Bacterial-mediated knockdown of tumor resistance to an oncolytic virus enhances therapy.

Oncolytic viruses (OVs) and bacteria share the property of tumor-selective replication following systemic administration. In the case of nonpathogenic bacteria, tumor selectivity relates to their ability to grow extracellularly within tumor stroma and is therefore ideally suited to restricting the production of bacterially produced therapeutic agents to tumors. We have previously shown the ability of the type 1 interferon antagonist B18R to enhance the replication and spread of vesicular stomatitis virus (VSV) by overcoming related cellular innate immunity. In this study, we utilized nonpathogenic bacteria (E. coli) expressing B18R to facilitate tumor-specific production of B18R, resulting in a microenvironment depleted of bioactive antiviral cytokine, thus "preconditioning" the tumor to enhance subsequent tumor destruction by the OV. Both in vitro and in vivo infection by VSVΔ51 was greatly enhanced by B18R produced from E. coli. Moreover, a significant increase in therapeutic efficacy resulted from intravenous (i.v.) injection of bacteria to tumor-bearing mice 5 days prior to i.v. VSVΔ51 administration, as evidenced by a significant reduction in tumor growth and increased survival in mice. Our strategy is the first example where two such diverse microorganisms are rationally combined and demonstrates the feasibility of combining complementary microorganisms to improve therapeutic outcome.

Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.

Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS(+)) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS(+) B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial-host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

Whole-brain traumatic controlled cortical impact to the left frontal lobe: Magnetic resonance image-based texture analysis.

This research assesses the capability of texture analysis (TA) derived from high-resolution (HR) T2-weighted magnetic resonance imaging to identify primary sequelae following 1-5 hours of controlled cortical impact mild or severe traumatic brain injury (TBI) to the left frontal cortex (focal impact) and secondary (diffuse) sequelae in the right frontal cortex, bilateral corpus callosum, and hippocampus in rats. The TA technique comprised first-order (histogram-based) and second-order statistics (including gray-level co-occurrence matrix, gray-level run length matrix, and neighborhood gray-level difference matrix). Edema in the left frontal impact region developed within 1 hour and continued throughout the 5-hour assessments. The TA features from HR images confirmed the focal injury. There was no significant difference among radiomics features between the left and right corpus callosum or hippocampus from 1 to 5 hours following a mild or severe impact. The adjacent corpus callosum region and the distal hippocampus region (s), showed no diffuse injury 1-5 hours after mild or severe TBI. These results suggest that combining HR images with TA may enhance detection of early primary and secondary sequelae following TBI.

Novel recombinant SARS-CoV-2 lineage detected through genomic surveillance in Wales, UK.

Recombination, the process whereby a segment of genetic material from one genome is inserted into another, producing a new chimeric genome, is an important evolutionary mechanism frequently observed in coronaviruses. The risks posed by recombination include the shuffling of advantageous mutations that may increase transmissibility, severity or vaccine escape. We present a genomic and epidemiological description of a new recombinant lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), XR, first identified in Wales. The Pathogen Genomics Unit (Public Health Wales, UK) sequences positive SARS-CoV-2 PCR tests using the ARTIC SARS-CoV-2 sequencing protocol. Recombinants were detected using an in-house pipeline and the epidemiological data analysed in R. Nosocomial cases were defined as those with samples taken after >7 days in hospital. Between February and March 2022, we identified 78 samples with highly similar genomes, comprising a BA.1-like 5' end, a BA.2-like 3' end and a BA.2-like spike protein. This signature is consistent with recombination and was defined as XR by Pangolin (PANGO v1.8). A total of 50 % of cases had a sample collected whilst in hospital and the first three cases were immunocompromised patients. The patient median age was 58 years (range: 4-95 years) and most of the patients were fully vaccinated against SARS-CoV-2 (74 % third dose/booster). Three patients died within 28 days of their sample collection date, one of whom had COVID-19 listed amongst ICD10 (International Classification of Diseases 10) coded causes of death. Our integrated system enabled real-time monitoring of recombinant SARS-CoV-2 for early detection, in order to rapidly risk assess and respond. This work highlights the importance of setting-based surveillance of recombinant SARS-CoV-2, as well as the need to monitor immunocompromised populations through repeat testing and sequencing.

Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen.

