Cell-Type-Specific Activation of the Oligoadenylate Synthetase–RNase L Pathway by a Murine Coronavirus

Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a 2′,5′-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway. Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not dependent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phosphodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types—neurons, astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macrophages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degradation was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for induction of an effective RNase L antiviral response.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Fecal Shedding of Zoonotic Food-Borne Pathogens by Wild Rodents in a Major Agricultural Region of the Central California Coast

Recent outbreaks of food-borne illness associated with the consumption of produce have increased concern over wildlife reservoirs of food-borne pathogens. Wild rodents are ubiquitous, and those living close to agricultural farms may pose a food safety risk should they shed zoonotic microorganisms in their feces near or on agricultural commodities. Fecal samples from wild rodents trapped on 13 agricultural farms (9 produce, 3 cow-calf operations, and 1 beef cattle feedlot) in Monterey and San Benito Counties, CA, were screened to determine the prevalence and risk factors for shedding of several food-borne pathogens. Deer mice (Peromyscus maniculatus) were the most abundant rodent species trapped (72.5%). Cryptosporidium species (26.0%) and Giardia species (24.2%) were the predominant isolates from rodent feces, followed by Salmonella enterica serovars (2.9%) and Escherichia coli O157:H7 (0.2%). Rodent trap success was significantly associated with detection of Salmonella in rodent feces, while farm type was associated with fecal shedding of Cryptosporidium and Giardia. Seasonal shedding patterns were evident, with rodents trapped during the spring and summer months being significantly less likely to be shedding Cryptosporidium oocysts than those trapped during autumn. Higher rodent species diversity tended to correlate with lower fecal microbial prevalence, and most spatiotemporal pathogen clusters involved deer mice. Rodents in the study area posed a minimal risk as environmental reservoirs of E. coli O157:H7, but they may play a role in environmental dissemination of Salmonella and protozoa. Rodent control efforts that potentially reduce biodiversity may increase pathogen shedding, possibly through promotion of intraspecific microbial transmission.     

Hibernation patterns of Turkish hamsters: influence of sex and ambient temperature

Turkish hamsters (Mesocricetus brandti) are a model organism for studies of hibernation, yet a detailed account of their torpor characteristics has not been undertaken. This study employed continuous telemetric monitoring of body temperature (T _b) in hibernating male and female Turkish hamsters at ambient temperatures (T _as) of 5 and 13 °C to precisely characterize torpor bout depth, duration, and frequency, as well as rates of entry into and arousal from torpor. Hamsters generated brief intervals of short (<12 h), shallow test bouts (T _b > 20 °C), followed by deep torpor bouts lasting 4–6 days at T _a = 5 °C and 2–3 days at T _a = 13 °C. Females at T _a = 5 °C had longer bouts than males, but maintained higher torpor T _b; there were no sex differences at T _a = 13 °C. Neither body mass loss nor food intake differed between the two T _as. Hamsters entered torpor primarily during the scotophase (subjective night), but timing of arousals was highly variable. Hamsters at both T _as generated short, shallow torpor bouts between deep bouts, suggesting that this species may be capable of both hibernation and daily torpor.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Design and synthesis of tricyclic cores for kinase inhibition

Interest in therapeutic kinase inhibitors continues to grow beyond success in oncology. To date, ATP-mimetic kinase inhibitors have focused primarily on monocyclic and bicyclic heterocyclic cores. We sought to expand on the repertoire of potential cores for kinase inhibition by exploring tricyclic variants of classical bicyclic hinge binding motifs such as pyrrolopyridine and pyrrolopyrazine. Herein we describe the syntheses of eight alternative tricyclic cores as well as in vitro screening results for representative kinases of potential therapeutic interest.     

Impact of participant and interventionist race concordance on weight loss outcomes

Objective:

We have previously shown that racial composition of behavioral intervention groups does not affect achieved weight loss. However, it is unclear if the race of the interventionist affects intervention outcomes. The objective of this analysis is to estimate the impact of race concordance between participant and interventionist on weight change in the initial weight loss phase (phase I) of the Weight Loss Maintenance trial (WLM).

Design and Methods:

A total of 1,685 overweight or obese adults (BMI 25‐45 kg/m²) who were taking medication for hypertension and/or dyslipidemia participated in phase I of the WLM trial. All participants received a 6‐month intensive behavioral intervention in groups of 15‐20 facilitated by a trained interventionist. The main outcome is change in weight at 6 months.

Results:

Participants were on average 55 years of age, 67% female and 44% African American (AA). Three of seventeen interventionists were AA, 14 were non‐AA. Seventy‐three percent of participants shared race concordance with the interventionist. There was a small but statistically significant difference in weight change of participants who were the same race as the interventionist (?5.84 kg, s.e. 0.17) as compared with those who were not race concordant (?5.04 kg, s.e. 0.33), a difference of 0.8 kg, (P = 0.04). The impact of concordance on weight change differed by race (i.e., interaction of race and concordance was significant, P = 0.02).

Conclusions:

In a post hoc analysis of a group‐based behavioral intervention, race concordance for non‐AA participants was associated with slightly greater weight loss. Race concordance was not associated with weight loss for AA participants.