Safety and efficacy of the tumor-selective adenovirus enadenotucirev, in combination with nivolumab, in patients with advanced/metastatic epithelial cancer: a phase I clinical trial (SPICE).

BACKGROUND: Novel combination therapies to overcome anti-PD-1 resistance are required. Enadenotucirev, a tumor-selective blood stable adenoviral vector, has demonstrated a manageable safety profile and ability to increase tumor immune-cell infiltration in phase I studies in solid tumors. METHODS: We conducted a phase I multicenter study of intravenous enadenotucirev plus nivolumab in patients with advanced/metastatic epithelial cancer not responding to standard therapy. Co-primary objectives were safety/tolerability and maximum tolerated dose and/or maximum feasible dose (MTD/MFD) of enadenotucirev plus nivolumab. Additional endpoints included response rate, cytokine responses, and anti-tumor immune responses. RESULTS: Overall, 51 heavily pre-treated patients were treated, 45/51 (88%) of whom had colorectal cancer (35/35 patients with information available were microsatellite instability-low/microsatellite stable) and 6/51 (12%) had squamous cell carcinoma of the head and neck. The MTD/MFD of enadenotucirev plus nivolumab was not reached, with the highest dose level tested (1×1012 vp day 1; 6×1012 vp days 3 and 5) shown to be tolerable. Overall, 31/51 (61%) patients experienced a grade 3-4 treatment-emergent adverse event (TEAE), most frequently anemia (12%), infusion-related reaction (8%), hyponatremia (6%), and large intestinal obstruction (6%). Seven (14%) patients experienced serious TEAEs related to enadenotucirev; the only serious TEAE related to enadenotucirev occurring in >1 patient was infusion-related reaction (n=2). Among the 47 patients included in efficacy analyses, median progression-free survival was 1.6 months, objective response rate was 2% (one partial response for 10 months), and 45% of patients achieved stable disease. Median overall survival was 16.0 months; 69% of patients were alive at 12 months. Persistent increases in Th1 and related cytokines (IFNγ, IL-12p70, IL-17A) were seen from ~day 15 in two patients, one of whom had a partial response. Among the 14 patients with matching pre-tumor and post-tumor biopsies, 12 had an increase in intra-tumoral CD8+ T-cell infiltration and 7 had increased markers of CD8 T-cell cytolytic activity. CONCLUSIONS: Intravenously dosed enadenotucirev plus nivolumab demonstrated manageable tolerability, an encouraging overall survival and induced immune cell infiltration and activation in patients with advanced/metastatic epithelial cancer. Studies of next-generation variants of enadenotucirev (T-SIGn vectors) designed to further re-program the tumor microenvironment by expressing immune-enhancer transgenes are ongoing. TRIAL REGISTRATION NUMBER: NCT02636036.

Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Quantitative polymerase chain reaction in the bioanalytical laboratory and technical and scientific considerations for nonclinical and clinical assay characterization, validation and sample analysis.

In this manuscript, the European Bioanalysis Forum reports back on their discussions on practical and scientific considerations related to bioanalytical applications of quantitative polymerase chain reaction. This publication follows an earlier publication in which the European Bioanalysis Forum recommends to consider principles of context of use when defining assay acceptance criteria for method validation criteria and sample analysis.

Patterns of syntrophic interactions in methanogenic conversion of propionate.

