Move Over, E. coli?

Escherichia coli was discovered over 130 years ago and has become the dominant model organism for microbiology, genetics, gene function and protein expression analysis and proteomics. In the past few decades it has become a workhorse in producing therapeutic recombinant proteins and antibodies, accounting for over 90% of bacterial bioprocessing. Reasons for this include its simple growth conditions, its loss of pathogenicity, the expansive understanding of its genetics and biochemistry (including full genome sequencing) and its fast doubling time of 20 minutes.

Recently, a group from Harvard, led by George Church, has pushed to replace E. coli with an alternate species, Vibrio natriegens. The chief reason for this change is the very fast doubling time of V. natriegens, as little as 10 minutes. They claim that for many experiments 90% of experimental time is due to bacterial growth. V. natriegens could potentially cut this in half. Other reasons for the change include complete genome sequencing, CRISPR technology and the ability to grow on cheap growth media. The group is currently analyzing gene mutants to see if even faster doubling times are possible.

Move Over, E. coli

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Low Endotoxin Recovery (LER) Using LAL

Low Endotoxin Recovery (LER) is the observation that, for many biological compounds, it is difficult to recover known endotoxin spikes. This has become the prevailing topic in the endotoxin field since the inability of LAL to detect endotoxin does not necessarily preclude a pyrogenic reaction in vivo. A recent publication by scientists at Eli Lilly highlight the technical problems involved with measuring LER and insights into its cause.

First, while the cause of LER has not been directly demonstrated, it is thought to be a function of the aggregation state of endotoxin monomers. Evidence of this is the observation that divalent cations and surfactants, which destabilize endotoxin aggregates, are usually involved in LER. This report shows that while cations and surfactants are essential in LER, they are not sufficient. Second, the paper shows the importance of a stringent testing protocol. The use of the verified USP <85> method for routine batch release testing resulted in acceptable endotoxin recovery (50-200%). However, changing small details such as mixing time and sampling scheme produced inconsistent results. Finally, they provide evidence that naturally occurring endotoxin (NOE) is not as susceptible to LER as a purified stock, indicating the importance of the supramolecular structure.

BioDtech has previously observed similar masking issues as a result of interactions between protein molecules and endotoxin. In response, we have developed the EndoPrep system to remove interfering proteins from samples and allow complete endotoxin recovery.

Endotoxin Recovery Using Limulus Amebocyte Lysate (LAL) Assay

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Inherited Microbiomes?

Previous work has shown that certain bacterial species are transferred from parents to children. A new study from the Max Planck Institute in Tubingen suggests that part of your microbiome is actually inherited. That is, there are specific genes that regulate which bacteria thrive. To come to that conclusion, scientists correlated genetic variations to bacterial species in 1126 pairs of twins, both identical and fraternal. They found dozens of species that, while environmentally acquired, propagate due to genetics. These include species involved in gastrointestinal disorders, blood pressure regulation and autoimmunity.

Twin Study finds that Gut Microbiomes Run in Families

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A Role for Endotoxin in Aggression and Depression

A link between depression and inflammation has been observed in many studies. Aberrant social behaviors, such as aggression, often present with depression and can be indicative of suicidal behavior.

A recent study addressed the association between stress-associated behaviors, such as aggression, and low-level inflammation induced by endotoxin challenge. They found that the addition of endotoxin-induced inflammation exacerbated depression in stress-induced mice but reduced signs of aggression. Specifically, endotoxin caused increases of IL-1β and 5-HT2A mRNA in the brain, increased serum corticosterone and increased TNFα in both the brain and liver. Stress alone did not cause these changes. However, the combination of stress and inflammation resulted in a reduction in the stress-induced changes in 5-HT and IL-1β. These results indicate that low-level inflammation can have significant impact on stress-induced behaviors, specifically reductions in aggression, which can lead to depression.

Low-dose lipopolysaccharide (LPS) inhibits aggressive and augments depressive behaviors in a chronic mild stress model in mice

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Bacteria Establish “Us vs Them”

It is well understood that many microbial species release amino acids and vitamins into the environment to benefit the population.  However, this behavior could be harmful if “outsider” species receive these molecules without contributing to the group. Scientists at the Max Planck Institute have observed the answer to this problem in biofilms. Using directed mutations, computer modeling and mass spectrometric imaging they created a new approach called “synthetic ecology”. Using this approach they found that “cooperating” bacteria create clusters and physically exclude “non-cooperating” bacteria. Notably, bacteria in liquid culture show no exclusion mechanisms. Given the significant health issues associated with bacterial biofilms, insight into their formation and persistence are crucial.