PURPOSE: The induction of systemic immune responses against antigenic targets that are over expressed by cancer cells represents a powerful therapeutic strategy to target metastatic cancer. We generated specific antitumor immune responses in a murine model of prostate cancer by oral administration of an attenuated strain of Salmonella typhimurium containing a plasmid coding for murine prostate stem cell antigen. MATERIALS AND METHODS: Trafficking of S. typhimurium SL7207 in the initial 10 hours after gavage feeding was determined using a bacterial lux expressing strain and live bioluminescence imaging. For vaccination trials male C57 BL/6 mice were gavage fed SL7207/murine prostate stem cell antigen expressing plasmid or controls twice at 2-week intervals. One week after the last feeding the mice were challenged subcutaneously with TRAMPC1 murine prostate carcinoma cells. Tumor dynamics and animal survival were recorded. RESULTS: Clearance of bacterial vector from animals was complete 9 hours after feeding. Delivery of vector transformed with a firefly luciferase reporter plasmid resulted in maximal eukaryotic reporter gene expression in splenocytes 48 hours after feeding. Induction of tumor protective immunity was achieved by feeding the mice murine prostate stem cell antigen expressing plasmid bearing bacteria and greater than 50% of immunized mice remained tumor free. No significant toxicity was observed. Induction of T-helper type 1 immune responses was determined by measuring interferon-γ produced by splenocytes from vaccinated mice. When adoptively transferred to naive animals, splenocytes from vaccinated mice prevented tumor growth in 66% of challenged animals. CONCLUSIONS: Endogenous prostate cancer antigen gene delivery using a bacterial vector resulted in breaking immune tolerance to murine prostate stem cell antigen and significant retardation of tumor growth.

Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors.

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. We show for the first time that oral administration of bacteria to mice resulted in their translocation from the gastrointestinal tract (GIT) with subsequent homing to and replication specifically in tumors. The commensal, nonpathogenic Bifidobacterium breve UCC2003 harboring a plasmid expressing lux fed to mice bearing subcutaneous (s.c.) tumors were readily detected specifically in tumors, by live whole-body imaging, at levels similar to i.v. administration. Reporter gene expression was visible for >2 weeks in tumors. Mice remained healthy throughout experiments. Cytokine analyses indicated a significant upregulation of interferon-gamma (IFN-gamma) in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B. breve feeding did not increase systemic levels of other commensal bacteria. The presence of tumor was not necessary for translocation to systemic organs to occur. These findings indicate potential for safe and efficient gene-based treatment and/or detection of tumors via ingestion of nonpathogenic bacteria expressing therapeutic or reporter genes.

Fine-scale foraging habitat selection by two diving central place foragers in the Northeast Atlantic.

Habitat selection and spatial usage are important components of animal behavior influencing fitness and population dynamic. Understanding the animal-habitat relationship is crucial in ecology, particularly in developing strategies for wildlife management and conservation. As this relationship is governed by environmental features and intra- and interspecific interactions, habitat selection of a population may vary locally between its core and edges. This is particularly true for central place foragers such as gray and harbor seals, where, in the Northeast Atlantic, the availability of habitat and prey around colonies vary at local scale. Here, we study how foraging habitat selection may vary locally under the influence of physical habitat features. Using GPS/GSM tags deployed at different gray and harbor seals' colonies, we investigated spatial patterns and foraging habitat selection by comparing trip characteristics and home-range similarities and fitting GAMMs to seal foraging locations and environmental data. To highlight the importance of modeling habitat selection at local scale, we fitted individual models to colonies as well as a global model. The global model suffered from issues of homogenization, while colony models showed that foraging habitat selection differed markedly between regions for both species. Despite being capable of undertaking far-ranging trips, both gray and harbor seals selected their foraging habitat depending on local availability, mainly based on distance from the last haul-out and bathymetry. Distance from shore and tidal current also influenced habitat preferences. Results suggest that local conditions have a strong influence on population spatial ecology, highlighting the relevance of processes occurring at fine geographical scale consistent with management within regional units.

Kymolyzer, a Semi-Autonomous Kymography Tool to Analyze Intracellular Motility.

The movement of intracellular cargo, such as transcripts, proteins, and organelles, is fundamental to cellular function. Neurons, due to their long axons and dendrites, are particularly dependent on proper intracellular trafficking and vulnerable to defects in the movement of intracellular cargo that are noted in neurodegenerative and neurodevelopmental disorders. Accurate quantification of intracellular transport is therefore needed for studying the mechanisms of cargo trafficking, the influence of mutations, and the effects of potentially therapeutic pharmaceuticals. In this article, we introduce an algorithm called "Kymolyzer." The algorithm can quantify intracellular trafficking along a defined path, such as that formed by the aligned microtubules of axons and dendrites. Kymolyzer works as a semi-autonomous kymography software application. It constructs and analyzes kymographs to measure the movement and distribution of fluorescently tagged objects along a user-defined path. The algorithm can be used under a wide variety of experimental conditions and can extract a diverse array of motility parameters describing intracellular movement, including time spent in motion, percentage of objects in motion, percentage of objects that are stationary, and velocities of motile objects. This article serves as a user manual describing the design of Kymolyzer, providing a stepwise protocol for its use and illustrating its functions with common examples. © 2020 Wiley Periodicals LLC Basic Protocol: Kymolyzer, a semi-autonomous kymography tool to analyze intracellular motility.