Methanogenesis is central to anaerobic digestion processes. The conversion of propionate as a key intermediate for methanogenesis requires syntrophic interactions between bacterial and archaeal partners. In this study, a series of methanogenic enrichments with propionate as the sole substrate were developed to identify microbial populations specifically involved in syntrophic propionate conversion. These rigorously controlled propionate enrichments exhibited functional stability with consistent propionate conversion and methane production; yet, the methanogenic microbial communities experienced substantial temporal dynamics, which has important implications on the understanding of mechanisms involved in microbial community assembly in anaerobic digestion. Syntrophobacter was identified as the most abundant and consistent bacterial partner in syntrophic propionate conversion regardless of the origin of the source culture, the concentration of propionate, or the temporal dynamics of the culture. In contrast, the methanogen partners involved in syntrophic propionate conversion lacked consistency, as the dominant methanogens varied as a function of process condition and temporal dynamics. Methanoculleus populations were specifically enriched as the syntrophic partner at inhibitory levels of propionate, likely due to the ability to function under unfavorable environmental conditions. Syntrophic propionate conversion was carried out exclusively via transformation of propionate into acetate and hydrogen in enrichments established in this study. Microbial populations highly tolerant of elevated propionate, represented by Syntrophobacter and Methanoculleus, are of great significance in understanding methanogenic activities during process perturbations when propionate accumulation is frequently encountered. Key points • Syntrophobacter was the most consistent bacterial partner in propionate metabolism. • Diverse hydrogenotrophic methanogen populations could serve as syntrophic partners. • Methanoculleus emerged as a methanogen partner tolerant of elevated propionate.

Applying context of use to quantitative polymerase chain reaction method validation and analysis: a recommendation from the European Bioanalysis Forum.

Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to a wide array of basic research purposes. COVID-19 infection testing has brought the three-letter PCR abbreviation into the vocabulary of billions of people, making it likely the most well-known laboratory test worldwide. With new modalities and translational medicine gaining importance in pharmaceutical research and development, PCR or more specifically, quantitative PCR (qPCR) is now becoming a standard tool in the (regulated) bioanalytical laboratory, driving the bioanalytical community to define best practices for method development, characterization and validation. In absence of specific guidance from health authorities, qPCR may be vulnerable to scope creep from pharmacokinetics (PK) assay validation as defined in bioanalytical method validation guidance/guidelines. In this manuscript, the European Bioanalysis Forum builds a rationale for applying context of use principles when defining requirements for qPCR assay performance and validation criteria.

Discovery, Optimization, and Biological Characterization of 2,3,6-Trisubstituted Pyridine-Containing M4 Positive Allosteric Modulators.

Herein we describe the discovery and optimization of a new series of 2,3-disubstituted and 2,3,6-trisubstituted muscarinic acetylcholine receptor 4 (M4 ) positive allosteric modulators (PAMs). Iterative libraries enabled rapid exploration of one-dimensional structure-activity relationships (SAR) and identification of potency-enhancing heterocycle and N-alkyl pyrazole substituents. Further optimization led to identification of the potent, receptor-subtype-selective, brain-penetrant tool compound 24 (7-[3-[1-[(1-fluorocyclopentyl)methyl]pyrazol-4-yl]-6-methyl-2-pyridyl]-3-methoxycinnoline). It is efficacious in preclinical assays that are predictive of antipsychotic effects, producing dose-dependent reversal of amphetamine-induced hyperlocomotion in rats and mice, but not in M4 knockout mice. Cholinergic-related adverse effects observed in rats treated with 24 at unbound plasma concentrations more than 3-fold higher than an efficacious dose in the hyperlocomotion assay were fewer and less severe than those observed in rats treated with the nonselective M4 agonist xanomeline, suggesting a receptor-subtype-selective PAM has the potential for an improved safety profile.

The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum.

BACKGROUND: Clostridium thermocellum utilizes a wide variety of free and cellulosomal cellulases and accessory enzymes to hydrolyze polysaccharides present in complex substrates. To date only a few studies have unveiled the details by which the expression of these cellulases are regulated. Recent studies have described the auto regulation of the celC operon and determined that the celC-glyR3-licA gene cluster and nearby manB-celT gene cluster are co-transcribed as polycistronic mRNA. RESULTS: In this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivo transcription of the celC-glyR3-licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription. CONCLUSIONS: Together these results reveal a mechanism by which manB is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, C. thermocellum is able to co-regulate both the celC and manB gene clusters in response to the availability of ß-1,3-polysaccharides in its environment.

Evolutionary Connectionism: Algorithmic Principles Underlying the Evolution of Biological Organisation in Evo-Devo, Evo-Eco and Evolutionary Transitions.

