl CH and CMe carbons, while 4^+ has about 1.5% of its spin at the four CMe carbons. The average distance between the unpaired electrons of 3^(2+) and 4^(2+) was obtained from the dipolar splittings of their thermally excited triplet states and, as expected, is significantly smaller for 3^(2+) (5.25 Å) than for 4^(2+) (5.63 Å). Rate constants for electron transfer between the hydrazine units of 3^+ and 4^+ in CH_2Cl_2 and CH_3CN were determined by dynamic ESR. The intervalence radical cations show charge transfer bands corresponding to vertical electron transfer between the ground state and the highly vibrationally excited electron-shifted state, allowing calculation of the parameters controlling electron transfer. Electron transfer parameters obtained from the CT bands using adiabatic energy surfaces which approximate the CT band shapes observed produce rate constants within experimental error of those extrapolated to room temperature from the ESR data for both 3^+ and 4^+ in both solvents, without using tunneling corrections. The effects of mixing of the electronic wave functions of the reduced and oxidized hydrazine units of 2^+ on d_(NN), the C(t-Bu)N,NA(Ar) twist angle, and the aryl nitrogen lone pair, aryl π twist angle which are observed by X-ray are close to those predicted from the position of the minima on the ET coordinate X of the adiabatic energy surface calculated from the CT band

Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

Background: The upper gastrointestinal tract plays a prominent role in human physiology as the primary site for enzymatic digestion and nutrient absorption, immune sampling, and drug uptake. Alterations to the small intestine microbiome have been implicated in various human diseases, such as non-alcoholic steatohepatitis and inflammatory bowel conditions. Yet, the physiological and functional roles of the small intestine microbiota in humans remain poorly characterized because of the complexities associated with its sampling. Rodent models are used extensively in microbiome research and enable the spatial, temporal, compositional, and functional interrogation of the gastrointestinal microbiota and its effects on the host physiology and disease phenotype. Classical, culture-based studies have documented that fecal microbial self-reinoculation (via coprophagy) affects the composition and abundance of microbes in the murine proximal gastrointestinal tract. This pervasive self-reinoculation behavior could be a particularly relevant study factor when investigating small intestine microbiota. Modern microbiome studies either do not take self-reinoculation into account, or assume that approaches such as single housing mice or housing on wire mesh floors eliminate it. These assumptions have not been rigorously tested with modern tools. Here, we used quantitative 16S rRNA gene amplicon sequencing, quantitative microbial functional gene content inference, and metabolomic analyses of bile acids to evaluate the effects of self-reinoculation on microbial loads, composition, and function in the murine upper gastrointestinal tract. Results: In coprophagic mice, continuous self-exposure to the fecal flora had substantial quantitative and qualitative effects on the upper gastrointestinal microbiome. These differences in microbial abundance and community composition were associated with an altered profile of the small intestine bile acid pool, and, importantly, could not be inferred from analyzing large intestine or stool samples. Overall, the patterns observed in the small intestine of non-coprophagic mice (reduced total microbial load, low abundance of anaerobic microbiota, and bile acids predominantly in the conjugated form) resemble those typically seen in the human small intestine. Conclusions: Future studies need to take self-reinoculation into account when using mouse models to evaluate gastrointestinal microbial colonization and function in relation to xenobiotic transformation and pharmacokinetics or in the context of physiological states and diseases linked to small intestine microbiome and to small intestine dysbiosis

Experimental study of skull bones reconstruction with the use of autografts and hydroxyapatitical gel

Introduction. To conduct reconstructive surgical interventions at pathological processes of skull base bone, defects of the temporal bone and dura mater, is especially complicated with the appearance of liquorrhea. This necessitates the improvement of operative technique and the use of new plastic materials. Objective: to study the features of meninges and skull bone defects plastic using autobone, fascia and HAP gel based on clinical observation, and macroscopic and microscopic experimental studies. Materials and methods: 36 mature nonlinear rats of both sexes up to 6 months old, weighing 190-250 g were divided into 2 groups. In the 1st (experimental) group there were 18 animals and autofragments of fascia and tibia with the addition of GAP gel were used for implantation; and in the 2nd group (comparison, n=18) the same plastic material without gel was used. The introduction of implant was carried out under general anesthesia in a standardized defect of skull frontal bones. The method of clinical observation, stereoscopic, common histological examination after staining with hematoxylin-eosin was applied. Results: The introduction of bone’s and fascia autografts into skull defect and dura mater provides the structural function of the damage tightly closing, preventing or stopping of liquorrhea, besides fascia contributes to vascularization of tissues in the healing zone. Additional administration of HAP ge, provides pronounced adhesion to bone tissue, gaps filling in the graft bed, creates a microenvironment for metabolism in the zone, shows a positive effect on osteogenesis, promotes calcification and bone reconstruction in the area of the defect. The results obtained also indicate the peculiarities of reparative processes in the bones of the skull, expressed in the dependence of bone repair and / or the development of fibrous tissue in the defect area and the density of adherence of the bone to the mother tissue, which is accompanied by improved bone restructuring under tight contact of the mother bone with the implanted material and the prevalence of fibrous tissue in case of their violation. This data confirm the effectiveness and promise HAPG use along with autofascia and autobone to close the defects of the meninges and skull bones and are important for improving reconstructive-restorative surgical interventions

Combining microfluidic networks and peptide arrays for multi-enzyme assays

This paper reports the use of microfluidic networks (μFNs) to both prepare peptide microarrays and carry out label-free enzyme assays on self-assembled monolayers (SAMs) of alkanethiolates on gold. A poly(dimethylsiloxane) (PDMS) stamp fabricated with microchannels is used to immobilize a linear array of cysteine-terminated peptides onto SAMs presenting maleimide groups. The stamp is then reapplied to the SAM in a perpendicular direction to introduce enzyme solutions so that each solution can interact with an identical linear array of immobilized peptides. The μFNs enable multiple enzyme−substrate interactions to be simultaneously evaluated at a submicroliter scale, while the use of SAMs enables the use of MALDI mass spectrometry (MS) to analyze the enzyme activities. This paper demonstrates applications of this system for assaying multiple kinases and for profiling the activities of kinases and phosphatases in human K562 cell extracts. The combination of μFN, SAMs, and MS detection provides a flexible platform for assaying enzyme activities in biological samples

ABO, D Blood Typing and Subtyping Using Plug-Based Microfluidics

A plug-based microfluidic approach was used to perform multiple agglutination assays in parallel without crosscontamination and using only microliter volumes of blood. To perform agglutination assays on-chip, a microfluidic device was designed to combine aqueous streams of antibody, buffer, and red blood cells (RBCs) to form droplets 30-40 nL in volume surrounded by a fluorinated carrier fluid. Using this approach, proof-of-concept ABO and D (Rh) blood typing and group A subtyping were successfully performed by screening against multiple antigens without cross-contamination. On-chip subtyping distinguished common A1 and A2 RBCs by using a lectinbased dilution assay. This flexible platform was extended to differentiate rare, weakly agglutinating RBCs of A subtypes by analyzing agglutination avidity as a function of shear rate. Quantitative analysis of changes in contrast within plugs revealed subtleties in agglutination kinetics and enabled characterization of agglutination of rare blood subtypes. Finally, this platform was used to detect bacteria, demonstrating the potential usefulness of this assay in detecting sepsis and the potential for applications in agglutination-based viral detection. The speed, control, and minimal sample consumption provided by this technology present an advance for point of care applications, blood typing of newborns, and general blood assays in small model organisms