Small-animal blood exchange is an emerging approach for systemic aging research.

We describe a small-animal blood exchange approach developed for aging research as an alternative to heterochronic parabiosis or plasma injections. In parabiosis, animals are surgically coupled, which has several disadvantages, including difficulty controlling experimental procedure, the effects of shared organs, environmental enrichment from jointly exploring the housing enclosure, involuntary exercise and an imprecise onset of blood sharing. Likewise, in plasma injections, the added volumes need to be small, and there is little flexibility in changing the relative contributions of ectopic to endogenous blood components. These factors complicate the conclusions and interpretations, including the identification of key mechanisms and molecular or cellular determinants. Our approach, where blood is exchanged between animals without them being surgically coupled, is less invasive than parabiosis. The percentage of exchanged blood or other exchanged fluids is known and precise. The age of plasma and cells can be mixed and matched at all desired relative contributions to the endogenous systemic milieu, and the onset of the effects can be accurately delineated. In this protocol, we describe the preparatory and animal surgery steps required for small-animal blood exchange in mice and compare this process with parabiosis and plasma injections. We also provide the design, hardware and software for the blood exchange device and compare automated and manual exchange methods. Lastly, we report mathematical modeling of the dilution of blood factors. The fluid exchange takes ~30 min when performed by a well-trained biomedical scientist; the entire process takes ~2 h.

Plasma dilution improves cognition and attenuates neuroinflammation in old mice.

Our recent study has established that young blood factors are not causal, nor necessary, for the systemic rejuvenation of mammalian tissues. Instead, a procedure referred to as neutral blood exchange (NBE) that resets signaling milieu to a pro-regenerative state through dilution of old plasma, enhanced the health and repair of the muscle and liver, and promoted better hippocampal neurogenesis in 2-year-old mice (Mehdipour et al., Aging 12:8790-8819, 2020). Here we expand the rejuvenative phenotypes of NBE, focusing on the brain. Namely, our results demonstrate that old mice perform much better in novel object and novel texture (whisker discrimination) tests after a single NBE, which is accompanied by reduced neuroinflammation (less-activated CD68+ microglia). Evidence against attenuation/dilution of peripheral senescence-associated secretory phenotype (SASP) as the main mechanism behind NBE was that the senolytic ABT 263 had limited effects on neuroinflammation and did not enhance hippocampal neurogenesis in the old mice. Interestingly, peripherally acting ABT 263 and NBE both diminished SA-ßGal signal in the old brain, demonstrating that peripheral senescence propagates to the brain, but NBE was more robustly rejuvenative than ABT 263, suggesting that rejuvenation was not simply by reducing senescence. Explaining the mechanism of the positive effects of NBE on the brain, our comparative proteomics analysis demonstrated that dilution of old blood plasma yields an increase in the determinants of brain maintenance and repair in mice and in people. These findings confirm the paradigm of rejuvenation through dilution of age-elevated systemic factors and extrapolate it to brain health and function.

Rejuvenation of three germ layers tissues by exchanging old blood plasma with saline-albumin.

Heterochronic blood sharing rejuvenates old tissues, and most of the studies on how this works focus on young plasma, its fractions, and a few youthful systemic candidates. However, it was not formally established that young blood is necessary for this multi-tissue rejuvenation. Here, using our recently developed small animal blood exchange process, we replaced half of the plasma in mice with saline containing 5% albumin (terming it a "neutral" age blood exchange, NBE) thus diluting the plasma factors and replenishing the albumin that would be diminished if only saline was used. Our data demonstrate that a single NBE suffices to meet or exceed the rejuvenative effects of enhancing muscle repair, reducing liver adiposity and fibrosis, and increasing hippocampal neurogenesis in old mice, all the key outcomes seen after blood heterochronicity. Comparative proteomic analysis on serum from NBE, and from a similar human clinical procedure of therapeutic plasma exchange (TPE), revealed a molecular re-setting of the systemic signaling milieu, interestingly, elevating the levels of some proteins, which broadly coordinate tissue maintenance and repair and promote immune responses. Moreover, a single TPE yielded functional blood rejuvenation, abrogating the typical old serum inhibition of progenitor cell proliferation. Ectopically added albumin does not seem to be the sole determinant of such rejuvenation, and levels of albumin do not decrease with age nor are increased by NBE/TPE. A model of action (supported by a large body of published data) is that significant dilution of autoregulatory proteins that crosstalk to multiple signaling pathways (with their own feedback loops) would, through changes in gene expression, have long-lasting molecular and functional effects that are consistent with our observations. This work improves our understanding of the systemic paradigms of multi-tissue rejuvenation and suggest a novel and immediate use of the FDA approved TPE for improving the health and resilience of older people.