Spatial variation in a top marine predator's diet at two regionally distinct sites.

In ecological studies it is often assumed that predator foraging strategies and resource use are geographically and seasonally homogeneous, resulting in relatively static trophic relationships. However, certain centrally placed foragers (e.g. seals) often have terrestrial sites for breeding, resting, and moulting that are geographically distinct, and associated with different habitat types. Therefore, accurate estimations of predator diet at relevant spatial and temporal scales are key to understanding energetic requirements, predator-prey interactions and ecosystem structure. We investigate geographic variation in the diet of grey seals (Halichoerus grypus), a relatively abundant and widely distributed central place forager, to provide insights into geographic variation in resource use. Prey composition was identified using scat samples collected over concurrent timescales and a multivariate approach was used to analyse diet from two contrasting habitats. Regional differences in prey assemblages occurred within all years (2011-2013) and all seasons (ANOSIM, all p<0.05), apart from in winter. Telemetry data were used to identify core foraging areas and habitats most likely associated with scat samples collected at the two haul-out sites. Regional differences in the diet appear to reflect regional differences in the physical habitat features, with seals foraging in deeper waters over sandy substrates showing a higher prevalence of pelagic and bentho-pelagic prey species such as blue whiting and sandeels. Conversely, seals foraging in comparatively shallow waters had a greater contribution of demersal and groundfish species such as cephalopods and flatfish in their diet. We suggest that shallower waters enable seals to spend more time foraging along the benthos while remaining within aerobic dive limits, resulting in more benthic species in the diet. In contrast, the diet of seals hauled-out in areas adjacent to deeper waters indicates that either seals engage in a more pelagic foraging strategy, or that seals can spend less time at the benthos, resulting in comparatively more pelagic prey recovered in the diet. The substantial differences in prey assemblages over a small spatial scale (<300 km) demonstrates the importance of using regionally-specific diet information in ecosystem-based models to better account for different trophic interactions.

Development of a luciferase-based reporter system to monitor Bifidobacterium breve UCC2003 persistence in mice.

BACKGROUND: Probiotics such as bifidobacteria have been shown to maintain a healthy intestinal microbial balance and help protect against infections. However, despite these benefits, bifidobacteria still remain poorly understood at the biochemical, physiological and especially the genetic level. Herein we describe, for the first time, the development of a non-invasive luciferase-based reporter system for real-time tracking of Bifidobacterium species in vivo. RESULTS: The reporter vector pLuxMC1 is based on the recently described theta-type plasmid pBC1 from B. catenatulatum 1 and the luxABCDE operon from pPL2lux 2. Derivatives of pLuxMC1, harbouring a bifidobacterial promoter (pLuxMC2) as well as a synthetically derived promoter (pLuxMC3) 3 placed upstream of luxABCDE, were constructed and found to stably replicate in B. breve UCC2003. The subsequent analysis of these strains allowed us to assess the functionality of pLuxMC1 both in vitro and in vivo. CONCLUSION: Our results demonstrate the potential of pLuxMC1 as a real-time, non-invasive reporter system for Bifidobacterium. It has also allowed us, for the first time, to track the colonisation potential and persistence of this probiotic species in real time. An interesting and significant outcome of the study is the identification of the caecum as a niche environment for B. breve UCC2003 within the mouse gastrointestinal tract (GI) tract.

Intracerebroventricular Administration of Amyloid ß-protein Oligomers Selectively Increases Dorsal Hippocampal Dialysate Glutamate Levels in the Awake Rat.

Extensive evidence supports an important role for soluble oligomers of the amyloid ß-protein (Aß) in Alzheimer's Disease pathogenesis. In the present study we combined intracerebroventricular (icv) injections with brain microdialysis technology in the fully conscious rat to assess the effects of icv administered SDS-stable low-n Aß oligomers (principally dimers and trimers) on excitatory and inhibitory amino acid transmission in the ipsilateral dorsal hippocampus. Microdialysis was employed to assess the effect of icv administration of Aß monomers and Aß oligomers on dialysate glutamate, aspartate and GABA levels in the dorsal hippocampus. Administration of Aß oligomers was associated with a +183% increase (p.

Molecular dissection of a bifidobacterial replicon.

The 2.1-kb cryptic plasmid pCIBAO89 from Bifidobacterium asteroides harbors a 1.4-kb segment which is sufficient for its autonomous replication. The segment is divided into two parts, the presumed replication origin, ori89, and the rep gene encoding the putative 41-kDa Rep89 replication initiation protein. This minimal replication region of pCIBAO89 was functionally dissected by transcriptional analyses as well as by DNA-binding studies, and the information obtained was exploited to create a number of Escherichia coli-Bifidobacterium shuttle vectors capable of transforming various bifidobacteria with an efficiency of up to 10(6) transformants/mug DNA.