The mechanisms of variation, selection and inheritance, on which evolution by natural selection depends, are not fixed over evolutionary time. Current evolutionary biology is increasingly focussed on understanding how the evolution of developmental organisations modifies the distribution of phenotypic variation, the evolution of ecological relationships modifies the selective environment, and the evolution of reproductive relationships modifies the heritability of the evolutionary unit. The major transitions in evolution, in particular, involve radical changes in developmental, ecological and reproductive organisations that instantiate variation, selection and inheritance at a higher level of biological organisation. However, current evolutionary theory is poorly equipped to describe how these organisations change over evolutionary time and especially how that results in adaptive complexes at successive scales of organisation (the key problem is that evolution is self-referential, i.e. the products of evolution change the parameters of the evolutionary process). Here we first reinterpret the central open questions in these domains from a perspective that emphasises the common underlying themes. We then synthesise the findings from a developing body of work that is building a new theoretical approach to these questions by converting well-understood theory and results from models of cognitive learning. Specifically, connectionist models of memory and learning demonstrate how simple incremental mechanisms, adjusting the relationships between individually-simple components, can produce organisations that exhibit complex system-level behaviours and improve the adaptive capabilities of the system. We use the term "evolutionary connectionism" to recognise that, by functionally equivalent processes, natural selection acting on the relationships within and between evolutionary entities can result in organisations that produce complex system-level behaviours in evolutionary systems and modify the adaptive capabilities of natural selection over time. We review the evidence supporting the functional equivalences between the domains of learning and of evolution, and discuss the potential for this to resolve conceptual problems in our understanding of the evolution of developmental, ecological and reproductive organisations and, in particular, the major evolutionary transitions.

The Low Noise Limit in Gene Expression.

Protein noise measurements are increasingly used to elucidate biophysical parameters. Unfortunately noise analyses are often at odds with directly measured parameters. Here we show that these inconsistencies arise from two problematic analytical choices: (i) the assumption that protein translation rate is invariant for different proteins of different abundances, which has inadvertently led to (ii) the assumption that a large constitutive extrinsic noise sets the low noise limit in gene expression. While growing evidence suggests that transcriptional bursting may set the low noise limit, variability in translational bursting has been largely ignored. We show that genome-wide systematic variation in translational efficiency can-and in the case of E. coli does-control the low noise limit in gene expression. Therefore constitutive extrinsic noise is small and only plays a role in the absence of a systematic variation in translational efficiency. These results show the existence of two distinct expression noise patterns: (1) a global noise floor uniformly imposed on all genes by expression bursting; and (2) high noise distributed to only a select group of genes.

Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain.

BACKGROUND: The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. RESULTS: In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell defense mechanisms. CONCLUSION: These results suggest the mechanisms of tolerance for the Populus hydrolysate-tolerant mutant strain of C. thermocellum are based on increased cellular efficiency caused apparently by downregulation of non-critical genes and increasing the expression of genes in energy production and conversion rather than tolerance to specific hydrolysate components. The wild type, conversely, responds to hydrolysate media by down-regulating growth genes and up-regulating stress response genes.

Industrial robustness: understanding the mechanism of tolerance for the Populus hydrolysate-tolerant mutant strain of Clostridium thermocellum.

BACKGROUND: An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v). CONCLUSION/SIGNIFICANCE: The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.

Kinetic modeling of batch fermentation for Populus hydrolysate tolerant mutant and wild type strains of Clostridium thermocellum.

The extent of inhibition of two strains of Clostridium thermocellum by a Populus hydrolysate was investigated. A Monod-based model of wild type (WT) and Populus hydrolysate tolerant mutant (PM) strains of the cellulolytic bacterium C. thermocellum was developed to quantify growth kinetics in standard media and the extent of inhibition to a Populus hydrolysate. The PM was characterized by a higher growth rate (µmax=1.223 vs. 0.571 h(-1)) and less inhibition (KI,gen=0.991 vs. 0.757) in 10% v/v Populus hydrolysate compared to the WT. In 17.5% v/v Populus hydrolysate inhibition of PM increased slightly (KI,gen=0.888), whereas the WT was strongly inhibited and did not grow in a reproducible manner. Of the individual inhibitors tested, 4-hydroxybenzoic acid was the most inhibitory, followed by galacturonic acid. The PM did not have a greater ability to detoxify the hydrolysate than the WT.