Skeletal muscle as an experimental model of choice to study tissue aging and rejuvenation.

Skeletal muscle is among the most age-sensitive tissues in mammal organisms. Significant changes in its resident stem cells (i.e., satellite cells, SCs), differentiated cells (i.e., myofibers), and extracellular matrix cause a decline in tissue homeostasis, function, and regenerative capacity. Based on the conservation of aging across tissues and taking advantage of the relatively well-characterization of the myofibers and associated SCs, skeletal muscle emerged as an experimental system to study the decline in function and maintenance of old tissues and to explore rejuvenation strategies. In this review, we summarize the approaches for understanding the aging process and for assaying the success of rejuvenation that use skeletal muscle as the experimental system of choice. We further discuss (and exemplify with studies of skeletal muscle) how conflicting results might be due to variations in the techniques of stem cell isolation, differences in the assays of functional rejuvenation, or deciding on the numbers of replicates and experimental cohorts.

The impact of long-term organic farming on soil-derived greenhouse gas emissions.

Agricultural practices contribute considerably to emissions of greenhouse gases. So far, knowledge on the impact of organic compared to non-organic farming on soil-derived nitrous oxide (N2O) and methane (CH4) emissions is limited. We investigated N2O and CH4 fluxes with manual chambers during 571 days in a grass-clover- silage maize - green manure cropping sequence in the long-term field trial "DOK" in Switzerland. We compared two organic farming systems - biodynamic (BIODYN) and bioorganic (BIOORG) - with two non-organic systems - solely mineral fertilisation (CONMIN) and mixed farming including farmyard manure (CONFYM) - all reflecting Swiss farming practices-together with an unfertilised control (NOFERT). We observed a 40.2% reduction of N2O emissions per hectare for organic compared to non-organic systems. In contrast to current knowledge, yield-scaled cumulated N2O emissions under silage maize were similar between organic and non-organic systems. Cumulated on area scale we recorded under silage maize a modest CH4 uptake for BIODYN and CONMIN and high CH4 emissions for CONFYM. We found that, in addition to N input, quality properties such as pH, soil organic carbon and microbial biomass significantly affected N2O emissions. This study showed that organic farming systems can be a viable measure contributing to greenhouse gas mitigation in the agricultural sector.

Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair.

CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

Utilizing Electronic Health Record Information to Optimize Medication Infusion Devices: A Manual Data Integration Approach.

Health information technology is increasingly utilized within healthcare delivery systems today. Two examples of this type of technology include the capture of patient-specific information within an electronic health record and intravenous medication infusion devices equipped with dose error reduction software known as drug libraries. Automatic integration of these systems, termed intravenous (IV) interoperability, should serve as the goal toward which all healthcare systems work to maximize patient safety. For institutions lacking IV interoperability, we describe a manual approach of querying the electronic health record to incorporate medication administration information with data from infusion device software to optimize drug library settings. This approach serves to maximize utilization of available information to optimize medication safety provided by drug library software.

Greenhouse gas fluxes from agricultural soils under organic and non-organic management--a global meta-analysis.