In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.

In vivo bacterial imaging without engineering; A novel probe-based strategy facilitated by endogenous nitroreductase enzymes.

The feasibility of utilising bacteria as vectors for gene therapy is becoming increasingly recognised. This is primarily due to a number of intrinsic properties of bacteria such as their tumour targeting capabilities, their ability to carry large genetic or protein loads and the availability of well-established genetic engineering tools for a range of common lab strains. However, a number of issues relating to the use of bacteria as vectors for gene therapy need to be addressed in order for the field to progress. Amongst these is the need for the development of non-invasive detection/imaging systems for bacteria within a living host. In vivo optical imaging has advanced preclinical research greatly, and typically involves engineering of bacteria with genetic expression constructs for luminescence (e.g. the lux operon) or fluorescent proteins (GFP etc.). This requirement for genetic modification can be restrictive, where engineering is not experimentally appropriate or technologically feasible (e.g. due to lack of suitable engineering tools). We describe a novel strategy exploiting endogenous bacterial enzymatic activity to specifically activate an exogenously administered fluorescent imaging probe. The red shifted, quenched fluorophore CytoCy5S is reduced to a fluorescent form by bacterial-specific nitroreductase (NTR) enzymes. NTR enzymes are present in a wide range of bacterial genera and absent in mammalian systems, permitting highly specific detection of Gram-negative and Gram-positive bacteria in vivo. In this study, dose-responsive bacterial-specific signals were observed in vitro from all genera examined - E. coli, Salmonella, Listeria, Bifidobacterium and Clostridium difficile. Examination of an NTR-knockout strain validated the enzyme specificity of the probe. In vivo whole-body imaging permitted specific, dose-responsive monitoring of bacteria over time in various infection models, and no toxicity to bacteria or host was observed. This study demonstrates the concept of exploiting innate NTR activity as a reporting strategy for wild-type bacteria using optical imaging, while the concept may also be extended to NTR-specific probes for use with other imaging modalities.

Structure of human brain fructose 1,6-(bis)phosphate aldolase: linking isozyme structure with function.

Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.

Bacterial systems for gene delivery to systemic tumors.

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. Here, we describe procedures for studying this phenomenon in vitro and in vivo for both invasive and noninvasive bacteria suitable for exploitation as tumor-specific therapeutic delivery vehicles, due to their ability to replicate specifically within tumors and/or mediate bacterial-mediated transfer of plasmid DNA to mammalian cells (bactofection).

Habitat-mediated dive behavior in free-ranging grey seals.

Understanding the links between foraging behaviour and habitat use of key species is essential to addressing fundamental questions about trophic interactions and ecosystem functioning. Eight female grey seals (Halichoerus grypus) were equipped with time-depth recorders linked to Fastloc GPS tags following the annual moult in southwest Ireland. Individual dives were coupled with environmental correlates to investigate the habitat use and dive behaviour of free-ranging seals. Dives were characterised as either pelagic, benthic, or shallow (where errors in location and charted water depth made differentiating between pelagic and benthic dives unreliable). Sixty-nine percent of dives occurring in water >50 m were benthic. Pelagic dives were more common at night than during the day. Seals performed more pelagic dives over fine sediments (mud/sand), and more benthic dives when foraging over more three-dimensionally complex rock substrates. We used Markov chain analysis to determine the probability of transiting between dive states. A low probability of repeat pelagic dives suggests that pelagic prey were encountered en route to the seabed. This approach could be applied to make more accurate predictions of habitat use in data-poor areas, and investigate contentious issues such as resource overlap and competition between top predators and fisheries, essential for the effective conservation of these key marine species.

The CAP-Gly of p150: one domain, two diseases, and a function at the end.

In this issue of Neuron, work from Moughamian and Holzbaur (2012) and Lloyd et al. (2012) reveals a role for p150 in initiation of retrograde transport at synaptic terminals. These studies also suggest how mutations of p150's CAP-Gly domain lead to both Perry syndrome and HMN7B disease.

Identification of iron-regulated genes of Bifidobacterium breve UCC2003 as a basis for controlled gene expression.

Iron is an essential growth factor for virtually all organisms. However, iron is not readily available in most environments and microorganisms have evolved specialized mechanisms, such as the use of siderophores and high-affinity transport systems, to acquire iron when confronted with iron-limiting conditions. In general these systems are tightly regulated to prevent iron-induced toxicity and because they are quite costly to the microbe. Because of this tight regulation we chose to explore the response of Bifidobacterium breve UCC2003 to iron limitation. Through microarray and complementation analyses we identified and characterized a presumed ferrous iron uptake system, encoded by bfeUOB, from B. breve UCC2003 and exploited its regulated transcription to develop an inducible expression system for use in bifidobacteria.