Transcriptional burst frequency and burst size are equally modulated across the human genome.

Gene expression occurs either as an episodic process, characterized by pulsatile bursts, or as a constitutive process, characterized by a Poisson-like accumulation of gene products. It is not clear which mode of gene expression (constitutive versus bursty) predominates across a genome or how transcriptional dynamics are influenced by genomic position and promoter sequence. Here, we use time-lapse fluorescence microscopy to analyze 8,000 individual human genomic loci and find that at virtually all loci, episodic bursting--as opposed to constitutive expression--is the predominant mode of expression. Quantitative analysis of the expression dynamics at these 8,000 loci indicates that both the frequency and size of the transcriptional bursts varies equally across the human genome, independent of promoter sequence. Strikingly, weaker expression loci modulate burst frequency to increase activity, whereas stronger expression loci modulate burst size to increase activity. Transcriptional activators such as trichostatin A (TSA) and tumor necrosis factor α (TNF) only modulate burst size and frequency along a constrained trend line governed by the promoter. In summary, transcriptional bursting dominates across the human genome, both burst frequency and burst size vary by chromosomal location, and transcriptional activators alter burst frequency and burst size, depending on the expression level of the locus.

Two frizzled planar cell polarity signals in the Drosophila wing are differentially organized by the Fat/Dachsous pathway.

The regular array of distally pointing hairs on the mature Drosophila wing is evidence for the fine control of Planar Cell Polarity (PCP) during wing development. Normal wing PCP requires both the Frizzled (Fz) PCP pathway and the Fat/Dachsous (Ft/Ds) pathway, although the functional relationship between these pathways remains under debate. There is strong evidence that the Fz PCP pathway signals twice during wing development, and we have previously presented a Bidirectional-Biphasic Fz PCP signaling model which proposes that the Early and Late Fz PCP signals are in different directions and employ different isoforms of the Prickle protein. The goal of this study was to investigate the role of the Ft/Ds pathway in the context of our Fz PCP signaling model. Our results allow us to draw the following conclusions: (1) The Early Fz PCP signals are in opposing directions in the anterior and posterior wing and converge precisely at the site of the L3 wing vein. (2) Increased or decreased expression of Ft/Ds pathway genes can alter the direction of the Early Fz PCP signal without affecting the Late Fz PCP signal. (3) Lowfat, a Ft/Ds pathway regulator, is required for the normal orientation of the Early Fz PCP signal but not the Late Fz PCP signal. (4) At the time of the Early Fz PCP signal there are symmetric gradients of dachsous (ds) expression centered on the L3 wing vein, suggesting Ds activity gradients may orient the Fz signal. (5) Localized knockdown or over-expression of Ft/Ds pathway genes shows that boundaries/gradients of Ft/Ds pathway gene expression can redirect the Early Fz PCP signal specifically. (6) Altering the timing of ds knockdown during wing development can separate the role of the Ft/Ds pathway in wing morphogenesis from its role in Early Fz PCP signaling.

Legal responsibility and accountability.

Shifting boundaries in healthcare roles have led to anxiety among some nurses about their legal responsibilities and accountabilities. This is partly because of a lack of education about legal principles that underpin healthcare delivery. This article explains the law in terms of standards of care, duty of care, vicarious liability and indemnity insurance.

Transcriptional bursting from the HIV-1 promoter is a significant source of stochastic noise in HIV-1 gene expression.

Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1's long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2-10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.

"My hip hurts": a collaboration of clinicians in the treatment of adhesive capsulitis to optimize patient care.

In order to achieve a positive outcome for individuals who are diagnosed with a "frozen joint" or adhesive capsulitits, a collaborative relationship among clinicians provides support and communication. The relationship should include the physical therapist, physician, and the pharmacist. Capsulitis, or capsular restriction, is often treated with anti-inflammatory agents, localized steroidal injections, directed physical therapy, or orthopedic visual inspection. The professional judgments of coordinating healthcare practitioners provide for assessments and plans that result in favorable outcomes with optimized recovery time.