It is anticipated that organic farming systems provide benefits concerning soil conservation and climate protection. A literature search on measured soil-derived greenhouse gas (GHG) (nitrous oxide and methane) fluxes under organic and non-organic management from farming system comparisons was conducted and followed by a meta-analysis. Up to date only 19 studies based on field measurements could be retrieved. Based on 12 studies that cover annual measurements, it appeared with a high significance that area-scaled nitrous oxide emissions from organically managed soils are 492 ± 160 kg CO2 eq. ha(-1) a(-1) lower than from non-organically managed soils. For arable soils the difference amounts to 497 ± 162 kg CO2 eq. ha(-1) a(-1). However, yield-scaled nitrous oxide emissions are higher by 41 ± 34 kg CO2 eq. t(-1) DM under organic management (arable and use). To equalize this mean difference in yield-scaled nitrous oxide emissions between both farming systems, the yield gap has to be less than 17%. Emissions from conventionally managed soils seemed to be influenced mainly by total N inputs, whereas for organically managed soils other variables such as soil characteristics seemed to be more important. This can be explained by the higher bioavailability of the synthetic N fertilisers in non-organic farming systems while the necessary mineralisation of the N sources under organic management leads to lower and retarded availability. Furthermore, a higher methane uptake of 3.2 ± 2.5 kg CO2 eq. ha(-1) a(-1) for arable soils under organic management can be observed. Only one comparative study on rice paddies has been published up to date. All 19 retrieved studies were conducted in the Northern hemisphere under temperate climate. Further GHG flux measurements in farming system comparisons are required to confirm the results and close the existing knowledge gaps.

Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources.

Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible with in vitro antibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the yeast surface display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation. Lysate-resident TfR was then combined with a mutagenic anti-TfR scFv library in a competitive, dissociation rate screen, and scFvs were identified with up to 4-fold improved dissociation rates on the surface of yeast. Importantly, although the lysates contained a complex mixture of biotinylated proteins, the engineered scFvs retained their TfR binding specificity. When secreted by yeast as soluble proteins, mutant scFvs bound to cell surface TfR with 3-7-fold improvements in equilibrium binding affinity. Although a known MP antigen was targeted for purposes of this study, employing biotin tagging as a means of antigen detection makes the lysate-based approach particularly flexible. We have previously shown that yeast display can be used to identify lead antibodies using cell lysate-resident MP antigens, and combined with this work showing that antibodies can also be quantitatively engineered using cell lysates, these approaches may provide a high-throughput platform for generation and optimization of antibodies against MPs.

Enhanced top soil carbon stocks under organic farming.

It has been suggested that conversion to organic farming contributes to soil carbon sequestration, but until now a comprehensive quantitative assessment has been lacking. Therefore, datasets from 74 studies from pairwise comparisons of organic vs. nonorganic farming systems were subjected to metaanalysis to identify differences in soil organic carbon (SOC). We found significant differences and higher values for organically farmed soils of 0.18 ± 0.06% points (mean ± 95% confidence interval) for SOC concentrations, 3.50 ± 1.08 Mg C ha(-1) for stocks, and 0.45 ± 0.21 Mg C ha(-1) y(-1) for sequestration rates compared with nonorganic management. Metaregression did not deliver clear results on drivers, but differences in external C inputs and crop rotations seemed important. Restricting the analysis to zero net input organic systems and retaining only the datasets with highest data quality (measured soil bulk densities and external C and N inputs), the mean difference in SOC stocks between the farming systems was still significant (1.98 ± 1.50 Mg C ha(-1)), whereas the difference in sequestration rates became insignificant (0.07 ± 0.08 Mg C ha(-1) y(-1)). Analyzing zero net input systems for all data without this quality requirement revealed significant, positive differences in SOC concentrations and stocks (0.13 ± 0.09% points and 2.16 ± 1.65 Mg C ha(-1), respectively) and insignificant differences for sequestration rates (0.27 ± 0.37 Mg C ha(-1) y(-1)). The data mainly cover top soil and temperate zones, whereas only few data from tropical regions and subsoil horizons exist. Summarizing, this study shows that organic farming has the potential to accumulate soil carbon.