Cooperating bacteria isolate cheaters

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Endotoxin in Nanoparticles

Endotoxin contamination in recombinant therapeutics is a widely discussed problem. A recent review highlights similar issues in the field of nanoparticles.

Nanomaterials are commonly contaminated with endotoxin due to their highly reactive surfaces combined with the ability of endotoxin to strongly bind both hydrophobic and positively charged areas. As with recombinant proteins, issues such as therapeutic inactivation, cytokine production, oxidative stress and cytotoxicity have been observed. Detection with LAL is non-optimal, but possible, with the gel clot assay being the most problematic. Serious interference with metal- or silica-containing nanoparticles has been observed. Finally, endotoxin removal is especially difficult with the typical problems encountered using ultrafiltration, size exclusion, two-phase extraction and ion exchange. Even affinity adsorption is problematic since endotoxin tends to bind to strongly to the microparticles. This “endotoxin masking” is a common problem with many therapeutic molecules.

Endotoxin Contamination: A Key Element in the Interpretation of Nanosafety Studies

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Bugs as Drugs

The role of gut bacteria has been well-established for diseases such as inflammatory bowel disease, diabetes and Alzheimer’s and has even been shown to play a role in depression, infertility, alcoholism and HIV progression. Starting in 2013, the US FDA began to regulate fecal transplantation as a way to re-establish healthy gut microbiota to patients with gastrointestinal disease or who have had their normal bacterial flora disrupted by antibiotics. These procedures has shown tremendous potential (reviews HERE and HERE). Recently, scientists from the Wellcome Trust Sanger Institute grew and catalogued 137 bacterial species, many previously “unculturable”, from the guts of healthy patients. This work will lay the foundation to determine the role that specific bacteria play in specific diseases as well as determining a proper combination of species required for treatment, potentially allowing fecal transplantation to be replaced by defined, active cultures in pill form. Additionally, for the first time this work demonstrated that 50-60% of bacteria in the intestinal microbiota for resistant spores, allowing for host-to-host transmission.

‘Bugs’ as Drugs: Harnessing novel gut bacteria for human health

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The LAL Assay May be Unsuitable for Blood

A recent publication in the Journal of Immunological Methods revisits a common problem with endotoxin detection in biological solutions. Using the Endosafe PTS (Charles River Laboratories) and the Kinetic-QCL LAL Assay (Lonza) with serum samples, they found that neither were accurate for determining endotoxin content due to the sequestration of endotoxin by serum proteins. Specifically, they found that this interaction caused an extended initial phase of the LAL reaction curve to the extent that a linear kinetic response was not established. The authors were not able to devise a heat/dilution protocol to overcome this sequestration. Additionally, they reported endotoxin contamination in several types of commonly used Vacutainer tubes.

BioDtech has also observed the significant issue of endotoxin neutralization by serum proteins and have developed the Endotoxin Sample Preparation (ESP™) kit to overcome this problem. ESP™ combines proper buffer conditions, heat and enzymatic digestion to allow accurate endotoxin detection in plasma and serum samples. The method has been used in human, mice, rat, rabbit and goat samples.

The Limulus Amebocyte Lysate assay may be unsuitable for detecting endotoxin in blood of healthy female subjects

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Alcohol Alters Gut Microbiome

Direct liver cell damage by alcohol has been well established as one of the primary causes of liver disease. Recent work by scientists at UC San Diego demonstrates that, in addition, alcohol can lead to liver disease by causing an imbalance in the gut microflora. Humans produce two natural broad-spectrum antimicrobial proteins called REG3B and REG3G that surveil gut mucosal bacteria and prevent overgrowth. Alcohol causes a down-regulation in the genes encoding for REG3B and REG3G leading to bacterial overgrowth, an increase in microbial translocation, immune activation and further cell damage, including the liver. Mice with REG3B/REG3G knockouts have more severe liver disease while mice with REG3G over-expression avoid liver damage. This work adds to the increasing importance of gut health on a wide range of disease.

Drinking Causes Gut Microbe Imbalance Linked to Liver Disease

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National Microbiome Initiative

Last Friday the White House announced that it was creating a National Microbiome Initiative to bring together scientists to study microbial diversity associated with the human body and our environment. The move will inject $121 million/year into microbiome projects from federal agencies as well as an additional $400 million from other institutions, including the Gates Foundation. In addition to monetary support, the NMI will foster collaborative studies, interdisciplinary research and comparative studies.

Announcing the National Microbiome Initiative

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